I WW al'. N l W ." 7W] . W: I I; — II [I H 1M I» 2: _3 ‘1' N MONOCYTiC LYSOZYME LEVELS lN MAUGNANT DESEASE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY NANCY R. JAEHN 1977 ABSTRACT MONOCYTIC LYSOZYME LEVELS IN MALIGNANT DISEASE BY Nancy R. Jaehn Intracellular monocytic lysozyme levels were measured from three groups of individuals: patients with untreated malignant disease, patients with benign disease, and normal individuals as a control group. The patients with untreated malignant disease had an elevation of their monocytic lysozyme content. The mononuclear leukocyte layer of cells was separated by the Ficoll-Hypaque procedure. A cell count was done on the product and a wright‘s stain was used to determine the percentages of monocytes and lymphocytes. The cell preparations were lysed by 5 cycles of freez- ing and thawing. The preparations were checked by phase contrast microscopy to determine that all cells were lysed. The lysozyme levels were measured by the lysoplate method at 25°C. The turbidimetric method was investigated and found to be unacceptable for the concentration at which 7 the cells had been suspended (10 monocytes per ml). MONOCYTIC LYSOZYME LEVELS IN MALIGNANT DISEASE By Nancy R. Jaehn A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1977 In memory of my parents, Harold and Clara Jaehn ii ACKNOWLEDGEMENTS I wish to express my appreciation to several indi- viduals who assisted me in this research project. Dr. Nikolay Dimitrov for the financial assistance of the Cancer Research Fund, Community Cancer Center, Saginaw, MI, which made this research possible, for his assistance in obtaining Specimens from his clinical oncology patients, and for his continuing guidance. Dr. Trevor Singh for his interest in my research and for his assistance in obtaining specimens from his clinical oncology patients. Mrs. Martha Thomas for her helpful suggestions in the preparation of this manuscript, and for her encouragement throughout my entire graduate degree program. Dr. John Dunkel for his thoughtful sharing of ideas and helpful suggestions. Dr. Jon Desguin, Dr. David Greenbaum, Dr. Leif Suhrland, and Dr. Larry Aronson for allowing me to ask their patients for their permission to donate their blood for my research. Mrs. Margaret Shick and the staff of Olin Health Center for their encouragement and donations of blood samples. Dr. Elliott F. Osserman for supplying the human standard for the early portion of my research, and for his helpful suggestions and encouragement. iii Mr. Thomas Denny for his assistance with the cell separation techniques, and interest in my work. Mrs. Louise Mueller for her thoughtful ideas and encouragement. iv TABLE OF CONTENTS INTRODUCTION . LITERATURE REVIEW. Lysozyme. Discovery. . Chemical Activity. Lysozyme Structure Multiple Forms Function . Monocytes-Macrophages in Neoplasia. Role in Non- Specific Cell-Mediated Immunity. . . . Lysozyme Concentrations in Activated Macrophages- Monocytes Review of Lysozyme Methodology. METHODS AND MATERIALS. Specimen Selection. Preparation of the Monocytes. Disruption of the Cell Membrane . . . Lysozyme Analysis by the Lysoplate Method . Lysozyme Analysis by the Turbidimetric Method . . . . . . . . . Egg- -White Standard vs. Human Standard . RESULTS AND DISCUSSION . Monocytic Lysozyme Levels as Measured by the Lysoplate Technique with Human Standard . . Serum Lysozyme as Measured by the Turbidi- metric Method. . . . . . . . SUMMARY AND CONCLUSIONS. \7 Page 10 12 12 12 15 17 19 21 21 39 44 Page APPENDICES . . . . . . . . . . . . . . . . . . . . . 45 A - REAGENTS. . . . . . . . . . . . . . . . . 45 B - INFORMED WRITTEN CONSENT FORM . . . . . . 46 REFERENCES . . . . . . . . . . . . . . . . . . . . . 47 VITA . . . . . . . . . . . . . . . . . . . . . . . . 52 vi Table A KOWVO‘UI LIST OF TABLES Background laboratory data of patients with malignant disease. . . . . . . . Background laboratory data for normal individuals . . . . . . Background laboratory data on patients with benign disease . . . . . Monocyte preparations . Lysoplate readings, malignant group . Lysoplate readings, normal grOup. Lysoplate readings, benign group. Summary of lysoplate determinations Serum lysozyme readings by the turbidi- metric method . . . . . . vii Page 26 27 28 30 31 32 34 '35 41 Figure LIST OF FIGURES Comparison of mean intracellular mono- cytic lysozyme in patients with untreated malignant disease, in normal individuals, and in patients with benign diseases. Lysoplate standard curve (with human standard) . . . . . . . Lysoplate standard curve (with egg-white standard) . . . . . . . . . . Lysoplate photograph. Monocyte cell preparation . Turbidimetric standard curve (egg-white standard) . . . . . . . . . . . . . . viii Page 22 23 25 37 38 40 INTRODUCTION Lysozyme was discovered in 1922 by Alexander Fleming when he observed that it caused lysis of some bacterial cell walls. He found lysozyme to be present in many tissues and secretions of the human body. Recent studies have investigated the possibility that certain polysaccharides of mammalian cells may be susceptible to enzymatic cleavage or substrate specific binding by lysozyme. E. F. Osserman has postulated that lysozyme may participate in the surveillance of membrane abnormalities, particularly those associated with neOplastic formation. Lysozyme may be a key mediator of the anti-tumor functions of macrOphages. The blood-borne precursors of tissue macrophages are monocytes. This research measured the lysozyme concentra- tions in human monocytes in patients with untreated malignant disease, patients with benign diseases, and normal indi- viduals as a control group. The primary objective was to determine if there is a significant relationship between intracellular monocytic lysozyme levels and malignant disease. The Ficoll-Hypaque technique was used to separate the mononuclear leukocyte layer from the other blood constituents. A cell count was done to determine the number of cells 1 2 present, and a Wright's stain differential was done to determine the percentages of lymphocytes and monocytes present. The cell preparations were lysed by 5 cycles of rapid freezing and thawing. Both the turbidimetric and the 1y50plate method were used to measure the lysozyme activity. LITERATURE REVIEW This literature review will be divided into three parts: (1) lysozyme - discovery, chemical activity, structure, multiple forms, and function; (2) monocytes- macrophages in neOplasia - role in non-specific cell mediated immunity, effect of Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum, and lysozyme concentrations in activated macrophages-monocytes; (3) lysozyme methodology - several observations regarding the turbidimetric and lyso- plate methods. Lysozyme Discovery Lysozyme was discovered by Alexander Fleming in 1922 when he observed that it caused lysis of some bacterial cell walls. He found lysozymes to be present in many tissues and secretions of the human body, such as tears, saliva, leukocytes, and skin. Fleming postulated lysozyme to be the natural defense agent of the human body. It was the first bactericidal substance he had studied that ful- filled a major requirement: that it be selectively more lethal to bacteria than to host cells (Fleming, 1922). Chemical Activity Lysozyme hydrolyzes the 3 1,4 glycosidic linkages between N-acetylglucosamine and N-acetylmuramic acid in bacterial cell walls. It may be of animal, plant, or microbial origin (Osserman, 1974). The oxygen atoms of the aspartic 52 and glutamic 35 residues of lysozyme specifically accomplish the hydrolytic cleavage (Phillips, 1967). Lysozyme is a small cationic protein molecule with a molecular weight of 14,000. Its optimum pH range is 6.0-6.5. It is heat stable at temperatures up to 52°C. Lysozyme specimens can be stored at -20°C for several weeks (Daniels, 1974). The lysozyme molecule is stable to repeated freezing and thawing (Gordon et a1., 1974; Perillie, 1968; Hansen and Andersen, 1973). Lysozyme Structure In 1965 Phillips and his co-workers produced an electron density map of lysozyme, and at the same time Jolles, P. (1964) in Paris and Canfield (1965) at the National Institutes of Health with their colleagues determined the amino acid sequence of the enzyme. This enabled them to identify the conformational features of the molecule by looking at the map in relation to the amino acid sequences. The lysozyme molecule consists of two immunologically active components. One immunologically active fragment consists of two peptides derived from the amino terminus 5 (residues 122—129) linked together by a disulfide bond. The other immunologically active fragment is derived from the region between residues 57-107 with two disulfide bonds. This also encompasses the area referred to as the "100p" of residues 60-83 (Arnon, 1969). Multiple Forms Only a single form of lysozyme was found in hen egg- white and goose egg-white as well as in normal human secre- tions and tissues. In the latter, lysozyme was purified from tears, leukocytes, plasma, milk, and placenta. The enzymes from the different sources were found to have the same chromatographic and electrophoretic behaviors, as well as the same amino acid sequences (Jolles, P., 1969). Multiple enzymes have been found in duck egg-white (Jolles, J., 1965), and in the tissues and secretions of patients with leukemia (Mouton, 1969). Immunologic methods can provide estimates of the approximate degree of sequence resemblance and conforma- tional similarity among the related lysozymes (Prager and Wilson, 1971). Function The function of lysozyme in higher vertebrates, other than its probable antibacterial role, is still not clear. The possibility has been suggested that lysozyme may have other important physiological functions, some of which may be related to specific enzymatic activities on 6 nonbacterial substrates, and others with its marked cationic property (Ambrose, 1966). It has also been postulated that lysozyme may be a key mediator of the anti-tumor functions of macrophages (Osserman, 1974). The presence of macrophages in tumors of man has been documented (Hamlin, 1968), and there is considerable evi- dence that these cells serve a major role in host defenses against neOplasia (Halpern, 1953; Old et al., 1961; Holterman et al., 1972). Since monocytes are the blood-borne precursors of macrophages (Osserman, 1966; Lessin and Bessis, 1972; Gordon et al., 1974), this research measured the level of lysozyme in peripheral blood monocytes. Clinical and experimental studies indicate that from 70 to 80% of the serum and tissue lysozyme is within or released from neutrophils and monocytes (Finch et al., 1974). Farhangi and Osserman (1974) have found that monocytes release their enzyme after synthesis, while granulocytes retain their lysozyme probably within lysosomes and release it only in the course of lysosomal disruption. This would imply that there may be two different cell populations (cells of the monocytic-macrOphage series and those of the granulocytic series) which utilize the same enzyme, lyso- zyme, in two distinctly different ways. That is, there may be specific biological functions of lysozyme as mediated by these different cell types. 7 The kidney plays a significant role in the degradation of lysozyme (Finch et al., 1974). Monocytes-Macrophages in Neoplasia Role in Non-Specific Cell- Mediated’lmmuni§X_ The role of large mononuclear leukocytes in host defense against ne0plastic cells is receiving increased attention. It has been suggested that such leukocytes are effectors in a primitive surveillance mechanism against aberrant cells (Holterman, 1973; Osserman, 1974). A phenomenon possibly related to a local amplification of this primitive surveillance mechanism in man is regres- sion of cutaneous neoplasms after recruitment of mononuclear inflammatory infiltrates at the tumor sites by induction of delayed type hypersensitivity reactions to chemical haptens (Klein, 1969), or by lymphokines (Holterman, 1974; Papermaster, 1974). Increased resistance to carcinogens and transplanted tumors after administration of various agents with a stimu- latory effect on the reticuloendothelial systerm (Yashphe, 1971) may be a reflection of a systemic activation of the same mechanism. This implies that large mononuclear cells are selectively cytotoxic for neoplastic cells. Zbar (1974) describes the current understanding of the cellular immune response which kills tumor cells when mediated by a non-specific stimulator such as Bacillus Calmette-Guerin (BCG), a bovine strain of Mycobacterium. 8 Bacillus Calmette-Guerin is a potent non-specific stimulator of the reticuloendothelial system which has caused tumor regression. Following the infection of the host, the host develops lymphocytes capable of recognizing the distinctive antigens of the tubercle bacillus. The lymphocytes react with the bacillus and produce a variety of potent molecules. Some of these may be directly cyto- toxic. If tumor cells are anatomically close to the bacteria, the tumor cells are killed by the activated macrophages and lymphotoxins. As a consequence of this reaction, immunity to specific tumor antigens may develop and, in turn, produce additional tumor cell death. Corynebacterium parvum has recently been found to have tumor resistance-inducing properties in experimental animals (Currie, 1970) and in preliminary studies with humans (Dimitrov, 1974). Activated macrophages are characterized by increased phagocytosis and increased content of lysosomal enzymes (Eisen, 1974). Since the large mononuclear cells of the peritoneum and other tissues, at least in part, are derived from the monocytes of the blood, one might expect that monocytes would also exhibit cytotoxic properties. Human blood mono- cytes were reported as cytotoxic to cells of a human osteo- sarcoma and a reticulum cell sarcoma in vitro. Activation of the monocytes by supernatant fluids of mitogen stimulated human lymphocytes markedly increased their cytotoxicity towards neoplastic cells (Holterman, 1974). 9 There does not seem to be any limit on the type of tumor susceptible to increased non-specific stimulation of host resistance. It would appear that tumors must be antigenic to be susceptible to such effects (Yashphe, 1971). Yashphe designates a non-specific stimulator as a material whose administration confers on the recipient the ability to respond, or respond more efficiently, to challenge with a non-cross reacting antigen, bacterium, or tumor as measured either by a heightened specific immune response or by increased resistance. According to Yashphe the best results of therapy against malignant disease may be based on the non-specific activation of the organism's responsive- ness to the tumor cells, rather than focusing attention on specific immunotherapy by means of tumor associated antigens. Lysozyme Concentrations in Acti- vated MacrophagesiMOnocytes Carson and Dannenberg (1965) reported lysozyme levels 1.4 times normal in peritoneal macrophages collected from tuberculous rabbits. Lysozyme levels in extracts of alveolar macrophages from rabbits vaccinated and challenged with tubercle bacilli of the BCG strain were 5 to 6 times normal (Oshima et al., 1961). The authors postulate that the high content of lysozyme in alveolar macrophages from normal rabbits may be related to the capacity of the cells to destroy some of the microorganisms which commonly invade the lower respira- tory tract. 10 The fact that lysozyme is elaborated by and released from macrophages is well documented (Myrvik et al., 1961; Cohn and Wiener, 1963). An increased production and/or release of lysozyme by macrOphages following BCG vaccina— tion has also been demonstrated (Cohn and Wiener, 1963; Myrvik et al., 1962; Carson and Dannenberg, 1965). Osserman (1974) has found that lysozyme alters the structure and in vitro behavior of certain transformed mammalian cells and postulates that lysozyme may be one if not the major mediator of the anti-tumor functions of macrophages. Review of Lysozyme Methodology Perillie and Finch (1974) and Hansen and Andersen (1973) comment on the lack of agreement between the two methods of analysis of lysozyme, and note the use of the two commonly employed standards, egg-white and human lysozyme. The method generally employed is a turbidimetric pro- cedure in which clearing of a suspension of Micrococcue Zysodeikticus occurs in prOportion to the concentration of lysozyme present in the test specimen (Osserman, 1966). There is also a radial diffusion (lysoplate) method (Osserman, 1966), in which heat-killed Micrococcus lyso- deikticus organisms are suspended uniformly in molten agar and poured into petri dishes. Wells are cut in the agar for the test samples. Zones of clearing develop in the gel as the result of bacterial lysis. The diameter of the ll cleared zone is proportional to the concentration of lyso- zyme in the test specimen. METHODS AND MATERIALS Specimen Selection Twelve patients with malignant disease were selected prior to their chemotherapy program. Eleven patients with a variety of benign conditions (defined as those con- ditions which are not malignant) were selected as a compari- son group. Fourteen individuals free of known disease were included as a control group. The groups were age and sex matched as closely as feasible. The patients and normal individuals did not have symptoms of renal disease. To eliminate this possibility serum BUN or creatinine levels were measured on each specimen. Preparation of the Monocytes Fifty milliliters of EDTA anticoagulated blood and 5 m1 of blood allowed to clot were drawn from each patient or control and were processed immediately. The collecting apparatus consisted of a 19-gauge butterfly needle and a 50 ml plastic syringe. A total white blood cell count and differential were done on each whole blood specimen. The mononuclear leukocyte layer of cells was obtained using the gradient centrifugation technique employing 12 13 Ficoll-Hypaque* according to the method of Boyum (1968). The 50 ml of EDTA anticoagulated blood were diluted 1:1 with 0.9% NaCl, pH 7.1. Seven milliliters of Ficoll- Hypaque solution were layered under 20 ml of the diluted blood. This was centrifuged immediately for 20 minutes at 650 g. The monocytes and lymphocytes appeared as a white ring on tOp of the Ficoll-Hypaque layer. The mono- cytes and lymphocytes were removed with a capillary pipette and placed in a 50 ml plastic centrifuge tube. The cells were washed three times in 0.9% NaCl. Centrifugation was at 300 g for 10 minutes for each wash. All plastic containers were used since monocytes adhere to glass. It was necessary to process rapidly and mix well since the cells tended to clump. Immediately following the final wash a cell count was done using the Coulter Fn automatic cell counter.** A minimum of three smears of the final product were made. Approximately equal amounts of the cell preparation and the patient's serum were mixed and spread on a glass slide. The serum is necessary to insure good staining of the cells. The slides were stained on an Ames Hematek automatic slide *** stainer which utilized a Wright's-Giemsa stain. * Ficoll, F-4375, Sigma Products, St. Louis, MO; Hypaque (50%), NDC 24-H65-4, Winthrop Labs, New York, NY. ** Coulter Electronics, Hialeah, FL. *** Ames Company, Elkhart, IN. 14 A minimum of three differentials were done fOr each cell preparation and the respective percentages of mono- cytes, lymphocytes, and granulocytes (if any) were determined. The cell preparation was then adjusted to the proper volume so that all specimens had a concentration of 107 monocytes/m1 of cell preparation. Several attempts were made to separate the monocytes from the lymphocytes. By utilizing the characteristic of adherence possessed by the monocytes, two techniques were investigated. The method of Brodersen and Burns (1973) involved incubating the mononuclear cell preparations in small glass beakers at 37°C on a shaker in a constant 5% CO2 environ- ment. After two hours the supernatant and nonadherent cells were decanted. The adherent monocytes were obtained by gentle scraping of the walls of the beakers. The second technique involved passing the cell prepara- tion through a column of glass beads according to the method of Rabinowitz (1965). The monocytes adhere to the glass beads and the lymphocytes pass through. Adherent cells are then released from the glass beads with 0.02% EDTA in Ca++ and Mg++ free buffered saline. In both of these instances the yield of monocytes obtained was inadequate. Generally these methods are successful when a full unit (500 ml) of blood is drawn. Since it was not possible to obtain this quantity of blood from patients with malignant disease, and Since it is well 15 documented that lymphocytes do not contain lysozyme (Flanagan and Lionetti, 1955; Gordon et al., 1974), it was decided not to attempt to remove the lymphocytes. Disruption of the Cell Membrane The method of choice was five cycles of rapid freezing at -20°C and thawing at room temperature (25°C). Ohta and Osserman (1972), Gordon et al. (1974), and Perillie (1968) all successfully utilized this method. Phase contrast microscopy was used to determine that all cells had been disrupted. Cell membranes can also be disrupted by means of sonication. A Branson model 140 sonicator* was utilized for this purpose. However, due to the high temperature (SO-90°C) which deve10ps within the Specimen during soni- cation, the lysozyme molecule was apparently destroyed. When lysozyme determinations were made on standard solutions following sonication, activity was lacking in the low con- centrations and greatly diminished in the higher concentra- tions. A cooling jacket for the specimen container may have alleviated the problem, but this was not available at the time of the experimentation. Lysozyme Analysis by the Lysoplate Method The radial diffusion or lysoplate method (Osserman, 1966) utilized heat-killed Micrococcus Zysodeikticus * Heat Systems Ultrasonics, Inc., Plainview, Long Island, NY. l6 organisms suspended uniformly in 1% molten agar. This mixture was poured into petri dishes. Wells were cut in the agar, and the test sample was added to the well. Recently the lysoplates became commercially available* and these plates were utilized in this research. A Sherwood pipette** with disposable plastic tips was used to deliver 10 ul of the lysed monocyte cell prepa- ration to the test wells. This must be done with care so all the specimen enters the well, and bubbles and splatters are avoided. All test specimens were run in triplicate. A reference enzyme solution was prepared by dissolving human lysozyme standard (urine)*** at a concentration of 1000 ug/ml in sodium phosphate buffer, pH 7.0. The reference enzyme solution was further diluted with sodium phosphate buffer to produce standards of the following concentrations (pg/ml): 150, 100, 50, 25, 15, 10, and 5. The plates were incubated at room temperature (25°C) for 18 hours. Zones of clearing develop in the gel as a result of bacterial lysis. The diameter of the cleared zones was measured to the nearest 0.5 mm. Indirect light was used to enhance the contrast and facilitate reading of the plates. The diameter of the cleared zones is related logarithmi- cally to the enzyme concentration. Using semilogarithmic * RADL, 3483, Worthington Biochemical Corp., NJ. icfi Sherwood Medical Industries, St. Louis, MO. *** LYHU92910, Worthington Biochemical Corp., NJ. 17 paper, the zone diameter (in mm) of each standard concen- tration was plotted on the linear axis. The standard enzyme activities (in ug/ml) were plotted on the log axis. The activities of the cell preparations were read from the standard curve prepared as above. This value was corrected for granulocyte contamina- tion (all test specimens had 10% or less granulocyte con- tamination). Several authors indicate that monocytes and granulocytes have approximately equal amounts of intra- cellular lysozyme (Senn et al., 1970; Hansen and Andersen, 1973; Ohta and Osserman, 1972), although their exact numerical values vary due to different methodologies and the use of different standards. For example, a specimen with 2% granulocytes and 34% monocytes (the remainder lymphocytes not containing lyso- zyme) with a lysoplate reading of 65 ug lysozyme/107 cells per ml would be corrected as g% x 65 = 61 ug lysozyme/107 cells per m1. Lysozyme Analysis by_the Turbidimetric Method The turbidimetric method utilizes a suspension of Micrococcus Zysodeikticus cells in sodium phosphate buffer, pH 6.2.* A clearing of the suspension occurs which is proportional to the concentration of lysozyme in the test specimen. * RLK #7978, Worthington Biochemical Corp., NJ. 18 Three milliliters of the Micrococcus Zysodeikticus substrate suspension were pipetted into a cuvette of the Gilford 240 Spectrophotometer.* Distilled water was used for the blank. The reaction was measured at 25°C, and at a wavelength setting of 550 nm. Three-tenths milliliter of serum or appropriate dilution of test specimen was added to the substrate. Absorbance readings were taken at 30 seconds and at three minutes. The change in absorbance (AA) over the two and one-half minute time interval was determined by the difference between the two absorbance readings. Worthington Biochemical Corporation supplies an egg- white standard for use with this procedure. The standard has a lysozyme concentration of 40 ug/ml. Appropriate dilutions were made with distilled water to provide the following recommended standards (in ug/ml): 20, 16, 12, 8, 4, and 2. The concentration of the standards was plotted on the horizontal axis, and the AA value was plotted on the vertical axis. The concentrations of the test specimens were then read from the curve. It is necessary to repeat on apprOpriate dilutions any specimens with lysozyme concentrations greater than 16 ug/ml, since the reaction drOps off rapidly at this point due to depletion of the substrate. Since the reaction is not linear over the two and one-half minute interval * Gilford Instrument Laboratories, Inc., Oberlin, OH. 19 (personal observation) specimens run on dilution are susceptible to increased error. In this project all sera were assayed by this method. Since the serum lysozyme values are generally under 15 ug/ml, this method provided the best sensitivity. Egg-White Standard vs. Human Standard The lysozyme standard is assayed by determining the rate of lysis of Micrococcua Zysodeikticus cell suspensions. One unit is defined as that which causes a decrease in turbidity of 0.001 per minute at 450 nm at pH 7.0 and 25°C. A wide range of activities are reported for pure lysozyme preparations under these conditions (Worthington Biochemical Corp., 1976). The Worthington egg-white standard assay is established in relationship to a high purity lysozyme standard defined at 12,000 units/mg protein. The egg-white standard used in this research had an activity of 9600 units/mg as assayed by Worthington. The Worthington human standard is isolated from the urine of patients with monocytic leukemia. The activity is approximately three times greater than the egg-white standard. The human standard used in this research had an activity of 33,500 units/mg as assayed by Worthington. Due to the high activity of the human standard, the substrate was depleted much too rapidly for use by the turbidimetric method. The 5 ug/ml human standard had a 20 AA of 0.074. The 10 pg/ml had a AA greater than 0.100, which indicates the substrate has been depleted. However, the human standard is an ideal choice for the lysoplate method since the 1y50plates have their best sensitivity from 25 to 500 ug/ml. Also, the human standard eliminated any differences in migration of the lysozyme in the lysoplates which may have been a factor in the use of an egg-white standard since their chemical conformations do vary. RESULTS AND DISCUSSION Monocytic Lysozyme Levels as Measured by the Lysoplate TeEhnique With Human Standard The results of the monocytic lysozyme levels of the patients with malignant or benign disease and the normal control individuals are illustrated in Figure l. The patients with malignant disease had a signifi- cantly higher monocytic lysozyme content (mean 70 ug/lO7 cells) than the normal individuals (mean 41 ug/lO7 cells) 7 or patients with benign diseases (mean 39 ug/lO cells). Ohta and Osserman (1972) obtained a mean lysozyme content of normal monocytes of 3.0 ug/106 cells, which is very similar to the present findings. They also utilized the lysoplate method and the human standard. Figure 2 is the lysoplate standard curve with human lysozyme standard. The human standard is the one of choice since it eliminates any question of variability of migra- tion due to the conformation of the molecule which occurs when the egg-white standard is used. It can also be noted from Figure 2 that the lysoplate method is useful for substances with relatively high con- centrations of lysozyme in the range of 25-150 ug lysozyme per ml. 21 22 120 110 100 on O \l O O" O 2’3 J 1 4} o ug lysozyme/107 monocytes (N O N O 10 Malignant Normal Benign Figure 7. Comparison of mean intracellular monocytic lysozyme in patients with untreated malignant disease, in normal individuals and in patients with benign diseases. Brackets indicate 95% confidence intervals. 150 100 50 N 01 g lysozyme/ml H U1 H O 23 Figure 2. 3 4 5 6 7 8 9 10 11 12 13 Zone Diameter (mm) Lysoplate standard curve with human standard. 24 For comparison purposes, Figure 3 is the lysoplate standard curve with egg-white lysozyme standard. It is very similar to the human lysozyme standard curve, as was also noted by Hansen and Andersen (1973). This is not what one would expect since the human standard is approxi- mately three times more active than the egg-white standard. Zucker et a1. (1970) explained this by noting that using the same enzyme standard, lysozyme activity determined with the turbidimetric method was shown to be about one-third of the activity found with the 1y50plate method. Tables 1, 2 and 3 list the results of additional laboratory tests which were done when the test specimens were drawn. The data are included to provide background information to aid in the evaluation of the test results. Table 1 lists the BUN (blood urea nitrogen) values which were obtained from the SMA-12 reports. Tables 2 and 3 list the creatinine values which I determined using the Jaffe reaction (Davidsohn and Wells, 1962). The purpose of the tests was to ascertain that renal function was within normal limits. A peripheral white blood count and differential were done on each specimen. Essentially the WBC counts were within normal limits, and the differentials were of normal percentage distribution. In the table the letter "C" refers to granulocytes, which is used as an inclusive term for neutrophils, basophils, and eosinophils. In a few patients with malignant disease there were occasional young neutrophils (myelocytes) present. 150 100 50 25 15 10 ug lysozyme/ml 1 2 3 4 5 6 7 8 9 10 11 12 13 Zone Diameter (mm) Figure 3. Lysoplate standard curve with egg-white standard. 26 Table 1. Background laboratory data of patients with malignant disease BUN Peripheral Peripheral Clinical Age/Sex (mg/d1) WBC/cu.mm. Differential Diagnosis 1. 41/M 10 6200 G62 L29 M9 CA of lung 2. 74/M 12 6800 G78 L19 M3 Squamous cell CA 3. 49/M 18 6300 G73 L15 M11 Hepatoma 4. 59/M 15 8500 G67 L26 M7 CA of prostate 5. 70/F 13 5400 G90 L8 M2 CA of breast 6. 62/M 19 7600 691 LS M4 CA of esophagus 7. 37/F 14 9800 G62 L34 M4 CA of breast 8. 59/M 10 9300 G75 L16 M9 CA of lung 9. 64/F 9 11500 G74 L20 M6 CA of colon 10. 64/F 17 4400 G64 L32 M4 CA of breast ll. 50/F 19 8890 G76 L19 M5 Broncho- genic CA 12. 64/F 8 9000 G88 L10 M12 Metastatic breastCA 13. 76/M 19 170,000 GS L95 M0 CLL 27 Table 2. Background laboratory data for normal individuals Peripheral Peripheral Age/Sex Creatinine (mg/d1) WBC/cu.mm. Differential l. ZO/F 0.6 7800 667 L29 M4 2. 27/M 1.1 6200 664 L31 M15 3. SS/M 1.2 6500 666 L29 M5 4. 50/F 0.5 6400 663 134 M4 5. 58/F 1.0 5000 659 L35 M6 6. 40/F 0.9 7400 653 L38 M9 7. 34/F 1.1 5100 G59 L33 M8 8. 44/F 1.2 8100 G70 L25 M5 9. 21/F 1.1 10600 685 L12 M3 10. 30/F 1.0 5000 658 L34 M4 11. 45/F 0.6 7000 659 L33 M8 12. 27/M 0.8 4000 650 L40 M10 13. 59/M 1.6 5300 677 L14 M9 14. 55/F 1.0 7500 662 L36 M2 28 Table 3. Background laboratory data on patients with benign disease Creati- Periph- Peripheral Age/ nine eral WBC Differen- Sex (mg/d1) /cu.mm. tial Disease 1. 65/M 1.0 11000 670 L20 M10 Coronary insuff. 2. 27/F 1.2 5200 661 L36 M3 Seborrhea derma- t1t1s 3. 24/F 1.4 6500 G67 L30 M3 Asthma 4. 63/F 0.6 7300 664 L28 M8 Allergies 5. 54/F 1.3 7400 663 L29 M8 Arthritis 6. 40/M 0.9 8700 674 L24 M2 Diabetes 7. 30/M 1.1 6500 676 L21 M3 Allergies 8. 23/F .2 10000 672 L22 M6 Crohn's disease 9. 40/F 0.8 7300 664 L32 M4 Hypothyroidism 10. 45/F 2.0 5000 658 L37 M5 Arteriosclerosis 11. 53/M 0.6 6100 653 L37 M10 Coagulopathy 29 Diagnosis, age and sex of each patient are also noted in the table. The SMA-lZ reports were essentially normal with one frequent exception: an elevated LDH (lactic acid dehy- drogenase). This is due to the increased cell destruction occurring as a result of the malignant disease process. Patient #13 in Table 1 is of interest. He was included because of his high concentration of lymphocytes. When the lysozyme level was determined on his cell prepara- tion it was 0 ug lysozyme by both the lysoplate and the turbidimetric methods. This substantiates the published data stating that lymphocytes do not contain lysozyme. Table 4 lists the volume of the final cell preparation and the Wright's stained differential results. The differ- entials listed are the average of a minimum of three dif- ferentials per cell preparation. From the data in Table 4 it can be seen that most of the granulocytes have been removed by the Ficoll-Hypaque process, leaving a product composed primarily of lympho- cytes and monocytes. The cell preparation volume was adjusted to 107 monocytes per ml in all specimens. Tables 5, 6 and 7 contain the triplicate readings for each lysozyme determination by the ly50plate method. The values are corrected to eliminate any granulocytic lyso— zyme that may have been present in the specimen. Table 8 is a summary table for the three groups indi- cating the mean for each group and the confidence intervals Table 4. 30 Monocyte preparations Cell Preparation Volume (ml) (107 monocytes/ml) Cell Preparation ‘Differential HHH NH O O hahud PHH awn th somwaxm-buNr-I NHO O‘OQ‘QGMthMNI-I‘ o o o o o o o o o o HOOCDVO‘U‘I-bMNI-i o o o o o o o o o o o QOOOOOOOCOOOOO COOOOOOOOOO o o o o o o o o o o o Malignant Group Normal Group Benign Group L74 L64 L68 L83 L77 L52 L73 L50 L80 L83 L72 L61 L88 L86 L81 L83 L86 L90 L92 L88 L80 L88 L89 L78 L76 L83 L83 L89 L86 L86 L73 L74 L86 L80 L85 L87 L91 M24 M34 M31 M11 M22 M39 M26 M40 M17 M12 M26 M29 M14 M11 M10 M21 M16 M11 M9 M8 M8 31 Table 5. Lysoplate readings, malignant group Lysozyme Zone Activity Corrected Average Diameter (pg/107 Lysozyme Lysozyme (mm) monocytes) Activity Activity 1. 10.0 64 59 10.5 85 78 67 10.0 70 64 2. 10.5 75 71 10.0 65 61 61 9.5 54 51 3. 11.0 89 86 12.0 133 130 115 12.0 134 130 4. 12.0 150 97 12.5 135 120 112 12.5 135 120 5. 10.0 68 65 11.0 96 92 83 11.0 96 92 6. 10.0 61 49 10.0 64 52 51 10.0 64 52 7. 9.5 51 49 10.0 57 55 51 9.5 51 49 8. 10.0 66 53 10.0 66 53 51 10.0 60 48 9. 11.0 106 90 12.0 156 133 110 11.5 126 107 10. 8.5 34 24 9.0 37 26 26 9.0 37 26 11. 9.5 47 44 9.0 45 42 44 9.5 47 44 12. 11.0 89 68 11.0 89 68 68 11.0 89 68 32 Table 6. Lysoplate readings, normal group Lysozyme Zone Activity Corrected Average Diameter (pg/107 Lysozyme Lysozyme (mm) monocytes) Activity Activity 1. 9.0 46 31 9.0 46 31 30 9.0 41 27 2. 8.0 31 29 8.5 37 34 35 9.0 46 43 3. 10.5 82 52 11.0 87 55 53 10.5 82 52 4. 10.0 56 43 10.0 56 43 46 10.0 68 52 5. 11.5 125 54 11.5 125 54 54 11.5 125 54 6. 10.5 84 59 10.5 84 59 59 10.5 84 59 7. 9.0 39 29 8.5 34 26 28 9.0 39 29 8. 8.5 37 31 8.5 37 31 31 8.5 37 31 9. 10.5 84 63 10.0 56 42 56 10.5 84 63 10. 8.5 37 22 8.5 31 18 21 8.5 37 22 11. 9.0 37 34 9.0 37 34 34 9.0 37 34 33 Table 6 (continued) Lysozyme Zone Activity Corrected Average Diameter (Hg/10 Lysozyme Lysozyme (mm) monocytes) Activity Activity 12. 10.0 68 43 10.0 84 53 50 10.5 84 53 13. 9.5 56 33 9.5 56 33 35 10.0 68 40 14. 11.5 125 51 11.0 100 41 47 11.5 125 51 34 Table 7. Lysoplate readings, benign group Lysozyme Zone Activity Corrected Average Diameter (pg/107 Lysozyme Lysozyme (mm) monocytes) Activity Activity 1. 10.5 84 69 10.0 56 46 54 10.0 56 46 2. 9 5 47 34 9 5 57 41 36 9 S 47 34 3. 8.5 37 29 8 5 37 29 29 8 5 37 29 4. 10.5 85 61 10.5 79 56 59 10 5 85 61 5. 10.0 56 44 10.0 56 44 44 10.0 56 44 6. 10.0 56 34 10.0 56 34 34 10.0 56 34 7. 8.5 37 10 8.5 37 19 20 9.0 46 23 8. 10.0 64 38 10.0 64 38 38 10 0 64 38 9. 10.5 84 50 10.5 84 50 47 10.0 68 41 10. 9 5 56 34 9.5 56 34 32 9 5 46 28 11. 8.5 37 33 9 5 46 41 36 8.5 37 33 Note: It was necessary to use two lot numbers of 1y50plates. A standard curve was constructed for each lot number. The curves varied slightly. Therefore a 10 mm zone will give a slightly different value depending upon which lot number was used. 35 Table 8. Summary of lysoplate determinations Malignant Normal Benign Group Individuals Group 67 30 54 61 35 36 115 53 29 112 46 59 83 54 44 51 59 34 51 28 20 51 31 38 110 56 47 26 21 32 44 34 36 68 50 35 47 x = 70 ?'= 41 '5 39 S = 19.2 95% simultaneous confidence intervals: < - s 9.59 - “l “2 48.41 < _ < 10.45 - “1 ”3 — 51.55 _ < - < 17.76 - ”2 “3 - 21.76 “1 = population mean of malignant group “2 = population mean of normal individuals population mean of benign group 36 calculated according to Scheffé simultaneous 95% confi- dence intervals. Since the population mean of the malignant group minus the population mean of the normal group in the 95% simul- taneous confidence intervals does not contain zero, it indicates that the population mean for the malignant group is larger than the population mean for the normal group. A similar method of interpretation is used for comparing the population mean of the malignant group minus the popu- lation mean of the benign group in the 95% simultaneous confidence intervals. Since the population mean of the normal group minus the population mean of the benign group in the 95% simul- taneous confidence interval does contain zero, it indicates that the population means of the two groups could be the same. The cell preparation data were also tested by the null hypothesis, which also concluded that the population mean of the malignant group was larger than the population mean of the normal group. The descriptive level at which we would reject the null hypothesis was less than .0005. Figure 4 is a photograph of a lysoplate showing the clear area of lysis due to the activity of lysozyme on the Micrococcus Zysodeikticus substrate. Figure 5 is a photograph of a Wright's stained smear of the monocyte preparation. 37 - ' . ( .‘f'\.‘r ' ‘ - ‘. Q. vi ' P a - do -) "‘ ’.,¢'i. ‘Wd'ffi' '0 ’- ‘ - . ' n . - Figure 4. Lysoplate photograph. 38 Figure 5. MOnocyte cell preparation. Wright's-Giemsa stain X 1000. .3. .0”. MI... I . .umr 9.1.4.» . J... m... a” . C.hl L“ n he.“ a w: ' we??? 33*- A. fit pi a . p. U Mn (fl .3 ' n: «41 " x. (a 6’6”." .11.. N». 39 Serum Lysozyme as Measured by the Turbidimetric Method The turbidimetric method is the one of choice for measuring specimens with lysozyme concentrations below 15 ug/ml as is usually the case with human blood serum. The egg-white lysozyme standard is used with this method because the human lysozyme standard is too active. The 5 ug/ml human standard has a AA of .074, and the 10 ug/ml has a AA of .112, which indicates the substrate has been depleted. Figure 6 is the standard curve for the turbidimetric method using an egg-white standard. Table 9 lists the data of the triplicate determina- tions of each specimen and the average ug/ml of lysozyme for the three readings is noted. Please note in regard to interpretation of the AA quantities that the values were read from the curve for its own substrate lot number. Two lots of substrate were used and the curves were slightly different. The serum lysozyme values for the three groups are very similar and do not seem to be affected by the malig- nant process. The means for the respective groups and the 95% simultaneous confidence intervals calculated according to Scheffé are as follows: 40 95% simultaneous con- Mean fidence intervals Malignant 12.5 -2.96 S “l - “2 S 5.56 Normal 11.5 -2.80 S “l - ”3 S 6.20 Benign 11.1 -3.93 S “2 - “3 S 4.73 S 4.21 “1 = population mean of malignant group “2 = population mean of normal group “3 = population mean of benign group Since each of the three confidence intervals contains a zero, it is possible that the population mean could be the same for each of the three groups. AA .110 .100 .090 .080 .070 .060 .050 .040 .030 .020 .010 .000 41 2 4 8 12 16 20 Lysozyme Activity (pg/ml) Figure 6. Turbidimetric standard curve with egg-white standard. 42 Table 9. Serum lysozyme readings by the turbidimetric method Malignant Normal Benign ug m AA ug/ml AA ug/ml 1. .064 .046 .073 .066 12.0 .049 8.5 .074 13.1 .065 .047 .073 2. .052 .074 .043 .054 9.8 .074 10.2 .047 8.2 .053 .074 .045 3. .076 .060 .046 .084 14;; f 2 .062 8.6 .047 8.3 .080 ° .061 .046 4. .079 .078 .059 .085 14.9 .079 14.0 .061 10.8 .081 .078 .060 s. .050 .100 .100 .054 9.6 .098 13.8 .098 13.2 .052 .099 .099 6. .042 .093 .060 .042 7.8 .093 12.9 .058 8.0 .042 .093 .059 7. .051 .062 .087 .050 9.3 .063 11.1 .088 11.5 .050 .062 .086 8. .059 .092 .095 .060 11.1 .090 12.6 .096 12.6 .060 .091 .094 9. .054 .110 .080 .054 10.0 .113 20.0 .080 10.6 .054 .111 .081 10. .037 .064 .083 .035 6.7 .065 9.0 .084 11.1 .036 .065 .084 11. .078 .046 .101 .081 14.7 .049 8.5 .103 14.4 .080 .047 .101 Table 9 (continued) 43 Malignant Normal Benign AA ug/ml AA ug/mI ug/ml 12. .096 .054 .100 18.0 .053 9.7 .098 .054 13. .048 .079 .048 8.9 .078 10.5 .048 .079 14. .083 083 11.5 :083 SUMMARY AND CONCLUSIONS The intracellular monocytic lysozyme level is elevated in patients with untreated malignant disease. Measurements were made using the lysoplate technique and a human lysozyme standard. The serum lysozyme level does not appear to be affected by the malignant disease process. Measurements were made using the turbidimetric method and egg-white lysozyme standard. We now know that monocytes and macrophages are selec- tively cytotoxic for some tumor cells, and the concentration of intracellular monocytic lysozyme in individuals with malignant disease is higher than in normal control indi— viduals. The most significant question now is: Is the monocytic-macrophage intracellular lysozyme the actual agent that is destroying tumor cells? Additional studies need to be conducted to determine the mechanism by which lysozyme may act on the tumor cell substrate. 44 . I'll [1'11 III! -I' III III ' III APPENDICES APPENDIX A REAGENTS Ficoll-Hypaque Solution Ficoll 9.54 gm Hot dist. water 106.00 ml * Hypaque (33.9%) 44.00 m1 Add hot distilled water to Ficoll. Bring to a boil. Remove from heat and allow to cool slightly. Add Hypaque (33.9%) and mix. Refrigerate. Stable for two weeks. Sodium Phosphate Buffer for Lysoplate Standard Diluent NaHZPO4-HZO 6.9 gm Dissolve sodium phosphate in one liter of distilled water. pH to 7.0. Refrigerate. * 50% Hypaque 30.0 ml dist. water 14.0 ml 45 APPENDIX B INFORMED WRITTEN CONSENT FORM A registered medical techologist will draw 55 ml of blood (approximately 2 fluid ounces) into a 60 ml plastic syringe by a single venipuncture technique. One kind of white blood cell, the monocyte, will be analyzed for its lysozyme content. The attendant discomfort and risk are the same as for a routine venipuncture. There is no direct benefit to the blood donor. However, the knowledge obtained from this research may aid in the diagnosis and treatment of disease. I understand that I am free to withdraw my consent and to discontinue my participation in the project. I understand the identity of the specimens will remain anonymous, and that the confidentiality of the reports will be maintained at all times. Should the results be published, the identity of the specimens will not be revealed. I freely consent to donate my blood specimen for this research. I understand all of the above statements and all of my questions concerning the procedure have been adequately answered. Signed Date Witness Physician signature 46 REFERENCES REFERENCES Ambrose, E. J.: The Biology of Cancer. Van Nostrand- Reinhold, Princeton, NJ, 1966. Arnon, R., and Sela, M.: Antibodies to a unique region in lysozyme provoked by a synthetic antigen conjugate. Proceedings of National Academy of Science, 62, (1969): 163-170. Bluming, A. Z., Vogel, C. L., Ziegler, J. L., Mody, N., and Kamya, 6.: Immunological effects of BCG in malig- nant melanoma: Two modes of administration compared. Annals of Internal Medicine, 76, (1972): 405-411. 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Journal of Laboratory and Clinical Medicine, 75, (1970): 83-92. VITA The author was born on July 2, 1942, in Saginaw, Michigan. In 1964 she received the Bachelor of Science degree in Biological Science from Michigan State University. She taught high school biology in Warren, Michigan, and in Medinah, Illinois. From January 1967 to January 1968 she served her medical technology internship at Hinsdale Sanitarium and Hospital, Hinsdale, Illinois. In 1968 she was employed as a registered technologist at Michigan State University, Olin Health Center Laboratory. From 1970 to 1974 she was employed as a staff technologist at the Holland Medical Center Laboratory, Holland, Michigan. In September 1974 the author began pursuing a graduate degree in Clinical Laboratory Science at Michigan State University. Since July 1974 she has been employed at Michigan State University, Olin Health Center Laboratory. 52 MICHIGAN STATE UNIVERSITY LIBRARIES 3 1193 03083 2491