hadn‘t-“n; _ ‘ I . r . WEB" [333752-3131 :iam [Infivcrxfigr This is to certify that the thesis entitled EVALUATION OF TRANSFER FACTOR ACTIVITY AND DELAYED-TYPE HYPERSENSITIVITY TO BRUCELLA WITH THE MOUSE FOOTPAD ASSAY ‘ ‘ presented by Virgil Duane Troyer has been accepted towards fulfillment of the requirements for M-S- degreein MICY‘ObIOIOQY & Public Health WW‘H CW" W Major professor 3 Date ‘3 " 1‘7 CI 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to Book drop to remove this checkout from your record. ‘Jl.lla‘! ll 1‘0} .. O l ‘ i EVALUATION OF TRANSFER FACTOR ACTIVITY AND DELAYED-TYPE HYPERSENSITIVITY TO BRUCELLA WITH THE MOUSE FOOTPAD ASSAY By Virgil Duane Troyer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1979 ABSTRACT EVALUATION OF TRANSFER FACTOR ACTIVITY AND DELAYED-TYPE HYPERSENSITIVITY TO BRUCELLA WITH THE MOUSE FOOTPAD ASSAY By Virgil Duane Troyer A footpad assay was developed to study delayed-type hypersen- sitivity to Brucella in mice treated with bovine and murine dialyzable transfer factor preparations. A nucleOprotein antigen, Brucellergen, elicited cutaneous delayed-type hypersensitivity reactions in infected rabbits and cattle, and positive footpad responses evident at 24 and 48 hours in infected mice. Histological studies of the footpads showed mixed PMN-mononuclear infiltrates at 24 hours and predominantly mononuclear infiltrates at 48 hours. Footpad testing with the antigen dilution used in these studies was shown to sensitize a proportion of mice as demonstrated by swelling in subsequent footpad tests. There was evidence of transfer activity in one transfer factor preparation from Brucella infected mice. Transfer activity was not consistently demonstrated in other murine transfer factor or whole cell preparations or in bovine transfer factor preparations. Possible explanations for these results are discussed. TABLE OF CONTENTS Page LIST OF TABLES ........................ v INTRODUCTION‘ ......................... l LITERATURE REVIEW ...................... 2 Delayed-Type Hypersensitivity ............. 2 Historical Background ............... 2 Induction of DTH .................. 3 In Vivo Expressions of DTH .. ............ 5 I3 Vitro Assays of DTH ............... l7 DElayed Hypersensitivity in Brucellosis ...... l9 Delayed Hypersensitivity in the Mouse ....... 22 Transfer Factor .................... 25 Historical Background ............... 25 Physical Properties of Transfer Factor ....... 26 Preparation of Transfer Factor ........... 28 Manifestations of Transfer Factor ......... 33 Animal Models ................... 44 MATERIALS AND METHODS .................... 49 Animals ........................ 49 Microorganisms ..................... 50 Immunization of Rabbits and Mice ............ 50 Tests for Delayed-Type Hypersensitivity ........ Sl Brucellergen Preparation .............. 5l Brucellergen Standardization ............ 52 DTH Tests in Cattle ................ 53 DTH Tests in Mice ................. 53 Preparation of Transfer Factor ............. 54 Bovine Peripheral Blood Transfer Factor ...... 54 Bovine Lymph Node Transfer Factor ......... 56 Murine Transfer Factor ............... 57 Assay of Transfer Factor Activity ........... 58 Histological Studies .................. 59 Preparation of Chemical Reagents ............ 59 Page RESULTS ............................ 6l Brucellergen Standardization in Rabbits ......... 6l DTH Response in Cattle to Brucellergen ......... 63 Footpad Reaction in Mice to Brucellergen ........ 63 Sensitization of Control Mice by Repeated Footpad Tests . 66 Histology of the Footpad Reaction ............ 68 Transfer Factor Activity in Mice ............ 68 DISCUSSION .......................... 76 BIBLIOGRAPHY ......................... 84 iv LIST OF TABLES Table Page l. Delayed Hypersensitivity Responses in Rabbits to Brucellergen ...................... 52 2. Delayed Hypersensitivity Responses in Cattle to Brucellergen ...................... 53 3. Delayed Hypersensitivity Response in Mice to Three Di- lutions of Brucellergen ................ 64 4. Delayed Hypersensitivity Response to Brucellergen in Strain l9 Infected Mice ................ 65 5. Delayed Hypersensitivity Response to Brucellergen in Strain 2308 Infected Mice ............... 55 6. Sensitization of Control Mice by Repeated Footpad Tests ......................... 57 7. Bovine Peripheral Blood Transfer Factor Activity in Mice .......................... 7o 8. Bovine Lymph Node Transfer Factor Activity in Mice . . . 71 9. Murine Transfer Factor Activity in Mice ........ 73 INTRODUCTION Transfer factor is a dialyzable moiety of lymphoid cell extracts capable of passively transferring delayed-type hypersensitivity from a sensitized individual to a non-sensitized individual. There is also evidence that immunity or resistance to diseases involving the cell mediated immune system may be transferred by transfer factor (95). Originally, descriptions of transfer factor activity were limited to human subjects, but recently a number of animal models have been deveIOped, including some involving cross-species transfer. Of par- ticular interest in this thesis are those involving the bovine and the mouse (67, 87, I30). The study outlined here was undertaken to develOp an assay for the demonstration of delayed-type hypersensitivity to Brucella in mice and subsequently to use that assay in a mouse model to study transfer of delayed hypersensitivity from sensitized bovine and murine animals using dialyzable transfer factor. This model would not only serve as a research tool to study theoretical aSpects of transfer factor, but also serve as a convenient jn_yixg_assay of the biological activity of transfer factor preparations before their use in other species or man. LITERATURE REVIEW Delayed Type Hypersensitivity Historical Background The first description of delayed-type hypersensitivity (DTH) is usually credited to Koch, although the phenomenon may have been first described by Jenner nearly a century earlier in his "reaction of immunity." Koch observed, in l890, that viable tubercle bacilli or culture filtrates (Old Tuberculin) injected subcutaneously evoked an inflammatory reaction in previously infected guinea pigs that was more intense than the reaction observed in uninfected animals. Man- toux described the reaction elicited by intradermal injection of tuberculin in l9lO. Thereafter the tuberculin reaction became a standard test in the detection of tuberculosis. (149) Several early studies indicated that DTH differed from other forms of hypersensitivity. Zinsser reported in l925 (l63) that DTH or anaphylactic reactivity could be selectively induced by different sensitization procedures, and that DTH was not passively transferred by serum antibodies. This work was supported by the histological studies of Dienes and Mallory (37) who demonstrated the slow develop- ment of a predominantly mononuclear infiltrate in the tuberculin re- action as opposed to the rapid development of a polymorphonuclear in- filtrate in anaphylactic skin reactions. The role of sensitized lymphoid cells in DTH was established by Landsteiner and Chase 3 (24, 9l) in l942, in a set of investigations that finally set DTH apart from serum-mediated immunologic reactions. They demonstrated the passive transfer of contact sensitivity and tuberculin sensitiv- ity in guinea pigs by peritoneal exudate cells, but not by serum. Subsequent investigations demonstrated that DTH could be in- duced to a variety of bacterial and non-bacterial antigens. Ig_ yjyg_manifestations of DTH other than the classic cutaneous reaction have also been demonstrated, including contact sensitivity, allo- graft rejection, and systemic reactions; and jg_yjtrg.assays that correlate to DTH have been developed (35, 43, I49). Induction of DTH DTH may be induced by active immunization or result from an infectious disease process. In either case DTH is induced by factors or processes that differ from those that normally induce the humoral immune response. DTH is an immune response in a number of infectious diseases, often of a chronic nature involving obligate or facultative intra- cellular organisms (lO7). The classic example is tuberculosis, though DTH has been detected in other bacterial, fungal, viral, and parasitic diseases (36, 43, l40, l59). A representative sample of these diseases and the corresponding skin test antigens is listed below: Disease Skin Test Antigen Tuberculosis PPD Brucellosis Brucellergen Lymphogranuloma Venereum (LGV) Frei test antigen Candidiasis Oidiomycin Coccidioidomycosis Coccidioidin Histoplasmosis Histoplasmin Disease Skin Test Antigen Blastomycosis Blastomysin Mumps Mumps antigen Leishmaniasis Montenegro test antigen The active induction of classical DTH is dependent on a number of factors, including the chemical nature of the antigen, vehicle for injection, route of injection, and the dose of antigen (43). Zinsser in his early work separating DTH from anaphylaxis (l63) demonstrated that injection of viable tubercle bacilli but not of cell-free ex- tracts would induce DTH. Others demonstrated that dead bacilli could also induce DTH, though the degree of sensitivity was less than with viable cells (l49). Most antigens inducing DTH are proteins or con- tain protein, while antigens composed of polysaccharide are ineffec- tual (43). Antigens of low potency, that are slowly absorbed tend to induce DTH preferentially (132). The reasons why certain antigen- types evoke DTH responses while others evoke humoral responses is still not fully understood (132). Though soluble proteins alone will not induce DTH, injection of these and other substances in certain adjuvants will evoke DTH reactivity. Dienes discovered in l926 that systemic DTH to egg al- bumin and horse serum could be induced by injecting the protein dir- ectly into a tuberculous focus (37). This and subsequent observa- tions that injection of antigens in liquid paraffin or vaseline would induce greater sensitivity, led to the development of complete Freund's adjuvant, a water-in-oil emulsion containing tubercle ba- cilli (l49). The route of injection and the dose of antigen have also been shown to affect induction of DTH. In general the intradermal injection 5 of antigen is more effective than injection by other routes (I32, 43, 32). Smaller doses of antigen favor induction of DTH, while larger doses may stimulate antibody production or even induce tolerance (43). Dvorak gt_al, (46), has repeatedly shown that cutaneous basophil hypersensitivity (CBH), formerly called "Jones-Mote" reactivity, may be induced by protein antigens injected with saline or mycobacterium- free adjuvants. Injection of these antigens in complete Freund's adjuvant induces classical DTH. Other differences in the induction and time course of CBH are discussed below. Contact sensitivity to simple chemical compounds, is usually induced by painting the sensitizing agent on the bare skin of a sub- ject. Contact sensitivity has also been induced however by intra- dermal injection without adjuvant, and by injection with Freund's adjuvant (l49). Ig_Vivo Expressions of DTH Cutaneous Reactivity Macroscopic Systemic DTH reactivity can be demonstrated locally by the in- tradermal injection of the specific antigen. Macroscopically the reaction is undetectable for several hours. Erythema and induration then appear at 6-12 hours, reaching a maximum at 24-48 hours (43, 47). Reactions in humans usually reach maximum intensity at 48 hours, whereas the reaction in some animals, such as guinea pigs (45, 46, l49) and mice (3l, 52), have been maximal at 24 hours. The reactions subside slowly, depending on the intensity at the peak, and severe reactions may show a central area of necrosis (43, l32). The delayed onset, late peak, and palpable induration are characteristics of the reaction that set DTH apart from antibody mediated hypersensitiv- ity reactions. Microscopic The microscopic picture of the DTH reaction is even more char- acteristic and usefull in distinguishing humoral from cell mediated DTH reactions. Dienes and Mallory (37) demonstrated early on that the DTH reaction was characterized by a predominantly mononuclear in- filtrate, whereas the humoral (anaphylactic) reactions were composed primarily of PMN's. This basic definition has held to today, though there has been considerable controversy regarding the relative pro- portions of mononuclear cell types, the presence and role of non- mononuclear cell-types, and the factors involved in the evolution of the local cutaneous DTH reaction. Some of this confusion is due to conclusions drawn from biopsies taken at different times after antigen injection and experiments done on different species. The guinea pig and man, for example, display similar macroscopic DTH reactions, although the histology of the reactions differs markedly (l49). Var- iations in preparation and staining of tissue samples has also yielded significantly different histological pictures (l49), in one case re- vealing a cell type not previously evident (45, 46). TWO investigators, Turk and Dvorak, have conducted extensive studies of the histology of the DTH reactions in guinea pigs and humans. Their descriptions and conclusions will be primarily re- viewed below. Some of the problems outlined above will be evident. Prior to Turk's studies in l965-l967 attempts were made to distinguish lymphocyte and monocyte cell types on morphological characteristics. It was felt however, that these distinctions were artificial and influenced by variations in the tissue section prepar- ation as well as variations in the tissue itself (l49). Turk and Diengdoh (150) developed a staining method to distinguish macro- phages from lymphocytes by the number of cytoplasmic granules stain- ing for acid phosphatase. They stained frozen sections with an acid phosphatase-methyl green pyronin stain. Macrophages and PMN's both contained numerous stained granules, but could be distinguished from each other by nuclear shape. Lymphocytes showed few or no acid phos- phatase staining granules. Using this staining technique, Turk §t_gl, (150) examined the histology of skin reactions in guinea pigs at 4, 8, 24, and 48 hour intervals after intradermal injection of a variety of antigens. They found that granulocytes predominated in the reaction at 4 hours, com- prising 60-70% of the perivascular infiltrate. After this time they formed 25% or less of the cells. At the peak of the reaction, 24 hours, macrophages formed 50-60% of the mononuclear cells in the in- filtrate, but by 48 hours they formed only 30% of the mononuclear cells, the remaining 70% being lymphocytes. A similar study was conducted on humans by Turk's group (ISI). Antigen was injected at two skin sites, which were biopsied at 24 and 48 hours respectively. The dilution of antigen, l:l0,000 Old Tubercu- lin, was chosen to yield minimal non-specific reactions and epidermal damage. Granulocytes were not seen to any extent. Turk attributed this to differences between guinea pigs and humans, but the times of biopsy and the use of a non-irritating dilution of antigen may have contributed to this observation. Acid-phosphatase staining cells, Turk's definition of macrophages, comprised only lO-l3% of the mono- nuclear perivascular infiltrate at both 24 and 48 hours. It was ob- served, as reported by others, that capillaries and venules were filled with mononuclear cells (l49). Dvorak and his colleagues developed a tissue fixation method designed primarily to preserve basophilic granulocytes (45, 46). Tissues were post-fixed in osmium tetroxide and lum, Epon-embedded sections were stained with Giemsa stain for light microscopic evalua- tion. Using this technique, Dvorak was able to demonstrate the presence of basophils, not detectable by normal tissue preparation methods, in certain types of DTH reactions. Ovorak's group used this tissue preparation method in extensive studies of classic DTH in guinea pigs and human subjects, partially to evaluate the presence of basophils in these reaction-types. Dis- tinctions between macrophage and lymphocyte mononuclear cells were made on a morphological basis, though frequently no distinction was made. In the guinea pig (45, 46), they found a perivascular infil- trate of mononuclear cells beginning at 6 hours and continuing in intensity to 24 hours. PMN's comprised only a minority of cells during any time in the reaction, though this was found to be depen- dent on a low dose of skin-test antigen. Basophils were found in significant numbers in these classic DTH reactions, particularly if the interval between immunization and skin test was relatively short. In one study (46), skin tests at l week post-immunization 9 contained l3-25% ba50phils, whereas skin tests at 6 weeks contained less than 7%. Dvorak §t_al,(47) conducted an extensive study of CBH, contact sensitivity, and classic DTH reactions in human subjects, using light and electron microscopy and immunoflourescent techniques. In the studies of classic DTH to several microbial antigens, biopsies were taken at 48 hours after antigen injection. As before, an in- tense perivascular infiltrate of mononuclear cells was observed, com- posed mostly of small or activated lymphocytes with a smaller pro- portion of macrophages. Basophils were observed in 6l% of the reactions, but in relatively low numbers in all but l9% of the re- actions. Neutrophils and eosinophils were noted,but in low numbers in most reactions. Mast cells at the reaction site exhibited vari- ably reduced numbers of cytoplasmic granules. This “degranulation” was much less extensive than that seen in antibody-mediated allergic skin reactions, but was a constant feature of the test reactions. Significantly new findings were also reported from this study (47) regarding the presence and consequences of enhanced vascular permeability in DTH reactions. These findings were described in detail in a serial biopsy study of contact dermatitis, though similar phenomena were seen in the classic DTH reactions biopsied at 48 hours. The microvasculature of the reaction sites exhibited compaction and congestion with tight intravascular packing of erythrocytes. Elec- tron microscopy often revealed large gaps between endothelial cells of the affected venules, through which plasma and less frequently erythrocytes were extravasated into the perivascular space. Inter- vascular deposition of fibrin, detected by flourescent antibody, ID was evident in nearly all reactions at 24 hours. It was suggested that vascular leakage of fibrinogen and other clotting factors led to this deposition of fibrin. It should be noted that immunoglobulin and complement (C3) deposits were not detected by immunoflourescent techniques in these reactions. From the evidence presented in these studies, Dvorak has pro- posed that mast cells and/or basophils are stimulated by lymphokines released at the test site from antigen-stimulated lymphocytes to re- lease a variety of vasoactive agents which mediate the observed vas- cular permeability changes (47). The possible role of mast cell pro- ducts in DTH skin reactions in the mouse has also been suggested by Gershon gt_al, (50). Few histological studies have been done on DTH reactions after 48 hours. Several studies of the tuberculin reaction in animals have shown typical tuberculous foci containing epithelioid cells, giant cells, and small lymphocytes. Some of these cells may arise as a consequence of tissue damaged during the immunological reaction (l49). Effector Cells In addition to the histological studies outlined above, which demonstrate the cell-types present at the reaction site, considerable research has been done to determine the origin and role of the "effec- tor" cells in the cutaneous DTH reaction. In a series of classic in- vestigations by McCluskey gt_al:(27, lll), it was demonstrated that the vast majority of infiltrating cells in the skin reaction site lacked specificity for the antigen used to elicit the reaction. When lymph node cells from specifically immunized donors, labeled with ll 3H-thymidine, were used to passively transfer DTH to non-reactive guinea pigs, less than 8% of the mononuclear cells at the test site were labeled. In other experiments, separate donors were immunized to non-cross reacting antigens and the donor cells of only one spec- 3H-thymidine ificity were labeled. Non-sensitized animals received labeled cells for one specificity and unlabeled cells for the other specificity. Subsequent skin tests by both antigens at separate sites revealed equivalent accumulation of the labeled cells at both test sites. When unsensitized recipients were repeatedly injected with 3H-thymidine before receiving unlabeled donor cells, the great major- ity of cells at the test site were labeled. These studies suggest that the DTH reaction is initiated by the arrival of a small number of specifically sensitized cells at the antigen injection site, but that these antigen specific cells are not in themselves the cells responsible for the macroscopic induration or the microscopic cellular infiltration. Using a modification of McCluskey's model, Lubaroff and Waksman demonstrated that a large percentage of the non-specific infiltrating cells were blood-borne bone-marrow derived cells (lOS, l06). They passively transferred DTH to unsensitized, thymectomized, heavily irradiated rats. Maximal reactions were only achieved when bone marrow cells, from normal or sensitized rats, were administered to the recipients prior to the lymph node cells from the sensitized donor (l05). Using an immunoflourescent technique to identify bone marrow and lymph node cells from genetically different donors, approximately 75% of the cells at the test site were shown to be from the bone marrow donor and approximately 25% from the sensitized 12 lymph node cell donor (106). A similar participation by non-sensi- tized bone marrow cells in the DTH response was reported by Youdim gt_al, (160) in immune irradiated mice that responded to antigen in- jection only after administration of normal bone marrow cells. Irra- diation had been shown to abrogate DTH reactivity, but not destroy the specific antigen-reactive cells. The role of a relatively few antigen-reactive lymphocytes in the DTH response is also indicated by results reported by David (35) and Bloom and Bennett (12) using the jg_yjtrg_macrophage migration inhibition assay. David initially demonstrated that as few as 2.5% peritoneal exudate cells from sensitized guinea pigs mediated the migration inhibition. Bloom and Bennett subsequently separated lymphocytes and macrophages from PEC suspensions and showed that as few as 0.6% purified sensitive lymphocytes would cause significant ‘ antigen-dependent macrophage migration inhibition, but that purified macrophages from sensitive guinea pigs exhibited no inhibition in the presence of antigen. The role of thymus-derived lymphocytes (T-cells) as effector cells in DTH responses is indicated by the failure of athymic mice to develop or display DTH reactivity (122, 161) and by the observed cellular proliferation in T-cell rich paracortical areas of lymph nodes following sensitization (149). In studies using local passive transfer of DTH with PEC in guinea pigs, Turk and Polak demonstrated initially that lymphocytes, but not macrophages were responsible for the transfer (152). Jaffer gt_al, (61) used the same model to demonstrate that T-cells and not B-cells were primarily responsible for the DTH transfer. They used a lymphocyte population containing 13 less than 5% B-cells, prepared by passing PEC through an anti-immuno- globulin column. Successful transfer was achieved by these T-cells as evaluated both macro- and microscopically. Attempts to transfer DTH with the column-retained B-cells, released by dextranase diges- tion of the support, could not be evaluated due to non-specific in- flammation induced by residues in the effluent. Youdim gt_al, demonstrated in a series of experiments in mice that treatment of sensitized lymphoid cells with anti-theta serum and complement, a treatment that lyses thymus-derived cells, abolished their capacity to transfer DTH to non-immune mice (160, 161). Immune thymocytes alone did not transfer DTH in these studies. Cutaneous Basophil Hypersensitivity Raffel and Newel (125) conducted investigations in 1958 on a delayed-type reaction induced by injecting antigen-antibody complexes in saline or mycobacterium-free adjuvants. They suggested that the sensitivity induced was not only different than Arthus and immediate- type hypersensitivity, but significantly different than classic DTH because of its early appearance (5-7 days) after immunization and disappearance at a time (16-20 days) when DTH reactions are reaching their maximum reactivity and antibody production is beginning. They suggested the term "Jones-Mote“ reaction, in reference to a report 20 years earlier by Jones and Mote (70) of a similar sensitivity in humans induced by repeated intradermal injection of rabbit serum proteins without adjuvant. Controversy continued regarding this distinction (148) until Dvorak §t_al, demonstrated that these re- actions could indeed be distinguished by extensive infiltration of l4 basophilic granulocytes in addition to mononuclear cells at the test site (45, 46). As noted above, Dvorak's detection of basophils was accomplished by a fixation technique that preserved the prev- iously undetected basophil. He suggested the term "cutaneous baso- phil hypersensitivity" (CBH) to denote the reaction. CBH was also fbund to be induced by injection of soluble proteins without myco- bacterial adjuvants, to display relatively non-indurated reactions with significantly less change in vascular permeability and fibrin deposition then seen with classic DTH, and to be resistant to car- rageenan injection, a treatment that markedly diminishes classic tuberculin DTH (44, 45, 46, 128). CBH resembles DTH in that the cutaneous reaction is delayed, reaching maximum intensity at 24-48 hours. CBH is also lymphocyte mediated, being sensitive to treatment by anti-lymphocyte serum (128) and being passively transferred by viable sensitized lymphocytes (46). The role of the basophil in the CBH reaction has not been es- tablished. Basophils do not bind or ingest specific antigen at the test site (46). Overt basophil degranulation by explosive release of granules has not been detected, though careful light- and electron- microscopic studies have shown evidence for a slow "piecemeal" re- lease of granules from basophils at the reaction site over a course of several days (42). Contact Skin Sensitivity Contact skin sensitivity has been induced in guinea pigs and man to a variety of simple chemical compounds, the most commonly 15 used being picryl chloride, dinitrochlorobenzene (DNCB) and dinitro- fluorobenzene (DNFB) (149). Contact skin sensitivity is responsible for a variety of skin allergies in man, such as to poison ivy, cos- metics, drugs, and synthetic chemicals (36). There is evidence that these simple compounds may act as haptens and attach to skin proteins, which act as carriers (l49). Sensitization can be accomplished by percutaneous application, intradermal injection, or injection by other routes if complete Freund's adjuvant is employed. The reaction, elicited by application of the sensitizing com- pound at a skin test site distant from that used for sensitization, is characterized by a delayed onset of erythema, induration, and occasional vesiculation. Maximum intensity is displayed at 24 hours in the guinea pig and 48-72 hours or longer in man. Microscopically, the cellular infiltrate is much like that seen with intradermally injected antigen, with the exception that the mononuclear cell in- filtrate extends into the epidermis (47, 149). Systemic Reactions Cutaneous DTH reactions, though elicited locally, represent a sys- temic hypersensitive state. When specific antigen is administered sys- temically, generalized hypersensitive reactions can be observed. Koch first observed this in 1890, when he saw a rise in temperature in tuber- culous human subjects after subcutaneous injection of Old Tuberculin (149) . This febrile reaction has been demonstrated in humans and animals by other investigators (3 1, 43, 149) and may be accompanied by other symptoms such as , prostration, malaise, irregular breathing, and lethargy. It has been 16 demonstrated that these reactions are neither examples of endotoxin fever nor anaphylactic shock (149). In highly sensitive subjects, inhalation of aerosols of the antigen may cause systemic reactions, often resembling an acute attack by the disease agent itself (113). Biological Significance of DTH DTH reactivity correlates closely to a number of cell-mediated immune phenomena. However, the role of the DTH reaction in these phenomena or the distinction between DTH and the phenomena has been the subject of much debate. For many years the allergic reactivity of DTH was thought to be responsible for cell mediated immunity (CMI) to infection. The role of DTH in CMI is supported by the emergence of DTH reactivity at a time when infecting microorganisms are being eliminated (10, 107, 160). Transfer of CMI and DTH are both achieved using lympho- cytes, and immunization techniques that induce antibody formation but not DTH confer no CMI protection (43, 149). Accumulating evidence however, indicates that DTH and CMI do not always correlate. Sensitive animals may be desensitized by re- peated injection of antigen and found to retain CMI to infection, though DTH can no longer be demonstrated. Immunization techniques that induce selectively DTH or CMI but not both, have offered strong evidence that the two mechanisms are indeed separate (59, 149, 162). It has been demonstrated that allograft rejection is mediated by an immunologically spedific reaction to surface antigens on cells <3f the grafted tissue. That DTH is involved in allograft rejection 17 is indicated by the extensive mononuclear infiltration into the grafted tissue (43, 149). Accelerated graft rejection can be pas- sively transferred by viable lymphoid cells (43, 149) or transfer factor (95), but not serum. Typical cutaneous DTH reactions can also be elicited by the intradermal injection of cells to an animal that previously received a graft from the cell donor (l49). Antibody is also induced by grafted tissue and may cause com- plement-dependent cytotoxicity for suspensions of target cells, but the role of antibody in direct graft rejection is thought to be minor. In fact specific antibody may in some instances enhance graft survival (43). The evidence for participation of DTH in auto-immune diseases is like that for allograft rejection: prominant mononuclear in- filtrates and passive transfer, in some diseases, with living lymphoid cells. The pathology and mechanisms of these diseases are however not well understood and the relative roles of cellular and humoral factors are not to date readily separated (135, 149). In Vitro Assays of DTH In vitro assays of DTH were developed when it was observed that sensitized lymphocytes release a number of factors (lymphokines) following stimulation with specific antigen in vitro. These lympho- kines mediated jn_vjtgg_effects on other cell-types including migra- tion inhibition of macrophages and PMN's; aggregation of macrophages; chemotactic attraction of PMN's, monocytes, and lymphocytes; stimula- tion of blastogenesis; cytotoxic changes; and activation of macro- Phages (35, 43, 133). Some of these activities correlate well with 18 l vivo DTH reactivity. They may also offer explanations for the mechanisms of the DTH reaction, though the functions measured 13_ yitrg_may not correspond exactly to the roles of these lymphokines in the more complex jg_yiyg_situation (35). Three jg_yjtrg_systems used most frequently to assay DTH, including DTH in recipients of transfer factor, are reviewed below. Macrophage Migration Inhibition Migration inhibition of macrophages is mediated by migration inhibition factor (MIF), a glycoprotein of 35,000-55,000 molecular weight, released by specifically sensitized lymphocytes incubated with antigen jg_yjtgg_(12, 35, 43). Since MIF production correlates well with DTH cutaneous reactivity, it has been assumed the factor is released from T-lymphocytes. Recent studies, however, have demon- strated that MIF is produced by both T- and B-cells (25). MIF production is assayed in laboratory animals by packing peritoneal exudate cells (PEC) into capillary tubes, incubating with and without antigen, and comparing the subsequent fan—like migration of macrophages from the end of the tube. For human studies, peri- pheral blood lymphocytes are usually incubated with antigen jn_vitro, the MIF is collected from the supernatant, and subsequently assayed on normal guinea pig PEC in capillary tubes (2, 35, 141). Leukocyte Migration Inhibition in Agarose Leukocyte»migration inhibition in agarose (LMIA) is an assay for the production of a lymphokine, leukocyte inhibition factor (LIF), which specifically inhibits the migration of PMN's. LIF can be separated from MIF in supernatants of antigen stimulated lymphocytes 19 by gel filtration, and does not inhibit migration of human monocytes or guinea pig macrophages (57, 133). LIF production is assayed by placing peripheral blood leukocytes, preincubated in vitro with anti- gen, in wells punched in agarose. Migration, or inhibition of migra- tion, of PMN's radially from the well is readily visualized (26). The assay correlates well with cutaneous DTH reactivity, requires fewer cells, and has been shown to be more sensitive than the MIF assay (26, 133). Blastogenesis Lymphocytes from sensitized animals or humans incubated with antigen jn_yjtgg_undergo morphological changes accompanied by in- creased protein, RNA, and DNA synthesis (141). These changes can be detected by measuring the incorporation of labeled precursors added to the cultures several days after stimulation with antigen. En- hanced DNA synthesis is measured by incorporation of 3 H-thymidine (141) and enhanced protein synthesis by incorporation of 3H-leucine (88). These lymphocyte stimulation assays correspond to the CMI status, and in general to the DTH status, of the test subjects (71, 88). Delayed Hypersensitivity in Brucellosis Brucellosis is an acute or chronic infectious disease caused by one of several species of Brucella. Each species of Brucella causes disease in a preferred domestic animal host, though the dis- ease is transmissable to man and a number of laboratory animals. The diseases are charaCterized in the primary animal host by abortion of pregnant females, low mortality, relatively nonapparent clinical 20 infections, and chronicity (113). Brucella is a facultative intracellular parasite, and stu- dies jn_!jtrg_and jn_yjyg_indicate that the host's ability to control the intracellular growth of the organism determines the course of the disease (59, 113, 121). The immune response to brucellosis is both humoral and cell mediated (114). DTH to bacterial antigens is a manifestation of the cell mediated response and will be considered here. DTH is displayed by most humans infected by Brucella, being absent in about 5% of culturally proven cases (114). Though the prevalence of DTH in infected domestic animals is not as well known, DTH has been demonstrated tn a number of experimentally infected dbmestic and laboratory animals including cattle (88), sheep (69), guinea pigs (8, 59, 68), rabbits (55, 142), and mice (107, 138). Experience has indicated that DTH is not usually induced by killed Brucella cells or fractions (8, 59) but requires actual multiplication of organisms within the host (113). Skin reactivity is usually de- tectable after one or more weeks of infection (8, 107, 113) and has been demonstrated to persist for years in humans (114) and for months in guinea pigs (8). The level of skin reactivity may diminish during acute stages of the disease, as demonstrated by Mackaness (107) in mice. In a serial study of DTH reactivity and growth of Brucella organisms in the spleens of infected mice, the level of reactivity initially rose and then diminished as the Brucella population con- tinued to increase. In the ensuing period of bacterial inactivation the DTH reactivity rose again and remained at a high level. A sim- ilar biphasic response has been seen in ECG-infected mice (10). 21 DTH to Brucella is usually assayed by intradermal injection of antigen. The reaction is highly specific for the genus, but species specificity is not detected by the individual skin test. Antigen preparations from a single species have elicited reactions in animals infected by homologous or heterologous species of Brucella, (8, 68, 142), though some investigators have found the homologous reaction to be stronger (8). The skin reaction generally is not diagnostically useful, since a positive reaction does not distinguish between past and recent exposure to the organism. Rather, serum agglutination titers are used extensively in human and animal diagnosis of bru- cellosis (114). A variety of antigen preparations have been used to elicit the cutaneous DTH reaction. Heat-killed suspensions of whole cells have been used in animals (59) but are not recommended fbr human use (114). Culture filtrates have also been used in animals (107). A variety of cell fractions prepared by physical or chemical disruption of cells and purification by extraction have been successfully used experi- mentally (1, 8, 9, 68, 138). A nucleoprotein fraction, Brucellergen, was developed by Huddleson (60) in the 1930's, and subsequently made available commercially fbr use in humans. Brucellergen is prepared by grinding ether-extracted Brucella organisms to a fine powder with subsequent purification by acid precipitation of the nucleoprotein components (60). Shortly after its development, Stahl (142) studied the chemical constitution and biological properties of the protein nucleate and of the separated protein and nucleic acid components. Protein composed about 70% of the whole protein nucleate and was shown to be the active component in eliciting the DTH cutaneous 22 reaction. The nucleic acid component contained guanine, adenine, and cytosine; and both pentose and desoxypentose sugars. The nucleic acid component did not elicit a DTH reaction. Antibody production in guinea pigs was stimulated by the intraperitoneal injection of relatively large amounts of either the protein nucleate or the puri- fied protein component, but not by injection of the nucleic acid. Ig_!jtrg_assays of DTH to Brucella have not been used exten- sively. Migration inhibition studies of macrophages and peripheral blood leukocytes have been conducted in laboratory animals and hu- mans (34, 35, 55, 138). Blastogenesis assays have been utilized ex- tensively in recent studies in experimental animal brucellosis (71, 88). Delayed Hypersensitivity in the Mouse For some time it was generally held that DTH sensitivity was not expressed by mice. Though there was available indirect evidence for DTH, investigators repeatedly failed to elicit typical cutaneous reactions by the intradermal injection of antigen (33, 52, 59). Crowle first demonstrated in 1959 that DTH reactivity could be eli- cited by intradermal injection of ovalbumin (30) or tuberculoprotein (31) in sensitized mice. That this was DTH was demonstrated by pas- sive transfer of the reactivity with lymphoid cells but not serum. The cutaneous reaction was also correlated to a number of other DTH reactions in mice (31). Both of these aspects have recently been confirmed (129). Crowle suggested that some of the previous problems arose from injection of relatively large volumes of antigen suspen- sion into mouse skin, which is very thin,and that such injections 23 may leak into subcutaneous tissues and fail to elicit the skin re- action (33). He describes a technique using careful intradermal in- jection of 0.01 ml of concentrated antigen into anesthetized mice under a stereo microscope (31). Few investigators other than Crowle have reported successful use of the skin test to detect DTH in mice, probably due to the development of the easier-to-use fbotpad assay. The footpad assay as originally described by Gray and Jennings in 1955 (52) to detect DTH in mice has been used predominantly since that time. Antigen is injected into the metatarsal pads in volumes of 0.01-0.05 ml using 26- to 30-gauge needles. The subsequent swell- ing at 24 to 48 hours is evaluated subjectively (31, 52) or measured by calipers (107, 129), fluid displacement (160, 118), or the weight of foot replicas (144). Values are usually expressed as a comparison of the antigen injected foot to the opposite foot, which is untreated or injected with antigen solvent alone. The macroscopic reaction, swelling and erythema, is often evi- dent by 6 hours and reaches maximum intensity by 24 hours (33, 72, 129). The 24 hour reaction is frequently more intense than the 48 hour reaction, and is usually reported (31, 52). The histologic reaction is much like that seen in other species, with a predomin- antly mononuclear infiltrate evident by 24 hours and more intense at 48 hours (33, 72, 129). Significant numbers of PMN's are seen early in the reaction and are often present at 24 hours (33) par- ticularly directly at the site of antigen injection (129) or with a preceding Arthus reaction (72). The time course and histologic picture presented above are evidence that the footpad reaction is a DTH reaction. Further 24 evidence is provided by the transfer of this reaction to other mice using lymphoid cells (33, 129, 160) and transfer factor (14, 130, 131). The footpad reaction has also been shown to correspond to the skin test reaction in the mouse (31, 130). Various explanations have been offered for the frequently ob- served discrepancy between skin test and footpad reactivity. Cer- tainly the footpad injection is technically easier to perform and the reaction is more evident, since skin test reactions in the mouse are not erythematous (31, 130). Crowle has reported that the foot- pad reaction may be more sensitive because the antigen is retained in the footpad longer than in the skin (33). The importance of anti- gen retention and use of particulate antigens in the mouse has been recently pointed out (72, 129). Gershon gt_al, (50) have proposed that the discrepancy is due to a significantly higher proportion of mast cells in the footpad than in the flank skin of the mouse. They present evidence that expression of DTH in the mouse is dependent on mast cell activity, and suggest that in the mouse, a species deficient in circulating baSOphils, these mast cells may substitute for the basophil activity proposed by Dvorak (47) in DTH reactions of other species. Other expressions of DTH have been demonstrated in mice, in- cluding contact sensitivity, systemic sensitivity, allograft rejec- tion, and reactivity in jg_yjtgg_assays (31, 33, 138). There is little doubt that DTH exists in mice, though its detection by intra— dermal injection of antigen may be difficult to demonstrate, and though the degree of sensitivity may be significantly less than that seen in man or guinea pigs (33). 25 Transfer Factor Historical Background The first apparent transfer of delayed-type hypersensitivity (DTH) was reported in 1909, in guinea pigs, using defibrinated blood (13). But it was not until 1942, that Landsteiner and Chase (24, 91) established the principle of passive transfer of DTH by using living cells from immune guinea pigs. Lawrence demonstrated trans- fer of DTH to non-sensitive humans in 1949 using viable blood leuk- ocytes from immunized humans (92), and subsequently observed that extracts of frozen and thawed leukocytes were as effective as viable cells (93). The term transfer factor (TF) was coined to designate the material responsible for the transfer of DTH, with the acknowledg- ment that more than one factor might be responsible (95). The find- ing that TF was dialyzable (98) greatly facilitated purification and identification of the components present in TF. Dialyzed transfer factor (TFd)* could now be prepared free of non-dialyzable leukocyte constituents, including transplantation antigens. Though TF activity was demonstrated repeatedly by Lawrence and others in experiments involving humans, the failure to demonstrate a similar phenomenon in animal models and the lack of a reliable jg_ xjtgg_model caused considerable skepticism (22, 108). Fifteen years after Lawrence first described TF, it was shown to have a therapeutic effect on patients with the Wiskott-Aldrich syndrome (140). This *In this review TF will refer to dialyzed or non-dialyzed trans- fer factor, TFd will refer specifically to dialyzed transfer factor. 26 and subsequent observations of benefit in other selected clinical problems sparked a renewed interest in TF research, resulting in in- creased activity in the analysis of TF, therapeutic trials, and attempts to develop animal models and jn_vitro assays fbr TF. Physical Properties of Transfer Factor Transfer factor is a soluble dialyzable moiety of leukocyte extracts, generally held to be less than 10,000 molecular weight F (95). The TF activity is resistant to enzyme treatment with ribo- nulcease or deoxyribonuclease, trypsin, and lysosomal hydrolases (76, 93, 95, 130), but is destroyed by pronase and snake venom phospho- diesterase (76, 130). Rifkind has recently demonstrated that mouse TFd activity, though resistant to monomeric ribonuclease, is sensi- tive to dimerized ribonuclease, an enzyme active against double- as well as single-stranded RNA (131). Other investigators have shown that a guinea pig TFd is destroyed by RNase III, an enzyme that speci- fically degrades double-stranded RNA, but is not affected by RNase enzymes active against single-stranded RNA (38). Transfer factor has been found to be quite stable under various storage conditions. It is labile to treatment at 56°C fbr 30 minutes but stable at 25°C or 37°C for as long as 6 hours (95). TF in frozen whole leucocyte preparations stored at -20°C for 4 years and subse- quently prepared as usual for TFd, was found to retain its activity (99). Lyophilized, TFd can be stored as a powder for at least 5 years at 4°C without loss of potency (99). The dialysate of crude leukocyte lysates contains a complex mixture of substances, including products of enzymatic breakdown of 27 cellular materials (22). It is probable that only some of these are responsible for the immunologic transfer activities, other substances being inactive or acting to suppress or modify these activities (20, 51). Though much work has been devoted to analyzing these substances in an effort to identify the active components, the material(s) re- sponsible for in_yiyg_activity have not to date been identified. Most investigations have shown the presence of ribose, poly- f . <0... peptides, and polynucleotides (6, 84, 95). Others have reported the presence of RNA bases, hexose, hypoxanthine, tyrosine, nico- tinamide, serotonin, histamine, ascorbate, and lipid phosphorous (20, 78, 80, 90, 131). Some of these components have been shown by selective elimination not to mediate the immunologic in vivo activity of TF, while others have been shown to be responsible fbr non-specific phenomena (19, 20, 80, 155). Lawrence demonstrated that TFd does not contain detectable immunoglobulin (95). He added either Bence-Jones protein or papain- digested gamma-globulin fragments to the lysate before dialysis in a procedure to monitor the integrity of the dialysis membrane. An- alysis of the dialysate by 10% trichloroacetic acid and by immunodif- fusion tests revealed no detectable albumin, alpha- or gamma-globulin, or other protein. The presence of antigenic substances is not expected in TF pre- parations that have been dialyzed. Attempts by Lawrence to raise antibody to TFd injected in Freund's adjuvant have been unsuccessful. Lawrence also demonstrated the failure of TFd to sensitize recipients to the HLA antigens of the TFd donor, as measured by either skin allograft rejection or humoral antibody determinations (95). This, 28 in view of the fact that TFd can transfer accelerated allograft re- jection from an actively sensitized donor (95), indicates the ab- sence of HLA antigens in the dialysate. Burger has also reported the absence of superantigen properties in guinea pig TFd (17). Several investigators have demonstrated serial transfer of DTH by TF (93, 79, 87). TF prepared from the cells of one individual transferred DTH reactivity to an anergic recipient and TF subsequently prepared from that recipient transferred DTH reactivity to a third individual. The dilutional aspects of these experiments argue against the role of antigen or immunoglobulin in TE preparations. Preparation of Transfer Factor am. Lawrence and associates established the basic methods for pre- paration of dialyzable transfer factor from human sources in their early work (99). Refinements of these techniques have been intro- duced by others (18, 141). There are six stages in the preparation procedure: (1) leukocyte preparation, (2) leukocyte lysis, (3) dial- ysis, (4) lyophilization, (5) reconstitution, and (6) filter sterili- zation prior to clinical use. As will be seen below there is con- siderable variation of technique in each of these stages, which has however not led to obvious differences in jn_vivo activity. Leukocyte Preparation Transfer factor donors are selected on the same criteria as donors for blood transfusion: good health, adequate hematocrit, free of blood-borne diseases and no evidence of hepatitis (99, 141). An additional criterion, the most important condition for successful 29 transfer, is that the donor have a marked degree of delayed cutan- eous reactivity to the antigen(s) under study (99, 141). This re- activity may be due to natural sensitization or elicited by active immunization. Human TF is usually prepared from peripheral blood leukocytes. Venous blood (up to 500 m1) is drawn into syringes or tubes contain- ing heparin or EDTA anticoagulant (49, 99, 141). A 500 m1 blood-donor bag (Fenwal Bag) may be more convenient fbr larger volumes of blood, but it has been shown that the leukocyte cell yield is significantly lower per volume of blood (141). Leukopheresis may be used when a large number of leukocytes are desired, as from a donor with a rare immunity. This procedure can be repeated on the same donor more fre- quently and yields a much higher number of leukocytes (49, 141), which are predominantly mononuclear (80). The safety, convenience, and efficiency of this process, in fact, makes it a more practical method of cell collection for all forms of TF (49). Leukocytes are separated from other blood components by one of several methods. Whole cell suspensions containing leukocytes, erythrocytes, and platlets are occasionally used (78), though the leukocyte preparations are usually more purified. Lawrence (99) and other investigators (104, 141) mix whole blood with high molec- ular weight substances, such as dextran, bovine fibrinogen fraction I, or polyvinylpyrrolidone, to accelerate erythrocyte sedimentation. The leukocyte-rich, erythrocyte-free plasma is then further processed. More recently whole blood has been layered over Hypaque-Ficoll density gradients to yield leukocyte suspensions containing 60-90% lympho- cytes and 10-30% monocytes, with few granulocytes (78). After 30 processing, the leukocytes are suspended in either distilled water (4, 80, 104) or saline (99, 141) prior to freeze-thaw lysis. Lysis of Leukocytes Lawrence demonstrated that TF could be released from leuko- cytes by distilled water lysis or by 7 to 10 cycles of freezing and thawing, in a dry-ice-alcohol bath and a 37°C water bath respectively (93). Since that time the freeze-thaw method has been universally used as a convenient, reproducible method to lyse leukocyte prepara- tions. This process releases intracellular DNA which forms a large gelatinous mass. DNAse and magnesium sulfate are added by most in- vestigators (49, 99), though not all (49, 80) to liquify this mass for easier handling. This treatment has been shown to not affect the ig_vivo activity of TF (93). The enzyme may be added before or after the freeze-thaw procedure. The resulting lysate contains TF activity and represents the preparation originally used by Lawrence to transfer DTH in humans. Dialysis Lawrence found in 1963 that dialysates of TF were as effective as the whole lysate in transferring DTH (98). This has been con- firmed by several groups of investigators (see 99) and TF preparation now usually includes dialysis, which yields a product free of the host of large molecular weight substances in cells, including immuno- globulins and HLA antigens (95, 99). TFd is thus more amenable to chemical characterization and represents a pharmacologically safer agent for clinical use. Various methods of dialysis have been employed. Though some 31 variation in fractionation profiles on gel chromatography has re- sulted, no change in i__yjyg_activity has been detected due to these variations (7). In methods employing dialysis tubing, the lysate is placed in- side a dialysis sac, avoiding contamination of the outside surface. Dialysis is then carried out against sterile distilled water (49, 99, 141), saline (99), NH4HCO3 (78), or tissue culture medium (4). The latter procedure was found to yield a preparation free of a factor found in water-dialyzed preparations to be toxic to lympho- cytes jg_vitrg_(4). Dialysis is usually done at 4°C with agitation for time periods of 18-24 hours. A procedure utilizing a vacuum dialysis apparatus (18) has been successfully used to prepare TFd. This process takes less time (4-8 hours) and eliminates the need to concentrate the dialysate by lyophilization and reconstitution. Ultrafiltration techniques have also been successfully utilized in the processing of large quantities of TFd (7, 54). LyOphilization, Reconstitution, and Filter Sterilization Preparations dialyzed into distilled water, saline, or NH4HC03 (see above) are concentrated by 1y0philization. The resultant powder can be conveniently stored at 4°C for an extended time (99), or re- constituted, filter sterilized and stored frozen until use (4, 80, 141). The powder may be reconstituted in water (4,99, 80) or saline (141) and can be reconstituted to an arbitrary lymphocyte-equivalent concentration (80). 32 m. Preparation methods for TF from animals do not vary greatly from those used for human TF with the exception of cell source and release of TF from the cells. Peripheral leukocytes have been utilized from animals, espec- ially in those species large enough to provide adequate volumes of blood (64, 67, 109). Other lymphoid organs however are frequently used as sources of animal TF. These include: lymph nodes (14, 15, 41, 86), spleen (130), peritoneal exudate (15, 41) and alveolar wash- ings (15). The release of TF from these cells has been achieved by several methods other than the freeze-thaw procedure of Lawrence, though this method remains in wide usage (67, 130). Burger found that TF activity was released from guinea pig (15) or rabbit (16) lymphoid cells when these cells were incubated in Hank's balanced salt solution at 37°C for four hours, with no control on the pH of the medium. Concomitant with decrease in pH and cellular viability, TF activity could be detected in the supernatant fluids and was found to decrease in the cellular material. Klesius has applied this procedure to bovine lymph node cells (86), and found it to be a practical method to produce large quantities of TF. The supernatant fluid can be di- alyzed and processed as any cell lysate preparation (15, 86). Jeter's group has also demonstrated the release of a TF-like activity into the plasma of guinea pigs treated jg_yjy9 with anti-lymphocyte serum (117). 33 Manifestations of Transfer Factor Specificity and De Novo Sensitization The question of dg_ggyg_sensitizationcrfan immunologically naive recipient by TF and the antigen specificity of that transfer has not been unequivocally resolved. A major problem in the investi- gation of these aspects of TF has been the predominant use of human subjects, whose history of disease and exposure to antigenic sub- stances and whose immunocompetance are often unknown (2, 137). Studies have also been criticized because of uncertainty regarding the specificity of test antigens, cross reactions of antigens, and the possibility of actively sensitizing TF recipients by skin testing befbre the administration of TF (49, 115, 141). The transfer of DTH to microbial antigens prevalent in the environment presents problems in that the TF recipient has likely been exposed to these, and sub- sequent conversion by TF may actually represent a non-specific stim- ulation of previously non-detectable reactivity (13, 21, 141). Evi- dence for this has been presented by Green gt_al, (53), who restored DTH reactivity in mice that had lost previously demonstrable DTH using a lymphoid cell lysate that did not transfer DTH to antigen- ically naive mice. Lawrence has cited two studies as evidence of d§_ngyg_sensitiza- tion and antigen specificity (95). In one study (127), TF from coccidioidin-sensitive California residents was prepared and admin- istered to residents of New York who had not traveled to an area en— demic fer coccidioidomycosis. Twenty-three of 27 recipients developed positive reactions. .These conclusions have however been criticized because several recipients also developed positive responses after 34 receiving TF from donors initially not showing reactivity, and because the recipients were pre-skin tested (49, 141). In another study, TF was prepared from an individual (A) who had been sensitized to the tissue antigens of individual (B) by re- peated skin grafts. Administration of this TF to individual (C) caused accelerated rejection of a skin graft from (B) but not of skin grafted, at the same time, from other individuals (95). This study is still regarded by many to be evidence for g§_ngyg.sensitiza- tion and antigen specificity in TF activity (11, 21, 49). Other studies have shown evidence for antigen specificity and gg_ngyg_sensitization both jn_yjyg_and jn_yitrg (see for example 41, 48, 104, 130, 139, 158, 164). TWO that do not suffer from the crit- icisms outlined earlier are the demonstration by Burger et_al, (21) of specific transfer of sensitivity to a neo-antigen, keyhole limpet hemocyanin, by TF preparations made from donors after but not befbre specific immunization of the donors, and the demonstration by Maurer (110) of DTH transfer using TF from donors sensitized to ethylene oxide-treated serum. TF recipients were not pretested by skin test in either study. Bloom has proposed that TF is not antigen-specific, but rather acts as a non-specific immunologic adjuvant (11). He cites the fact that TF recipients rarely demonstrate reactions to all of the specifi- cities of the donor, occasionally showing reactivity to specificities for which the donor is negative, and a theoretical difficulty in ex- plaining the ability of such low-molecular-weight molecules to carry specific information for all antigens for which successful transfer has been reported. Other investigators have suggested that TF restores 35 defects in non-specific expression of DTH rather than the antigen- specific recognition phase of the DTH response (90). Demonstration of TF activity in a guinea pig model (154) and an jg_yitrg_lymphocyte stimulation assay (19, 28) has proved to be independent of TF donor specificity and in fact dependent on recipient (or recipient-lymphocyte donor) reactivity. The jg_vitro activity has also been demonstrated in dialyzable extracts from nonlymphoid as well as lymphoid organs (153). A number of other antigen-inde- pendent phenomena, both jg_vjtrg_and jn_yjyg, have been attributed to TF or fractions of TF (20, 75, 78, 80). The available evidence indicates that TF can be antigen spec- ific, transferring the reactivities of the TF donor, but that TF probably represents a mixture of specifically and non-specifically acting moieties (11, 97, 108). Part or all of these components may contribute to the cell mediated immune response as evidenced by clin- ical improvement,conversionof’skin DTH reactivity, and change in other immunologic parameters (89, 104). In Vivo Manifestations As outlined above TF has a number of manifestations i vivo and jg_yjtgg, demonstrating antigen specificity and non-specificity. The originally described manifestation, by which TF is functionally defined, is the transfer of delayed cutaneous hypersensitivity ex- hibited by the TF donor to a recipient previously anergic to the specificities transferred. The transferred DTH appears in the re- cipient as early as four hours, and generally by 18 hours after TF administration. Sensitivity has been shown to persist for months to 36 two years. Successful transfer requires marked sensitivity in the TF donor and is lymphocyte—equivalent dose dependent (95). Transfer of sensitivity to bacterial (93, 155), fungal (23, 67, 130), viral (96), parasitic (86, 109), and chemical (21, 155) antigens has been demonstrated. Transfer of DTH skin reactivity has been accomplished by both local techniques (intradermal injection of TF followed 24-96 hours later by antigen at the same site) and systemic techniques (intra- dermal, intramuscular, subcutaneous, or intravenous injection of TF followed 24-96 hours later by intradermal antigen injection at a separate site) (99). The local technique requires a ten-fold lower TF dose, but the systemic technique is more meaningful because the possible contribution of non-specific local inflammatory responses to TF is excluded from the DTH reaction site (99). The DTH reactivity in TF recipients demonstrated by intradermal injection of antigen, has been confirmed by jg_yjtgg_assays performed on cells of TF recipients. Conversion has frequently been demon- strated by macrophage migration inhibition assay (5, 7, 79, 104), but inconsistently by lymphocyte blastogenesis (49, 86, 95). A number of antigen-independent phenomena have been reported in recipients of TF. These include increase in PHA responsiveness, mixed lymphocyte culture responsiveness, ability to respond to active sensitization with dinitrochlorobenzene (DNCB), and E-rossette forma- tion (97, 75). These have been primarily demonstrated in recipients who exhibited deficiencies in cell mediated immunity. Kirkpatrick and Smith (78) have demonstrated an antigen-independent chemotactic activity in TFd for PMN's and monocytes. The activity was demonstrated 37 both jn_yjyg_and in 11522: Gottlieb gt al, found that TFd fraction- ated on Sephadex G-10 contained a fraction that elicited an antigen- independent intradermal reaction resembling DTH (51). Further purif- ication of this fraction revealed two active sub-fractions, one of which augmented existing recipient sensitivity to an antigen when injected intradermally with the antigen. Vandenbark and Burger (155) reported a similar activity in a Sephadex G-25 fraction which was also active in transferring cutaneous sensitivity. They were however able to separate these two activities by modification of the frac- tionation procedure. Transfer Factor Therapy Since the early 1970's, when Levine and Spitler demonstrated clinical improvement in patients with the Wiskott-Aldrich Syndrome fol- lowing administration of TF (140), there has been a great deal of interest in the treatment of other diseases of the cellular immune system with TF. Dialyzable TF is especially useful in this.regard, since it does not contain viable cells or antigenic material, and thus avoids the possibility of the often fatal graft-versus-host reaction seen with bone marrow transplantation and can be adminis- tered repeatedly without sensitizing the recipient to donor leuko- cyte antigens (141). Dialyzable transfer factor (TFd) can also be conveniently stockpiled and administered when needed. There is a low frequency of adverse reactions to TFd administra- tion, even when large doses have been given over a long period of time (49). Local inflammation and mild pyrexia and malaise may occurr (49) and there is one report of transient immunosuppression 38 following TFd administration (77). Possible hazards of TFd therapy include the transmission of hepatitis, induction of autoimmunity due to TFd-donor sensitivity to HLA antigens of the recipient, and initia- tion of an overwhelming reaction following a sudden conversion of DTH reactivity in patients with disseminated infection or malignancy (141). TF has been administered in the treatment of patients with congenital and acquired immunodeficiencies; chronic infections caused by mycobacteria, fungi, and viruses; and a variety of malig- nancies. An attempt will not be made here to review all of the clin- ical trials of TF treatment of these syndromes. Representative ex- amples in each area will be cited. Several recent review articles are available (49, 96, 97, 100). Infectious Diseases The bulk of data concerning TF treatment of infectious dis- eases has been accumulated from trials on patients with chronic mucocutaneous candidiasis. Patients with this disease display an array of clinical and cell-mediated immune abnormalities (5, 78, 141). This may account for the variations in reported success rates, though most investigators report better than 50% of treated patients demonstrate some clinical improvement (95, 108, 124). Clinical im- provement is generally preceded or accompanied by correction of the observed immunological abnormalities (5, 104, 108), an indication that TF functions to convert the patients to immunological compe- tency. Studies by Kirkpatrick (78, 81) and others (104) have shown 39 that TF will not effect clinical improvement without prior or con- comitant anti-fungal drug therapy. After initiation of TF therapy, drug therapy may be terminated, and TF functions to maintain clinical remission. A study by Littman §t_al:(104) provides evidence that the clinical response is antigen specific. Several interesting investiga- tions in mucocutaneous candidiasis patients with thymic deficiencies, have demonstrated the need fbr thymus-derived cells in recipients of k TF. No response was seen in these patients when TF alone was admin- istered, but clinical and immunological improvements were seen when a i: s“ .i'lf-I m‘ 3 fetal thymus tissue was transplanted prior to or concomitant with the TF (5, 81). Patients with chronic disseminated coccidioidomycosis display abnormalities in cell mediated immunity much like those seen with chronic mucocutaneous candidiasis. The cumulative experience of a number of investigators in treatment of this disease-state with TF has recently been reported (23). Thirty of 49 patients with chronic coccidioidomycosis refractory to Amphotericin therapy who were treated with TF, showed evidence of clinical improvement. Ampho- tericin treatment was continued during TF administration. Significant conversion of skin test reactivity and migration inhibition were also noted. Successful treatment of patients with progressive tuberculosis refractory to chemotherapy has been reported. Rubinstein gt_al,(136) demonstrated sustained clinical recovery from tuberculosis after TF treatment in a patient displaying marked cutaneous skin reactivity to tuberculin and normal macrophage and leukocyte migration inhibi- tion reactivity, but low T-cell numbers and abnormalities in other 4o jn_yitrg_lymphocyte functions. Rocklin (134) reported a similar successful treatment of a tuberculous patient with an undiagnosed cellular immune deficiency. There are several reports of successful treatment of viral in- fections with TF (97, 96). Children with giant-cell measles pneu- monia and with subacute sclerosing panencephalitis (SSPE) have been treated with TFd from rubeola-immune donors, with subsequent clinical f and immunological improvement. Neonatal herpes infection and cases I of herpes zoster in immunosuppressed patients have likewise been 1 treated. Earlier reports showed that disseminated vaccinia could be treated with whole viable leukocytes, indicating this disease could be treated with TF. Some of the beneficial effects seen may be due to enhanced interferon levels, which have been demonstrated following TFd administration (74). Lawrence has noted that caution should be exercised in treat- ing viral infections located in certain areas of the body (97). Local inflammatory responses have been detected at viral infection sites fbllowing systemic TFd administration. In cases such as cytomegalo- virus retinitis, serious damage to the affected tissue could result. The studies of Klesius' group, though not involving trials in humans, should be noted. Using a bovine TFd preparation, Klesius has transferred partial protection to coccidiosis, a parasitic dis- ease, in calves (86) and mice (87). The same preparation failed to confer protection to rabbits, as did a similar TFd prepared from rabbits (86). Liburd et_a1, have demonstrated a similar phenomenon using treatment of rats with rat TF (103). 41 Immune Deficiencies Transfer factor has been administered to patients with a var- iety of deficiencies of the cellular immune system, in the hope that TF would reconstitute the deficient components and render the patients immunocompetant. As noted above, the earliest experimental therapy in this area was carried out by Levin and Spitler (140, 141) on patients with the Niskott-Aldrich Syndrome. This syndrome, an x- 1inked disease, is characterized by recurrent pyogenic infections, eczema, splenomegaly, thrombocytopenia, and failure to display DTH immune responses in_gjyg_or jg_!jtgg_(49, 140). Since that time more than 30 patients with this condition have been treated. A good clin- ical response, as evidenced by freedom from infections, regression of splenomegaly, and clearing of eczema, has been recorded in about 50% of these patients (49, 100, 140). Appearance of DTH skin reactivity and MIF responsiveness have also been noted (49, 141) and appear to correlate with the clinical response. Maintenance of the improved clinical and immunological conditions is dependent on continued TF administration to these patients. Reports of beneficial effects following TF therapy in other immunodeficiency conditions are less extensive. Clinical and immuno- logical improvement have been noted in patients with Swiss-type agammaglobulinemia (141), ataxia teleangiectasia (95, 100), a monocyte defect (2), and a variety of other combined immunodeficiency dis- eases (95, 100). Transfer factor may also have a role in the treatment of infec- tions in immunosuppressed patients. Lawrence has recently reviewed this subject (97). Response to TF therapy has been seen in patients 42 with internal immunosuppression as a result of disseminated intra- cellular infections and secondary to diseases such as sarcoidosis, lymphocytic leukemia, established cancer, kwashiorkor, and Hodgkin's disease (97) though the response in the last syndrome has been quite low (22). Several reports have also indicated benefit by TF admin- istration to patients on external immunosuppressive therapy (97). These have included patients with Hodgkin's disease, rheumatoid ar- thritis, disseminated lupus erythematosus, and a patient undergoing renal transplantation. Malignancy There is extensive evidence from studies in animals and tumor- bearing patients that cellular immunity is involved in the anti-tumor response of the host (95). It is assumed that there are specific antigenic determinants, "tumor-associated antigens," on the surface of malignant cells to which the host's lymphocytes mount a response. This response may be adversely affected by suppressor cells, block- ing antibodies or antigen-antibody complexes, and by the inability of the host immune system to adequately contain a tumor (49). The rationale for use of TF in anti-tumor therapy is that TF from donors displaying CMI to specific tumors will induce specific anti-tumor activity in the recipient. This has been evidenced by lymphocytic and monocytic infiltration and regression or control of certain tumors (97, 100, 156). TE has been prepared from household contacts, who have been found to display tumor specific immunity to certain malignancies (49, 100, 156). Cancers with suspected viral association might be effectively treated by TF from donors previously exposed to the viral agent (49). 43 A list of some malignancies that have shown clinical or immuno- logical benefit from TF therapy is included below. TWO tumors treated most frequently are melanoma and osteogenic sarcoma (49). Tumor Type References melanoma 49 156 97 80 osteogenic sarcoma 49 156 97 100 breast carcinoma 49 95 100 nasopharyngeal carcinoma 49 156 Wilms' renal cell carcinoma 49 156 lymphosarcoma 49 156 neuroblastoma 49 rhubdomyosarcoma 156 leiomyosarcoma 156 epidermoid carcinoma 156 adenocarcinoma 156 alveolar sarcoma 80 lymphocytic leukemia 80 vulvar carcinoma 49 Reported results vary greatly, and may be attributed to the in- herent variations in patient health status, tumor load, and concur- rent anti-tumor therapy (49). Clinical improvement has ranged from total regression to arrest of metastasis. Concurrent immunological improvement has been measured by conversion or enhancement of DTH skin reactivity, MIF release, and lymphocyte-mediated cytotoxicity as well as non-specific parameters (49, 156). TF may be optimally used as an adjunct to other anti-tumor therapy, such as surgery, radiotherapy, and chemotherapy. TF is more effective in treatment or maintenance of patients with minimal tumor-load, which can be accomplished by surgical removal of macro- scopic tumor (49, 100). Radiotherapy and chemotherapy have numerous undesirable side affects. TF therapy may be instituted to directly remedy one of these, transient immunosuppression, and indirectly remedy others by decreasing the need for these treatment modalities (49). 44 Recent studies by Pizza et_al, (120), are of interest, in that they may provide a means of large scale TF production for use in therapy of malignancy and other diseases. These investigators have demonstrated the in vitro production of a biologically active TF specific for transitional cell carcinoma of the bladder (TCCB) by continuous lymphoblastoid cell lines induced jg_vitro by TFd from TCCB patients. Ifl_vivo immunologic activity of this jn_vitro pro- I,» duced TF has been demonstrated, but studies in clinical application are still in progress. Animal Models The development of a reliable animal model for TF would con- L tribute much to the study and characterization of TF. Controlled experiments could be performed on large numbers of genetically sim- ilar animals whose prior exposure to antigenic substances and dis- ease are more fully known. ProCedures such as the injection of tumor cells or induction of disease for TF protection studies and the external manipulation of immune systems todetermine the role(s) of TF could be performed. Recent studies showing evidence for activ- ity of animal TF in humans (66) provides an interesting possibility of large scale production in domestic animals of specific TF for human use. The transfer of DTH by living whole cells was demonstrated in animals (24, 91) befbre the same phenomenon was demonstrated in humans (92). Repeated attempts to confirm Lawrence's work with cell lysates (TF) in animals however produced conflicting results (13). Jeter et_al, showed evidence of transfer of DTH to 45 2,4-dinitrochlorobenzene (DNCB) in guinea pigs by extracts of dis- rupted cells in 1954 (63). Other investigators reported confirma- tion of these results, but Bloom and Chase by their studies refuted transfer of DTH in the guinea pig model by preparations other than viable leukocytes (l3). Burger and Jeter later reported that DTH to chemicals (DNCB and DNFB) could be transferred in guinea pigs by dialyzable supernatant fluids from sensitive leukocyte cultures in- cubated without antigen (15). The authors suggested that earlier I .. L difficulties may have been due to loss of TF from cells into the suspending medium during preparation (62). These observations were fbllowed by isolated reports of TF activity in primates (95, 109) and rodents (95, 103). In recent years the number of animal models re- ported has expanded rapidly. These reports are organized below by species. Non-Human Primates Transfer of DTH by TFd to two antigens, tuberculin (PPD) and Schistosomal extracts, has been demonstrated by the group of Maddison (109) in Rhesus monkeys. Transfer was seen using TFd derived from monkey or human leukocytes. Attempts to assess protection to infec- tion by these preparations, however, yielded equivocal results. Dumonde (39) has reported conflicting results in Rhesus monkeys using KLH antigen. Steele et_al, (143) demonstrated TFd activity in three other species of monkeys using either human or monkey TFd. Human TFd has also been shown to be active in monkeys by other inves- tigators (80, 95, 143) using a variety of antigens. Klesius has shown a similar phenomenon using bovine TFd (83). 46 m The bovine has been shown by several investigators to be a prom- ising model and source of TFd. Klesius has prepared large amounts of TFd from bovine lymph node cells incubated jg_yjtrg, Using this pre- paration, his group has demonstrated transfer of DTH to calves (85, 86), rabbits (86), mice (87), dogs (83), and Rhesus monkeys (83). DTH to PPD, KLH, diptheria toxoid, and coccidia antigens was trans- ferred. These results have correlated with jg_yitgg_lymphocyte stim- ulation studies and in some species with partial protection to in- fection by the coccidia species of that host (86, 87). Demonstration of activity in alcohol precipitation fractions has been also reported (85). Jeter gt_al,(29, 67) have reported the transfer of DTH to cattle by TFd prepared from peripheral leukocytes of actively sen- sitized bovine donors. DTH to PPD and coccidioidin was transferred. This TFd has been reported to show jg_vivo activity in guinea pigs (102) and humans (66). Dogs Jeter has reported successful systemic transfer to dogs of DTH to tuberculin by TFd prepared from sensitized dogs (64). Tomar has reported less success systemically, but reported local transfer, though the specificity of the phenomenon was suspect (145, 147). Guinea Pigs Most early attempts to develop an animal model involved guinea pigs (13). The problems associated with these attempts were outlined above. In addition to the early reports cited above, successful 47 transfer of DTH to DNCB and PPD to guinea pigs by guinea pig TFd has recently been reported by Jeter gt_al.(65, 102). It was initially hoped the guinea pig could serve as an animal model to assay human TF activity. Attempts were unsuccessful until the apparent development by several investigators (154, 157) of a technique using antigen primed guinea pigs. The obvious need for antigen priming, injection of antigen with TF, and subsequent demon- strations of non-donor specificity however indicated this was not a true model for TF activity (154). Recent reports indicate that pur— ified preparations of human TFd may transfer DTH to guinea pigs with no antigen priming (101, 102). Rabbits Klesius gt al.have demonstrated the transfer to rabbits of DTH to coccidian and PPD antigens by bovine TFd and to coccidian antigen by rabbit TFd (86). Protection to infection by the coccidial parasite (Eimeria stiedai) was not afforded by either preparation. Burger gt_al, (16) demonstrated rabbit-to-rabbit transfer of DTH using a TFd prepared by the technique developed earlier for guinea pig TFd (15). Rats Partial immunity in rats to infection by the coccidial para- site (E, nieschulzi) was induced by Liburd gt_al, (103) using TFd prepared from lymphoid tissues of immune rats. 48 N122. Crowle demonstrated in 1959 that DTH could be transferred to mice by whole lymphoid cells from immunized mice (30). Transfer with TF has not been accomplished in the mouse until recently, but the mouse could provide a convenient, inexpensive model and assay fbr TF activity. Rifkind g__al, (130, 131) have demonstrated that TFd prepared from spleen cells of imunized mice will transfer DTH to recipient mice. The transfer demonstrated specificity for Coccidioides, Candida, and Mycobacterial antigens. Lawrence's group has confirmed mouse-to- mouse transfer using TFd prepared from lymph node cells (14). The possible use of the mouse as an assay for TFd prepared from other species is indicated by recent reports. Both Rifkind's (119) and Lawrence's (14) group have reported transfer to mice using human TFd. Klesius has reported a similar transfer with bovine TFd (87). MATERIALS AND METHODS Animals Albino ICR female adult mice were purchased initially from Spartan Research Animals, Inc., Haslett, Michigan, and later from Harlan Industries, Inc., Cumberland, Indiana. All mice used in a single experiment were purchased from one source. Mice weighed 25- 30 grams at use. New Zealand white female rabbits weighing 8-10 pounds (Spartan Research Animals, Inc., Haslett, Michigan.) were used in procedures to standardize the skin test antigen (Brucellergen). Holstein heifers used in these experiments were part of a brucellosis research project being conducted at Michigan State Uni- versity, under a grant from the United States Department of Agricul- ture. The animals were housed in an isolation barn. Non-infected controls were kept separated from infected cattle. Heifer #221, #230, and #249 were 2-year old non-infected heifers. Heifer #221, a negative control animal, had not been actively or pas- sively immunized. Heifer #230 and #249 were immunized at about 5 months of age bysubcutaneous injection of 5 ml/each of the commercially available attenuated Brucella abortus strain 19 vaccine (Colorado Serum Co., Denver, Colorado). This treatment was repeated 3 months later. Heifer #7, a pregnant test animal, had received 1.6 x 109 viable 49 SO peripheral mononuclear leukocytes from heifer #230 thirty days preced- 5 ing challenge with Brucella abortus strain 2308 (9.5 x 10 cells). Subsequent active Brucella infection was demonstrated by rise in agglutination titers and isolation of the organism from placental membranes at partuition. Microorganisms Two strains of Brucella abortus were used in these experiments. I Strain 19, an attenuated vaccine strain, was subcultured from the com- mercially supplied vaccine (Colorado Serum Co., Denver, Colorado) and maintained on Trypticase Soy agar (TSA) (Difco, Detroit, Mich.) slants. Strain 2308, a fully virulent non-C02 requiring strain, was maintained as a stock culture on TSA slants in our laboratory. This strain was checked for maintenance of smooth characteristics before use by the oblique lighting method of Henry (56). Organisms used for immuniza- tion of mice or rabbits were prepared by washing 48-hour TSA slants with peptone-saline (0.1% peptone-0.85% NaCl) and diluting the suspen- sion to a reading of 78% light transmittance at 650 nm on a Perkin- Elmer spectrophotometer, Model 139 (Coleman, Maywood, 111.). This reading has been determined to equal 1 x 109 bacteria/ml. The sus- pension was further diluted in peptone saline to the desired concen- tration and the actual cell count established by plate counts on TSA. Immunization of Rabbits and Mice Rabbits STl and 74H were immunized by intravenous injection of 1.0 ml of a 1:100 dilution of the growth washed from a 48-hour TSA slant culture of Brucella abortus strain 2308, according to the methods originally described by Huddleson (60) for the standardization 51 of Brucellergen. In the experiments outlined here, this was equival- 7 ent to 5.8 x 10 organisms per dose. Rabbits from a previous project, which had been infected 1 year earlier with Brucella abortus strain 6 2308 (l x 10 organisms), were also skin tested to assay antigen activity. Mice were immunized by intraperitoneal injection of (unless 6 to 5.3 x 106 viable organisms in 0.5 or otherwise noted) 4.8 x 10 1.0 m1 peptone saline. Both strain 2308 and strain 19 were used in respective experiments. Tests for Delayed-Type Hypersensitivity Brucellergen Preparation Brucellergen was prepared as originally described by Huddleson (60). Smooth Brucella abortus strain 2308 was grown at 37°C on liver infusion agar prepared as described by McCullough (114) in flat 32- ounce bottles. The growth at 72 hours was washed from the surface of the agar with distilled water and the cells recovered by centri- fugation. The cell pellets from 27 bottles were combined and extracted with three successive changes of anhydrous ether (total 1500 ml) over a 30 day period to remove lipids. The cells were dried ig_!aggg_over H2304 at 37°C and subsequently ground to a fine powder in a ball mill for seven days. One gram of this powder was suspended in 200 m1 of phosphate buffered distilled water (pH 7.0) and allowed to stand at 4°C overnight (17 hours). Insoluble material was removed by centri- fugation, and the protein nucleate was precipitated from the super- natant at pH 4.0 by addition of 1:2 glacial acetic acid. Precipita- tion was allowed to continue for 24 hours at 4°C. The precipitate 52 was recovered by centrifugation, resuspended in 100 m1 cold distilled water, and redissolved at pH 6.8 by addition of l N NaOH. Insoluble material was again removed by centrifugation. The precipitation at pH 4.0 and resolution at pH 6.8 was repeated twice more. Each centri- fugation was done at 3,000 rpm (2,000 x g) on a IEC-PR 6000 Centrifuge (Damon/IEO, Needham Hts., Mass.). The final precipitate was centri- fuged in graduated centrifuge tubes so that the volume could be esti- mated. The precipitate was redissolved at approximately 1% (vol/vol) in distilled water at pH 6.8. Phenol was added to a concentration of 0.5% and the solution was filter sterilized (0.2 pm) (Nalgene, Rochester, N.Y.). Brucellergen Standardization The allergic potency of the Brucellergen preparation was de- termined by skin tests in sensitized rabbits, the method previously used to standardize Brucellergen for human use (60, 114). An aliquot of the 1% stock solution was adjusted with l N HCL until a fine par- ticulate precipitate of the antigen was visible. Serial dilutions of this suspension were made in sterile phenolized saline (0.5% phenol- 0.85% NaCL) and tested by intradermal injection of 0.1 m1 into separ- ate skin sites on the shaved abdomen of the rabbits. In these exper- iments, the 1% stock solution was taken to be a 1:100 starting dilution. Non-sensitized rabbits were included as controls. Reac- tions at the injection sites were read at 24, 48, and occasionally 72 hours by two right-angle measurements of the diameter of indura- tion and erythema using Vernier calipers (supplied by T. H. Conger, D.V.M.). Although erythema was noted, the measurement of palpable 53 induration was used as indication of a DTH reaction. DTH Tests in Cattle DTH skin tests in cattle were applied in the lateral cervical area by intradermal injection of 0.1 m1 of a 1:1,000 dilution of Brucellergen, prepared as described above. Hair was clipped from the test area before injection. Induration was measured at 48 and 72 hours in the manner described for rabbits. DTH Tests in Mice DTH was tested in mice which had been actively immunized by Brucella infection or passively immunized with TFd, by the footpad assay as originally described by Gray and Jennings (52). Injections were made into the ventral pads of the hind feet using a 30-gauge needle and a 1 ml tuberculin syringe calibrated in 0.01 ml incre- ments. Preliminary experiments indicated that injection of 0.05 ml but not 0.025 ml occasionally caused excessive trauma. The latter volume was therefore used throughout. Brucellergen, prepared as described for the rabbit skin tests, was injected into one footpad. An equal volume of phenolized saline, acidified and diluted in the same manner as the antigen, was injected into the opposite footpad. Reactions were read at 24 and 48 hours both by subjective evaluation and by measurement of footpad swelling using Vernier calipers. The dorsoventral thickness of each foot was measured three times and the result expressed as the difference between the mean of these three measurements for the antigen-injected foot and the mean of the three measurements for the saline-injected foot (test minus control). Sub- jective evaluation was limited to the descriptions below: 54 negative(-) No observable difference between test and control foot slight (sl) Observable, but insignificant swell- ing in test foot plus (+) Observable difference in swelling be- tween test and control foot. Positive reaction Preparation of Transfer Factor Dialyzable transfer factor (TFd) was prepared from three sources: bovine peripheral blood cells, bovine lymph node cells, and murine spleen cells. Bovine Peripheral Blood Transfer Factor TFd was prepared from the peripheral blood mononuclear leuko- cytes of heifer #230, a strain 19 hyperimmunized animal displaying positive DTH skin reactivity to Brucellergen. Blood (1,500 ml) was collected from the jugular vein into flasks containing a 10% EDTA (J. T. Baker Chemical Co., Phillipsburg, N.J.) solution. The blood was centrifuged in 250 ml centrifuge bottles at 2300 rpm (1,400 x g) (IEC-PR 6000 centrifuge) for 30 minutes at 4°C. The clear plasma layer was decanted and the leukocyte-rich buffy coat layer was care- fully aspirated and mixed in an equal volume of EDTA-phosphate buffered saline (EPS). This suspension was centrifuged at 2,000 rpm (800 x g) for 15-20 minutes and the buffy coat layer aspirated and mixed with EPS as before. Ten to 12 ml of the buffy coat suspension was carefully layered over 3 ml of Ficoll-Hypaque solution in plastic screw-cap tubes (Corning, Corning, N.Y.), and centrifuged at 2,000 rpm for 30 minutes at room temperature. This method of density—gradient centrifugation yields a distinct white layer at the Ficoll-Hypaque- 55 supernatant interface consisting of greater than 95% mononuclear leu- kocytes. Erythrocytes and granulocytes are centrifuged through the Ficoll-Hypaque gradient to the bottom of the tube. The mononuclear cell layer, and part of the Ficoll-Hypaque layer if it contained cells (indicated by cloudiness), was carefully removed and mixed with phosphate buffered saline (PBS) (Gibco, Grand Island, N.Y.). The cells were washed free of Ficoll-Hypaque by two cycles of centrifuga- tion and resuspension in PBS. Cell viability was determined by try- pan blue exclusion (123), cell counts were made with a hemacytometer, and an air-dried smear was stained by Wright's stain for a differen- tial cell count. The cells were then sedimented by centrifugation at I 1,300 rpm (350 x g) and resuspended in sterile distilled water to a total volume of 8.5 ml. This suspension was frozen and stored at -20°C. A total of 2.2 x109 viable mononuclear cells were isolated from 1,500 m1 of blood. The cell lysate was prepared by adding 1 mg/ml bovine pancreatic DNase (Nutritional Biochemical Co., Clevelend, Ohio) and 75 mg/ml Mg 504'7H20 to the cell suspension and submitting the suspension, in plastic screw-cap tubes, to 10 freeze-and-thaw cycles in a dry ice-90% alcohol bath and 37°C water bath respectively. Cell lysis was confirmed by observing a wet mount microscopically. The lysate was dialyzed against 25 volumes of sterile distilled water using 7/8" dialysis tubing (A. H. Thomas Co., Philidelphia, Pa.). Dialysis was carried out at 4°C with gentle agitation provided by a magnetic stirring bar. The dialysate was replaced at 24 hours with fresh sterile distilled water and dialysis was continued another 24 hours. The dialysates were then combined, shell frozen, and 56 lyophilized (New Brunswick Lyophilizer, New Brunswick, N.J.). The lyophilized powder from an original suspension of 2 x 109 viable mononuclear cells was dissolved in 6.0 ml saline. The prepar- ation was filter steriliZed (0.22 pm) (Millipore, Bedford, Mass.) and stored at -20°C until use. Bovine Lymph Node Transfer Factor TFd was prepared from lymph nodes collected from heifer #7 at ta necropsy post-partuition. The major lymph nodes, stored at -20°C since necropsy (one month), were thawed, trimmed of extraneous fatty tissue, and coarsely minced with scissors. Weighed portions were mixed with saline and ground to a fine slurry using a Waring blender. E This slurry was expressed through a metal screen to remove gross material and diluted to a measured total volume with additional sal- ine to contain 0.1 grams lymph node/ml. The lymph node slurry was pipetted to screw-cap plastic tubes (6-7 ml per tube), 25 mg/ml MgSO4-7H20 and a few crystals of DNase were added to each tube, and the cells were further lysed by 10 freeze-thaw cycles as before. The lysates were combined and dialyzed against approximately 10 volumes of sterile distilled water for 24 hours at 4°C with slight agitation. Because of the large volumes of lysate and dialysate involved, a second 24 hour dialysis was not done. The dialysate was shell frozen and lyophilized as before. The lyophilized powder was reconstituted in sterile distilled water to yield a final concentration of 0.5 gram-equivalents/ml in one prepar- ation and 0.1 gram-equivalents per ml in a second preparation. A gram-equivalent is defined as the lyophilized powder obtained from one 57 gram of original lymph node material. Experience in our lab indicates that 1 gram of freshly harvested lymph node will yield roughly 1x109 cells. One gram of lymph node prepared as above yielded ap- proximately 200 mg of lyophilized powder. The TFd preparations were filter sterilized (0.20 pm) (Nalgene) and stored at ~20°C until use. Murine Transfer Factor TFd was prepared from the spleen cells of sensitized mice by the method of Rifkind et_al, (130). Lot #1 was prepared from mice used for the initial evaluation of the footpad test and reinfected with strain 19 three weeks before spleens were harvested. Lot #2 and Lot #3 were prepared from mice infected four weeks earlier with strain 2308 and strain 19 respectively. These mice had been footpad tested for DTH 21 days after infection, and only those mice showing DTH reactivity were used. Lot #4 was prepared from mice that had been sensitized by repeated footpad tests. A negative control TFd was prepared from unsensitized mice. Spleens were aseptically collected and kept in cold M-l99 tissue culture medium (Gibco, Grand Island, N.Y.) during processing. Initially (Lot #1) cells were dispersed by expressing coarsely minced spleens through a Ten Broeck glass tissue grinder. Since this treatment could conceivably release transfer factor activity from the cells, a modified procedure was used in subsequent preparations (Lots #2, 3, 4). Spleen cells were dispersed by injection of 1 ml M-l99 into the spleen, fbllowed by manipulation with forceps to tease the cells free from the spleen capsule. The spleen cells were further dispersed by gently drawing the suspension several times 58 through a 23-gauge needle. The cells were washed three times in M-199 by centrifugation, 1,000 rpm (200 x g), and resuspension. Viability was determined by trypan blue exclusion and cell count de- termined using a hemacytometer. The cell suspensions were adjusted to 5 x108 cells/ml in M-199 and stored frozen at -20°C. Lysates were prepared as before by 10 freeze-thaw cycles follow- ing the addition of 50 mg/ml MgSO4-7H20 and a few crystals of DNase. a, The lysates were dialyzed at 4°C against 10 volumes of sterile dis- tilled water with slight agitation. The dialysate was replaced at 24 hours with fresh sterile distilled water and dialysis was contin- ued another 24 hours. To avoid filter sterilization of the small volume of final reconstituted product, the dialysates were filter sterilized (.22 pH) (Millipore) prior to shell freezing and lyophil- ization. The lyophilized powder was reconstituted with sterile dis- tilled water to the same volume as the original lysate, therefore 8 spleen cell equivalents/m1. One ml of M-199 con- containing 5 x 10 taining 5 x 108 spleen cells yielded approximately 50 mg of lyophilized powder. Reconstituted TFd preparations were stored at -20°C. Assay of Transfer Factor Activity TFd was administered to mice by intraperitoneal injection, the route used by both Rifkind (130) and Klesius (87). Doses were de- termined by cell-equivalents for bovine peripheral-blood TFd and murine spleen-cell TFd, and by gram-equivalents for the bovine lymph- node TFd. Whole viable cells were administered to mice by intraper- itoneal injection of bovine cells and by intravenous injection of murine cells. 59 Initial experiments indicated that large doses of TFd were toxic to mice, causing death in some. Dose-response studies however clearly revealed that the toxicity was due to the magnesium sulfate concentration in the preparations. Mice could be killed by a single intraperitoneal dose of 20 mg MgSO4'7H20. Subsequent large TFd doses were administered by several injections over a period of time to avoid this toxic effect. This problem was not encountered when normal doses of TFd were administered. Recipient mice were tested for DTH reactivity using the footpad assay 48 hours, 72 hours, or 7 days following the administration of “TFd or whole cells. The footpad test was usually repeated one week later. Mice were not footpad tested before receiving TFd or whole cells. Histological Studies Footpads submitted for histological study were fixed in formalin. Paraffin-embedded, hematoxylin-and-eosin stained sections were prepared by the Animal Health Diagnostic Laboratory at Michigan State Univer- sity, East Lansing, Michigan. Both saline- and antigen-injected foot- pads were examined. Preparation of Chemical Reagents EDTA-phosphate buffered saline (EPS) 3.58 gm EDTA - trisodium salt (Sigma Chemical Co., St. Louis, M0.) 9.0 gm NaCl 1.08 gm KH2P04 1,000 ml distilled H20 The solution was filter sterilized (0.20 pm) (Nalgene) and adjusted to pH 7.2 - 7.4 before use. 60 Ficoll-Hypaque Density Gradient 72.8 gm Ficoll 400 (Pharmacia, Piscataway, N.J.) 480 m1 Hypaque (sodium diatrizoate) 25% aqueous solution (Winthrop Labs., New York, N.Y.) 720 ml distilled H20 Specific gravity was determined to be 1.076 to 1.079 using a hydrometer (Arthur Thomas Co., Philadelphia, Pa.) and the solution was filter sterilized (0.20 pm) (Nalgene) and stored at 4°C. RESULTS Brucellergen Standardization in Rabbits The allergic potency of the Brucellergen preparation was assayed by intradermal injection of serial dilutions of the preparation into rabbits infected with Brucella abortus strain 2308. TWo rabbits (STl and 74H) were skin tested 30 days after infection, as outlined orig- inally by Huddleson for standardization of Brucellergen for human use (60). They were skin tested again 7 weeks after infection. TWo rabbits infected 1 year earlier and two non-infected rabbits were also tested. The results are shown in Table l. The rabbits infected for 30 days showed positive, indurated DTH responses at 1:1,000 and 1:4,000 dilutions, though the reactions at 48 hours were less than the 5 mm standard set by Huddleson (60). The reactivity of the rabbits infected a year earlier was stronger, showing positive reactions at 1:8,000 and reactions of 5.0 mm at the 1:4,000 dilution. Skin tests applied to STl and 74H at 7 weeks fol- lowing infection showed enhanced reactivity. Positive reactions were seen to persist for 72 hours, when measurements were taken at that time. In addition to palpable induration, erythema and edematous swelling were frequently noted in infected rabbits, indicating concur- rent Arthus-type hypersensitivity. 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Typ- ical indurated DTH responses were seen in the immunized cattle, but not in the non-immunized cattle, when examined at 48 and 72 hours. The results are shown in Table 2. Table 2. Delayed Hypersensitivity Responses in Cattle to Brucellergen Diameter of Induration Cattle 48 hours 72 hours Immunized2 #230 19.5 mm 14.3 mm #249 12.2 nm 8.2 11111 Non-Immunized #221 0 0 #10 0 0 1Measured 48 and 72 hours after intradermal injection of 1:1,000 Brucellergen (0.1 ml). 2Brucella abortus Strain l9 vaccinated. Footpad Reaction in Mice to Brucellergen The footpad assay was used to test the DTH response of infected mice to Brucellergen. Antigen was injected into the footpad of the right hind foot and the antigen solvent (phenolized saline) was in- jected into the left footpad. At 24 and 48 hours, and in one study 64 at 6 hours, the swelling of the two footpads was compared and mea- sured. Values were expressed as swelling of right footpad minus swelling of left footpad (test minus control). Table 3 shows the results obtained using 3 dilutions of Bru- cellergen. The 1:500 dilution eliCited maximal swelling in infected mice and no response in control mice. This dilution was therefore used throughout this study in the footpad assay. In preliminary k studies, a 1:100 dilution was found to cause non-specific footpad swelling in non-sensitized mice. The 1:4,000 dilution elicited only weak reactions in infected mice. Table 3. Delayed Hypersensitivity Response in Mice to Three Dilu- ; tions of Brucellergen1 Antigen Footpad Reaction at 24 Hours2 ”'1“t'°" 1:500 1 1,000 1 4,000 Infected3 Mice 59(10/10) 52 (8/9) 15 (1/5) Non-Infected Mice 4 (0/7) 7 (0/2) 4 (0/5) 1Eiicited by injection of 0.025 ml antigen dilution into hind footpad. 2Expressed as the mean footpad swelling, in 0.01-mm increments, of all mice tested (number positive reactions/total mice tested). Values for 1:500 and 1:1,000 combined from two experiments. 3Infection with Brucella abortus strain 19 or 2308. The footpad reactions of strain 19 infected and strain 2308 infected mice are shown in Tables 4 and 5 respectively. The footpad assay was applied to these mice at 21 days after infection, a time when Brucella-infected mice have been shown to exhibit strong DTH 65 Table 4. Delayed Hypersensitivit Response 'U) Brucellergen in Strain l9 Infected Mice 2 Mice3 Footpad Reaction at 6 hours 24 hours 48 hours Group A Infected 58 (4/6) 54 (6/6) 30 (6/6) Non-Infected 17 (0/6) -3 (0/6) -3 (0/6) Group B Infected not done 39 (ll/ll) 15 (8/11) Non-Infected not done 6 (0/6) 7 (0/6) 7'? 1Eiicited by injection of 0.025 ml antigen (1 500) into hind footpad. 2Expressed as the mean footpad swelling, in 0.01-mm incre— ments,)of all mice tested (number positive reactions/total mice tested . cells/mouse. cells/mouse. 001 3Group A mice infected with 1.8 x 10 Group 8 mice infected with 5.3 x 10 Table 5. Delayed Hypersensitivity Response to Brucellergen in Strain 2308 Infected Mice Footpad Reaction2 at Mice 24 hours 48 hours Infected3 51 (23/24) 40 (22/24) Non-Infected 4 (0/14) 3 (0/14) lEiicited by injection of 0.025 ml antigen (1:500) into hind footpad. 2Expressed as the mean footpad swelling, in 0.01-mm incre- ments,)of all mice tested (number positive reactions/total mice tested 3 6 6 Infected with 4.8 x 10 - 5.0 x 10 cells/mouse. 66 reactivity (107). Good DTH responses, characterized by swelling and erythema at 24 and 48 hours, were seen in mice infected by either strain of Brucella. Swelling persisted in positive reactions for 48 hours, although the swelling at 48 hours was quantitatively less than at 24 hours. In one group where reactions were measured at 6 hours, significant swelling was observed in 4 of 6 mice tested, possibly due to an Arthus-type reaction. A small degree of swelling was also evi- dent at 6 hours in non-infected controls, a non-specific early re- sponse also reported by Youdim gt_al, (160). The macroscopic findings correspond to those reported fbr footpad DTH reactions by others (33, 72, 129). Sensitization of Control Mice by Repeated Footpad Tests When the footpad test was repeatedly applied to mice that had received no other treatment, it was noted that a number of mice showing no reactivity in the initial (1°) test, showed positive reactions in a second (2°) footpad test one week later. When data was compiled from several experiments (Table 6), it was evident that the 1° footpad test had sensitized a considerable number of mice. An even greater proportion of the mice showed reactivity in a third (3°) consecutive footpad test, applied 1 week after the 2° test. These reactions were of equal intensity and could not be distinguished from the reactions displayed by infected mice. Since antigen had been injected into the same footpad in both 1° and 2° tests, two experiments were done in which the opposite footpad was injected with antigen in the 2° test. The results (Table 6) show that not only was a response elicited in the opposite 67 _Pc eo .mucoemcocw esipo.o cw .mcso; «N am mcwppmzm ueguooe come on» we ummmmcaxm .m_a>cooee xooz _ cc aaaoaac ee_e one? 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No footpad reactivity was demon- strated one week later by a second footpad test at 1:4,000. In an- other experiment, mice were primed by subcutaneous injection of 0.1 ml of a 1:2,000 dilution of Brucellergen, a total antigen dose equiv- alent to that used in the footpad assay (0.025 ml of 1:500 Brucel- lergen). No response was seen in 10 of 12 mice when footpad tested one week later. TWo mice showed reactions with quantitative values of 20 and 34 respectively. Histology of the Footpad Reaction Histological studies of the footpad reaction in mice sensitized by either infection or repeated footpad testing were done on sections of the footpads taken at 24 and 48 hours after antigen injection. A marked cellular infiltrate was seen in both reactions. At 24 hours the infiltrate was composed of PMN's and mononuclear cells in approx- imately equal numbers. At 48 hours mononuclear cells were predominant, with few PMN's seen. Transfer Factor Activity in Mice The TFd preparations described in Materials and Methods were used in an attempt to transfer DTH to Brucella from sensitized ani- mals to non-sensitized mice. Transfer was also attempted in several experiments using whole cells, collected and isolated in the same 69 manner as the cells used for preparation of TFd. These cells were not lysed, but injected as intact viable cells. The results of transfer experiments using bovine (#230) per- ipheral leukocytes are shown in Table 7. Preliminary experiments using whole cell preparations in a small number of mice showed no footpad reactivity in recipient mice at the 1° test applied 72 hours after administration of the cells. Positive responses were seen in two of these recipient mice at a 2° footpad test one week later. In an attempt to induce a greater response, a larger dose (3 x in8 cells) was administered. No significant response was seen in the recipient mouse at the 1° or 2° footpad test. To test the possibility that an interval of more than 72 hours was necessary for transferred whole cells to render the recipient mice DTH reactive, an experiment was done in which the 1° footpad test was not applied until 7 days after injection of cells. In this set of experiments, using a larger number of mice, there was no apparent evidence of DTH transfer. Experiments using several doses of TFd prepared from bovine (#230) per- ipheral leukocytes also failed to demonstrate transfer of DTH to re- cipient mice in either the 1° footpad test at 72 hours or the 2° test one week later (Table 7). The results of transfer experiments using bovine lymph node TFd are shown in Table 8. TFd was administered to mice in doses ranging from 0.5 to 0.005 gram-equivalents of original lymph node material. There was no demonstration of transfer of DTH as assayed by the 1° footpad test at 2, 3, or 7 days following administration of TFd. The apparent transfer demonstrated by the 2° footpad test in one experiment was not confirmed in an experiment using a larger 70 Table 7. Bovine Peripheral Blood Transfer Factor Activity in Mice.1 f j. (.0 2 Time Footpad Reactions4 Dose TESt 1° test 2° test Done Whole Cells 1.5 x 107 72 hr. 4 (0/3) 30 (2/3) control -7 (0/1) 8 (0/1) 3 x 108 72 hr. 7 (0/1) 18 (0/1) control -3 (0/1) 0 (0/1) i x 107 7 days 5 :_2.7 (1/15) 8 1_4.4 (4/15) control 0 1.2.2 (1115) 15 :,3.7 (5/15) TFd 1 x 108 72 hr. 5 :_3.2 (0/6) Not Done 5 x 107 o :_l.7 (0/10) i4 :,4 3 (3/9) 5 x 106 3 :,2.3 (0/10) 25 :_3.6 (7/10) control 5 :_1.5 (0/10) 42 :_10.9 (6/10) 1 Elicited by injection of 0.025 ml antigen (1:500) into hind footpad. 2Dose of whole cells or cell-equivalents of TFd administered by intraperitoneal injection to test mice. Controls received no cells or TFd. 3Interval between administration of TFd and injection of antigen for primary test. Secondary test applied one week following primary test. 4Expressed as the mean fbotpad swelling at 24 hours, in 0.01-mm increments, of all mice tested :_standard error (number positive reactions/total mice tested). 71 1 Table 8. Bovine Lymph Node Transfer Factor Activity in Mice The3 F t dR t' 4 TFdz 1. 00 pa eac ions Dose Test 0 0 Done 1 test 2 test 0.50 72 hr. 2 :_4.7 (0/3) 1 :_1.8 (0/3) 0.05 6 i 2.1 (0/4) 14 1 6.5 (4/4) 0.005 2 1 4.7 (0/4) 24 :_10.3 (2/4) control -3.: 2.9 (0/4) -7 :_4.0 (0/4) 0.05 7 days -2 i 3.6 (0/10) 0.005 1 i 2.2 (0/10) Not Done control 0 :_3.8 (0/5) 0.50 48 hr. 0 i 2.2 (0/10) 38 :_6.1 (9/10) 0.10 4 :_2.0 (0/9) 25 :_5.5 (7/9) 0.05 0 :1.0 (0/10) 34 1 7.4 (8/10) 0.005 5 :_2.7 (0/10) 23 :_7.4 (3/10) 0.05 heat 4 :;2.6 (0/9) 26 :_7.6 (4/9) treated 5 :_1.5 (0/10) 42 :_10.9 (6/10) control 1 Elicited by injection of 0.025 ml antigen (1:500) into hind footpad. 2Dose of gram-equivalents administered by intraperitoneal in- jection to test mice. Controls received no TFd. 3Interval between administration of TFd and injection of antigen for primary test. Secondary test applied one week following primary test. 4Expressed as the mean footpad swelling at 24 hours, in 0.01-mm increments, of all mice tested i standard error (number positive reactions/total mice tested). 72 number of mice. One preparation was heated at 56°C for 30 minutes, a treatment reported to destroy transfer factor activity (95). Mice receiving this preparation showed no significantly different reactiv- ity at either the 1° or 2° footpad test. Since the failure to demonstrate transfer of DTH in these exper- iments could be attributed to difficulty of cross species transfer and/ or the unknown DTH status of heifer #7, transfer factor was prepared from Brucella infected mice showing strong footpad reactivity to Bru- r cellergen. Only those mice showing positive reactions were used as I TFd donors; the mean footpad reaction being 61 overall and less than 50 in only one preparation (Lot #3). The results of experiments using these TFd preparations are shown in Table 9. i Experiments using a TFd preparation (Lot #1) from strain 19 in- fected mice showed some evidence of DTH transfer activity. In one ex- periment using doses of 5 x 107 and 5 x 106 cell-equivalents, statis- tically significant (student's t test, P §_.01) DTH reactivity was detected by the 1° footpad test on mice receiving 5 x 107 cell-equival- ents, but not on mice receiving 5 x 106 cell-equivalents. No signifi- cant difference was demonstrated in the DTH reactivity of test and control mice in the 2° footpad test applied one week later, though the relative number of "positive" reactions, determined by subjec- tive evaluation, was higher in mice that had received TFd. In a 8 cell- second experiment, a 5-fold higher dose of TFd (2.5 x 10 equivalents) was administered in an attempt to induce greater reactivity in the 1° footpad test. No significant difference be- tween test and control animals was demonstrated, however, by the 1° test. A single mouse in the TFd treated group showed reactivity (value 51) in the 2° test, but the mean reactivity was not 73 Table 9. Murine Transfer Factor Activity in Mice1 - 3 Preparation* T1Te Footpad Reactions4 and 2 Test 0 0 Dose Done 1 test 2 test TFd Lot #1 Prep. #1 5 x 107 48 hr. 15 i 3.3 3/8) 29 i 5.4 (5/5) 5 x 10° 7 i 3.4 (1/7) 25 18.7 (4/5) control -1 :_3.0 (0/8) 19 :_8.7 (2/5) 2.5 x 108 48 hr. 4 1 2.3 (0/3) 25 i 15.3 (1/3) control 10 11.5 (0/2) 18 30.7 (0/2) TFd Lot #1 Prep. #2 5 x 107 48 hr. 5 i 2.3 (0/7) 55 i 9.5 (577) control -3 :_1.8 (0/12) 29 :_6.2 (8/12) TFd Lot #2 5 x 107 48 hr. 1 :15 (0/9) 29 _+_ 5.5 (5/9) 5 x 10° -1 i 2.5 (0/10) 38 :8.3 (8/10) 5 x 105 3 11.9 ‘(0/9) 35 i 7.9 (5/9) 5 x 107 -2 :15 (0/10) 22 _+_ 5.3 (5/10) heat treated control -3 i 1.8 (0/12) 29 i 5.2 (8/12) *Lot #1 prepared from strain 19 infected mice; Lot #2 prepared from strain 2308 infected mice; Lot #3 prepared from strain 19 infected mice; Lot #4 prepared from non-infected mice sensitized by repeated footpad tests. Negative TFd prepared from non-sensitized mice. 74 Table 9. (Continued) ' 3 . 4 Preparation* T4Te Footpad Reactions 0:222 33:: 1° test ‘ 2° test TFd Lot #3 5 x 107 48 hr. 1 i 3.3 (0/7) 33 _+_ 9.2 (4/7) control 2 i 2.5 (0/7) 34 i 5.3 (5/7) TFd Lot #4 5 x 107 48 hr. -1 _t 2.8 (0/7) 42 i 7.5 (5/7) control 2 .t 2.5 (0/7) 34 i 5.0 (5/7) Whole Cells Lot #4 5 x 107 5 days 0 i 2.5 (on) control 7 :_2.6 (0/7) Negative TFd Prep. 5 x 107 48 hr. 3 .t 2.3 (0/10) 30 :4.0 (7/10) control -3 :_l.8 (0/12) 29 :_6.2 (8/12) 1Elicited by injection of 0.025 ml antigen (1:500) into hind footpad. 2 Dose of whole cells or cell-equivalents 0f TFD administered by intraperitoneal injection to test mice. Controls received no TFd. 3Interval between administration of TFd and injection of antigen for primary test. Secondary test applied one week following primary test. 4Expressed as the mean footpad swelling at 24 hours, in 0.01-mm increments, of all mice tested :_standard error (number positive re- actions/total mice tested). 75 significantly different between test and control animals. A second TFd preparation was made from an aliquot of the non- dialyzed cell lysate that had been stored at -20°C. Mice receiving 5 x 107 cell-equivalents of this preparation did not show evidence of DTH transfer in the 1° f00tpad test. Strong DTH reactions were seen in the 2° footpad test in 6 0f 7 mice that had received TFd. A TFd preparation (Lot #3) from a second group of strain 19 in- fected mice failed to transfer DTH to recipient mice as assayed by 1° and 2° f00tpad tests (Table 9). Similarly, a preparation (Lot #2) from strain 2308 infected mice administered at several doses failed to transfer DTH. In the latter experiment, a group of mice received a heat treated (56°C for 30 minutes) TFd preparation. These mice showed no significant difference in reactivity in the footpad tests. Transfer of DTH was also attempted using spleen cell prepara- tions (Lot #4) from mice that had been sensitized by repeated footpad testing. These donor mice showed strong reactivity in a footpad test applied one week earlier (mean 85, range 68-130). One aliquot of the spleen cells was injected intravenously as intact viable cells and a second aliquot was used to prepare TFd. Mice receiving either of these two preparations failed to demonstrate transfer of DTH (Table 9). A TFd preparation from spleen cells of non-sensitized mice was administered as a negative control. The f00tpad reactivity of mice receiving this preparation was not significantly different when com- pared either to non-treated controls or to mice, in a concurrent ex- periment, that had received TFd (Lot#2) from Brucella infected mice (Table 9). DISCUSSION A model for testing the 0TH status of mice to Brucella anti- gens was developed to measure the transfer of DTH from sensitized animals to non-sensitized mice with TFd. It was hoped this model could be used as a research tool to study transfer factor. The mouse, a readily available and easily maintained animal, would also provide a convenient in yiyg_assay of the biological activity of TFd prepar- ations before their use in other animals. The ability to use the Brucellergen preparation to assay DTH was demonstrated by typical DTH reactions to intradermal injection of the antigen in sensitized rabbits and cattle, and by positive foot- pad reactions in mice. N0 macroscopic reactions were seen in unsen- sitized animals. The reactivity in rabbits 30 days after infection with Brucella was not as great as that reported in the standardization techniques of Huddleson (60, 114), where 5 mm of induration was usually seen with the 1:8,000 dilution. It is of interest that rabbits in- fected nearly a year earlier showed better DTH responses than the 30- day infected rabbits. The enhanced DTH response seen in rabbits 7 weeks after infection may indicate that maximum DTH reactivity is not attained by 30 days. The footpad assay, using Brucellergen as the test antigen, was shown to be a convenient measure of DTH reactivity to Brucella in mice in accordance with earlier reports using other antigen preparations 76 77 (107, 138). The DTH response was characterized macroscopically by swelling and erythema at 24 and 48 hours and microscopically by a mixed PMN-mononuclear infiltrate in the 24-hour reaction which was replaced by a predominant mononuclear component at 48 hours. An Arthus-type reaction may also have been displayed as evidenced by the swelling at 6 hours and the PMN component at 24 hours. This macro- scopic and histological picture in the f00tpad of a DTH reaction pre- ceded by an Arthus reaction has recently been described by Katsura gt al.(72). Since humoral antibody production is part of the immune response in brucellosis, Arthus reactivity in infected mice could be i expected. Positive reactions were readily evident by subjective observa- tion, and this method could be used where exact measurements are not required or where large numbers of mice are being used. Although measurement of the reaction with calipers is more tedious, consistent measurements can be achieved and may be more useful in analysis of experimental data. An unexpected result was the apparent DTH sensitization of mice by footpad testing with the Brucellergen preparation. Earlier stu- dies indicated that an antibody response could be stimulated in guinea pigs by the injection of relatively large amounts of Brucel- lergen (142), and occasionally in man by a single Brucellergen skin test, whether that test was positive or negative (60, 112). Repeated skin tests using normal human doses (1:8,000 to 1:10,000) did not re- sult in positive skin tests (60). Stimulation of antibody production by repeated skin tests has also been demonstrated in this study in rabbits using a 1:1,000 dilution of Brucellergen. A DTH response 78 was not observed in these rabbits. Mice however displayed a delayed-type footpad reaction, char- acterized by swelling and erythema, following repeated footpad test- ing with Brucellergen. About 40% of the negative control mice showed a reaction one week after the first footpad test and most of the mice showed reactions one week after the 2° test. The time course of the reaction resembled a DTH reaction with swelling at 24 and 48 hours. Histologically the 24 hour reaction contained a mixed PMN-mononuclear infiltrate with a much more predominant mononuclear component at 48 hours. The sensitization was shown to be more than a local phenomenon by the ability to elicit the reaction in the opposite foot in the 2° footpad test. A weak antibody titer (:_1/20) was occasionally seen in serum drawn one week after the 1° test. A number of mice showing strong footpad reactions at the 2° test had no detectable antibody. A rise in the antibody titers was seen following repeated footpad testing (data not shown). The discrepancy between these results and results in other ani- mal species is probably due to the high concentration of antigen used in the mouse footpad assay. Though the total antigen in 0.025 ml of a 1:500 dilution is equivalent to that in 0.1 ml of a 1:2,000 dilu- tion, the mouse itself is significantly smaller than these other Species. DTH was not induced or detected in an experiment using a 1:4,000 antigen dilution in the f00tpad test. It should also be noted that the intradermal route of antigen injection has been found to be the most efficient route for DTH sensitization in the mouse (32) and that injection into the footpad probably mimics intradermal in- jection. In one experiment, subcutaneous injection of an equivalent 79 amount of Brucellergen used in the footpad test (0.1 m1 of a 1:2,000 dilution) failed to significantly sensitize mice as assayed by the normal f00tpad test ten days later. Whether the delayed reaction described above is a classic tuberculin-type DTH reaction can not be ascertained. It is possible that this is an example of cutaneous basophil hypersensitivity (CBH). The early appearance (7 days) of the reaction and the induction by in- jection of a non-viable protein antigen without adjuvant, in fact Fk favors this interpretation. In one experiment (data not shown) no footpad reaction was seen in control mice that had been footpad tested 2 months previously. Though this is a relatively long inter- é val, it does indicate the sensitivity is not long lasting, a charac- teristic of CBH. Dvorak gt_al, (45, 46) have reported that basophils are not detected by the tissue preparation methods used in this study. In experiments using the footpad assay to evaluate transfer of Brucella DTH from sensitized animals to nonsensitized mice, there was evidence of transfer activity in one TFd preparation, Lot #1, prepared from strain 19 infected mice. Administration of this TFd to mice provided the only evidence of transferred sensitivity detect- able in the 1° footpad test. Mice having received 5 x 107 cell- equivalents 48 hours earlier displayed weak, but statistically sig- nificant responses in the 1° test compared to control mice. Subse— quent experiments using a higher dose of the same preparation and a second preparation from the same cell source did not show significant difference in 1° test footpad reactivity of test and control mice, although there was evidence of enhanced reactivity in test mice in 80 the 2° footpad test, especially in those that received preparation #2. Enhanced reactivity in the 2° footpad test was also evident in TFd- or whole cell-recipient mice in several other experiments. One of these involved mice given bovine (#230) peripheral leukocytes and another involved mice given bovine (#7) lymph-node TFd. Evidence for transfer of DTH provided by the 2° f00tpad test must however be eval- uated with caution in light of the demonstrated sensitization of mice by the 1° footpad test. A number of unknown variables are involved in this sensitization. A possible explanation of the enhanced re- activity seen in some of these cases is that TFd treatment may in fact have an adjuvant effect on the sensitization induced by the 1° footpad test. Although this would represent a TF activity proposed by some authors (11), it would not be the classic activity of TF this study has attempted to demonstrate; that is the conversion of DTH reactivity in antigen-naive mice demonstrated by the 1° footpad test a short time (48-72 hours) following TFd treatment. As mentioned above, this type of reactivity was seen in mice receiving one murine TFd preparation, but not in mice receiving other murine or bovine TFd and whole cell preparations. The failure to consistently demonstrate transfer of DTH was disappointing in light of several recent reports of successful trans- fer of DTH to mice. Klesius gt_al, (87) have transferred sensitivity to PPD using a bovine lymph-node TFd preparation. Rifkind gt_al, (130, 131) have transferred sensitivity to Coccidioides, Candida, and Mycobacteria with a murine TFd prepared in the same manner as the murine TFd in this study, and Lawrence's group has reported transfer of sensitivity to several antigens using a murine lymph-node TFd 81 preparation (14). Successful mouse-to-mouse transfer with whole lymphoid cells has also been reported (33, 129, 160), although we have found no report of transfer of DTH to Brucella by either whole cells or transfer factor. Several explanations may be offered for the failure to consis- tently demonstrate transfer. Cross-species transfer, though reported by Klesius, has in general been difficult to demonstrate. TF donors I ’3" also need to display strong DTH reactivity, an unknown factor in heifer #7. Both of these problems should not be factors in the ex- periments involving mouse-to-mouse transfer with TFd prepared from mice displaying strong (mean value 61) DTH reactivity. The authors cited above have not, however, reported the DTH reactivity of TFd- donor animals, so direct comparisons can not be made. Methods of preparation may be questioned, especially in the bovine preparations which represented modifications of previously re- ported techniques. Methods of cell collection and isolation were evaluated by the viability of the cell suspensions after processing. Whole cell or cell-equivalent TFd doses were then calculated as viable cell numbers, on the assumption that viable cells had retained their transfer factor moieties. The failure to transfer DTH with either whole bovine mononuclear peripheral cells or whole murine spleen cells indicates that the primary problem, at least, is not due to methods involved in preparation of TFd after the harvesting of cells. The doses of TFd used were in accord with those reported by others (87, 129, 130, 160). Doses both higher and lower than these were employed, ranging from 2.5 x 108 cell-equivalents to 5 x 105 82 cell-equivalents. Youdim at al, (160) demonstrated that 51(107, but not 1 x 107 mouse spleen cells, would transfer DTH to Listeria. This (5 x 107) is the TFd and whole cell dose regularly employed by Rifkind gtal.(129, 130). Primary footpad tests were applied 48 hours, 72 hours, and 7 days f01lowing TFd administration. Classically, transferred DTH is demonstrable in humans within 48 hours (95). Rifkind gt al, (130) demonstrated transferred DTH in mice as early as 24 hours after TFd administration. The use of the footpad assay to test DTH conversion may be questioned. Although others have used this assay to test DTH to Brucella antigens (107, 138) and the evidence in this study indicates that DTH reactivity is responsible far the f00tpad swelling, a com- prehensive study of the use of the footpad assay in mice sensitized to Brucella has not been conducted. Such a study would be beneficial in the interpretation of the negative transfer data presented above. One factor in the experiments reported here that is not present in any of the reports of successful DTH transfer to mice cited above, is the use of Brucella antigens. Klesius g_.al. have reported trans- fer of reactivity to Brucella antigens in mice as assayed by an in. yitrg_lymphocyte stimulation assay (82) but not by footpad reactivity (Klesius-personal communication). A significant non-specific reac- tivity has been noted under certain conditions in this assay (82). Brucella organisms and the imnune response they elicit may be significantly different than other organisms for which successful DTH transfer has been achieved. The role of these differences has not been resolved in this study. Spleen cells were harvested for TFd 83 preparation at a time following Brucella infection when f00tpad reac- tivity has been shown to be maximal (107). This may not have been, however, the optimal time in the immune response to prepare TFd from the donor animals. It is conceivable that a suppressor moiety or suppressor cell stimulating moiety could have been present in cells collected at this time. The role of recipient priming by environ- mental exposure to microbial antigens prior to administration of TFd may be important in comparing transfer of DTH to relatively rare organisms such as Brucella to transfer of DTH to more commonly en- countered organisms such as Candida or Mycobacteria. It should also be noted that mice are relatively resistant to Brucella infection, showing few signs of disease when infected with doses that cause serious illness in some other species. The mouse, therefore, may not be an optimal model to study certain aspects of brucellosis, among these transfer of DTH reactivity. Transfer of DTH to Brucella was not consistently demonstrated by the methods or preparations used in this study. It should be noted that initial studies in transfer factor research have often yielded negative data, until new methods or models were developed (13, 62, 95). Modification of one or several aspects of the pro- cedures used here might lead to unequivocal demonstration that DTH to Brucella can be transferred using TFd in mice. BIBLIOGRAPHY 10. BIBLIOGRAPHY Alton, G. G., and L. M. Jones. 1967. Laboratory Techniques in Brucellosis. World Health Organization Monograph. Series No. 55. Geneva, Switzerland. Arala-Chaves, M. P., A. Silva, M. T. Porto, A. Picoto, M. T. F. Ramos, and H. H. Fudenberg. 1977. In_Vitro and In_Vivo Studies of the Target Cell for Dialyzable Leukocyte Extracts. Evidence :2; Recipient Specificity. Clin. Immunol. Immunopath. 85430- Arnason, B. G., and B. H. Waksman. 1963. The Retest Reaction in Delayed Sensitivity. Lab. Invest. 123737-747. Ascher, M. S., W. J. Schneider, F. Valentine, and H. S. Lawrence. 1974. In_Vitro Properties of Leukocyte Dial zates Containing Transfer Factor. Proc. Nat. Acad. Sci. (USA . 2151178-1182. Ballow, M., and L. R. Hyman. 1977. Combination Immunotherapy in Chronic Mucocutaneous Candidiasis. Synergism Between Transfer Factor and Fetal Thymus Tissue. Clin. Immunol. Immunopath. 8:504-512. Baram, P., L. Yuan, and M. M. Moska. 1966. Studies on the Transfer of Human Delayed-Type Hypersensitivity. I. Partial Purification and Characterization of Two Active Components. J. Immunol. 91:407-420. Basten, A., S. Croft, and J. Edwards. 1976. Experimental Studies of Transfer Factor, pp. 75-84. Ig_M. Ascher, A. Gottlieb, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clinical Applications. Academic Press Inc., N.Y. Benedict, A. A., and S. S. Elberg. 1953. Cutaneous Hypersen- sitivity in Brucellosis. J. Immunol. 19:152-164. Bhongbhibhat, N., S. Elberg, and T. H. Chen. 1970. Character- ization of Brucella Skin-Test Antigens. J. Infect. Dis. 122: 70-82. Blanden, R. V., M. J. Lefford, and G. B. Mackaness. 1969. The Host Response to Calmette-Guerin Bacillus Infection in Mice. J. Exp. Med. 12931079-1107. 84 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 85 Bloom, 8. R. 1973. Does Transfer Factor Act Specifically or as an Immunologic Adjuvant? N. Eng. J. Med. 113908-909. Bloom, 8. R., and 8. Bennett. 1966. Mechanism of a Reaction In_ Vitro Associated with Delayed-Type Hypersensitivity. Science 1“—_5_3_:80-82. Bloom, 8. R., and M. W. Chase. 1967. Transfer of Delayed-Type Hypersensitivity. A Critical Review and Experimental Study in the Guinea Pig. Progr. Allergy 193151-255. Brummer, E., N. Bhardwaj, L. Foster, and H. S. Lawrence. 1978. A Mouse Model f0r Studying the Transfer of DTH with Human TFd and Murine TFd. Abstr. of the 3rd. International Symposium on Transfer Factor. J. Clin. Hematol. Oncol. 83106. Burger, D. R., and W. S. Jeter. 1971. Cell-Free Passive Trans- fer of Delayed Hypersensitivity to Chemicals in Guinea Pigs. Infection and Immunity 43575-580. Burger, D. R., W. S. Cozine, and D. J. Hinrichs. 1971. The Passive Transfer of Chemical Hypersensitivity in Rabbits. Proc. Soc. Exp. Biol. Med. 13631385-1388. Burger, D. R., R. M. Vetto, and A. Malley. 1972. Transfer Factor from Guinea Pigs Sensitive to Dinitrochlorobenzene: Absence of Superantigen Pr0perties. Science 11531473-1475. Burger, D. R., R. M. Vetto, and A. A. Vandenbark. 1974. Prepar- ation of Human Transfer Factor: A Time Saving Modification for Preparing Dialyzable Transfer Factor. Cell. Immunol. 143332-333. Burger, D. R., A. A. Vandenbark, P. Finke, J. E. Nolte, and R. M. Vetto. 1976. Human Transfer Factor: Effects on Lymphocyte Transformation. J. Immunol. 1113782-788. Burger, D. R., A. A. Vandenbark, D. Daves, W. A. Anderson, Jr., R. M. Vetto, and P. Finke. 1976. Human Transfer Factor: Frac- tionation and Biological Activity. J. Immunol. 1123789-796. Burger, D. R., A. A. Vandenbark, P. Finke, and R. M. Vetto. 1977. Qg_Novo Appearance of KLH Transfer Factor Following Immunization. Cell. Immunol. 293410-413. Burnet, F. M. 1974. Transfer Factor. A Theoretical Discussion. J. Allergy and Clin. Immunol. 5431-13. Catanzaro, A., and L. Spitler. 1976. Clinical and Immunologic Results of Transfer Factor Therapy in Coccidioidomycosis, pp. 477-494. IQ_M. Ascher, A. G. Gottlieb, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clincial Applications. Academic Press Inc., N.Y. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 86 Chase, M. W. 1945. The Cellular Transfer of Cutaneous Hyper- sensitivity to Tuberculin. Proc. Soc. Exp. Biol. Med. 993134- 135. Chess, L., and S. F. Schlossman. 1977. Human Lymphocyte Sub- populations. Adv. Immunol. 993213-241. Clausen, J. E. 1971. Tuberculin-Induced Migration Inhibition of Human Peripheral Leukocytes in Agarose Medium. Acta. Allergolog- ica 26:56-80. Cohen, S., R. T. McCluskey, and B. Benacerraf. 1967. Studies on the Specificity of the Cellular Infiltrate of Delayed Hyper- sensitivity Reactions. J. Immunol. 993269-273. Cohen, L., R. Holzman, F. Valentine, and H. S. Lawrence. 1976. Requirement of Pre-Committed Cells as Targets for the Augmenta- tion of Lymphocyte Proliferation by Leukocyte Dialysates. J. Exp. Med. 1593791-804. Cozine, W. S., W. S. Jeter, R. N. Ferebee, T. C. Soli, and R. E. Reed. 1976. Transfer of Tuberculin Hypersensitivity in Cattle with Dialysates of Leukocytic Extracts. Fed. Proc. (Abstr.). 35:338. Crowle, A. J. 1959. Delayed Hypersensitivity in Mice: Its Detection by Skin Tests and Its Passive Transfer. Science 1993 159-160. Crowle, A. J. 1959. Delayed Hypersensitivity in Several Strains of Mice Studied with Six Different Tests. J. Allergy. 30:442-459. Crowle, A. J. 1962. Factors Which Affect Induction of Delayed Hypersensitivity to Protein Antigens in Mice. J. Allergy 99: 458-467. Crowle, A. J. 1975. Delayed Hypersensitivity in the Mouse. Adv. Immunol. 993197-264. Darlington, R. W., and M. Scherago. 1960. The Ig_Vitro Sensi- tivity to Brucellergen of Leukocytes from Guinea Pigs Experiment- ally Infected with Brucella abortus. J. Infect. Dis. 1993 106-110. David, J. R., and R. R. David. 1972. Cellular Hypersensitivity and Immunity. Inhibition of Macrophage Migration and the Lympho- cyte Mediators. Progr. Allergy 193300-449. Davis, D. 0., R. Dulbecco, H. Eisen, H. S. Ginsberg, W. 8. Wood, Jr., and M. McCarty. 1973. Microbiology, 2nd. ed., Harper and Row, Hagerstown, Md. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 87 Dienes, L., and T. B. Mallory. 1932. Histological Studies of Hypersensitive Reactions. Part I. The Contrast Between the Histological Responses in the Tuberculin (Allergic) Type and the Anaphylactic Type of Skin Reactions. Amer. J. Path. 93689-700. Dressler, 0., and S. Rosenfeld. 1974. On the Chemical Nature of Transfer Factor. Proc. Nat. Acad. Sci. (USA) 2134429-4434. Dumonde, D. C., M. R. Mazaheri, J. Kremastinou, G. Scalise, A. Hamblin, and S. J. Zuckerman. 1976. Acquisition of Mixed Cell Migration Reacivity by Actively Sensitized and Transfer-Factor Treated Rhesus Monkeys. pp. 117-127. lg_M. Ascher, A. Gottlieb, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clinical Applications. Academic Press Inc., N.Y. Dunnick, W., and F. H. Bach. 1976. Guinea Pig "Transfer Factor" 19_Vitro: Physiochemical Properties and Partial Purification, pp. 185-195. Ig_M. Ascher, A. Gottlieb, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clinical Applica- tions. Academic Press Inc., N.Y. Dunnick, W. A. and F. H. Bach. 1977. Specificity and Structural Analysis of A Guinea Pig Transfer Factor-Like Activity. J. Immunol. 11931944-1950. Dvorak, A. M., M. C. Mihm, and H. F. Dvorak. 1976. Degranula- tion of Basophilic Leukocytes in Allergic Contact Dermatitis Reactions in Man. J. Immunol. 1193687-695. Dvorak, H. F. 1974. Delayed Hypersensitivity, pp. 291-345.- IQ_B. W. Zweifach, L. Grant, and R. T. McCluskey (eds.), The Inflammatory Process, vol. III. Academic Press Inc., N.Y. Dvorak, H. F. 1976. Cutaneous Basophil Hypersensitivity. J. Allergy Clin. Immunol. 993229-240. Dvorak, H. F., A. M. Dvorak, B. A. Simpson, H. B. Richerson, S. Leskowitz, and M. J. Karnovsky. 1970. Cutaneous Basophil Hyper- sensitivity. II. A Light and Electron Microscopic Description. J. Exp. Med. 1993558-582. Dvorak, H. F., B. A. Simpson, R. C. Bast, and S. Leskowitz. 1971. Cutaneous Basophil Hypersensitivity III. Participation of the Basophil in Hypersensitivity to Antigen-Antibody Complexes, De- layed Hypersensitivity, and Contact Allergy. Passive Transfer. J. Immunol. 1923138-148. Dvorak, H. F., M. C. Mihm, A. M. Dvorak, R. A. Manseau, E. Mor- gan, and R. B. Colvin. 1974. Morphology of Delayed Type Hyper- sensitivity Reactions in Man. 1. Quantitative Description of the Inflammatory Response. Lab. Invest. 913111-130. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 88 Eichberg, J. W. 1976. Cellular Immunity in Gnotobiotic Pri- mates Induced by Transfer Factor. Cell. Immunol. 993114-119. Freedman, R. S., J. T. Wharton, F. Rutledge, and J. G. Sinkovics. 1978. Transfer Factor and Possible Applications in Gynecology. Am. J. Obstet. Gynecol. 1993572-584. Gershon, R. K., P. W. Askenase, and M. D. Gershon. 1975. Re- quirement for Vasoactive Amines for Production of Delayed-Type Hypersensitivity Skin Reactions. J. Exp. Med. 1993732-747. Gottlieb, A. A., K. Saito, S. Surcliffe, L. A. Foster, N. Tamaki, G. Maziarz, C. Sutherland, and B. Brennessel. 1977. Biochemical Analysis of Dialyzable Leukocytic Extracts. J. Reticuloendo- thelial Soc. 913403-416. Gray, 0. F., and P. A. Jennings. 1955. Allergy in Experimental Mouse Tuberculosis. Am. Rev. Tuberc. Pulm. Dis. 993171-195. Green, J. A., J. Williams, and H. B. Levy. 1977. Specific Restoration of Delayed Hypersensitivity by Lymphoid Tissue Ex- tracts. J. Immunol. 11931936-1943. Grob, P. J., J. F. Reymond, M. A. Hacki, and M. Frey-Wettstein. 1976. Some Physio-Chemical and Biological Properties of a Transfer Factor Preparation and Its Clinical Application, pp. 247-262. 99_M. Ascher, A. Gottlieb, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clinical Applications. Academic Press Inc., N.Y. Heilman, D. H., H. E. Weimer, and C. M. Carpenter. 1960. Tissue Culture Studies on Bacterial Allergy in Experimental Brucellosis. II. The Cytotoxicity of Nucleoprotein Fractions of Brucellae. J. Immunol. 993258-267. Henry, B. S. 1933. Dissociation in the Genus Brucella. J. Infect. Dis. 993374-402. Hoffman, P. M., L. E. Spitler, M. Hsu, and H. H. Fudenberg. 1975. Leukocyte Migration Inhibition in Agarose. Cell. Immunol. 193 21-30. Hoffman, P. M., L. E. Spitler, and M. Hsu. 1976. Leukocyte Migration Inhibition in Guinea Pigs. I. Correlation with Skin Test Reactivity and Macrophage Migration Inhibition. Cell Immunol. 21:358-363. Holland, J. J., and M. J. Pickett. 1958. A Cellular Basis of Immunity in Experimental Brucella Infection. J. Exp. Med. 1993 343-359. Huddleson, I. F. 1943. Brucellosis in Man and Animals. The Commonwealth Fund. N.Y. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 89 Jaffer, A. M., G. Jones, E. J. Kasden, and S. F. Schlossman. 1973. Local Transfer of Delayed Hypersensitivity by T Lymphocytes. J. Immunol. 11131268-1269. Jeter, W. S. 1973. Discussion Paper: Subcellular Transfer of Activity. Ann. N.Y. Acad. Sci. 9993406-407. Jeter W. S., M. M. Tremaine, and P. M. Seebohm. 1954. Passive Transfer of Delayed Hypersensitivity to 2,4-Dinitrochlorobenzene in Guinea Pigs with Leukocyte Extracts. Proc. Soc. Exp. Biol. Med. 993251-253. Jeter, W. S., T. C. Soli, and R. E. Reed. 1976. Transfer of Delayed Hypersensitivity to Tuberculin in the Dog with Dialyzable Leukocytic Extracts. Abstracts of Annual Meeting, A. S. M., p. 81. Jeter, W. S., A. Paquet, Jr., R. N. Ferebee, G. Olson, and F. Roinestead. 1976. Correlation of 99_Vivo and 99_Vitro Activ- ity of Guinea Pig Transfer Factor, pp. 431-436. In M. Ascher, A. Gottleib, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clinical Applications. Academic Press Inc., N.Y. Jeter. W. S., R. Kibler, and C. A. Stephens. 1977. Oral Admin- istration of Dialyzable Transfer Factor to Tuberculin to Human Volunteers. Abstr. 14th. Annual Nat'l. Meeting. J. Reticulo- endothel. Soc. 99346a. Jeter, W. S., R. E. Reed, 1. C. Soli, and J. Cramer. 1977. Transfer Factor to 99ccidioides imnitis in Cattle. PP. 359-363. 99_L. Ajello (ed.), Coccidioidomyco§isz Current Clinical and Diagnostic Status. Symposia Specialists, Fla. Jones, L. M., R. Diaz, and A. G. Taylor. 1973. Characterization of Allergens Prepared from Smooth and Rough Strains of Brucella melitensis. Br. J. Exp. Pathol. 993492-508. Jones, L. M., and J. Marly. 1975. Titration of a Brucella Protein Allergen in Sheep Sensitized with Brucella melitensis. Ann. Rech. Veter. 93173-178. Jones, T. 0., and J. R. Mote. 1934. The Phases of Foreign Protein Sensitization in Human Beings. N. Eng. J. Med. 9193 120-123. Kaneene, J. M., D. W. Johnson, R. K. Anderson, R. D. Angus, and C. C. Muscoplat. 1978. Specific Lymphocyte Stimulation in Cattle Naturally Infected with Strains of Brucella abortus and Cattle Vaccinated with Brucella abortus Strain 19. Am. J. Vet. Res. 993585-589. Katsura, K., K. Nakano, Y. Kabara, and I. Uesaka. 1977. Cell Mediated and Humoral Immune Responses in Mice. I. Necessary 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 90 Conditions for the Detection of Delayed-Type Hypersensitivity. Int. Archs. Allergy Appl. Immun. 993152-161. Katsura, Y., K. Inaba, T. Izuma, and I. Uesaka. 1977. Cell- Mediated and Humoral Immune Responses in Mice. II. Sensitizing Conditions for Delayed-Type Hypersensitivity. Int. Archs. Allergy. Appl. Immun. 993329-340. Khan, A. 1978. Transfer Factor in Viral Diseases. Lancet i: 328-329. Kirkpatrick, C. H. 1975. Properties and Activities of Transfer Factor. J. Allergy Clin. Immunol. 993411-421. Kirkpatrick, C. H., and D. Rifkind. 1974. Meeting Report. Workshop on Basic Properties and Clinical Applications of Trans- fer Factor. Cell. Immunol. 193165-168. Kirkpatrick, C. H., and J. I. Gallin. 1975. Suppression of Cellular Immune Responses Following Transfer Factor: Report of a Case. Cell. Immunol. 193470-474. Kirkpatrick, C. H., and T. K. Smith. 1976. The Nature of Transfer Factor and Its Clinical Efficacy in the Management of Cutaneous Disorders. J. Invest. Dermatol. 993425-430. Kirkpatrick, D. H., and T. K. Smith. 1976. Serial Transfer of Delayed Hypersensitivity with Dialyzable Transfer Factor. Cell. Immunol. 993323-327. Kirkpatrick, C. H., L. B. Robinson, and T. K. Smith. 1976. The Identification and Significance of Hypoxanthine in Dialyzable Transfer Factor. Cell. Immunol. 993230-240. Kirkpatrick, C. H., E. A. Ottenson, T. K. Smith, S. A. Wells, and J. F. Burdick. 1976. Reconstitution of Defective Cellular Immunity with Foetal Thymus and Dialyzable Transfer Factor. Long-Term Studies in a Patient with Chronic Mucocutaneous Can- didiasis. Clin. Exp. Immunol. 993414-428. Klesius, P. H. 1978. Bovine Dialyzable Transfer Factor: Stim- ulation of Antigen-Specific DTH in C5781 Mice. Abstracts of the 3rd Int'l. Symposium on Transfer Factor. J. Clin. Hematol. Oncol. 93106. Klesius, P. H., T. Kramer, 0. Burger, and A. Walley. 1975. Passive Transfer of Coccidian Oocyst Antigen and Diptheria Toxoid Hypersensitivity in Calves Across Species Barriers. Transplt. Proc. 93449-452. Klesius, P. H., F. Kristernen, J. V. Ernst, and T. T. Kramer. 1976. Bovine Transfer Factor: Isolation and Characteristics, pp. 311-319. 99 M. Ascher, A. Gottleib, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clinical Applications. Academic Press Inc., N.Y. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 91 Klesius, P. H., and H. H. Fudenberg. 1977. Bovine Transfer Factor: 99_Vivo Transfer of Cell-Mediated Immunity to Cattle with Alcohol Precipitates. Clin. Immunol. Immunopathol. 93 238-246. Klesius, P. H., and F. Kristensen. 1977. Bovine Transfer Fac- tor: Effect on Bovine and Rabbit Coccidiosis. Clin. Immunol. Immunopathol. 93240-252. Klesius, P. H., D. F. Qualls, A. L. Elston, and H. H. Fudenberg. 1978. Effects of Bovine Transfer Factor (TFd) in Mouse Cocci- diosis (Eimeria ferrisi). Clin. Immunol. Immunopathol. 193214- 221. Klesius, P. H., T. T. Kramer, A. I. Swan, and C. C. Christen- berry. 1978. Cell-Mediated Immune Response after Brucella abortus Sl9 Vaccination. Am. J. Vet. Res. 993883-886. Krohn, K., P. Grohn, M. Horsmanheimo, and M. Virolainen. 1976. Fractionation Studies on Human Leukocyte Dialyzates. Demonstra- tion of Three Components with Transfer Factor Activity. Med. Biol. 993334-340. Krohn, K., A. Uotila, J. Vaisanen, and P. Grohn. 1977. Studies on the Chemical Composition and Biological Properties of Transfer Factor. Z. Immun-Forsch. Immunobiol. 1993395-411. Landsteiner, K., and M. W. Chase. 1942. Experiments on Transfer of Cutaneous Sensitivity to Simple Compounds. Proc. Soc. Exp. Biol. Med. 993688-690. Lawrence, H. S. 1949. The Cellular Transfer of Cutaneous Hypersensitivity to Tuberculin in Man. Proc. Soc. Exp. Biol. Med. 913516-522. Lawrence, H. S. 1954. The Transfer in Humans of Delayed Skin Sensitivity to Streptococcal M Substance and to Tuberculin with Disrupted Leukocytes. J. Clin. Invest. 993219-230. Lawrence, H. S. 1969. Transfer Factor, pp. 145-175. 99_H. S. Lawrence and M. Landy (eds.), Mediators of Cellular Immunity. Academic Press Inc., N.Y. Lawrence, H. S. 1974. Transfer Factor in Cellular Immunity, pp. 239-350. 99 The Harvey Lectures, Series 68. Academic Press Inc., N.Y. Lawrence, H. S. 1975. Transfer Factor and Cellular Immunity to Viral Infection, pp. 135-152. 99_M. Pollard (ed.), Perspectives in Virology. Academic Press Inc., N.Y. Lawrence, H. S. 1977. Transfer Factor in Transplantation Immu- nobiology. Transplt. Proc. 931319-1326. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 92 Lawrence, H. S., S. Al-Askari, J. David, E. C. Franklin, and B. Zweiman. 1963. Transfer of Immunological Information in Humans with Dialysates of Leukocyte Extracts. Trans. Assoc. Amer. Phys. 99384-91. lawrence, H. S., and S. Al-Askari. 1971. The Preparation and Purification of Transfer Factor, pp. 531-546. 99_Bloom and Glade (eds.), 99_Vitro Methods in Cell Mediated Immunity. Academic Press Inc., N.Y. Levin, A. S. 1975. Transfer Factor Therapy: Current Status. Southern Med. Journal 9931465-1467. Lewis, 0., M. Stafford, R., Kibler, and W. S. Jeter. 1978. Transfer of Delayed Hypersensitivity from Humans to Guinea Pigs without Antigen Priming. Abstr. of the Annual Meeting. A.S.M. p. 64. Lewis, 0. E., M. E. Stafford, R. Kibler, T. C. Soli, and W. S. Jeter. 1978. Interspecies Transfer of Delayed Hypersensitivity by Purified Transfer Factor Preparations. Fed. Proc. (Abstr). 37:1365. Liburd, E. M., H. F. Pabst, and W. 0. Armstrong. 1972. Trans- fer Factor in Rat Coccidiosis. Cell. Immunol. 93487-489. Littman, B. H., R. E. Rochlin, R. Parkman, J. R. David. 1978. Transfer Factor Treatment of Chronic Mucocutaneous Candidiasis: Requirement for Donor Reactivity to Candida Antigen. Clin. Immunol. Immunopath. 9397-110. Lubaroff, D. M., and B. H. Waksman. 1968. Bone Marrow as a Source of Cells in Reactions of Cellular Hypersensitivity. I. Passive Transfer of Tuberculin Sensitivity in Syngeneic Systems. J. Exp. Med. 19931425-1435. Lubaroff, D. M., and B. H. Waksman. 1968. Bone Marrow as Source of Cells in Reactions of Cellular Hypersensitivity. II. Identification of Allogenic or Hybrid Cells by Immunoflourescence in Passively Transferred Tuberculin Reactions. J. Exp. Med. 19931437-1449. Mackaness, G. B., 1964. The Immunological Basis of Acquired Cellular Resistance. J. Exp. Med. 1993105-120. Mackie, R. M. 1975. Comment: Transfer Factor 1975. Brit. J. Dermatol. 993107-110. Maddison, S. E. 1975. Dialyzable Transfer Factor. Southern Med. Journal 9931545-1551. Maurer, P. H. 1961. Immunologic Studies with Ethylene Oxide- Treated Human Serum. J. Exp. Med. 19931029-1039. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 93 McCluskey, R. T., B. Benacerraf, and J. W. McCluskey. 1963. Studies on the Specificity of the Cellular Infiltrate in Delayed Hypersensitivity Reactions. J. Immunol. 993466-477. McCullough, N. B. 1949. Laboratory Tests in the Diagnosis of Brucellosis. Am. J. Pub. Health 993866-869. McCullough, N. B. 1970. Microbial and Host Factors in the Pathogenesis of Brucellosis, pp. 324-345. 99_S. Mudd (ed.), Infectious Agents and Host Reactions. Saunders Co., Phila- delphia, Pa. McCullough, N. B. 1976. Immune Response to Brucella, pp. 304- 311. In N. R. Rose and H. Friedman (eds.), Manual of Clinical ImmunoTEgy. A.S.M. Neidhart, J. A., N. Christakis, E. N. Metz, S. P. Balcerzak, and A. F. LoBuglio. 1978. Skin Test Conversion Following Transfer Factor. A Double-Blinded Study of Normal Individuals. J. Allergy Clin. Immunol. 993115-118. O'Dorisio, M. S., J. A. Neidhart, F. 8. Daniel, S. P. Balcerzak, and A. F. LoBuglio. 1976. Identification of Hypoxanthine as the Major Component of a Chromatographic Fraction of Transfer Factor. Cell. Immunol. 993191-202. Paquet, A., Jr., G. 8. Olson, and W. S. Jeter. 1976. 99_Vitro Activity of Guinea Pig Transfer Factor Released into Plasma. Infec. Immun. 193290-297. Pearson, L. 0., J. W. Osebold, and P. C. Wagner. 1971. A De- vice for Measuring the Volume of Footpad Swelling from Delayed Hypersensitivity Reactions in Mice. Lab. Anim. Sci. 913591-593. Peterson, E. A., J. A. Frey, M. Dinowitz, and D. Rifkind. 1976. Transfer of Delayed Hypersensitivity to Mice with Human Immune Cell Extracts, PP. 387-395. 99_M. Ascher, A. Gottleib, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clin- ical Applications. Academic Press Inc., N.Y. Pizza, 6., O. Viza, C. Boucheix, and F. Corrado. 1976. 99_ Vitro Production of a Transfer Factor Specific for Transitional- Cell Carcinoma of the Bladder. Br. J. Cancer. 993606-611. Pomales-Lebron, A., and W. R. Stineberg. 1957. Intracellular Multiplication of Brucella abortus in Normal and Immune Mono- nuclear Phagocytes. Proc. Soc. Exp. Biol. Med. 99378-83. Pritchard, H., and H. S. Micklem. 1972. Immune Response in Congenitally Thymus-Less Mice. I. Absence of Response to Oxazolone. Clin. Exp. Immunol. 193151-161. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 94 Phillips, H. J. 1973. Dye Exclusion Tests for Cell Viability, pp. 406-408. 99_P. F. Kruse and M. Patterson (eds.), Tissue Culture Methods and Applications. Academic Press Inc., N.Y. Rabinowitz, Y. 1973. Nonenzymatic Dissociations. Leukocyte Cell Separation on Glass, pp. 25-29. 99_P. Kruse and M. Pat- terson (eds.), Tissue Culture Methods and Applications. Academic Press Inc., N.Y. Raffel, S., and J. M. Newel. 1958. The "Delayed Hypersensitiv- ity" Induced by Antigen-Antibody Complexes. J. Exp. Med. 1993 823-841. Ralston, 0., and S. Elberg. 1961. Intramonocytic Destruction of Brucella: Potentiating Effect of Glycine on Intracellular Lysozyme Activity. J. Infect. Dis. 199371-80. Rapaport, F. T., H. S. Lawrence, J. W. Miller, D. Pappagianis, and C. E. Smith. 1960. Transfer of Delayed Hypersensitivity to Coccidioidin in Man. J. Immunol. 993358-367. Richerson, H. B., H. F. Dvorak, and S. Leskowitz. 1970. Cutaneous Basophil Hypersensitivity. I. A New Look at the Jones-Mote Reaction, General Characteristics. J. Exp. Med. 1993546-557. Rifkind, D., J. Frey, E. A. Peterson, and M. Dinowitz. 1976. Delayed Hypersensitivity to Fungal Antigens in Mice. I. Use of the Intradermal Skin and Footpad Swelling Tests as Assays of Active and Passive Sensitization. J. Infect. Dis. 1993 50-56. Rifkind, 0., J. Frey, E. A. Peterson, and M. Dinowitz. 1977. Transfer of Delayed Hypersensitivity in Mice to Microbial Anti- gens with Dialyzable Transfer Factor. Infect. Immun. 193258-262. Rifkind, D., J. A. Frey, and M. Dinowitz. 1977. Susceptibility of Murine Transfer Factor to Dimerized Ribonuclease A. Infect. Immun. 193920-922. Robbins, S. L., and Angell. 1976. Basic Pathology, 2nd. edi- tion, pp. 167-209. W. B. Saunders Co., Philadelphia, Pa. Rocklin, R. E. 1974. Products of Activated Lymphocytes: Leukocyte Inhibitory Factor (LIF) Distinct from Migration Inhi- bitory Factor (MIF). J. Immunol. 11931461-1466. Rocklin, R. E. 1975. Use of Transfer Factor in Patients with Depressed Cellular Immunity and Chronic Infectidn. Birth Defects. 193431-435. Rose, N. R. 1974. Autoimmune Diseases, pp. 347-399. In B. W. Zweifach, L. Grant, and R. T. McCluskey (eds.), The InfTEmmatory Process. Academic Press Inc., N.Y. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 95 Rubinstein, A., J. Melamed, and D. Todescu. 1977. Transfer Factor Treatment in a Patient with Progressive Tuberculosis. Clin. Immunol. Immunopathol. 8339-50. Salaman, M. R. 1978. An Investigation into the Antigen- Specificity of Transfer Factor in Its Stimulatory Action on Lymphocyte Transformation. Immunol. 353247-256. Sandok, P. L., R. D. Hindsill, and R. M. Albrech. 1971. Migration Inhibition of Mouse Macrophages by Brucella Antigens. Infec. Immun. 13516-518. Slavin, R. G. , and J. E. Garvin. 1964. Delayed Hypersensitiv- ity in Man: Transfer by Lymphocyte Preparations of Peripheral Blood. Science 145. 52- 53. Spitler, L. E., A. S. Levin, D. P. Stites, H. H. Fudenberg, B. Pirofsky, C. 5. August, S. R. Stiehm, W. H. Hitzig, and R. A. Gatti. 1972. The Wiskott-Aldrich Syndrome: Results of Trans- fer Factor Therapy. .J. Clin. Invest. 5133216p3224. Spitler, L. E, A. S. Levin, H. H. Fudenberg. 1973. Human Lymphocyte Transfer Factor, pp. 59-106. In H. Busch (ed. ), Methods in Cancer Research, vol. 8. Academic Press Inc. ., N. Y. Stahl, W. H. 1939. A Study of the Protein-Nucleates of the Species of the Genus Brucella. I. Chemical Constitution of the Protein Nucleates. II Biological Properties of the Protein Nucleates. Michigan Agricultural Experiment Station Technical Bulletin 168. Steele, R. W., J. W. Eichberg, R. L. Hebering, J. J. Eller, S. S. Kalter, and W. T. Knicker. 1976. Transfer of Cellular Reactiv- ity to 3 Non-Human Primate Species with Human and Baboon Trans- fer Factor, pp. 371- 380. In M. Ascher, A. Gott1eib, and C. Kirkpatrick (eds. ), Transfer Factor: Basic Properties and Clin- ical Applications. Academic Press Inc. , N. Y. Strobe], G., and H. J. Staab. 1975. A New Reliable Method fer Evaluation of Footpad Swelling as an Experimental In Vivo Immune Phenomenon. Int. Arch. Allergy Appl. Immunol. 1932933302' Tomar, R. H. 1976. Local Transfer of Delayed Hypersensitivity to Dogs by Human Transfer Factor, p. 234. In M. Ascher, A. Gottleib, and C. Kirkpatrick (eds. ). Transfer Factor: Basic Properties and Clinical Applications. Academic Press Inc., N.Y. Tomar, R. H., R. Knight, and M. Stern. 1976. Transfer Factor: Hypoxanthine is a Major Component of a Fraction with 1n_Vivo Activity. J. Allergy Clin. Immunol. 583190-197. Tomar, R. H., and J. Terzian. 1977. Reactions in Canine Skin with Human Leukocyte Lysates. Proc. Soc. Exp. Biol. Med. 1563 247-250 96 148. Turk, J. L. 1967. Delayed Hypersensitivity. North-Holland Research Monographs. North-Holland Pub. Co., Amsterdam. 149. Turk, J. L. 1975. Delayed Hypersensitivity. North-Holland Pub. Co., Amsterdam. 150. Turk, J. L., C. J. Heather, and J. V. Diengdoh. 1966. A Histochemica1 Analysis of Mononuclear Cell Infiltrates of the Skin with Particular Reference to Delayed Hypersensitivity in the Guinea Pig. Int. Arch. Allergy 253278-289. 151. Turk, J. L., E. J. Rudner, and C. J. Heather. 1966. A Histo- chemica1 Analysis of Mononuclear Cell Infiltrates of the Skin. 11. Delayed Hypersensitivity in the Human. Int. Arch. Allergy 30:248-256. 152. Turk, J. L., and Polak, L. 1967. Studies on the Origin and Reactive Ability 1Q_Vivo of Peritoneal Exudate Cells in Delayed Hypersensitivity. Int. Arch. Allergy 513403-416. 153. Uotila, A., A. Hamblin, D. C. Dumonde, and K. J. E. Krohn. 1978. The Effect of Transfer Factor on Lymphocyte Transformation. Com- parison of Augmentation by Dialysates of Leukocytes and Lymphoid and Non-lymphoid Organs. Int. Archs. Alergy Appl. Immun. 513 210-220. 154. Vandenbark, A. A., D. R. Burger, and R. M. Vetto. 1977. Human Transfer Factor Activity in the Guinea Pig: Absence of Antigen Specificity. Clin. Inmunol. Inmunopathol. 5:7-16. 155. Vandenbark, A. A., D. R. Burger, D. L. Dreyer, G. D. Daves, Jr., and R. M. Vetto. 1977. Human Transfer Factor: Fraction by Electrofocusing and High Pressure, Reverse Phase Chromatography. J. Immunol. 1153636-641. 156. Vetto, R. M., D. R. Burger, J. E. Nolte, and A. A. Vandenbark. 1976. Transfer Factor Immunotherapy in Cancer, pp. 523-535. 1Q_M. Ascher, A. Gottleib, and C. Kirkpatrick (eds.), Transfer Factor: Basic Properties and Clinical Applications. Academic Press Inc., N.Y. 157. Welch, T. M., R. Triglia, L. E. Spitler, and H. H. Fudenberg. 1976. Preliminary Studies on Human "Transfer Factor" Activity in Guinea Pigs. Clin. Immunol. Immunopathol. 53407-415. 158. Wilson, G. B., T. M. Welch, D. R. Knapp, A. Horsmanheimo, and H. H. Fudenberg. 1977. Characterization of Tx, an Active Subfraction of Human Dialyzable Transfer Factor. I. Identif- ication of the Major Component in TFg, a Precursor of TFx, as Hypoxanthine. Clin. Immunol. Immunopathol. 53551-568. 159. World Health Organization Technical Report, Series 519. 1973. Cell-Mediated Immunity and Resistance to Infection. World Health Organization, Geneva. 160. 161. 162. 163. 164. 97 Youdim, S., 0. Stutman, and R. A. Good. 1973. Studies of Delayed Hypersensitivity to L, monocytogenes in Mice: Nature of Cells Involved in Passive Transfers. Cell. Immunol. 5398-109. Youdim, S., 0. Stutman, and R. A. Good. 1973. Thymus Depend- ency of Cells Involved in Transfer of Delayed Hypersensitivity to Listeria monocytogenes in Mice. Cell. Immunol. 53395-402. Youmans, G. P., and A. S. Youmans. 1969. Recent Studies on Acquired Immunity in Tuberculosis. Curr. Top. Microbiol. Immunol. 153129-178. Zinsser, H. 1925. Bacteria1 Allergies and Tissue Reactions. Proc. Soc. Exp. Biol. Med. 22335-39. Zuckerman, K. S., J. A. Neidhart, S. P. Balcerzak, and A. F. LoBuglio. 1974. Immunologic Specificity of Transfer Factor. J. Clin. Invest. 513997-1000. - 111111111 1242 1' l 8 7 1 3 o 3 9 2 1 3 I“ "I! n" N”| " Ill " "I H"