M‘-‘.~_‘—

 

:AEhAsPEchs AND INTERSP‘ECTES TRANSFERS oE
DELAYED TYPE HYPERSENSITWHY. TO GUINEA Pies,
AND THETR PERTTONEAL EXUDATE cELLs wmA

' RNA EXTRAGTED FROM LYMPHOID TISSUES FROM

GUENEA PIGS AND CATTLE

Thesis for the Degree of M; .S. ‘ '
- MIC-HTGAN STATE umvmsfiv' ~
SHANG WANG
.1973

0-... o - .."’"""”‘V‘
'

 

mu " ‘..-

 

 

ABSTRACT

INTRASPECIES AND INTERSPECIES TRANSFERS OF DELAYED TYPE HYPERSENSITIVITY
TO GUINEA PICS AND THEIR PERITONEAL EXUDATE CELLS WITH RNA EXTRACTED
FROM LYMPHOID TISSUES FROM GUINEA PICS AND CATTLE

by

Shang Wang

Ribonucleic acid extracts were made from normal (N-RNA) and ECG-
infected (S-RNA) guinea pigs and cattle. The RNA was extracted from the
spleen and lymph nodes of guinea pigs and from the lymph nodes of cattle.

260/OD280 = 2 were used. The concentration was

adjusted on the basis that 1 mg RNA/ml OD260 = 20. To allow passive

sensitization, 108 PEG in 2 ml medium and 500 ug RNA in 0.1 ml were mixed

Only RNA extracts with OD

-with gentle agitation and incubated at 379C in 5% C02 for 60 min. Cells
were centrifuged and washed twice. The criterion of passive tuberculin
sensitization by RNA in vitro of normal guinea pig peritoneal exudate cells
(PEG) was the inhibition of PEG migration radially or from capillary tubes
by 10 ug tuberculin (PPD)/ml.

Both guinea pig and bovine S-RNA conferred sensitivity in vitro to
normal guinea pig PEC; N~RNA did not. This was interpreted as intraspecies
and interspecies in vitro passive sensitization.

Normal guinea pigs were inoculated intraperitoneally with their own
(autologous), washed PEC after incubation with guinea pig S-RNA. Migration
of peritoneal cells collected 25 days later was inhibited by PPD. This

was interpreted as intraspecies in vivo passive sensitization.

Normal guinea pigs were inoculated intraperitoneally with autologous,
washed PEC after incubation with bovine S-RNA or bovine N-RNA. Three days
later, 10 TU PPD injected intradermally elicited 18-25 mm reactions (delayed
type only) on guinea pigs whose cells had been incubated with bovine S-RNA;
none on guinea pigs whose cells had been incubated with bovine N-RNA. From
the same guinea pigs nine days after autologous cell injections, there was
inhibition of migration in vitro by PPD of PEG from guinea pigs whose cells
had been incubated with bovine S-RNA; little or none with bovine N-RNAo The
transfer of PPD sensitivity to guinea pigs and to their PEC by inoculation
of autologous washed PEC incubated with Bovine S-RNA was interpreted as

interspecies in vivo passive sensitization.

INTRASPECIES AND INTERSPECIES TRANSFERS OF DELAYED TYPE HYPERSENSITIVITY
TO GUINEA PIGS AND THEIR PERITONEAL EXUDATE CELLS WITH RNA EXTRACTED
FROM LYMPHOID TISSUES FROM BUINEA PICS AND CATTLE

by

20

Av

Shang Wang

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

1973

O
06% ACKNOWLEDGEMENTS
The author expresses his sincere appreciation to Dr. Virginia H.
Mallmann for her guidance and to the group of the Tuberculosis Project
at Michigan State University for their assistance throughout this study.

Particular gratitude is also extended to my parents for their

continuous encouragement to me in my education.

LIST OF ll‘ABI—LE S O O O O O O O 0
INTRODUCTION. . . . . . . . .
L ITE RATURE RE VIEW . . . . . .

MATERIALS AND masons .' . . .

Sensitization of guinea pigs

Sensitization of cattle.
RNA extraction . . . . .

TABLE OF

Prestimulation and collection of
Passive sensitization of guinea pig PEC in vitro by RNA extracts

Passive sensitization of guinea pigs by injection of autologous

CONTENTS

'0 0

peritoneal exudate cells (PEC).

washed PEC after incubation in vitro with RNA.
Capillary tube test for migration-inhibition . . . .
Spot test for migration-inhibition .

Skin tests . . . . . . .

Passive sensitization tests.

FE SUIJI1S O O O O O 9 O O O O 0

Passive transfer of delayed sensitivity in vitro
to normal guinea pig PEC by RNA extracted from

guinea pigs. . . . . .

O

and

BCG-

O O O O O
in vivo
infected

Passive transfer of delayed sensitivity to normal guinea pig
PEC in vitro with RNA extracts from ECG-infected steers. . .

Passive sensitization in vivo of normal guinea pigs by
inoculation of autologous PEC after incubation in vitro
with RNA from BCG~infected steers.

DISCUSSION. 0 O 0 O O O O O O

L ITE RAE'EIJ RE C ITE D a o o o o o 0

iii

Page

18

18
18
18
19
20

20
21
21
22
22

23

23

36

45

51

LIST OF TABLES

'Table Page

1. Migration inhibition tests of normal guinea pig
peritoneal exudate cells (PEG), after incubation with RNA
extracts from spleens and lymph nodes of ECG-infected
guinea pigs (S-RNA) or normal guinea pigs (N-RNA)cu1tured
with and without Purified Protein Derivatives (PPD)................. 24

2. Migration inhibition tests of peritoneal exudate cells (PEG)
from ECG-infected guinea pigs prior to ribonucleic acid
extraction from spleen and lymph nodes............................... 25

3. Migration inhibition tests of peritoneal exudate cells (PEG)
from a normal guinea pig after incubation in vitro with RNA
extracts from the spleens and lymph nodes from BCG-infected
guinea pigs (GP-I or GP-II, TABLE 2) cultured with and with-
out Purified Protein Derivatives (PPD)............................... 26

4. Migration inhibition tests of peritoneal exudate cells (PEG)
from a normal guinea pig (GP-III) injected 25 days previously
with autologous PEG after incubation with RNA extracts from
the spleens and lymph nodes of BOG-infected guinea pigs
(GP-T and GP-II) cultured with and without Purified Protein
Derivatives (PPD)............................... ...... ..... ..... ..... 27

5. Migration inhibition tests of leucocytes from a normal steer (#56)
and two BOG-infected steers (#36 and #51) prior to ribonucleic
acid extractions of lymph nodes...................................... 29

6. Migration inhibition tests of normal guinea pig (GP-Sb-l)
peritoneal exudate cells (PEG) incubated with the RNA extracts
from the lymph node of a normal steer (NB-56-RNA) cultured
with and without Purified Protein Derivatives (PPD).................. 3O

7. Migration inhibition tests of normal guinea pig (GP-56-2)
peritoneal exudate cells (PEG) incubated with the RNA extracts
from the lymph node of a normal steer (NB—56-RNA) cultured
with and without Purified Protein Derivatives (PPD).................. 31

8. Migration inhibition tests of normal guinea pig (GP-36-l)
peritoneal exudate cells (PEG) incubated with the RNA extracts
from the lymph nodes of a normal steer (NB-56-RNA) or ECG-infected
steer (SD~36-RNA) cultured with and without Purified Protein
Derivatives (PPD).................................................... 32

9. Migration inhibition tests of normal guinea pig (GP-36-2)
peritoneal exudate cells (PEG) incubated in vitro with the RNA
extracts from the lymph nodes of normal steer (NB-56-RNA) or
EGG-infected steer (SB-36-RNA) cultured with and without
Purified Protein Derivatives (PPD)................................... 33

iv

 

luau"- I' alga .,

Table
10.

11..

12.

13.

14.

15.

16.

17.

18.

Page

Migration inhibition tests of normal guinea pig (GP-Sl-l)

peritoneal exudate cells (PEC) incubated in vitro with the RNA

extracts from the lymph nodes of normal steer (NB-56-RNA) or

ECG-infected steer (SB-51-RNA) cultured with and without

Purified Protein Derivatives (PPD)................................... 34

Migration inhibition tests of normal guinea pig (GP-51-2)

peritoneal exudate cells (PEC) incubated in vitro with the

RNA extracts from the lymph nodes of normal steer (NB-56-RNA)

or ECG-infected steer (SB-Sl-RNA) cultured with and without

Purified Protein Derivatives (PPD)................................... 35

Summary of TABLES 6-11 of migration tests to detect passive

sensitization of normal guinea pig PEC in vitro by RNA extracts

from lymph nodes of a normal steer (NB-56-RNA) or from BGG-

infected steers (SB-36-RNA or SB-51-RNA)............................. 37

Migration inhibition test of peritoneal exudate cells (PEC)

from normal guinea pigs (GP-56-l and GP—56-2) nine days after
intraperitoneal injection of autologous PEC incubated pre-

viously with the RNA from normal steer (NB-56-RNA) cultured

with and without Purified Protein Derivatives (PPD).................. 38

Migration inhibition tests of peritoneal exudate cells (PEC)

from normal guinea pigs (GP-36-l and GP-36-2) nine days after
intraperitoneal injection of autologous PEC incubated pre-

viously with the RNA of EGG-infected steer (SB-36-RNA) cultured

with and without Purified Protein Derivatives (PPD).................. 39

Migration inhibition tests of peritoneal exudate cells (PEC)

from normal guinea pigs (GP-Sl-l and GP-51-2) nine days after
intraperitoneal injection of autologous PEG incubated pre-

viously with the RNA of ECG-infected steer (SB-Sl-RNA) cultured

with and without Purified Protein Derivatives (PPD).................. 40

Summary of TABLES 13, 14 and 15 of migration tests to detect

passive sensitization of normal guinea pig PEC in vivo by RNA

extracts from lymph nodes of a normal steer (NB-56-RNA) or from
ECG-infected steers (SB-36-RNA or SB~51-RNA)......................... 41

Results of skin tests of guinea pigs three days after intra-

peritoneal injection of autologous PEC incubated with the RNA from
normal steer (NB-56-RNA) or ECG-infected steer (SB-36-RNA and SB-

51~RNA) observed at 24 hrs, 48 hrs and 72 hrs........................ 43

Summary of TABLES 12, 16 and 17 of migration inhibition and skin

tests to detect passive sensitization in vitro and in vivo of

normal guinea pigs by RNA extracts from lymph nodes from a normal

steer (NR-56~RNA) and from two ECG-sensitized steers (SB-36-RNA

and SB-Sl-RNA)..... ...... ........... ...... ........................... 44

INTRODUCTION

A tuberculin reaction has been the classical example of delayed type
sensitivity. Its immunologic status and role in tuberculo-immunity and
pathogenesis has been controversial. It can not be transferred in serum
with antibody-mediated immunity and sensitivity. The classic work by
Landsteiner and Chase (88) nearly thirty years ago demonstrated that
immunologically-specific sensitivity can be transferred to man and guinea
pigs with homologous white cells, later by Lawrence (89) by transfer factor
extracted from white cells. Very few cells were required and the passively
acquired sensitivity persisted too long for transfer factor to be anti-
bodies. It was not inactivated by DNase, RNase or trypsin.

It is reasonably well established now that delayed sensitivity is
mediated by thymus-dependent small lymphocytes. When delayed sensitivity
has been induced, the sensitive lymphocyte possesses immunologically
specific receptor sites. The tuberculin-sensitive lymphocyte reacts with
tuberculin and releases effector molecules. The effector molecules are
not immunoglobulins and, with exception of transfer factor, probably are
not immunologically specific in their action. They may attract, activate
or immobilize macrophages, thereby contributing to the "localizing" im-
munity of tubercle formation or a delayed type skin reaction. The effector
‘ molecules do not passively confer long-term delayed sensitivity in vivo.
They produce effects on cells detectable in vitro, referred to as in vitro
correlates of delayed sensitivity, such as the migration-inhibition factor
(MIF). The immunologic role of macrophages is more controversial.

There are numerous reports on the transfer of antibody-producing

capacity by RNA and RNA-antigen complexes extracted from lymph nodes, spleen,

liver and peritoneal exudate cells. There have been fewer transfer of
delayed sensitivity. The RNA was extracted from lymph nodes, thoracic
duct lymphocytes or peritoneal exudate cells.

This is a report of intraspecies and interspecies transfer of tuber-
culin sensitivity with RNA extract from lymphoid cells. The sensitivity
was detected by inhibition of migration in vitro of normal guinea pig
peritoneal exudate cells and by intradermal tests of guinea pigs. The
RNA extracts were made from lymph nodes and spleens of guinea pigs and
from lymph nodes of cattle infected with viable, attenuated Mycobacterium

bovis strain Bacille de Calmette—Guerin (BCG).

LITERATURE REVIEW

The genus fiycobacterium includes a variety of organisms ranging from

 

the pathogenic Species such as M. tuberculosis to the sapr0phytic nonpatho-

 

genie species such as M. phlei. The three species of the genus Mycobacterium
primarily responsible for tuberculosis of mammals and birds are M. tuberculo-
_§i§, fl. bgyis, and M. avium for which man, cattle and chickens are the pri-
mary but not exclusive hosts, respectively. As the incidence of tuberculosis
due to the three species has decreased, the incidence of disease and sensi-
tivity due to the anonymous mycobacteria has been recognized in man (140)
and in cattle (108). Cross reacting antigens and other similarities con-
tribute to difficulties in differentiating among the infecting species.
The common properties linking these organisms include their acid-fastness
and the similarity of the basic structure of peptoglycans, mycosides, and
mycolic acids of their cell walls, and the degree of homology found around
their DNA. There are antigenic determinants common to some or all, but
relatively few specific £01 a species (138).

The pathogenic mycobacteria are facultative intracellular parasites.
They survive or even multiply within macrophages generally capable of
killing many other bacteria. Other facultative intracellular parasites

include species of the genera Salmonella, Pasteurella, Brucella and

 

 

lggflggjg, and some fungi and protozoan. The number which survive or are
destroyed are dependent on such factors as on the number and virulence of
the organisms, the portal of entry and the species and condition of the
animal infected (65, 66). The facultative intracellular parasites are able
to survive substances in or from phagocytic cells, including the hydrolytic

enzymes in the lysozymes. The surviving intracellular bacilli may multiply

and may kill the phagocytic cells by which they were ingested. When delayed
type sensitivity and a increased macrophage activity, now called cell medi-
ated immunity, developes, the infection may be limited and granulomata

or tubercles formed (130, 131, 135).

Killed mycobacterial cells induce delayed sensitivity which wanes with
time, and there is less increased phagocytic activity than with viable at-
tenuated cells (173, 174, 175). Attenuated strains of mycobacteria have
been selected by serial passage through unnatural hosts or on artificial
culture media. The Bacille de Calmette-Guerin (BCG), is a strain of g.
bovis attenuated by serial culture on bile-containing media in 1909.

This strain has been and is still used to immunize humans against tubercu-
losis. It is also used extensively in experimental animals to study
mechanisms of delayed sensitivity and cellular immunity.

The consistent immune response in tuberculosis is not the production
of circulating antibody, nor that the immunity and sensitivity can be
passively transferred with serum. The most consistent response is the
development of delayed type sensitivity to tuberculin which can be trans-
ferred by lymphoid cells, transfer factor or peritoneal exudate which con-
tains macrophages and some lymphocytes (16, 24, 27, 71, 88, 116, 118, 147,
158, 159, 177, 178).

I The role of macrophages in the induction of delayed sensitivity has
not been conclusively resolved. Macrophages in the liver, spleen, lymph
nodes and peritoneum readily ingest and digest the effete host cells and
molecules and most foreign proteins and cells (115). However, some foreign
materials may be retained for weeks or months within macrophages (70).
Labelled bovine serum albumin persisted in macrophages in the spleen and

liver phagocytes of rabbits for periods of up to 130 days (22). Although

macrophages can readily digest may antigens, they are probably incapable
of producing antibody gamma globulin (120, 128).

Macrophages may be activated by some factors which are produced by
lymphocytes during blast cell formation in response to antigens, or non-
specific stimulation (11, 33, 72, 112, 117, 122, 125, 146)'and release of
molecules (61) such as migration inhibitory factor (11, 12, 15, 17, 38, 39,
41, 42) macrophage aggregation factor (95), chemotactic factor (172), skin
reactive factor (164), blastogenic factor (43, 166), transfer factor (92),
lymph node permeability factor (145, 180, 181), monocytogenic hormone (182),
cytotoxic factor (44, 63, 64, 139), interferon (67, 179) and complement (126).

Activation causes many changes in macrophages. Some of the changes
include the size of cell (10, 29), its process (23), Golgi apparatus (30),
the number of lysosomes (23, 29, 30), lysosomal enzymes (29, 35, 73),
'mitochondrial enzymes (10, 34, 35), phagocytosis (10, 18, 35, 36, 69, 74,
75, 79, 101, 149, 159, 162) and mitotic rate (84). Some changes may con-
tribute to the ability ofnmcrOphages to destroy bacteria, others may not.

The number of defense cells, especially macrophages, increases _in a
tuberculous lesion or delayed sensitivity reaction. In the delayed hyper-
sensitivity reaction, circulating monocytes (macrophages) emigrate from
the blood stream into the granulomatous lesion where they may proliferate
(86, 167, 168, 169). In a host in which delayed hypersensitivity has de-
veloped, the tubercle bacilli or thin products will cause local macrophage
activation, proliferation, and immunity to occur at a faster rate and to a
greater degree than in normal host (52, 105, 106). The monocytogenic
humeral factor (182) from lymph nodes apparently causes the bone marrow to
produce or release macrophages. It may also contribute to local macrophage

accumulation, activation, and proliferation. After inoculation of tubercle

bacilli, macrophages not only are directly or indirectly sensitized by
the bacilli and its tuberculin-like products, they also contribute to
immunity by an increasing ability to destroy the bacilli (101).

Whether the macrophage responds in an immunologically specific manner
after the induction of delayed sensitivity and cell mediated'immunity is
controversial. Macrophage cytophilic immunoglobulins and non-immunoglobulin
specific receptor sites on macrophages have been reasonably well established.
There isas clear evidence for adherence of soluble immune complexes to
macrophages (4). Whether any of these are a critical part of delayed
sensitivity has not been resolved. It is stated in the review (163) on macro-
phage cytophilic antibodies that "while evidence accumulates that some
form of ce11~bound antigen specific material plays a role in delayed hyper-
sensitivity, there is little to suggest that it is identical to serum
Inacrophage-cytophilic antibody.” In regard to cell mediated immunity,
it states:

”This is manifested by an increase in the phagocytic
and bactericidal abilities of macrOphages and usually
arises as the result of infection by an organism
capable of intracellular multiplication such as
flgcobacterium tuberculosis or Listerig'monocvtogenes.
Infection with such an organism stimulates the ap-
pearance of a new population of small lymphocytes.
These cells migrate rapidly to the site of infection,
where, after interaction with antigen, they are able
to influence the surrounding macrophages so that they
are more capable of combatting the invading organism.
The induction of this lymphocyte population and its '
further interaction with antigen are immunologically
specific. However, the modification of macrophages
as a result of this interaction is nonspecific.
Stimulated macrophages exhibit enhanced phagocytosis
and destruction of any antigen presented to them.

For this reason it is improbable that a macrophage-
cytOphilic antibody mediates these changes in macro-
phage behavior.”

 

 

 

Cellular immunity such as the acquired cellular resistance in
tuberculosis, was defined by Dannenberg in 1968 (36) as a state in which
macrophages have been activated, proliferated, and possess an increased
capacity to destroy tubercle bacilli. Initially, the blood is the major
source of macrophages in these lesions (56, 86, 150, 151, 152, 167, 168,
169) but, as chronicity is established, local proliferation may become
more important (86, 87, 152). A lymph node permeability factor (145, 180,
181) is present in lymphocytes or macrophages or both in the lesion site
of inflammation of tuberculosis. This factor attracts more cells to the
area and increases the blood supply which provides more oxygen and nutrients
for cells (100, 101, 102). If inflammation is severe, the fibrin formed
tends to fix the bacilli locally (100, 101).

The following is from a recent report by Dannenberg et a1. (37):

"The following concept of the pathogenesis of
tuberculosis is proposed. Large numbers of
macrOphages accumulate at the site of the bacilli,
which stimulate these macrophages to develop high
levels of microbicidins and digestive enzymes. DH
and interaction with lymphocytes are intimately
involved in the local antimicrobial immunity so
developed. In a typical lesion such as local immunity
does not develop rapidly enough to be effective
immediately, and the macrophages are killed by the
high concentration of bacilli and their tuberculin-
1ike products. New macrophages enter and distribute
the bacillary load among them. These cells can
develop microbicidins and enzymes even more quickly
than the initial cells, because of the presence of
increasing hypersensitivity in the host and probably
other, as yet unknown, factors. If this second group
of macrophages dies before the bacilli are eliminated
or inhibited, a third group of macrophages ingests
them. The sequence continues through subsequent
groups. Because of the ever increasing speed of
deve10ping cellular immunity, one of these groups will
succeed in overcoming the bacilli. Then the lesion
will regress and heal."

The lymphocytes which become sensitive to tuberculin (ll, 33, 117,
125) can passively transfer delayed hypersensitivity to a normal host
(16, 24, 27, 28, 62, 88, 90, 97, 98, 99, 113, 118, 177, 178). Both macro-
phage activation and macrophage proliferation may be direct effects of
tuberculin, or more probably indirect effects mediated by factors (listed
previously) released from sensitive lymphocytes.

Cellular immunity is a combination of both immunological specific
factors and nonspecific factors. The major effect observed is mainly non-
specific in nature, because such immune macrophages have an increased
capacity to destroy and inhibit many types of bacteria (46, 104, 105, 107).
Cellular immunity is an immunological mechanism specifically engendered and
recalled, but is nonspecific in effect.' The systemic protection afforded
by cellular immunity is relatively minor but it is sufficient to increase
the resistance of the host against a variety of unrelated microorganisms
(104, 105, 106, 107).

A characteristic of delayed hypersensitivity in man is the delayed skin
reaction. When an antigen is injected intradermally into a previously
sensitized animal, the typical delayed reaction begins to appear after four
hours, may reach a peak at twenty-four hours, and may fade after forty-
eight hours. It is grossly characterized by induration, erythyma, and
occasionally necrosis. The histology of the delayed reaction has been
studied by numerous investigators (28, 49, 56, 86, 87, 113, 136, 137, 170,
171). These observations suggest that tubercle bacilli and their components
in the local lesion stimulate both the development of epithelioid cells and
an increase in their enzymes, and that this increase is associated with

bacillary destruction.

There are two reasons for long duration of the hypersensitivity to
tuberculin. The first is its immunological nature. Some lymphoid cells
have tlie "memory” for the antigen to which they previously reacted. The
second reason is that the bacilli or their antigens may persist in the
caseous center of "closed" lesions (114, 133, 154). The occasional escape
of bacilli or their products from the lesions maintains hypersensitivity
to tuberculin.

Landsteiner and Chase (88) found that viable blood leukocytes were
effective in the transfer of the ability to develop a delayed sensitivity
skin reaction to tuberculin and to streptococcal antigens. Lawrence (89)
has transferred the sensitivity with extracts of leukocytes prepared by
freezing and thawing or by osmotic lysis and named the active material

from blood leukocytes transfer factor. It can be inactivated by heating

 

at 560C for 30 min. It is dialyzable and its molecular weight is estimated
at less than 10,000. It is not immunoglobulin. It is resistant to RNase.
It passively confers cutaneous reactivity and converts normal lymphocytes
in vitro and in vivo to antigen-responsive state. The sensitivity is
immunologically specific. Transfer factor differs mechanistically in two
ways from the effector molecules of the in vitro correlates from sensitive
lymphocytes: transfer factor is mechanically or chemically extracted from
cells without antigen present and the factor then confers an ability to
the recipient to respond to only the appropriate antigen; the effector
molecules are elaborated from sensitive lymphocytes by contact with the
antigen to which they are sensitive and the effector molecules nonspecifically
affect other cells or their activities.

One of the widely used tests of the in vitro correlates to detect

delayed sensitivity is the MIF test, the inhibition of migration of macrophages

.10

on a glass surface. It is elaborated by the sensitized lymphocytes when
stimulated by specific antigen. With certain exceptions such as phyto-
hemagglutinin (FHA) (85, 121) and conconavalin A (8, 127, 156, 157), the
lymphocytes produce them in vitro only, or at least more of them, when
stimulated by the same antigen as that with which active sensitization of
lymphocytes was induced in vivo. Rich and Lewis observed nearly 40 years
ago (134) that the migration of splenic or lymphocytic cells would be
inhibited if these cells from sensitized animals were placed with specific
antigen in vitro. George and Vaughan developed a more quantitative system
(57) to study this in 1962. By modifications of the technique, Bloom and
Bennett and as well as some other investigators (ll, 12, 15, 38, 39, 41,
42) have indicated that cellular immunity involves both a sensitized
lymphocyte population and a normal macrophage population. They (36) estimated
that one lymphocyte is sufficient to affect indirectly the migration of
about 1,000 macrOphages. The MIF is heat stable at 560C for 30 min, and a
nondialyzable macromolecule of an estimated molecular weight between 35,000
and 55,000. It is generally accepted that MIF and the other in vitro cor-
relates are probably not immunoglobulins, DNA or RNA.

It is well accepted that antibody-mediated immunity and sensitivity can
be transferred by RNAs and/or antigenFRNA complexes. These molecules are
a part of the immune system but their functions at-the cellular level are
not yet defined (2). The RNAs have been extracted from a variety of cells
from a variety of speCies of animals. The transfer of the immune capacity
has been tested in vivo and in vitro. The kinds of antigen have been quite
varied. It has not been clearly resolved that the systems for different
antigens and different cells are the same. The literature on the passive
transfer of antibody production by RNA extracts will be reviewed first,

then on delayed sensitivity and cell mediate immunity.

11

Sterzl and Hrubesova (155) first reported that a ribonucleoprotein
extract (RNA) from the spleens of rabbits injected two days before with

Salmonella_paratyphi B gave rise to antibody production in adult rabbits

 

and in 5-day old rabbits. The latter was evidence that more than only
antigen had been transferred. The 5-day old rabbits did not respond to
antigen only. The RNA extracts passively conferred the ability to produce
antibodies. Subsequently, this was confirmed in X-irradiated rabbits (53).

In 1957 Garvey and Campbell (55) found S35 labelled albumin complexed
with RNA extracted from liver cells after rabbits were injected with the
albumin. The complex was 200 times more effective in guinea pigs than the
original antigen. The dissociated complexes were not immunogenic. A
secondary response was enhanced by the antigen-RNA complexes in rabbit
lymph node cultures (68) and by RNA extracts from mouse peritoneal exudate
cells in syngenic mice (5, 6). The antigen-nucleoprotein complexes behaved
as a ”super antigen" and appeared to associate readily with soluble RNA (141).
These findings promoted the suggestion that the RNA was RNA-antigen complexes
transferred from antigen stimulated macrophages to antibody producing cells,
thereby initiating an immune response.

ffhe immunologic active RNA may be antigen-free(48, 77, 78, 80, 119,
142). Sharp (148) and Fishman (48) presented suggestive evidence that RNA
is passed from a macrophage to a lymphocyte by means of cytoplasmic ap-
lpendages. Fishman has described the synthesis of T2 bacteriOphage antibody
by lymph node cells in vitro when normal cells are cultured with macrophages
(47) or RNA from macrOphagcs (48) which have been incubated with T2 phages.
The response is sensitive to ribonuclease. Weiss and Fishman (176) have
also suggested that the active RNA is of higher molecular weight because

the RNA from rabbit macrophages, obtained from the MAK II (168) and MAK III

12

(23 to 288) of MAK column (109, 153) must be active in Stimulating anti-
T2 antibody synthesis.

Several investigators have shown that RNA related to antibody produc-
tion has many characteristics of messenger RNA and that the mRNA is coming
from antigen stimulated lymphocytes rather than macrophages (78).

It has been suggested that antibody synthesis is dependent on the
production of a species of RNA (103). This RNA is inhibited by actinomycin
D (l65)and it is relative unstable to turnover(93). The light and heavy
chains of the gamma globulin molecule are synthesized on separate classes
of polysomes (7, 143). Since these chains are different sizes it is probable
that there is unique messenger RNA code for each type of chain.

Cohen and workers (25, 26) used RNA extract from the spleens of mice
immunized with sheep erythocytes to convert a small portion of the spleen
cells of normal mice to cells forming hemolytic antibody. The extract was
sensitive to RNase and insensitive to pronasc, trypsin and amylase. The
most active RNAs sedimented in the 8-123 region of sucrose gradient, sug-
gesting a molecular weight of approximately 1-3 x 105, too small to serve
as an intact messenger RNA for antibody globulin which has a minimum weight
of 160,000(45). It is still possible that the smaller RNA may represent
a portion of a larger RNA which has broken down during isolation.

Friedman et al. (54) found that the RNA obtained from T2-infected
macrophages contained antigenic components of the bacteriophage which could
be detected by canplement fixation. Askonas and Rhodes (5, 6) also demon-
strated RNA extracted from mouse peritoneal cells after the uptake of 1131
hemocyanin caused the production of antibody directed against hemocyanin
injected in normal mice. The RNA preparationcontained.significant amounts

of radioactivity reflecting the presence of antigen.

.13

Gottlieb et a1. (58) reported that RNAs derived from two sets of
peritoneal exudate cells (PEC) exposed to R17 and T2 phage annealed to
])NA. The ability of the bound RNA to elicit antibody formation against
T2 was measured. The two RNAs recovered from cells infected with either
antigen were identical. Thus, the RNAs were not messenger RNAs.

In the review of antigen-RNA interactions by Gottlieb and Schwartz
(60) a comparison is given between crude or bulk-RNA extracts and a minor
component, an RNA-protein complex, designated as RNP. The latter was un-
affected by DNase. It could be extracted from macrophages whether exposed
to antigens or not. It was immunogenic only if the macrOphages had been
exposed to antigen. The RNP fraction was equal in immunogenicity to that
of crude or bulk RNA extracts. Only fragments of antigen bound strongly
to the RNP molecule and antigen-RNP complexes could not be dissociated by
the methods to dissociate antigen-RNA complexes. The table given by
Gottlieb and Schwartz is as follows:

CHARACTExISTICS OF ANTIGEN-RN? AND ANTIGEN—RNA INTERACTIONS

 

 

 

Criteria Antigen-RNP Antigen—RNA
Source of RNA Unique to macrOphages Any cell homogenate
Favored by Mg2+ ion No Yes
Abolished by EDTA No Yes
Abolished by hot phenol No Yes
Location in RNA profile on Only on RNP molecule Broadly dispersed

polyacrylamide gel
electrophoresis

Antigen in complex Always fragmented Native

 

 

The ribonucleoprotein complex does not contain the base sequence infor-
mation sufficient to code for antibody globulin. The ribonucleoprotein complex

has an 820 of 1.8, suggesting a molecular weight of about 12,000, of which

14

approximately 28% is protein (59) and a relatively high guanine-cytosine
content of 58% (58). The small size would exclude a messenger role for
this RNA, since it is too small to code for either the light (molecular
weight 25,000) or the heavy (molecular weight 50,000) chains of gamma
globulin.

Therefore, there appears to be two different types of immunogenically
active RNA from macrophages. The immunogenicity of macrophage RNA has been
attributed to the presence of an antigen component derived from antigenic
processing (5, 6, 54, 58), as well as to unique informational properties of
the macrophage RNA (1, 26, 48). Thus, there are probably two different
cell types. They have been summarized by Bishop and Gottlieb (14) in the

following table:

 

 

RNA specifying

 

Antigen-RNA allotypic
complex determinants
Type of globulin produced 78, 19S(?) 198 only
Sensitivity to RNAse l : 15 l : 75
Antigen present yes no
Sensitivity to actinomycin no yes
Location in sucrose gradient heavy fractions light fractions
Contains messenger RNA no possibly

 

Five ways in which RNA may affect the immune response of cells are as
follows:

1. The information for synthesizing immunoglobulin is
transferred by information RNA (iRNA) which is free
of antigen (48, 77, 78, 80, 119, 142). Some in-
vestigators have suggested that this kind of iRNA
is messenger RNA (mRNA) (78).

15

2. RNA may be serving a protective role. Friedman and
some other investigators (54, 58) have found that RNA
preparations contain antigencn:fragments of antigen.
The complex is resistant to low concentrations of
RNase and trypsin and it may preserve antigenic de-
terminants as they are transported through the cir-
culation to immunocompetent areas such as spleen
and lymph nodes.

3. RNA may serve as an adjuvant . Braun et a1. (19)
have demonstrated that polynucleotides and oligo-
nucleotides can serve as nonspecific stimulators
of the immune response and amplify the response to
antigenic stimuli. Youmans (183) has also indicated
that mycobacterial RNA may function as an adjuvant
for induction of delayed hypersensitivity.

4. RNA may be an "activator” or ”derepressor." Braun
suggested that the macrophage combines a processed
antigen with a nonspecific RNA which is assumed to
be capable of turning on all of stem cells into
antibody forming lymphocytes (19, 20).

5. Bishop and Gottlieb (14) have suggested that it is
possible that ribonucleoprotein complex is not re-
spoinsible for induction of the immune response,
but may serve to regulate the amount of antigen
available to the definitive antibody producing cell.

The reports on the passive transfer of delayed sensitivity and cel-
lular immunity by RNA extracts are few. Mannick and Egdahl (110) pas-
sively conferred sensitivity to rabbit lymph node cells in vitro with
RNA extracts from lymph nodes from rabbits sensitized by skin homografts.
The in vitro cells elicited a positive reaction in homograft donors, not
in nondonors. No sensitivity was transferred by extracts from nonsensitive
rabbit lymph nodes.

Fong et a1. (50, 51) have reported that cultured macrOphages from
rabbits immunized with BCG were resistant to the effects of virulent
tubercle,bacilli, and brucella but that normal macrophages were susceptible.

This resistance could be transferred by the microsome fraction derived from

the immunized cells or by RNA extracted from this fraction. The ability to

16

transfer resistance was abolished by ribonuclease.

Incubation of normal rabbit lymph node or spleen cells with RNA ob-
tained from lymph nodes draining the site of a rejected homograft ap-
parently converted cells into a state of transplantation immunity, as
detected by their ability to elicit a positive skin reaction in recipients
(110) or to cause a more rapid rejection of a skin transplant in recipients
(111). The uptake of RNA by host cells, mostly histiocytes, is probably
a requirement for induction of cellular resistance (3, 51, 110, 111).

The migration of human macrophages from lymph node tissue cultures
from tuberculosis patients is inhibited by the purified protein derivative
(PPD) of tubercle bacilli. When nonsensitive cells were incubated with an
RNA extracted from lymph nodes of human donors sensitive to PPD, histo-
plasmin, or both, the migration of these cells was specifically inhibited
by the respective antigen (39, 160, 161). The migration-inhibition effect
could be transferred to normal C57Bl/6J spleen cells by RNA extracted from
lymph nodes and spleens of immunized mice which had rejected sarcoma-l
tumors (94).

The conversion of nonsensitive cells to sensitive cells by an RNA
extract is highly sensitive to ribonuclease. Although it has not been
conclusively excluded that the conversion to a state of delayed hyper-

. sensitivity may be the result of active immunization by antigen or antigen
:fraagnierlts the possibility exists that the active RNA may be a messenger
mechanism which confers sensitivity. This phenomenon has also been found

in vitro in guinea pigs (81) and rabbits (9). In addition, RNA from the
lymph nodes of guinea pigs sensitive to PPD, brain antigen,CKEDNP-oligolysine
and E~DNh-oligolysine conferred on nonsensitive guinea pigs lymphoid cells

the ability to transfer to nonsensitive guinea pigs (82) cells the ability

17

to have a delayed hypersensitivity skin reaction to the appropriate antigen.
Nonsensitive guinea pigs peritoneal exudate cells were incubated in vitro
with RNA extracts from a rhesus monkey with delayed hypersensitivity to

PPD only, or PPD and coccidioidin. The peritoneal exudate cells were
inhibited in their migration from capillary tubes according to the presence
of the apprOpriate antigen. Conversion of nonsensitive guinea pig peritoneal
exudate cells were sensitized with monkey RNA extracts (123). The growth

of tumor isograft in inbred mice is inhibited by intraperitoneal injec-

tions of syngeneic spleen incubated in vitro, with RNA extracted from

guinea pig immunized with the same mouse tumor (132).

Paque and Dray have also indicated that RNA extracted from primate
lymphoid tissue of rhesus monkeys with skin-test sensitivity to keyhole
limpet hemocyanin (KTN), PPD or coccidioidin can convert nonsensitive human
peripheral leukocytes to sensitive leukocytes. In the presence of specific
antigen the sensitive leukocytes release MIF which inhibited the migration
of nonsensitive guinea pig macrophages (124).

The purpose of this research reported in this thesis to passively sensi-
tize normal guinea pig cells in vitro with NA extracts from BOG-infected
guinea pigs and cattle and then to attempt to passively sensitize normal
guinea pigs by injection of autologous cells incubated in vitro with guinea
pig and bovine-RNA. If this were possible, it would then be feasible to
use the model to study the role of the donor RNA in passive sensitization and
to develop a test from lymphoid tissue to aid in detection of bovine tuber-

culosis.

MATERIALS AND METHODS

Sensitization of guinea pigs. Random-bred albino guinea pigs from a
closed colony since 1961 were inoculated subcutaneously in the cervical

area with viable attenuated Mycobacterium bovis strain Bacillus de Calmette-

 

Guerin (BCG) in Freund's incomplete adjuvant (Difco Laboratories, Detroit
Michigan). Viable BCC were suspended at an optical density reading of

0.15 at 620 nm, approximately 108 cells/m1. Each guinea pigs was inoculated
with approximately 1 x 107 cells in 0.5 m1 of adjuvant.

Sensitization of cattle. Tuberculin negative, 6 to 9 month-old steers

 

from a tuberculosis-free herd were inoculated subcutaneously in the left
brisket with 0.5 mgm wet weight (10 mgm/ml) viable BCG in 0.5 ml incomplete
Freund's adjuvant. After euthanization approximately 5 months later
tissues mere removed and stored at -7OOC until RNA extraction. Prior to
euthanization, the steers were tuberculin sensitive by caudal and cervical
tests, and in vitro tests. Control steers were negative.

RNA extraction. Lymph nodes and spleens of ECG-infected and normal

 

guinea pigs, and lymph nodes of BOG-infected and normal cattle were excised
and if the RNA extractions were not made immediately, stored at -7OOC un-
til they were to be used. The RNA extraction was by a modification of the
procedure described by Scherrer and Darnell (144). Tissues were homogenized
with a Waring blender at 40C in distilled phenol containing 0.1% 8-hydroxy-
quinoline (J.T. Baker Chemical Co., Phillipsburg, N.J.) saturated with

0.01 M sodium acetate (J.T. Baker Chemical Co., Phillipsburg, N.J.), 0.5%
sodium dodecyl sulfate (K & K Laboratories, Inc., Plainview, N.J.), and

4 ug/ml of polyvinyl sulfate (Eastman Organic Chemicals, Rochester, N.Y.).
An equal volume of sodium acetate used to saturate phenol was added to the

homogenate with 0.5 mg of bentonite (Fisher Scientific Co., Fairlawn, N.J.)

18

19

per m1. Particular care was taken to use 10-12 m1 of the aqueous phenol
extraction buffer and an equal volume of sodium acetate containing 0.5 mg

of bentonite/ml for each gram of tissue used. This mixture was heated to
600C with intermittent stirring for 10 min and rapidly cooled to 10°C in

an ice water bath. The phenol and aqueous phases were separated by centri-
fugation at 40,000 X g at 40C for 10 min. The aqueous phase containing RNA
was carefully removed with a pipet. An equal amount of fresh buffer
saturated phenol was added to the aqueous phase and the extraction procedure

/OD ratio 2. After the last

repeated three times or until the OD 280

260
extraction, the aqueous phase was precipitated with 95% ethanol and stored

in the freezer overnight. The RNA precipitate was centrifuged at 40,000 X g
at 40C for 15 min. The pellet was dissolved in 0.3 M sodium acetate (pH 5.1)
and treated with 10 ug deoxyribonuclease (DNase, Worthington Biochemical
'Corporation, Freehold, N.J.) in 0.1 ml of aqueous RNA extracts for 30 min

at 370C. Deexyribonuclease was removed by re-extraction of treated RNA

with phenol and by alcohol reprecipitation of the RNA extracted from phenol
acetate buffer as described above. The RNA was reprecipitated at least

three times with three parts of 95% ethanol at ~700C and stored for use as

a pellet under ethanol at -7OOC. The RNA preparations used had an OD260/

OD ratio of 2.0.

Prestimulation and collection of peritoneal exudate cells (PEC). Randon
bred albino guinea pigs were inoculated intraperitoneally through the linea
alba with 5 m1 sterile light mineral oil (E.H. Sargent and Co.). Four days
later, the animals were inoculated intraperitoneally with 40 ml of Hank's
Balanced Salt Solution (BSS) (Microbiological Associates Inc., Bethesda,
Maryland) containing 6 units of heparin (Scientific Products, Evanston,

Illinois) per ml. The peritoneal exudate was removed aseptically with a

20

sterile syringe and centrifuged 500 X g for 10 min at 4°C. The sedhnented
PEC were washed and centrifuged three times in cold BSS without heparin.
They were resuspended in TC 199 medium (Microbiological Associates Inc.,
Bethesda, Maryland).

Passive sensitization of guinea pig PEC in vitro by RNA extracts. The
RNA extracts were dissolved in a minimum of B33 (0.3-1.0 m1) and the con-
centration was adjusted on the basis that 1 mg of RNA/m1 has an 0D260 of 20.
The RNA, 500 ug in 0.1 ml B38 was added to washed PEC, 108 cells in 2 m1 of
TC 199 medium. The mixture was incubated 60 minutes at 370C in a 5% carbon
dioxide incubator with intermittent shaking. Washed PEC were incubated
with BSS as controls. After incubation, the RNA treated and RNA untreated
PEC were centrifuged at 140 X g for 10 min. -The supernatant fluid was
discarded. The peritoneal exudate cells were resuspended in TC 199 medium
containing 0.2% L-glutamine (Grand Island Biological Co., Grand Island, N.Y.
14072) and l % inactivated fetal bovine serum (Microbiological Associates
Inc., Bethesda, Maryland) and 5% inactivated guinea pig serum (Grand Island
Biological Co.). The cells were used immediately in capillary tube or spot
tests for migration-inhibition.

Passive sensitization of guinea pigs by injection of autologous washed

PEC after incubation in vitro with RNA. The RNA extracts were dissolved in

 

a minimum of B88 (0.3-1.0 ml) and the concentrations adjusted on the basis
of 1 gm of RNA/ml yields an OD260 of 20. The PEC from each guinea pig
were suspended in B88 and mixed with RNA extracts after adjusting the
concentration of peritoneal exudate cells to 108 cells/500 ug of RNA. The

mixtures were incubated at 370C in 5% CO2 for 60 minutes with gentle shaking,

centrifuged and the cells washed twice with BSS.

21

Cells were resuspended, 108/ml B88, and each guinea pig was inoculated
intraperitoneally with its own (autologous) cells only. The guinea pigs
had not been skin tested before receiving their own RNA-treated cells.
The closed colony has had no natural sensitivity during the past 12 years.
Capillary tube test for migrationvinhibition. Capillary tubes (0.5-
0.9 x 75 mm) were filled with PEC suspensions, plugged at one end with
sterile paraffin and centrifuged at 55 X g for 15 min. At the interphase
of medium and cells, the capillary tubes were cut bydiamondfxfixmg placed
in a sterile petri dish and anchored with sterile paraffin. Two ml medium
TC 199 containing 0.2% of L-"lutamine, 5% guinea pig serum and 10% of fetal
bovine serum with or without 10 ug PPD/m1 (Parke-Davis, Detroit, Michigan)
was added to each petri dish. They were incubated at 370C in 5% carbon
dioxide for 24 hrs. After incubation, the dishes were placed on a projection
microscope and the areas of cell migration (migration index) was calculated
by the methods of George and Vaughn (1962) using the formula:

M I _ Average area of migration with antigen X 100
° ' Average area of migration without antigen

 

Spot test for migration—inhibit~on. The spot test was developed and

 

 

modified in our laboratory (129) as follows:

When the cells were ready to be tested, they were suspended in TC 199
at approximately 108 cells/ml, and drawn into a % m1 syringe with a 25 gauge
needle. One drop was placed at the center of each cover slip in a petri
dish and incubated at room temperature 15 minutes. The cover slips were
rinsed carefully in BSS to remove nonadherent cells and placed in a small,
plastic petri dish (35 x 10 mm). To each petri dish, 2.0 m1 of TC 199 with

2% L—glutamine, 5% guinea pig serum and 10% fetal bovine serum with or

without 10 ug PPD (Parke-Davis)/m1,

22
The vertical and horizontal diameters of each spot was measured under
the light microscope 10X objective with an ocular micrometer and the average
recorded. The chambers were incubated at 370C, 5% CO2 for 24 hours. The
diameters were measured, and the increase in average diameter recorded as

migration. The migration index was calculated as for the capillary test.

Skin tests. Guinea pigs were skin tested with 0.1 ml containing 10 TU

 

PPD-S (Parke Davis). The sites of inoculation were observed 15 minutes,
2, 24, 48 and 72 hours later. The size of the reactions was recorded in mm
of erythema and/or edema.

Passive sensitization tests. Tests were made to determine if passive

 

sensitization occurred under the following circumstances:

1. Pooled RNA from ECG-infected guinea pigs to normal
guinea pig .EC in vitro measured by MIF tests
(Table 1).

2. Individual guinea pig RNA from ECG-infected guinea
pigs which were sensitive to PPD in vitro prior to
extraction (Table 2) to normal guinea pig PEC in
vitro measured by MIF tests (Table 3).

3. Autologous cells after incubation with RNA from BCG-
infected guinea pigs to normal guinea pig in vivo
measured by MIF tests 25 days later (Table 4).

4. Bovine RNA from BOG-infected cattle which were
sensitive to PPD in vitro prior to extraction
(Table 5) to (a) normal guinea pig PEC in vitro
as measured by MIF (Tables 6-12); (h) to normal
guinea pigs in vivo by their autologous cells as
measured by MIF nine days later (Tables 13-16) and
by skin tests three days later (Table 17).

RESULTS

Passive transfer of delayed sensitivity in vitro and in vivo to normal
Ugineagpig PRC by RNA extracted from ECG-infected guinea pigs. The results
from five capillary tube tests on PEC from each test group of guinea pigs to
detect in vitro transfer of sensitivity are given in Table 1. The average
of migration of PEG incubated in B38 and no PPD was 65.0 i 1.4 and repre-
sented a hundred percent migration (100.0 : 2.2)° The migration of the PEC,
which had been incubated previously with RNA extracted from a BOG-infected
guinea pig (S-RNA) was inhibited by PPD (M.I. = 23.5.: 2.2); there was
little or no inhibition without PPD (M.I. = 95.4 i 1.4). The migration of
PEC incubated previously with the RNA which had been extracted from the
spleen and lymph nodes of a normal guinea pig (N~RNA) was not inhibited
by PPD (M.I. = 91.3 i 3.7) or without PPD (M.I. = 92.3 i 2.2). The migra-
tion of normal PEC was not inhibited by PPD (M.1. = 96.1 i 3.9).

The migration of PEG from two ECG-infected guinea pigs, prior to ex-
traction of the RNA above, was inhibited by PPD (M.I. = 48.3 i 0.80, and
M.T. = 36.9 i 1.8, Table 2). The migration of normal PEC was inhibited by
PPD when previously incubated with RNA from either guinea pig (M.I. = 53.6
.i 1.6 and M.I. = 38.3 i 1.3, Table 3); the migration was not inhibited
without PPD (M.I. = 102.4 i 1.1; N.I. 96.1 i 1.4). The migration of PEC
previously incubated with 888 without RNA as controls was not inhibited by
' PPD (M.I. = 103.4 3: 1.5) or without PPD 01.1. = 100 i 0.8).

The migration of PEC from a guinea pig which 25 days previously had
received its own cells incubated in vitro with the RNA extracts from BCG-
infected guinea pigs was inhibited by PPD (M.I. = 44.5 i 2.9) and not without

(M.I. = 100 i- l.8, Table 4).

woumaflewucooo

meow uozU

mDHm> wowmausosp
cofluSHom unm woodmamm mxcmzm

 

 

 

~.th.mm a.HH¢.mm s.me.Ha «.mfiw.ma m.mflg.om . , N.NHd.ooH Axe xmeaH coaamuwaz
a.aha.mn m.owo.mo a.NH¢.am a.HHo.oo m.mfiw.mo a.HHo.mo Amanm>
mewkHuso uSOSuHBV
mmmuo><
N.Nww.mm m.oHH¢.ws o.sHHo.as N.NHW.Na . a.sHH#.eNH N.NHo.ooH Axe xmseH coaamamaz
e.HHn.mH o.HHHa.Hm o.HHHw.ma a.HHo.oo m.HHHo.Nm a.HHo.me Amaamp Ham gauze

ommwm><

 

 

ezoo me me so 092 so m
qH ms m.mm oo ooHs no a
Na Ho me om . . nmoaa mo m
NH cm mm mm me me N
ma . as 00 No 00 Ho H
mam «em-m mam aze-z cam mmmm was“ sumflgaamo
+ + +
«E -m ea .2 mmm

 

 

 

.Aommv,mm>aum>nuoo gaseous emauausa uuoeaua cam ease eausafiso seem-zy1maae macamw Haasoc
no m¢zmnmv qua wocwsm wouoowcHawom we moved Lmfika use mcoofiom Eoum muomuuxo «2m sues cowumnsoCH
poumm .Aummv mHHoo oumwsxm HmoGOuwsomimHQIwocflsm Hwfiuoc mo mummu COHUHQHLCH coHumHmwz .H mqm<e

 

 

25

TABLE 2. Migration inhibition tests of peritoneal exudate cells (PEC)
from ECG-infected guineagpigs prior to ribonucleic acid extraction
from spleen and lymph nodes.

Guinea Pig #1 (QP-I)

 

 

 

 

 

 

 

 

 

a BSS
Capillary tube BSS with PPD
l 58 . 30
2 61 29
3 63 ' 28
4 56 g 28
5 NDb CONC
Average 59.5i4.9 28.8:O.5
Migration Index (Z) 100.Qi8.3 48.3:p.8
Guinea Pig *2 (GP-IT),
BSS
Capillary tube BSS with PPD
1 3O 12
2 33 12
3 33 10
4 25 9
5 28 11
6 ND 12
Average 29.8il.4 11.Q:O.5
Migration Index (Z) 100.Qi4.6 36.&tl.8

 

 

aHanks Balanced Salt Solution
bNot done

c .
Contaminated

26

econ uozn

coquHOm uHmm wmoamamm exammw.

 

 

 

 

m.HHa.wm a.HHa.ea e.HHa.mm H.HH¢.NOH m.HHa.moH m.oHo.ooH Ase xmecH acaumamaz
N.th.om m.HHw.Hm m.HHd.Hm o.HHw.sm a.HHw.ma m.oflw.mm mmmum><
am am mm mm OOH as a
mm as aoz ooH ma em m
mm we we mm mm am N
mm ma an em HoH .aa a
mam fiHH-moe<zm mam AH-eoVazm mam mmmm . mama sumflflnemw
+ + + + + +
AHH-moVazm omm AH-movazm 0mm mmm 0mm
+ + +
one 0mm one

 

 

 

.mmmmv mo>Hum>Hpom egououm onWHusm usosuwz use LuwB mmHSuHDU

AN mamas .HH-mo Ho H-muv wwaa seesaw

@muommewnwom Eoww moves seemfi was memoHQm ozu Eoum muomwuxo szm :uHB ouuw> CH coflumnsocH noumm
awe mosasmleEuoc m Eoum mUmmV mHHoo mumwsxo HmmGOuwnmm mo muwou cowuwnwncfl cowumuwwz .m mqmfia

27

TABLE 4. Migration inhibition tests of_peritoneal exudate cells (PEC)
fggm a normal guinea pig (GP-III) injected 25 days previously with agtolggpus
PECC after incubation with (PM extracts from the spleens and lymph nodes
of ECG-infected guinea_pig§ (UP-l and GP—II) cultured with and
without Purified Protein Derivatives (PPD).

 

 

 

 

 

Capillary tube PEC without antigen PEC with PPD
1 50 . 25
2 47 ' 26
3 50 20
4 52 19
5 48 20
Average 49.4:O.9 22.Qil.4

Migration Index (Z) 100.Qil.8 ' 44.5i2.9

 

 

28

Passive transfer of delayed sensitivity to normal guinea pig PEC in
vitro with RNA extracts from BCG-infected steers. Prior to RNA extractions
from bovine lymph nodes, the migration of blood leucocytes from steers #36
and #51 infected with BCG were inhibited by 10 ug of a tuberculo-protein (138)
(M.I. = 19.5 i 9.5 and 20.0.: 4.0), the migration of blood leucocytes from
a normal steer was not inhibited (M.I. = 100.0 f 4.8, Table 5).

The M.I. of PEG from normal guinea pigs previously incubated with BSS
or RNA from a normal steer (NB-56-RNA) with or without PPD was 91.6 i 3.0
and 100.0,: 12.4 respectively for GP-56-l (Table 6); 100.5.i 4.8 and 100.0
.i 3.2 respectively for GP-56-2 (Table 7). The M.I. with the cells incubated
with RNA from a normal steer (NB-56-RNA) with and without PPD were 99.1 i
6.2 and 91.9 i 6.7 for GP-56-l (Table 6); 8627 i 7.1 and 92.5 i 11.2 for
GP—56~2 (Table 7).

The migration of PEG previously incubated with RNA extracted from BCG-
infected steers,_§B-36~RNA and SB-Sl-RNA, was inhibited by PPD. The results
are given in Table 8 and Table 9 for_§B-36-RNA and in Table 10 and 11 for
-§B-51-RNA. The migration of PEC from a normal guinea pig (GP-36-1) pre-
viously incubated with SB-36-RNA were inhibited by PPD (M.I. = 53.6 i 3.1,
Table 8) and was not inhibited without PPD (M.I. = 96.1 i 1.3). The migra-
tion of PEG from another guinea pig (GP-36-2), previously incubated with
§B-36-RNA, was inhibited with PPD (M.I. = 5.1 i 1.8); not without PPD
(M.I. = 97.6 i 2.7, Table 9).

The migration of PEG from GP-51-1 previously incubated with SB-Sl-RNA
was inhibited with PPD and not without PPD (M.I. = 4.8 i 2.1 and M.I. =
102.9.i 3.9, respectively, Table 10). The migration of PEG from another
normal GP (GP-51~2) previously incubated with §B-51-RNA was inhibited with

PPD (M.I = 4.7 i 1.4); not without PPD (M.I. = 117.1 i 2.5, Table 11).

aowumnaocw mawuaw uomm ego mo noumfimww aw omwouocH

n

00m Scum wmuomuuxo awmuoumnofisouopsu «m

 

 

Ase xmecH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

:sHodN oéwodS m.aHm.2 m.SHo.oS imawwdm Ramadan ceaumuwaz
53.2 06.362 0.2.9.8 0.2.3.2: 5.2.3.2: o.mHo.mS mwfimfi.
0m omq 0mm ONH ooq o¢m om oaq ooq ONH Odd omm oo 06% . owm 00H omd omm N
ON 0mm oom omH on Omm OH 0mm owm om omd omm qu cam 00¢ OHM 0mm Owe H
mmHQ.us «N ML 0 mmHQ mun am u: o mmHQ my: em n: o mmHQ mun am u: o MMHQ mu: «m us 0 ammHQ mu: am H: o nonemso
am-m ea 02 am-m we 02 sa~-m 4 .ma 02 poem
Hmw Eonm mmuhoooaoq ems Eoum mouNoooaoA c ems Eoum moumoooaoq
ALmHmB mesh kn wmuuoaou mummav
.mmwoc LQE>H mo meowuomnuxo oflom oHoHoscoan cu Hownm.~am% was omwv
museum wouommcHuwom oBu was momfiv nooum Hmewoc m Eoum moumooosofi mo momma cofluwnwncfl cowuwnwwz .m mummfi

30

meow uoze
woumcflamuaooo

cowumesocw mewnsw uomm ecu mo.uouoEmwv cw mmmouoaHn
cowusHom uHmm woocmHmm mxammm

 

 

 

 

 

 

 

 

N.aflH.mm n.0Hm.Hm o.mHa.Hm q.NHHo.ooH Ase xmecH coHumusz
m.HHo.aN s.HHw.NN s.oHN.NN o.mH~.aN ammum><
m.NN m.HmH aOH om aeH mmH m.m~ m.mmH moH m.NN aeH m.HaH a
m.om m.moH maH m.HN m.moH saH ax zoo m.soH om m.saH m.sNH m
m.em m.NoN osH m.mm m.mmH NsH NN NHH cmH woe ozoo esH N
m.e~ moH m.HaH mH owH NGH Hm mMH sHH om m.eaH m.aoH H
eeHa was am a; o eaHe as: am as 0 mean was am a; o neeHm as; am “a o passage
mam + :. azm-em-mz new + . mmmm uoam
aza-©m-mz mam A

 

 

 

.Nammv mo>wum>fluom awouonm onmHuam uDOSoHB was :UHB onSuHso
Amzm:©mumzv wooum HmEHoc m mo oboe naema oeu Eowm muomnuxo 42m we“ suwz woumnsocH
mommv mHHmo oumwaxo awesouwuoa mHaomumuv mwmlsmcMsm HmEnoc mo umou cowuwnwficw cowuwmez .o mummfi

 

 

31

meow uozHo
vmumaHEMucooo

dowuwnaocw wownap uomm ecu mo HoumEmHv aw mmmmuocHa

cowusHom uHmm voocmHmm mxcmzm

 

 

 

 

 

 

 

 

H.mws.sw ~.HHHw.Na m.¢ww.ooH ~.mwo.ooH Ave amecH coaumuwaz
e.HHN.om o.mww.HN H.HHa.mN m.ohw.mm mmmum>a
NH mmH moH m.HN oeH m.mmH m.mN NNH m.eaH mz 200 m.omH a
oz zoo msH m.HN meH m.eaH HN m.HaH m.HsH m.m~ m.ssH mmH m

m.NN m.eaH asH mH HwH ooH mm amH m.HmH am m.msH m.eaH N
Hm m.waH m.ssH mm m.Hom m.msH ma ‘aeH saH eaz ozoo m.NmH H
eeHg mug am as o eaHo mu: am a; o eeHm was «a a; o neer was am we 0 passage
mam + n. azm-omumz mam + . mmmm uoam
aze-om-mz mam

 

 

 

.mammv ms>wum>wwom aHouonm wmflmHuom usozowz was SUHB wonauHSU
«zmnomnmzv pooum HmEuoc m we woos :QEMH ego Eoum muomwuxo 42m ago suwz woumnsocfi
Homes mHHaU «seesaw Hmmaouaummlawwem-aoelaaa mmcHamlHa2po: uo momma coHuanHecu coHumuaHz .s mum<e

 

 

32

vmumcHEmucooo

meow uoznH

ous> wcmeusosm

 

 

 

 

 

H.mHs.mm m.HHH.om w.HH¢.ma a.HHo.ooH Hxv smecH coHamHMaz
©.Hnw.sm s.oflw.aa m.oHo.Hm n.0nw.Hm HmsHm>
mew%auso usonuHBV
ammum>¢
H.mna.mm m.aHHN.Hw w.HHa.wa a.HHd.ooH Axe xaecH coaamuwuz
e.HHm.sN m.mwm.ma a.owo.Hm n.0Hw.Hm HamsHm> HHm euazv
owmum>¢
200 cm nz am e.
ozoo as nnz mm m
Hm we cm on a
on «we as on. m
mm om mm mm N
mm mm mm Hm H
Qmm + I. ms 02 I. can + mm 02 menu humHHHmmu
azm-om-mm 42m-om-mm aze-em-mm. azm-om-nm

 

 

 

ofimmmv mm>fluw>HH®Q wauOHm UmHWHHHAm UDOLMHS UCM £uw3 UQHSuHSU AanMIQMImmV .Hmwwum

 

wmuoomcwvoum no fimzmnomnm%%mooum HmEhoc m we memos :QEAH oLu Eonm muomuuxo 42m oLu suHB UmuNfiSUCw

 

(domme mHHmo mumesxm HmmCOuHummlflH-omumoV mHe mwcHsa,Hmauoc mo mummu coHananH coHumusz .w MHm<H

33

meow quo

woumcwfimucoon
sowumnsocw wcwusw uomm onu mo nmuoEmwc aw ammouoch

 

 

 

 

 

 

 

 

m.HHH.m s.NHa.sm ~.mnd.ooH m.mHo.ooH Ase xmecH eoHumumHz
a.ond.H m.oHH.aH o.ona.aH o.oflo.aH ammumsa
H m.moH m.NoH m.om m.me msH m.mH m.msH mmH m.oN m.awH aeH, m
m.H mmH m.mmH one nzoo mom aH n.0sH m.smH mH «NH omH a
N st me aH qu moH aH m.msH n.0mH m.mH m.OsH NmH m
o me mmH aH m.HmH m.N©H NN m.meH m.qu m.HN amH m.N©H N
m.o NoH m.H©H mH m.osH m.NmH m.mH esH m.emH m.mH NmH m.NaH H
eeHa was «a we 0 eeHm an: am as o emHm was am a; o ummHm was as as o panameo
mam + I. azm-om-mm new + I. - azm-om-mz scam
aze-om-mm aza-®m-mz

 

 

 

.Aomav.mm>uum>Humo chuoum amauHusm “soauaz was LuHa amuseHsoldazm-om-mwwlummum emuomuca-oom
no ~¢zmn©mnmzv umoum HmEnos wo memos LQEMH osu Eoum muomnuxo <zm one Lows ouuw> Cw couscous“
. mommv mHHoo oumwsxo HmoCOuwuomldNnomnmov wee mocwawlfimanoc mo mumou cowuflnwscfl cowummwwz .m mam<9

 

 

34

meow uOZQ
cowumnnocfi mewusw uoam ecu mo nonmawwp aw mmmmuoch

 

 

 

 

 

 

 

 

H.an.a m.mhm.NoH m.wHHm.m~H s.oHnd.ooH Axe xmacH coaumumHz.

0.0Hm.H H.HHw+mN m.mHm.mm o.mHo.mN ammum>4

m.o m.mHN mHN om m.smm m.saH m.oa mmH m.qu Hm sow msH m

m.N m.NmH owH Hm m.sam m.eHN Dz . .oz age Hm OmH aHH N

H m.moN m.mo~ m.mN n.0mm HHN om moH m.smH NN m.oaH m.on H

sane use am a; o eaHm mu; am we c page as: am we 0 wanna mu: am p: o “engage

9E + I ETHmImm PE + I «5-352 ”.on
«zeIHn-mm . aZMIem-mz

 

 

 

.Ammmv mw>Huw>Hme cHouonm vowwflndm uaoxufis was suwz @oMSUHsoadzmIHmummv nooum wouoomcwnuom
no Admeomumze nooum Hughes we motes gefihH ecu Eonm muowuuxo «2m onu Low? ouuw> CH onmQSUCH
Aommv mHHmo oumwsxo Hmocobwuoa AHIHmImUMTmHQ moseswlamanoc mo mumou cowuwnwzcfl coflummwwz .oH mume

snow uOZQ

woumaHEMucoop
cowumnsocH mewusv uoam ecu mo woumEmww aw ammouoch

 

 

 

 

 

 

 

 

o.HHN.o m.NHH.NHH o.HHNw-HNH o.NHHo.ooH HNV xmooH cowomumaz
N. IH.H o.oHo.NN N.NHN.oN o.mHo.mN momum><
o m.NNH m.NNH oN m.NoH m.oNH oz zoo m.mNH m.oN HoH m.ooH m
m.H m.NoH ooH oN m.moH m.omH oz zoo NNH m.Nm NNN m.ooH a
H m.ooH m.NaH oN ooH HoH m.Nm m.mNH moH m.oN m.moH moH m
H oNH oNH ooz ozoo mHN om NNH NaH m.oH HmH m.oNH N
N m.HoH m.omH m.NN m.NoH ooH m.mN oNH m.omH oN m.NNH m.mmH H
Neoo who oN so o NoHo moo aN no o oooo moo oN N: o aNNHo as: «N oo o nonsmoo
PE + I 32-3-5 PE + I azmIomImz ooam
axe-Hm-om aze-om-oz

 

 

 

.dmmmv mo>woe>wwoo aflouonm wowmwnsm usosuNB was toNB wonaoaso a<zmIHmnmmV “meow wouoowCHuoom

 

no A<zmuomumZv pooow HmEuo: mo mowoc soewH ego 50pm muomuuxo 42m ope noHB ouuw> CH moumn:ocw

 

.4ommm mHHoo oumwsxo HmoSOuHuom.ANIHmImUVImHQ.meowslesEpoc mo womou cowuwnwscw cowummwwz

.HH mumdfi

The results from Tables 6-11 are summarized in Table 12. There was
passive sensitization of normal guinea pig PEC in vitro by RNA extracted
from lymph nodes of BCG-infected steers.

Passive sensitization in vivo of normalgguinea pigs by inoculation of
autologous PEC after incubation in vitro with RNA from BCGLinfected steers.
The migration of PEC from two guinea pigs (GP-56-1 and GP-56-2) nine days
after they had received autologous PEC after incubation in vitro with RNA
from a normal steer (NB-56-RNA) was not inhibited with or without PPD. The
M.I. indices were 111.4 : 5.0, 100.0 : 4.3 and 87.6 i 7.1, 100.0 i 4.9,
respectively, (Table 13).

The migration of PEG from two guinea pigs (GP-36-1 and GP-36-2)nine
days after they received autologous cells after incubation in vitro with
RNA from a BCG-infected steer (fiB-36-RNA) was inhibited by PPD (M.I. =
9.6 i 3.1 and M.I. = 15.6 i 1.8) and not inhibited by PPD (M.I. = 100.0
‘i 4.1 and 100.0 i 9.0, Table 14).

The migration of PEG from two guinea pigs (GP-Sl-l and GP-51-2) nine
days after they had received autologous cells after incubation in vitro

with RNA from a BCG-infected steer (SB-Sl-RNA) was inhibited by PPD

(M.I. 16.0 i 2.9 and M.I. = 20.8.: 3.7) and not inhibited by PPD

(M.I. 100.0 + 4.3 and 100.0 i 3.2, Table 15.

The results of Tables 13, 14 and 15 are summarized in Table 16. The
guinea pigs were passively sensitized in vivo, as detected by migration -
inhibition tests, by RNA extracted from lymph nodes from BCG-infected cattle,
not from a normal steer.

Skin tests with 10 TU PPD three days after guinea pigs received autologous

cells incubated with RNA from normal bovine RNA elicited no detectable

37

TABLE 12. Summary of TABLES 6-11 of migration tests to detect_passive

sensitization of normal guineagpiggPhC in vitro by RNA extracts from

lymph nodes of a normal steer (NB-Sh-RNA) or from BCG-infected steers
- (SB-Bh—RNA or SB-Sl-RNA).

 

 

Migration Average

 

 

Guinea Pig PEC without PPD PEC with PPD 'iigration Indeij)
GP—SG-l 22.3 i 1.7 24.0 i 1.5 99.1 i 6.28a
GP-56-2 . 21.5 i 6.6 20.2 i 1.6 86.7 i 7.1*
GP-36-1 49.8 i 0.7 27.8 i 1.6 53.6 i 3.1**b
GP-36-2 19.1 i 0.5 1.0 i 0.4 5.1 i- 1.8**
GP-Sl-l 28.8 i 1.1 1.3 i 0.6 4.8 i 2.1%
GP-51-2 27.6 :t 0.6 1.1 i 0.3 4.7 i 1.4%

 

 

aThe migration index was the percentage difference between the migration of
the PEC incubated with BSS and with PPD.

bThe migration index was the percentage difference between the migration of
PEC incubated with NB-56-RNA and with SB-36-RNA or SB-Sl-RNA.

8
3

meow uozQ

cowumnsoaw wcwusw uomm men mo HouoEmwv a“ ammouomHm

 

 

 

 

 

 

 

 

H.NHo.No o.ano.ooH o.mwa.HHH m.owo.ooH HNV xmooH scoomoooz
m.HHo.oH H.HHN.HN o.HHw.HN o.oHo.oH oomoo>a
oH mam on m.oH m.ooN HNN oN on ooN m.NH oom m.on m
mH on moN oN m.ooN m.oNN oN mom mNm NH moo oom 8
oz oz oz oN oNN omN NN NNN mHm m.oH m.mHo mom N

m.oH m.omm oNN m.oN m.on oom oz oz ooz m.HN omm m.NHN N

m.NN ooN o.mNN m.oN m.ooN mNN 8N oHN aoN .HN oom mNm H

ozoo moo «N on o ooHo moo oN oo o ozHo moo 8N oo o mooHo moo «N as o oooEmoo
ooo + NuomINo ooo + HIomnoo zoom
NuomIoo Huomuoo

 

 

 

.Aoooo mo>oom>oo6o coooooo ooooouso

 

usosoNB was Sofia @oNSuHso AszmIomxmzv nooom HmEuoc Eoum 42m osu sow? MHw50H>me

 

woumnaocN 0mm mnemoHOuammfloHRVUoofldw HmoGOanoQMNueH zooms exam mafia Awsomsmw was Hammumwv

 

wwwa moewsm.amanoc Eoum hommv mHHoo mommsxo HmoCOoNuoa mo momma cowuwawxaw cowumnwwz .mH mum<H

39

meow uozn

cowuwnsocw wawusw uoam one wo HouoEme aw ommoNoCHm

 

 

 

 

 

 

 

 

N.HH6.NH o.ono.ooH H.NHo.o_ H.ono.ooH HNV smoaH ecoomoooz
a.oHN.N N.HHN.oN N.oHo.N o.oHN.oN womo6><
o N.NoN N.HoN N.NH N.NoN NoN N HHN ooN oz oz oz N
N N.oNN N.NNN N.NH N.ooN NNN . oz oz oz HN oNN oNN a
a N.ooN N.NoN oz oz ooz N . HNN ooN NN aoN . HNN N
N NNN ooN NH ooN HNN N.H N.NNN HNN oN NHN NNN N
N NaN ooN oN ooN oNN N.N NNN N.NoN NH NNN oNN H
zooo moo «N oo o oooo moo 4N no o ono moo 4N oo o uzzHo moo aN no o ooooaoo
ooo + NIoNINo ozo + - HIoNuoo ooom

N-oN-oo H-oN-oo

 

 

 

.Ammme mo>woo>euom cNouonm wowmwnsm
uSOLuNB was news woNSUHsoldmzmIomnmmv zmoum wouoowcwuoum mo wzm owe Luw3 NHm50w>onm
wmumnsocw 0mm msqonoHQmmo eowfixfioi.Hmoc0uwuoamzucH zooms wmmw mafia Amuomumw was HIQMIWWV
mwfia moownwuHmEpoc Boom Aummv mHHmo oumwsxo Hmocouwnom1mo momma cowuwnwscH coHomwwwz .¢H m4m<H

 

 

 

4O

aoflquSUCH wcHusw uomm use mo umuoEme aw ommmuode

 

 

 

 

 

 

 

 

N.NHN.oN N.NHo.ooH o.NHo.oH N.ono.ooH HNV meoH acoomzooz
N.oHN.o N.owo.HN o.oHN.N o.ono.oN 6Nmo6>a
N.o N.NNN NNN oN N.HoN N.HaN H N.NoN N.HoN NH NNN NoN N
N.N N.NNN oNN N.oN NNN N.¢NN N.N N.NoN ooN N.oH NHN N.NNN a
o oNN oNN N.HN N.NNN NNN N.N ooN N.NoN N.oN N.NoN NNN N
N ooN NNN 4N N.oNN N.NHN N.o N.HoN NNN NN N.HHN N.NNN N
N.o NNN N.NNN NN N.HNN N.ooN a .NNN NNN NN NHN NNN H
ono moo 4N no o ooHo moo 8N 6o o Immo 6oz oN go o mooHo moo 4N no o “mosaoo
ooo + NIHmdo $5 + HIHmInHo ooom
N-HN-oo HIHN-oo

 

 

 

.Aam%v mo>Num>Nuom eaouowm woamauom

 

usonuaz was wuNB wousuHSU nwzmI IHmsmwu nooum woooomwNIwum mo wzm use seas hamsoN>oHQ

 

woomDSocN 0mm msoonOuss mo coflooowew HmosOanomsquH noomw mmdw mafia ANIHmImU wow HIHmImmv

 

mafia momwsw amazon Eoum dummy mHHoo wowwoxo HwoCOunom mo mumoo cowuwnwscw coHumeHS .mH mummH

TABLE 16. Summary of TABLES l3, l4 and 15 of migration tests to detect

passive. sensitization of normal guinea pig PEC in vivo by RNA extracts

from lymph nodes of a normal steerg(NB-56-RNA) or from BCG-infected
steers @li-Iib-RNA or SB-Sl-RNA).

 

M igr a t ion Ave rage

 

 

Guinea Pig PEC without PPD PEC with PPD Migration Indexfl.)
GP-56-1 19.3 i 0.8 ' 21.5 i 1.0 ' 111.4 i 5.0
GP-56-2 21.7 :1: 1.1 19.0 i 1.5 87.6 i 7.1
GP-36-l 20.8 i 0.9 2.0 i 0.7 9.6 i 3.1
GP-36-2 20.5 i 1.8 3.2 i 0.4 15.6 _+_ 1.8
GP-Sl-l 20.6 i 0.9 3.3 i 0.6 16.0 i 2.9
GP-51-2 . 21.6 i 0.7 4.5 i 0.8 20.8 i 3.7

 

 

42
reactions. All guinea pigs which received autologous cells after incubation
with RNA from BCG-infected steers had 18-25 mm reactions at the site of
inoculation of 10 TU of PPD at 24, 48 and 72 hours. There were no detectable
reactions 15 minutes or 2 hours after the PPD injection. The results are
given in Table 17.
The results of the in vitro and in vivo passive sensitizations of guinea

pigs by bovine RNA are summarized in Table 18.

3

aonon mEoLuhzo mo Hoodoo one on Cowman won umamow m wwwafivo

.coNuomom ofinmuoouow oz

3

Hmo>aoIozzmoo oNN oe oH zoos ooomoo enema

 

 

 

 

 

 

zN HoooN oN No HaHVNN NN No HNHVNN NN N-HN-oo
NN HovoH oN zN AoHooN NN zN uHoHVNN NN N-oN-No
mz mz mz mz mz mz xH m m NIomImo
xm «H qH x8 0H om xm ON ON HIHnImO
xm «H «H xm @H om Nm ma wH HIomImU
mz mz mz mz mz mz m2 mz nmz HIomImo
as V 38 25 9.8 9.8 H88
mmoconLH conoH Cowman mmocxowae conou donou mmocxowsa .coflwon Cowman wwm mocfino
maoeumnm mEowm «Eowoznm wEowm maosokum mEowm
we mm one we on: em

 

 

 

.mp3 Nu wow on: we awn: qm um wm>nomno
A<zmIHmImm wow «zmIomImmwinooum woooochIoum no ~<zmIomIsz nooum HmENoa Scum wzm one nuNB woumnnoCH

 

0mm maomoHOosm mo coHuoowcH HmoCOowumemzucH poems mmww mwuwo mafia mmcwmm mo mm%Wou awxm mo wuHDmmm

.NH mmme

44

TABLE 18. Summary of TABLES 12J 16 and 17 of migration inhibition and
skin tests to drtect passive sensitization in vitro and in vivo of normal

guinta pi-{s hy_lli‘.‘;\~£:;txacts fret-.1 lymph nodes from a 110113.111 steer (Nli-bé-RNA)
and from two BCG-sensitized steers (SB-36-RNA and SB~51-RNA).

 

 

 

In vivo sensitization
by injection of pas--
sively sensitized

 

 

In vitro sensitization autologous cells

Guinea Pig Migration of PEG Migration of PEC Skin Reaction
GP-56-l 99.1'i 6.2 111.4‘i 5.0 none
GP-56-2 86.7.: 7.1 87.6.: 7.1 none
GP-36-1 53.6 i 3.1 9-6.i 3.1 20 mm
GP-36-2 ‘ 5.1 i 1.8 15.6,: 1.8 25 mm
GP—Sl-l 4.8 i 2.1 16.0.: 2.9 20 mm
GP-51-2 4.7.: 1.4 20.8.: 3.7 25 mm

 

 

DISCUSSION

The purpose of this research was to passively sensitize guinea pigs
and their PEC to PPD with homologous RNA and to determine if bovine RNA
would passively sensitize normal guinea pigs and their PEC. If so, it
could provide a model to study the kind and role of RNA in delayed sensi-
tivity reactions and a means of detecting tuberculous herds by tests of
lymph nodes collected at abattoirs at the tune of slaughter.

The results obtained by passive sensitization with homologous RNA were
similar to the results reported by Jureziz et a1. (81) of the inhibition
by antigen of migration in vitro of PEG from normal guinea pigs after
incubation with RNA extracted from the spleens and lymph nodes of sensitive
guinea pigs. .The migration of PEG was inhibited by PPD after incubation
with RNA from a donor infected with viable BCG, not after incubation with
' RNA extracts from normal guinea pigs. Jureziz et al. used a pool of lymph
nodes and spleens from randomly bred guinea pigs for RNA extractions and
tested cells from single guinea pigs to which the results shown in Table 1
are similar. The results of transfers with separate RNA extracts from
lymph nodes and spleens of two guinea pigs infected with BCG are given in
Table 3. There was inhibition of migration of normal PEC by PPD after
passive sensitization by each of the RNA extracts. The amount of inhibition
of PEG from the donor prior to transfer (Table 2) was greater by GP-II than
GP-I. The number of animals is too few to draw any conclusions regarding
the relative amount of sensitivity transferred.

The RNA extracts used by Jureziz et a1. (82) were from svneenic guinea
pigs, and incubated with syngenic but not autologous lymphoid cells, which

were injected into guinea pigs of the same syngenic strain. Passive

45

46
sensitization required epinephrine. They suggested it increased the
phagocytic capacity of lymphoid cells. In our experiments to transfer

sensitivity in vivo, normal guinea pigs received washed autologous PEC

 

after incubation in vitro with RNA. Because the PEC contain predominantly
macrophages, no epinephrine was used.

The use of autologous cells for passive sensitization in vivo made it
unnecessary to have syngenic strains to avoid histoincompatibility. It did
create some operational difficulties because with RNA the animal can not
be killed to obtain the PEC which are incubated with RNA and returned to
the animal. The care required during the collection usually resulted in
too few cells to make adequate tests by the capillary tube test for MIF.
The radial migration test requires fewer cells than the capillary test and
results from previous studies (108, 129) with bovine le11cc>c37tezs
indicated the "Spot” test was equally as good or better than the capillary
test.

There was passive sensitization 25 days after injection of autologous
PEC cells when they had been incubated with the RNAs from BCG-infected
guinea pigs (Table 4). This indicated that ”immune-RNA" would transfer
sensitivity not only to normal exudate cells in vitro, but that washed cells
after incubation with the RNA also transferred sensitivity to normal guinea
pigs. This was interpreted as an in vivo intraspecies passive sensitization.

In vitro and in vivo interspecies transfer also occurred. There was
some inhibition of normal PEC from guinea pigs in vitro by RNA from the
normal steer (NB-56~RNA) (Tables 6 and 7). Therefore, cells with NB-56-RNA
were used as controls (Tables 8, 9, 10 and 11) when determining the M.I. of
PEC incubated with RNA from BCG-infected steers. This was indicated in

Table 12 which summarized the results.

47

When guinea pigs were inoculated with washed autologous cells after
incubation with RNA there were strong skin reactions to PPD three days later
(Table 17) and inhibition of migration by PPD of their PEC nine days later
(Tables 13-16) when the RNA was from BCG-infected cattle. What effect the
skin test at three days may have had on the subsequent M.Il test at nine
days was considered. There is an unresolved, long term controversy as to
the effect of tuberculin testing an actively sensitized animal. It may in-
crease, decrease or not affect subsequent skin tests or serologic tests.
Little or nothing has been reported on the effect on passively sensitized
animals. In our laboratory, we have found that skin testing cattle infected
with BCG decreases the M.I. 3-20 days after the skin test. Therefore, it
seemed probable that in a passively sensitized animal, a skin test would more
probably cause a decrease than an increase in M.I. Because there is a
chance of the losing the guinea pig when collecting PEC, we elected to skin
test first. It is possible that the skin test could activate lymphocytes
and macrophages other than locally at the site of the test and cause the
cells in the PEC to be more active in vitro. It might decrease the number
of responsive cells in the peritoneal cavity, or have no effect. This
should be determined.

Many reports have indicated that immunogenic RNA is sensitive to
RNase (76, 110, 160, 161). RNase-treated extracts do not confer sensitivity
'on normal cell populations, suggesting that intact RNA has some role in
the transfer (161). It is of great importance therefore, in the whole process
of RNA extraction to prevent the degradation of RNA. Little biological
activity in RNA preparation is lost if stored at -200C or less, either in
alcohol or in aqueous solution because low temperature decreases the enzymatic

. . . . . . o
act1v1ties of RNase (2). In this experiment, samples were stored under -70 C

48

and extractions made at 40C except in the steps of denaturing protein by
hot phenol at 56-6000 for 10 min. High concentration of RNA may compensate
for small losses of RNA by RNase. Most investigators use 100-1000 ug of
RNA/lO7-108 cells in a volume of 0.1-5.0 ml (2). We used 500 ug of RNA/108
cells in 2 ml of medium.

Several inhibitors of RNase have been used in the phenol mixtures.
Polyvinyl sulfate (PVS) has been reported to be an inhibitor of RNase by
Bernfield et a1. (13). Addition of 8-hydroxyquinoline to the phenol improved
the yield of RNA, decreased the protein contamination, and lessened but
did not eliminate, the action of RNase (83). Bentonite can absorb RNase on
its surface but it must be removed immediately because RNase absorbed on
bentonite still has some enzymatic activities. The possible contamination
of DNA can be eliminated by use of DNase.

The RNA preparation in this experiment was extracted by hot phenol at

/OD

-280 ratio equal to or greater than 2.

least three times or until the OD260
Some of the initial attempts at extraction were unsuccessful but no difficulty
was encountered thereafter. Under this condition, DNA and the protein are pre-
sumably removed (144). However, slight amount of contaminations have been
reported. Herscowitz and Stelos (76) found that RNA preparations having an

/OD ratio of 2 contained 2 to 10 ug of protein/800 ug of RNA, as

OD260 280
measured by the method of Lowry et a1. (96) using bovine serum albumin as
standard, and 8 to 12 ug of DNA/800 ug of RNA, as measured by the diphenylamine
reaction (21) using slamon sperm DNA. These contaminations are presumed to
be negligible.

Redistilled phenol was saturated with sodium acetate which prevents

phenol from crystallizing under 40C. Phenol saturated in buffer probably is

a better denaturing agent of protein than phenol alone. Although phenol may

49

extract RNA, the subsequent purification can result in degradation of RNA
and destroy its activity. Significant amounts of active RNA may be lost
in the phenol-buffer interphase during extraction (2).

Autologous peritoneal cells were incubated in vitro with RNA and then
washed before injection into the same gUinea pig. The interaction of PEC
with RNA before injection was believed to increase the probability of passive
sensitization by the RNA. The cells were washed to remove free RNA. It
was presumed that RNA was incorporated by some of the PEC cells but no tests
were made to determine if RNA were only adsorbed to the surface of the PEC
cells. Such studies will be necessary to precisely determine the roles of
RNA and the PEC in the passive transfer. The need of epinephrine to increase
phagocytosis by lymphoid cells does imply the need of phagocytosis for the
passive transfer (82).

Peritoneal exudate cells are very susceptible to specific inhibition
of migration in vitro and it has been suggested that migration inhibition
is the one of the best methods to correlate delayed hypersensitivity (57).
There are some difficulties such as an insufficient number of peritoneal
exudate cells to run capillary tube test; partial stacking of cells rather
than monolayer migration; variable diameters of capillary tubes, the eveness
of the cut and closeness to the glass when immobilized with paraffin; lack
of uniformity of the glass surface on which cell migrate; an air bubble at
the mouth of capillary tube; unequal pH changes in each chamber during incu-
bation. The ”spot” test lessens some of the problems but not all. In
the author's opinion, it is simpler, more convenient, and more reproducible
than capillary tube technique but no comparative tests were made in this

study.

50

The slight inhibitory effect on PEC by N-RNA without PPD or by PPD
without RNA is probably due to non-Specific release of effector molecules
from lymphocytes (124) or the RNA extracts may contain small amounts of
phenol or alcohol toxic for PEC.

Whether transfer factor occurs in bovine cells has not been determined.
It has been found in a number of species. The following differences be-
tween transfer factor and RNA extract do not exclude that passive sensiti-
zation in vivo was by transfer factor: RNA extracts are inactivated by
RNase sensitivity, transfer factor is not; RNA is nondialyzable and has a
sedimentation value between 88 to 128 suggesting a molecular weight greater
than 80,000 (160); transfer factor is dialyzable and molecular weight is
less than 10,000 (92). However, the hot phenol extraction of RNA probably
excludes the transfer factor. A

The degree of specificity of sensitivity transferred has not been
determined yet. Tlie 'Hnmmne-RNA" from either guinea pigs or steers did
passively transfer delayed hypersensitivity to guinea pigs and their peri-
toneal exudate cells from normal guinea pig. It provides a model for further
study of the role of RNA in delayed sensitivity reactions and the possibility

of an in vitro test to detect tuberculosis.

10.

11.

12.

LITERATURE CITED

Adler, F. L., M. Fishman, and S. Dray. 1966. Antibody formation
initiated in vitro. III. Antibody formation and allotypic
specificity directed by ribonucleic acid from peritoneal exudate
cells. J. Immunol. _91:554.

Adler, F. L. 1972. Meeting report: RNA in the immune response. Cell.
Immunol. .§:363.

Alexander, P., E. J. Delorme, L. D. G. Hamilton and J. G. Hall. 1968.
Stimulation of anti-tumor activity of the host with RNA from
immune lymphocytes, p. 527. I3 O.J. Plescia and W. Braun (eds),
Nucleic acids in immunology. Springer-Verlag, New York.

Arend, W. P., and M. Mannik. 1972. In vitro adherence of soluble
bmmune complexes to macrOphages. J. Exp. Med. 136:514.

Askonas, B. A. and J. M. Rhodes. 1965. Immunogenicity of antigen-
containing ribonucleic acid and preparation from macrophages.
Nature (London) 205:470.

Askonas, B. A. and J. M. Rhodes. 1965. Is antigen associated with
macrophage RNA?, p. 503. .Lg J. Sterzl (ed), Molecular and cellular
basis of antibody formation. Academic Press, New York.

Askonas, B. A., and A. R. Williamson. 1966. Biosynthesis of immuno-
globulins on polysomes and assembly of the IgG molecule. Proc.
Roy. Soc. B. 166:232.

Beckert, W. H., and M. M. Sharkey. 1970. Mitogenic activity of the
jack bean (canavalin ensiformis) with rabbit peritoneal blood
lymphocytes. Int. Arch. Allergy Appl. Immunol. 22s337.

Bell, C., and S. Dray. 1969. Conversion of non-immune spleen cells
by ribonucleic acid of lymphoid cells from an immunized rabbit to
produceCYM antibody of foreign light chain allotype. J. Immunol.
103:1196.

Bennett, W. E., and Z. A. Cohn. 1966. The isolation and selected
properties of blood monocytes. J. Exp. Med. 123:145.

Bennett, B., and B. R. Bloom. 1967. Studies on the migration inhibitory
factor associated with delayed-type hypersensitivity: Cytodynamics
and specificity. Transplantation .52996.

Bennett, B. and B. R. Bloom. 1968. Reactions in vivo and in vitro

produced by a soluble substance associated with delayed-type
hypersensitivity. Proc. Nat. Acad. Sci. U.S.A. _52:756.

51

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

52

Berfield, P., J. S. Nisselbaum, B. J. Berkeley, and R. W. Hanson.
1960. Chemical and phsyicochemical nature of macropmolecular
polyanion on their interaction with human serum B-lipoproteins.
J. Biol. Chem. .23§:2852.

Bishop, D. C., and A. A. Gottlieb. 1970. Macrophages, RNAs and the
immune response. Current Topics in Microbiol. Immun. 5131.

Bloom, B. R., and B. Bennett. 1966.' Mechanism of a reaction in vitro
associated with delayed-type hypersensitivity. Science 153:80.

Bloom, B. R., and M. W. Chase. 1967. Transfer of delayed-type hyper-
sensitivity. A critical review and experimental study in the
guinea pie. Prog. Allergy .lg:151.

Bloom, B. R., and B. Bennett. 1968. Migration inhibitory factor '

associated with delayed-type hypersensitivity. Fed. Proc. 22513.

Boyden, S. V. 1958. The immunological response of antigen of the
tubercle bacillus, some experimental aspects. Prog. Allergy .§:l49.

Braun, W., M. Nakano, J. Jaraskova, Y. Yajima, and L. Jimenez. 1968.
Stimulation of antibody-forming cells by oligonucleotides of known
composition, p. 347. lg O.J. Plescia and W. Braun (eds), Nucleic
acids in immunology. Springer-Verlag, New York.

Braun, W., and E. P. Cohen. 1968. On the role of nucleic acid in
antibody formation, p. 152. 13 C.C. Thomas (ed), Regulation of the
antibody response. C.Co Thomas, Publisher, Springfield, Illinois.

Burton, K. 1965. A study of the conditions and mechanism of the
diphenylamine reaction for the colorimetric estimation of deoxy-
ribonucleic acid. Biochem. J. IQ;:315.

Campbell, D. H., and J. S. Garvey. 1963. Nature of retained antigen
and its role in immune mechanisms. Advanc. Immunol. 33261.

Carr, I. 1967. The cellular basis of reticuloendothelial stimulation.
J. Pathol. Bacteriol. Qfiz323.

Chase, M. W. 1945. The cellular transfer of cutaneous hypersensitivity
to tuberculin. Proc. Soc. Exp. Biol. ‘50:124.

Coe, J. E., J. D. Feldman, and S. J. Lee. 1966. Immunologic competence
of thoracic duct cells. I. Delayed hypersensitivity. J. Exp. Med.
123:267. '

Cohen, E. P., and J. J. Parks. 1964. Antibody production by nonimmune
spleen cells incubated with RNA from immunized mice. Science
144:1012.

Cohen, E. P., R. W. Newcomb, and L. K. Crosby. 1965. Conversion of
nonimmune spleen cells to antibody forming cells by RNA: Strain
specificity of the response. J. Immunol. ng583.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

53

Cohen, S., R. T. McClus key, and B. Benacerraf. 1967. Studies on the
specificity of the cellular infiltrate of delayed hypersensitivity
reactions. J. Immunol. 98: 269.

Cohn, Z.A., and B. Benson. 1965. The differentiation of mononuclear
phagocytes. Morphology, cytochemistry, and biochemistry. J. Exp.
Med. 121:153.

Cohn, Z. A., M. E. Fedorko, and J. G. Hirsch. 1966. The in vitro dif-
feration of mononuclear phagocytes. V. The formation of macrophage
lysosomes. J. Exp. Med. 123:757.

Cohn, Z. A., and E. Parks. 1967. The regulation of pinocytosis in
mouse macrophages. II. Factors inducing vesical formation. J. Exp.
Med. 125:213.

Cohn, Z. A., and E. Parks. 1967. The regulation of pinocytosis in
mouse macrophages. IV. The immunological induction of pinocytotic
vesicles, secondary lysosomes, and hydrolytic enzymes. J. Exp. Med.
125:1091.

Cowling, D. C., D. Quaglino, and E. Davidson. ’1963. Changes induced
by tuberculin in leucocyte cultures. Lancet ,2:1091.

Dannenberg, A. M. Jr., M. S. Burstone, P. C. Walter, and J. W. Kinsley.
1963. A histochemical study of phagocytic and enzymatic functions
of rabbit mononuclear and polymorphonuclear exudate cells and a1-
veolar macrophages. 1. Survey and quantitation of enzymes and state
of cellular activation. J. Cell Biol. 11:465.

Dannenberg, A. M. Jr., 0. T. Meyer, J. R. Esterly, and T. Kambara. 1968.
The local nature of immunity in tuberculosis, illustrated histo-
chemically in dermal BCG lesions. J. Immunol. 100:931.

Dannenberg, A. M. Jr. 1968. Cellular hypersensitivity and cellular
immunity in the pathogenesis of tuberculosis: Specificity, systemic
and local nature, and associated macrophage enzymes. Bacteriol. Rev.
32:85.

Dannenberg, A. M. Jr., M. Ando, and K. Shima. 1972. Macrophage accumu-
lation, division, maturation, and digestive and microbicidal capacities
in tuberculous lesions. IV. The turn over of macrophages and its
relation to their activation and antimicrobial immunity in primaryBCG
lesions and those of reinfection. J. Immunol. 102:1109.

David, J. R., S. Al-Askari, H. S. Lawrence, and L. Thomas. 1964.
Delayed hypersensitivity in vitro. 1. The specificity of inhibition
of cell migration by antigens. J. Immunol. 223264.

David, J. R., H. S. Lawrence, L. Thomas. 1961. Delayed hypersensitivity
in vitro. 11. Effect of sensitive cells on normal cells in the pres-
ence of antigen. J. Immunol. 23;274.

4'.\
O

41.

440

45.

46.

47.

48.

49.

50.

51.

52.
53.

54.

54

David, J. R., and P. Y. Paterson. 1965. In vitro demonstration of
cellular hypersensitivity in allergic encephalomyelitis. J. Exp.
Med. 122:1161.

David, J. R. 1966. Delayed hypersensitivity in vitro: Its mediation
by cell-free substances formed by lymphoid cell-antigen interaction.
Proc. Nat. Acad. Sci. (Wash.) 56:72.

David, J. R. 1968. Macrophage migration. Fed. Proc. 321:6.

Dumonde, D. C., W. J. Howson, and R. A. Wolstencroft. 1968. Fifth
international symposium on immunopathology, p. 263. Schwabe, Basal.

Dumonde, D. C.,W. J. Howson, and R. A. Wolstencroft, G. S. Panayi,
M. Matthew, J. Morley, and W. T. Howson. 1969. Lymphokines:
Non-antibody mediators of cellular immunity. Nature (London)
224:338.

Edelman, G. M., and J. R. Galley. 1962. The nature of Bence-Jones
proteins. Chemical similarities to polypeptide chains of myeloma
globulins and normal d—globulins. J. Exp. Med. ll6;207.

Elberg, S. S., P. Schneider, and J. Fong. 1957. Cross-immunity between
Brucella melitensis and Mycobacterium tuberculosis. Intracellular
behavior of Brucella melitensis in monocytes from vaccinated animals.
J. Exp. Med. .lggz545.

 

 

 

Fishman, M. 1961. Antibody formation in vitro. J. Exp. Med. 114:837.

Fishman, M., and F. L. Adler. 1963. Antibody formation initiated in
vitro. 11. Antibody synthesis in X-irradiated recipients of dif-
fusion chambers containing nucleic acid derived from macrophages
incubated with antigen. J. Exp. Med. 117:595.

Flax, M. H., and J. B. Caulfield. 1961. Ultrastructural changes in
contact dermatitis in the guinea pig. Fed. Proc. 29:263.

Fong, J., D. Chin, and S. S. Elberg. 1963. Studies of tubercle bacillus-
histiocyte relationship. VI. Induction of cellular resistance by
ribosomes and ribosomal RNA. J. Exp. Med. 118:371.

Fong, J., D. Chin, and S. S. Elberg. 1964. Studies of tubercle bacillus-
histiocyte relationships. VIII. Comparative study of cellular re-
sistance induced by Brucella and mycobacteria. J. Exp. Med. 120:885.

Forbes, I. J., and G. B. Mackaness. 1963. Mitosis in macrOphages.
Lancet 2:1203.

Friedman, H. P. 1959. Transfer of agglutinin formation by immune lymph
node cell fractions. Fed. Proc. Fed. Soc. Exp. Biol. l§;567.

Friedman, H. P., A. B. Stavitsky, and J. M. Solomon. 1965. Induction
in vitro of antibodies to phage antigens in the RNA extract employed.
Science 149:1106.

55.

56.

57.

58.

59.

60.
61.
62.

63.

64.

65.

66.

67.

68.

55

' 3
Garvey, J. S. and D. H. Campbell. 1957. The retention of S 5-

labelled bovine serum albumin in normal and immunized rabbit
liver tissue. J. Exp. Med. 105:361. .

Gell, P. G. H., and I. T. Hinde. 1951. The histology of the tuberculin
reaction and its modification by cortisone. Brit. J. Exp. Pathol.
32:516.

George, M., and J. H. Vaughan. 1962. In vitro cell migration as a
model of delayed hypersensitivity. Fed. Proc. _§:514.

Gottlieb, A. A., V. R. Glisin, and P. Doty. 1967. Studies on macro-
phage RNA involved in antibody production. Proc. Nat. Acad. Sci.
,5Z:l849.

Gottlieb, A. A., and D. S. Straus. 1969. Physical studies on the light
density ribonucleoprotein complex of macrophages cells. J. Biol.
Chem. 244:3324.

Gottlieb, A. A. and R. H. Schwartz. 1972. Antigen-RNA interaction cell.
Immunol. 5g34l. '

Gowans, J. L., and D. D. McGregor. 1965. The immunological activities
of lymphocytes. Prog. Allergy _gzl.

Gowans, J. L. 1967. Life-span, recirculation, and transformation of
lymphocytes. Intern. Rev. Exp. Pathol. 551.

Granger, G. A., and W. P. Kolb. 1968. Lymphocyte in vitro cytotoxicity:
Mechanisms of immune and nonimmune small lymphocyte mediated target
L cell destruction. J. Immunol. 101:111.

Granger, G. A. 1969. Varied effects of lymphocyte products ranging fran
destruction of target cells to activation of lymphocytes and macro-
phages, p. 321. 53 H.S. Lawrence and M. Landy (eds), Mediators of
cellular immunity. Academic Press, New York.

Gray, D. P., and C. Cheers. 1967. The steady state in cellular immunity.
I. Chemotherapy and superinfection in murine tuberculosis. Aust.
J. Exp. Biol. Med. Sci. .45z407.

Gray, D. F. and C. Cheers. 1967. The steady state in cellular immunity.
II. Immunological complaisance in murine petussis. Aust. J. Exp.
B101. I‘ledo SCio 3.22417.

Green, J. A., S. R. Cooperhand, and S. Kibrick. 1969. Immune specific
induction of interferon production in cultures of human blood
lymphocytes. Science 164:1415.

Halliday, W. J., and J. S. Garvey. 1964. Some factors affecting the
secondary immune reSponse in tissue cultures containing Hydro-
cortison. J. Immunol. .25z757.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

56

Halpern, B. N., B. Benacerraf, and J. F. Delafresnay. 1957. C. C.
Thomas (ed), Springfield, Illinois.

Haurowitz, F.- 1960. Immunochemistry. Ann. Rev. Biochem. .22:609.

Haxthausen, H. 1948. Studies on the role of the lymphocytes as
"Transmitter” of the hypersensitiveness in allergic eczema. Acta.
Derm. Vener Stockh. ‘2l:275.

Heilman, D. H., and W. McFarland. 1966. Inhibition of tuberculin-
induced mitogensis in cultures of lymphocytes from tuberculous
donors. Intern. Arch. Allergy _igz58.

Heise, E. R., Q. N. Myrvik, and E. S. Leake. 1965. Effect of Bacillus
Calmette-Guerin on the levels of acid phosphatase, lysozyme and
cathepsin in rabbit alveolar macrophages. J. Immunol. ‘25:125.

Heller, J. H. 1960. The reticulo-endothelial system (RES). Ann.
Acad. Sci. (New York) 85:1.

Heller, J. H. 1960. .1Q Reticulo-endothelial structure and function.
J. H. Heller (ed), Ronald Press Co., New York.

Herscowitz, H. B., and P. Stelos. 1970. The induction of bacteriophage-
neutralization antibody by mouse spleen immunogenic RNA. J. Immunol.
105:771.

Jacherts, D., and J. Drescher. 1970. Antibody response in rhesus monkeys
and guinea pigs to inoculation with RNA derived from antigenically
stimulated cell-free systems. 'J. Immunol. 104:746.

Jacob, F., and J. Monod. 1961. Genetic regulatory mechanisms in the
synthesis of proteins. J. Mol. Biol. ,5:318.

Jenkin, C., and B. Benacerraf. 1960. In vitro studies on the inter-
action between mouse peritoneal macrophages and strains of Salmonella
and Escherichia coli. J. Exp. Med. 112:403.

Johnson, H. G., and A. G. Johnson. 1971. Regulation of the immune
system by synthetic polynucleotides. II. Action on peritoneal exudate
cells. J. Exp. Med. 133:649.

Jureziz, R. E., D. E. Thor, and S. Dray. 1968. Transfer with RNA
extracts of the cell migration inhibition correlate of delayed
hypersensitivity in the guinea pig. J. Immunol. 101:823.

Jureziz, R. E., D. E. Thor, and S. Dray. 1970. Transfer of the delayed
hypersensitivity skin reaction in the guinea pig using RNA-treated
lymphoid cells. J. Immunol. 105:1313. .

Kirby, K. S. 1968. Isolation of nucleic acids with phenolic solvents,
p. 87. _13 L. Grossman and K. Moldave (eds),Methods in enzymology
XII. Academic Press, New York.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

57

Khoo, K. K., and G. B. Mackaness. 1964. Macrophage proliferation in
relation to acquired cellular resistance.‘ Aust. J. Exp. Biol.
Med. Sci. 342:707.

Kornfeld, S., and R. Kornfeld. 1969. Solubilization and partial
characterization of a phytohemagglutinin receptor site from
human erythrocytes. Proc. Nat. Acad. Sci. U.S.A. [63:1439.

Kosunen, T. U., B. H. Waksman, M. H. Flax, and W. S. Tihen. 1963.
Radioautographic study of cellular mechanisms in delayed hyper-
sensitivity. I. Delayed reactions to tuberculin and purified
proteins in the rat and guinea pig. Immunol. .§:276.

Kosunen, T. U. 1966. Origin and specificity of mononuclear cells in
delayed hypersensitivity reactions. Nature (London) 211:1418.

Landsteiner, R., and M. W. Chase. 1942. Experiments on transfer of

cutaneous sensitivity to simple compounds. Proc. Soc. Exp. Biol.
(N.Y.) 42:688. '

Lawrence, H.S. 1954. The transfer of generalized cutaneous hypersen-
sitivity of the delayed tuberculin type in man by means of the
constituents of disrupted leucocytes. J. Clin. Invest. 335951.

Lawrence, H. S. 1959. The transfer of hypersensitivity of the delayed
type in man, p. 279. lg H.S. Lawrence (ed), Cellular and humoral
aspects of the hypersensitive states. Hoeber~Harper, New York.

Lawrence, H. S. 1960. 13 G.E.W. Wolstenholme and M. O'Conner (eds),
Cellular aspects of immunity, p. 243. Ciba Found. Symposium,
Little, Brown and Co., Boston.

Lawrence, H. S. 1969. III. Transfer factor, p. 179. .12 H.S. Lawrence

and M. Landy (eds), Mediators of cellular immunity. Academic Press,
New York.

Lazda, V. A., and J. L. Starr. 1965. The stability of messenger ribo-
nucleic acid in antibody synthesis. J. Immunol. .2§:254.

Likhite, V., and A. Sehon. 1972. Cell-mediated tumor allograft
immunity: In vitro transfer with RNA. Science 175:204.

Lolekha, 8., S. Dray, and S. P. Gotoff. 1970. Macrophage aggregation
in vitro: A correlate of delayed hypersensitivity. J. Immunol.
104:296.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.
Protein measurement with the folin phenol reagent. J. Biol. Chem.
193:265.

Lubaroff, D. M., and B. H. Waksman. 1967. Delayed hypersensitivity:

Bone marrow as a source of cells in delayed skin reaction. Science
157:322.

98.

99.

100.

101.

102.

103.

104.

1.06 O

107.

108.

109.

110.

111.

112.

113.

58

Lubaroff, D. M. and B. H. Waksman. 1968. Bone marrow as source of
cells in reactions of cellular hypersensitivity. I. Passive
transfer_of tuberculin sensitivity in syngeneic system. J. Exp.
Med. 128:1425.

Lubaroff, D. M. and B. H. Waksman. 1968. Bone marrow as source of cells
in reactions of cellular hypersensitivity. II. Identification of
allogeneic or hybrid cells by immunofluorescence in passively trans-
ferred tuberculin reaction. J. Exp. Med. 128:1437.

Lurie, M. B. 1950. Native and acquired resistance to tuberculosis.
Amer. J. Med. 2;591.

Lurie, M. B. 1964. Resistance to tuberculosis: Experimental studies
in native and acquired defensive mechanisms. Harvard University
Press, Cambridge, Mass.

Lurie, M. B., and A. M. Dannenberg, Jr. 1965. Macrophage function in
infectious disease with inbred rabbits. Bacteriol. Rev. _22:466.

Mach, B., and P. Vassalli. 1965. Biosynthesis of RNA in antibody-
producing tissues. Proc. Nat. Acad. Sci. (Wash.) .54:875.

Mackaness, G. B. 1964. The immunological basis of acquired cellular
resistance. J. Exp. Med. 120:105.

Mackaness, G. B. 1967. The relationship of delayed hypersensitivity
to acquired cellular resistance. Brit. Med. Bull. _23:52.

Mackaness, C. B. and R. V. Blanden. 1967. Cellular immunity. Prog.
Allergy .llz89.

Mackaness, G. B. 1968. Editorial: The immunology of antituberculous
immunity. Amer. Rev. Resp. Dis. 213337.

Mallmann, V. H., W. L. Mallmann, and Robinson, P. 1964. Relationship
of atypical bovine and porcine mycobacteria to those of human origin.
Health Lab Sci. .lzll.

Mandell, J. D. and A. D. Hershey. 1960. A fractionating column for
analysis of nucleic acids. Ann. Biochem. .lz66.

Mannick, J. A. and R. H. Egdahl. 1962. Ribonucleic acids in trans-
formation of lymphoid cells. Science 137:976.

Mannick, J. A. and R. H. Bgdahl. 1964. Transfer of heightened immunity
to skin homograft by lymphoid RNA. J. Clin. Invest. égg2166.

Marshall, W. H. and K. B. Roberts. 1963. Tuberculin induced mitosis
in peripheral blood leukocytes. Lancet_l:773.

McCluskey, R. T., B. Benacerraf, and J. N. McCluskey. 1963. Studies
on the specificity of the cellular infiltrate in delayed hypersensi-
tivity reaction. J. Immunol. .29:460.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

 

McCune, R. M., F. M. Feldmann, M. P. Feldmann and W. McDermott. 1966.
Microbial persistence. I. The capacity of tubercle bacilli to
survive sterilization in mouse tissues. J. Exp. Med. 123:445.

Metchnikoff, E. 1899. Etudes sur la resorption des cellules. Ann.
Inst. Pasteur .13:737.

Millman, I. 1961. Nonspecific resistance to tuberculosis. Amer.
Rev. Resp. Dis. .83:668.

Mills, J. A. 1966. The immunologic significance of antigen induced
lymphocytes transformation in vitro. J. Immunol. .21:239.

Mitchison, N. A. 1954. Passive transfer of transplantation immunity.
Proc. Roy. Soc. B. 142:72.

Mitsuhashi, S., S. Surashige, M. Kawakami, and T. Jojima. 1968.
Transfer agent of immunity. I. Immune ribonucleic and which induced
antibody formation to Salmonella flagella. Jap. J. Microbiol.
12:261.

 

Mosier, D. E. 1967. A requirement for two cell types for antibody
formation in vitro. Science 158:1573.

Nowell, P. C. 1960. Phytohemagglutinin: An initiator of mitosism
cultures of normal human leukocytes. Cancer Res. .29;462.

Oppenheim, J. J. 1968. Relationship of in vitro lymphocyte trans-
formation to delayed hypersensitivity in guinea pigs and man.
Fed. Proc. “21:21.

Paque, R. E., and S. Dray. 1970. Interspecies transfer of delayed
hypersensitivity in vitro with RNA extracts. J. Immunol. 105:1334.

Paque, R. E., and S. Dray. 1972. Monkey to human transfer of delayed
hypersensitivity in vitro with RNA extracts. Cell. Immunol. 5330.

Pearmain, C., R. R. Lycette, and P. H. Fitzgerald. 1963. Tuberculosis
induced mitosis in peripheral blood leucocytes. Lancet .1:637.

Perlmann, P., H. Perlmann, H. Miller—Eberhard, and J. A. Manni.» 1969.
Cytotoxic effects of leukocytes triggered by complement bound to
target cells. Science 163:937.

Powell, A. E., and M. A. Leon. 1970. Reversible interaction of human

lymphocytes with the mitogen concanavalin A. Exp. Cell. Res.
62:315.

Pribnow, J. P., and M. S. Silverman. 1967. Studies on the radio-
sensitive phase of the primary antibody response in rabbits. I. The
role of the macrophage. J. Immunol. 2§j225.

Quadri, S. K. and V. H. Mallmann. 1969. A simple in vitro test for
delayed sensitivity by specific inhibition of macrophage migration.
(unpublished).

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

60

Raleigh, G. W., and G. P. Youmans. 1948. The use of mice in experimental
chemotherapy of tuberculosis. I. Rationale and review of the liter-
ature. .1. Infect I)is. ffi;:l97. '

Raleigh, G. W., and G. P. Youmans. 1948. The use of mice in experimental
chemotherapy of tuberculosis. II. Pathology and pathogenesis. J.
Infect. Dis. _§g:205.

Ramming, K. P., and Y. H. Pileh. 1970. Mediation of immunity to tumor
isografts in mice by heterologous ribonucleic acid. Science 168:492.

Rees, R. J. W., and P. D. Hart. 1961. Analysis of the host-parasite
equilibrium in chronic murine tuberculosis by total and viable
counts. Brit. J. Exp. Pathol. 42:38.

Rich, A. R., and M. R. Lewis. 1932. The nature of allergy in tubercu-
losis as revealed by tissue culture studies. Bull. Johns Hopkins
Hosp.-.§Q:115.

 

Rich, A. R. 1951. la C.C. Thomas (ed), The pathogensis of tuberculosis.
Springfield, Illinois.

Roitt, I. M., H. E. H. Jones, and D. Doniach. 1962. Mechanism of tissue
damage in human and experimental autoimmune thyroiditis. l3 Mechanisms
of cell and tissue damage produced by immune reactions, p. 174-183.
Grune and Stratton, New York.

Rose, N. R., J. H. Kite, and F. K. Doebbler. 1962. Experimental auto-
immune thyroiditis, p. 161-173. lg Mechanism of cell and tissue
damage produced by immune reactions. Grune and Stratton.

Roszman, T. L., V. H. Mallmann, N. L. Mallmann, and D. O'Reilly. 1968.
Immunologic response evoked by mycobacterial components. Amer. Rev.
Resp. Dis. .91:103.

Ruddle, N. H. and B. H. Waksman. 1968. Cytotoxicity mediated by soluble
antigen and lymphocytes in delayed hypersensitivity. 111. Analysis
of mechanism. J. Exp. Med. 128:1267.

Runyon, E. H. 1959. Anonymous mycobacteria in pulmonary diseases.
Med. Clin. N.A. ‘£;:273.

Saha, A., J. S. Garvey, and D. H. Campbell. 1964. The physiochemical
characterization of the ribonucleic acid—antigen complex persisting
in the liver of immunized rabbits. Arch. Biochem. 105:179.

Saito, R., S. Kurashige, and S. Mitsuhashi. 1969. Serial transfers of
immunity through immune RNA. Jap. J. Microbiol. .13:122.

Scharff, M. D., A. L. Shapiro, and B. Ginsberg. 1967. The synthesis,
assembly, and secretion of gamma globulin polypeptide chains by
cells of a mouse plasma cell tumor. Cold Spr. Harb. Symp. Quant.
Biol. .32:235.

 

144 o

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

158.

61

Scherrer, K. and J. E. Darnell. 1962. Sedmentation characteristics
of rapidly labelled RNA from HeLa cells. Biochem. Biophysiol. Res.
Comun. .Z:486.

Schield, H. 0., and D. A. Willoughby. 1967. Possible pharmacological
mediators of delayed hypersensitivity. Brit. Med. Bull. 23:46.

Schrek, R. 1963. Cell transformations and mitoses produced in vitro
by tuberculin purified protein derivative in human-blood cells.
Amer. Rev. Resp. Dis. [81:734.

Sever, J. L. 1960. Passive transfer of resistance to tuberculosis
through use of monocytes. Proc. Soc. Exp. Biol. (N.Y.) 103:326.

Sharp, J. A. and R. G. Burwell, 1960. Interaction of macrophages
and lymphocytes after skin homografting on challenge with soluble
antigens. Nature (London) 188:474.

Shilo, M. 1959. Nonspecific resistance to infections. Ann. Rev.
Microbiol. .13:255.

Spector, W. G. and A. W. J. Lykke. 1966. The cellular evolution of
inflammatory granulomata. J. Pathol. Bacteriol. 22;163.

Spector, W. G. 1967. Histology of allergic inflammation. Brit. Med.
Bull. .g;:35.

Spector, W. G., A. W. J. Lykke, and D. A. Willoughby. 1967. A quanti-
tative study of leucocyte emigration in chronic inflammatory granu-
lomata. J. Pathol. Bacteriol.' 23;101.

Spirin, A. S. 1963. Some problems concerning the macromolecular structure
of ribonucleic acids, p. 301. 13 J.M. Davidson and W.E. Cohn (eds),
Progress in nucleic acid research, Vol. 1. Academic Press, New York.

Steenken, W., Jr. 1961. The persistance of tubercle bacilli in caseous
lesions in experimental animal (guinea pig). Amer. Rev. Resp. Dis.
83:550.

Sterzl, J., and M. Hrubesova. 1956. The transfer of antibody formation
by means of nucleoprotein fractions to non-immunized recipients.
Folia. Biol. (Praha) .g:21.

Summer, J. B. 1919. The globulins of the jack bean, Canavalia ensiformis.
J. Biol. Chem. .31:137.

Summer, .. B., and S. F. Howell. 1936. Identification of the hemag-
glutinin of the jack bean with concanavalin A. J. Bacteriol. 32g277.

Suter, E. 1961. Passive transfer of acquired resistance to infection
with Mycobactcrium tuberculosis by means of cells. Amer. Rev. Resp.
Dis. _83:535.

 

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

170.

171.

172.

62

Suter, E., and H. Ramsier. 1964. Cellular reactions in infection, p. 117.
_13 F.J. Dixon, Jr. and J.H. Humphrey (eds), Advances in immunology,
Vol. 4. Academic Press, New York, London. '

Thor, D. E. 1967. Delayed hypersensitivity in man: A correlate in
vitro and transfer by an RNA extract. Science 157:1567.

Thor, D. E., and S. Dray. 1968. The cell-migration-inhibition correlate
of delayed hypersensitivity: Conversion of human nonsensitive lymph
node cells to sensitive cells with an RNA extract. J. Immunol.
101:469.

Thorbecke, G. J., and B. Benacerraf. 1962. The reticuloendothelial
system and immunological phenomena. Prog. Allerg .§:559.

Tizard, I. A. 1971. Macrophage-cytophilic antibodies and the functions
of macrophage-bound immunoglobulins. Bacteriol. Rev. .35:365-378.

Turk, J. L. 1969. IV. Biological activities of lymphocyte products,
p. 301. lg H.S. Lawrence and M. Landy (eds), Mediators of cellular
immunity. Academic Press, New York.

Uhr, J. W. 1963. Actinomycin D: Its effect on antibody formation in
vitro. Science 142:1476. '

Valentine, F. T. and H. S. Lawrence. 1969. Lymphocyte stimulation:
Transfer of cellular hypersensitivity to antigen in vitro. Science
165:1014.

Volkman, A., and J. L. Gowans. 1965. The production of macrophages in
the rat. Brit. J. Exp. Pathol. 36350.

Volkman, A., and J. L. Gowans. 1965. The origin of macrophages from
bone marrow in the rat. Brit. J. Exp. Pathol. .&§:62.

Volkman, A. 1966. The origin and turnover of mononuclear cells in
peritoneal exudates in rats. J. Exp. Med. 124:241.

Waksman, B. H. 1959. A comparative histopathological study of delayed
hypersensitivity reactions, p. 280. .12 G.E.W. Wolstenholme and
M. O'Conner (eds), Cellular aspects of immunity. Ciba. Found.
Symp. Little, Brown and Co., Boston.

Waksman, B. H. 1962. Tissue damage in the "delayed” (cellular) type
of hypersensitivity, p. 146. 13 P.L. Grabar and P. Miescher (eds),
Mechanisms of cell and tissue damage produced by immune reactions.
Grune and Stratton, New York.

Ward, P., H. G. Remold, J. David. 1970. The production by antigen-
stimulated lymphocytes of a leukolactic factor distinct from migra-
tion inhibitory factor. Cell. Immunol. Il:162.

173.

174.

175.

176.

177.

178.

179.

180.

181.

182.

183.

UJ

Weiss, D. W. 1959. Vaccination against tuberculosis with non-living
vaccines. I. The problem and its historical background. Amer.
Rev. Resp. Dis. 88:340.

Weiss, D. W. 1959. Vaccination against tuberculosis with non-living
vaccines. I. The problem and its historical background. Amer. Rev.
Resp. Dis. .8Q:495.

Weiss, D. W. 1959. Vaccination against tuberculosis with non-living
vaccines. I. The problem and its historical bacground. Amer. Rev.
Resp. Dis. 88:676.

Weiss, H. K., and M. Fishman. 1972. Formation of antibody in rabbit
Spleen cell cultures stimulated with immunogenic RNA. J. Immunol.
108:1172.

Wesslen, T. 1952. Passive transfer of tuberculin hypersensitivity by
viable lymphocytes from the thoracic duct. Acta. Tuberc. Scand.
26:38.

Wesslen, T. 1952. A histological study of the tuberculin reaction in
animals with passively transferred hypersensitivity. Acta. Tuberc.
Scand. _88:125.

Wheelock, E. F. 1965. Interferon-like virus-inhibition induced in
human leukocytes by phytohemagglutinin. Science 149:310.

Willoughby, D. A., B. Boughton, and H. D. Schild. 1963. A factor
capable of increasing vascular permeability present in lymph node
cells. Immunol. .8:484.

Willoughby, D. A., W. G. Spector, and B. 3oughton. 1964. A lymph node
permeability factor in the tuberculin reaction. J. Pathol. Bacteriol.
87:353.

Willoughby, D. A., E. Coote, and W. G. Specter. 1967. A monocytogenic
humoral factor released after lymph-node stimulation. Immunol.
12:165.

Youmans, G. P., and A. S. Youmans. 1972. The effect of mycobacterial
RNA on the primary antibody response of mice to bovine and globulin.
J. Immunol. 109:217.

ER

 

"lllllljfillfll’fllflllflililllmllfllflylllll“