The effect of adjuvants on biosynthesis of 19S and 7S antibody against bacteriophage ΦX174 in the guinea-pig

Guinea-pigs given a single injection of bacteriophage ΦX174 produced 19S antibody 8 days later. The levels of 19S antibody attained were not significantly different in animals injected with bacteriophage in water-in-oil emulsions with and without added mycobacteria, in bentonite or in saline alone. On the other hand, while both 7S γ₁- and γ₂-immunoglobulin antibodies were present 21 days after a single injection of bacteriophage ΦX174 in saline alone, the addition of water-in-oil emulsions to the injection mixture led to enhanced levels of 7S antibody and the addition of mycobacteria to these emulsions increased 7S antibody levels still further. Delayed hypersensitivity reactions were detected by corneal testing in a proportion of the guinea-pigs injected with bacteriophage in water-in-oil emulsions whether or not mycobacteria were also present in the mixture. Guinea-pigs immunized with bacteriophage in saline showed negative corneal tests.     

Cell-mediated responses to heterologous and homologous thyroglobulin in guinea-pigs immunized with heterologous thyroid extracts

Immune cellular responses and circulating antibodies to heterologous and homologous thyroglobulin have been studied in two groups of guinea-pigs immunized with human or bovine thyroid extract in Freund's complete adjuvant. In animals immunized with human thyroid extract, the in vitro [2-¹⁴C]thymidine incorporation by lymphocytes and the inhibition of peritoneal exudate cell migration in the presence of human thyroglobulin were earlier and more marked than those to bovine thyroglobulin as observed in animals immunized with bovine thyroid extract. In the two groups of guinea-pigs no significant difference was found regarding the production of circulating antibodies.

Moreover cellular cross-reaction to homologous thyroglobulin could be detected in animals immunized with human but not in those immunized with bovine thyroid extract. Serological and cellular cross-reactions between human and bovine thyroglobulin were present in both groups of guinea-pigs.

Finally a significantly higher incidence of thyroid inflammatory lesions was found in the human thyroid extract immunized animals. The role of cell-mediated immune responses in initiating tissue damage in experimental thyroiditis is discussed.

     

Immunopathological studies on thyroid immunity. IX. Thyroid and renal amyloidosis in thyroglobulin immunized rabbits

In serial studies of immunopathologic changes in an animal model of thyroiditis 52 rabbits were immunized with either bovine, porcine or human thyroglobulin (Tg) in Freund's complete adjuvant, while another 47 animals served as noninjected or adjuvant injected controls. The immunized animals were divided into two groups, one receiving an initial series of only three Tg injections while the other received, in addition, challenging injections over an 8-week period. The immunized animals were killed over a period of 6-34 months after the last Tg injection, and untreated controls were killed at comparable ages. In Tg immunized animals, lymphocytic thyroiditis was encountered in 25 per cent and thyroid amyloid in 17 per cent; glomerular amyloid was encountered in 44 per cent with diffuse lesions in 8 per cent, nodular lesions in 17 per cent and a mixture of the two in 19 per cent. That the thyroid and glomerular hyaline deposits contained amyloid was shown by various histochemical criteria, as well as by the presence of typical fibrils on electron microscopy. Immunohistochemical studies indicated that the amyloid was predominantly of the AA type. Rabbits receiving challenging Tg injections, in addition to the initial series, showed only thyroiditis and nodular glomerular lesions most of which were amyloid. Whilst the vast majority of rabbits with lymphocytic or amyloidotic responses showed both thyroid and renal lesions, a small percentage of animals showed only a lesion of one or the other of these two organs. It is of interest that the thyroid and renal amyloid lesions, described for the first time with induced thyroglobulin immunity, were not detected in other earlier short term investigations.     

The dual necessity for delayed hypersensitivity and circulating antibody in the pathogenesis of experimental allergic orchitis in guinea-pigs

1. Guinea-pigs injected with testis autoclavate (WT) in incomplete adjuvant develop antibody but no delayed hypersensitivity or testicular lesions. These animals fail to develop delayed hypersensitivity or a lesion when subsequently injected with antigen in complete adjuvant. Animals similarly treated, and then given repeated injection of immune cells from animals treated with whole testis and complete adjuvant, develop both delayed hypersensitivity and the characteristic orchitis.

2. Guinea-pigs injected with a purified fraction (designated H) with incomplete adjuvant fail to develop either circulating antibody, delayed hypersensitivity or a testicular lesion. Animals subsequently injected with this antigen and complete adjuvant, develop delayed hypersensitivity but no circulating antibody or testicular lesion. Three of the five animals similarly treated and given repeated injections of serum containing anti-testis antibody also develop orchitis.

3. A sequential study of fourteen animals killed at daily intervals after they were injected with WT and complete adjuvant, showed that antibody was first detected in the testis on the same day as delayed hypersensitivity first appeared. Circulating antibody was not detected until 2 days later. The specific testicular antigen showed first signs of disappearing on the 10th day, by which time histological evidence of orchitis was first detected.

4. Antibody was not detected in the testes in any animal in the absence of delayed hypersensitivity.

     

Induction of delayed hypersensitivity by dinitrophenylated lymphocytes

Erythrocytes, serum proteins and both living and killed lymphocytes from the peripheral blood of guinea-pigs were dinitrophenylated and then injected intraperitoneally into either the donor or other guinea-pigs. Animals sensitized by intradermal DNCB and unsensitized normal guinea-pigs served as positive and negative controls respectively. Eleven to 14 days after injection, the animals were tested with topically applied DNCB and the intensity of the reactions assessed by the local exudation of bovine serum albumin labelled with radio-iodine ([¹³¹I]BSA.)

Sensitization was achieved with dinitrophenylated living autologous lymphocytes, to a lesser degree with homologous lymphocytes, but not with erythrocytes, serum proteins or killed lymphocytes.

     

Antigen-Induced Bronchial Anaphylaxis in Actively Sensitized Guinea-Pigs Pattern of Response in Relation to Immunization Regimen

This work studies the temporal development of the acute anaphylactic bronchoconstriction in guinea-pigs sensitized to ovalbtiin by different regimens, including IgE-antibody promoting ones. The results show that guinea-pigs sensitized with low amounts (1-10μg) of ovalbumin together with alum produce the most pronounced bronchospasm when challenged with an intravenous injection of a low dose of antigen. Examination of the antibody classes by PCA technique shows that guinea-pigs sensitized with small amounts of antigen together with alum produce IgE and IgG₁ antibodies. However, in sera from animals immunized with large amounts of antigen, only IgG₁ antibodies could be detected.     

Induction of Autoimmunity to Adrenal Gland

Rabbits that were immunized with homologous and heterologous adrenal homogenates in complete adjuvant responded with complement-fixing heat-stable autoantibody primarily directed against adrenal. Rabbits responded with autoantibody production against cytoplasm of adrenal cortex and of ovarian and testicular cells as detected by immunofluorescence. Antibody, with sedimentation similar to 7S γ globulin, was directed against a heat-labile antigen in rabbit, guinea-pig and rat adrenal. Antibody responsible for reactions with the antigens in the complement fixation and immunofluorescent tests was absorbed by an ultra-centrifugal sediment of adrenal or ovary.

Histological evidence of adrenalitis was present only in those rabbits immunized with heterologous adrenal.

Guinea-pigs immunized with heterologous adrenal homogenates developed more extensive adrenal infiltrates than did guinea-pigs immunized with homologous adrenal. Antibody from guinea-pigs immunized with heterologous adrenal reacted with autologous organ antigens as well as with rabbit and rat adrenal. Skin tests with homologous adrenal revealed no evidence for delayed-type hypersensitivity.

The detection of autoantigens in foetal rabbit adrenal and ovary suggests that rabbits should be tolerant to such antigens. The greater efficacy of immunization with heterologous adrenal in eliciting autoantibody and autoimmune adrenalitis may be a feature of experimental autoimmunity induced by those organ-specific antigens to which the animal is tolerant. The presence of autoantibody in the serum, bound γ globulin at the site of adrenal lesions and the absence of delayed-type hypersensitivity to homologous adrenal suggest that the humoral immune mechanisms play a role in experimental autoimmune adrenalitis.

     

Immunogenicity of heterologous Fc and Fab immunoglobulin fragments in rabbits, guinea-pigs and rats

Rabbits, guinea-pigs and rats were immunized with various heterologous 7S and 19S immunoglobulins from each other and man, and the antisera obtained were studied by immunoelectrophoresis.Rabbits produced antibodies against the Fc and the Fab fragments of the immunoglobulin injected, while guinea-pigs and rats only produced anti-Fc antibodies.The fact that guinea-pigs and rats only respond to the specific determinants of each class of immunoglobulin provides a simple method for the preparation of class-specific antisera.     

Infectivity of a strain of Cryptosporidium found in the guinea-pig (Cavia porcellus) for guinea-pigs, mice and lambs

Cryptosporidiosis was diagnosed in guinea-pigs bred by a commercial laboratory supplier on histological examination of the intestine. Oral transmission to laboratory guinea-pigs aged up to 16 weeks and to infant mice, with gut contents containing oocysts, was successful, but the organism failed to infect adult mice. From day 5 post-inoculation (pi) in guinea-pigs, infection of the ileum was associated with villous stunting and fusion, and with infiltrates of macrophages and other mononuclear cells, and eosinophils. Some guinea-pigs died; others were depressed and anorectic, with diarrhoea or watery caecal contents. Mouse infections were subclinical and caused no significant pathological changes. By contrast, a bovine Cryptosporidium isolate infected infant mice but failed to infect young guinea-pigs. Guinea-pigs and infant mice excreted oocysts in faeces after a prepatent period of 3 to 4 days. Some guinea-pigs excreted oocysts for up to 2 weeks, but excretion in mice lasted only about 4 days. Infection of guinea-pigs by contact with a contaminated environment occurred, with excretion of oocysts between 17 and 27 days after exposure. An indirect fluorescent antibody test (IFA) showed that antibody was present by day 17 pi with infected bowel contents, but none was detected in the guinea-pigs exposed to the contaminated environment. The IFA test demonstrated a serological relationship between the guinea-pig isolate and a bovine strain used to infect gnotobiotic lambs. Transmission electron microscopy of intestine from infected guinea-pigs and mice showed that more than one schizont generation occurred. The first consisted of 8 merozoite packets attached to enterocytes, but many packets of 2 or 4 merozoites of the second or subsequent generations were apparently released into the gut lumen. Fixed microgametocytes contained lipid vacuoles and had microneme-like structures in their cytoplasm. Oocysts and sporocysts were also identified, with sporulation occurring within the parasitiphorous vacuole. A sparse infection was established in 1 of 2 12-day-old specific pathogen-free lambs by day 3 pi, but no oocysts were detected in its caecal contents or those of a second lamb killed 4 days later.     

Interaction between 'sensitized lymphocytes' and antigen in vitro. 3. Release of soluble mediators by particulate antigen

Guinea-pigs immunized with sheep red cells (SRC) in Freund''s complete adjuvant (FCA) developed delayed type hypersensitivity to a SRC urea extract 7 days after sensitization and showed combined Arthus and delayed reactivity from the 14th day on. Animals immunized by an intracardiac injection of SRC in saline and skin tested 4–5 days later with SRC urea extract, responded with an erythematous and indurated lesion reaching its peak 24 hr after testing and resembling a typical delayed hypersensitivity reaction. Lymphocytes from lymph nodes, peritoneal exudate and peripheral blood of guinea-pigs immunized with SRC in FCA released skin reactive factor (SRF) and macrophage migration inhibitory factor (MIF) when cultured for 24 hr with SRC. No SRF was formed in the absence of divalent cations in the cultured medium. No SRF or MIF was produced by lymphocytes of guinea-pigs immunized by the intracardiac route and cultured under identical conditions. SRF obtained after in vitro exposure of sensitized lymphocytes to SRC was eluted from Sephadex G-200 in the region of serum albumin. Lytic anti-SRC antibody of the IgM class was liberated into the supernatant of spleen cultures, after intracardiac immunization and antibody of the IgG class was produced by lymph node cultures after immunization with SRC in FCA, in both cases in the absence of antigenic stimulation in vitro. It is considered improbable that the latter antibody is involved in the release of soluble mediators.     

The immune response of guinea-pigs to type II collagen: poor cross-reactivity with homologous type II collagen accounts for resistance to collagen-induced arthritis.

In order to determine the susceptibility of guinea-pigs to collagen-induced arthritis (CIA), Hartley and Strain 13 guinea-pigs were immunized with heterologous or homologous type II collagen. None of the animals developed CIA. Because immunity to type II collagen plays a critical role in CIA, we characterized the guinea-pig's immune response to determine the basis for this resistance. Guinea-pigs develop cellular and humoral reactivity to heterologous type II collagen similar to that of CIA-susceptible rats. The reactions distinguish type I from type II collagen but not among several heterologous type II collagens. The cell-mediated immune (CMI) response was specific for determinants on the primary amino acid structure of collagen, whether native or denatured collagen was used for immunization; however, the humoral response was specific for the form of the molecule used for immunization. Guinea-pigs differ from CIA-susceptible rats in that immunization with homologous or heterologous type II collagen fails to induce significant cross-reactive immunity with the homologous antigen. A transient arthritis could be induced in animals immunized with heterologous type II collagen by injecting them intra-articularly with heterologous but not with homologous type II collagen. Our results show that the disparity between immunity to type II collagen and the susceptibility to develop CIA in guinea-pigs is due to their poor cross-reactive immune response to autologous type II collagen.     

Ascitic starch phagocytosis in experimental guinea-pig peritonitis.

Phagocytosis of starch granules in ascitic fluid was sought in guinea-pigs 1 to 10 days after i.p. injection of a suspension of starch powder. Starch phagocytosis occurred in 75.9% of control animals with free peritoneal fluid. It probably represents a nonspecific reaction to the particulate nature of starch granules. Guinea-pigs sensitized to starch by nuchal inoculation of an emulsion of Freund''s adjuvant and starch showed no increase in frequency or intensity of ascitic starch phagocytosis beyond that seen in control animals. Since only sensitized animals develop granulomatous peritonitis, ascitic starch phagocytosis is thus not indicative of granulomatous hypersensitivity peritonitis. This experimental model suggests that examination of ascitic fluid is not a reliable clinical investigation to establish a diagnosis of human granulomatous starch peritonitis.     

Immune responses to thyroglobulin in experimental allergic thyroiditis

Guinea-pigs immunized with homologous thyroglobulin in complete Freund's adjuvant were observed for lesions in the thyroid gland, and for development of specific delayed type skin hypersensitivity. Their blood leucocytes were examined for specific sensitivity in vitro by determining the inhibition of migration in the presence of thyroglobulin. Serum was tested for humoral antibodies by the passive haemagglutination technique. There was a significant correlation between the development of thyroiditis and the intensity of the skin reactions. No relationship was observed between the presence of thyroiditis and leucocyte sensitivity nor between thyroiditis and circulating antibodies. On the other hand serum antibody titres were correlated to the degree of leucocyte sensitivity. Thyroglobulin inhibited the migration of cells from normal animals in the presence of plasma from thyroglobulin-sensitized animals. The activity of sensitized plasma disappeared when diluted 1:10 in normal plasma irrespective of the serum antibody titre. Guinea-pigs immunized with thyroglobulin or kidney antigen in complete Freund's adjuvant and tested for skin or leucocyte hypersensitivity showed specific but no cross reactivity to the two preparations.     

Experimental immune inflammation in the synovial membrane: II. The origin and local activity of inflammatory cells

Synovitis was produced in guinea-pigs previously immunized with bovine γ-globulin and Freund's complete adjuvant, by injection of bovine γ-globulin and tuberculin PPD into the knee joints. In animals given prior intravenous injections of tritiated thymidine and colloidal carbon, up to 40 per cent of the local accumulations of mononuclear cells 48 hours after antigen challenge were tritium-labelled as seen by autoradiography. These inflammatory cells are considered to be of haematogenous origin. A smaller percentage of cells was carbon-labelled. The percentage of tritium-labelled cells decreased with duration of the granuloma following antigen challenge. In other animals, synovial inflammatory cells were labelled by intra-articular injection of tritiated thymidine at different times following the establishment of synovial inflammation by injection of antigen. The highest proportion of cells labelled was 17 per cent, but when label was administered as late as 60 days after the inflammatory stimulus, only about 3 per cent of cells were labelled. Grain counts of cells labelled by local tritium administration or by short-term incubation of slices in vitro showed approximately 30–100 grains per nucleus. Sequential study of locally labelled synovia showed that such high-grain cells were still present 12 days after tritium administration, suggesting that at least some of the mononuclear cells were relatively long-lived.     

Production of rheumatoid factor in adoptively immune guinea-pigs after challenge with Treponema pallidum.

Guinea-pigs of inbred strains 2 and C4D were infused with various concentrations (1 x 10(8) to 4 x 10(8) of syngeneic nylon wool-purified Treponema pallidum-immune T lymphocytes (TPI-T) and challenged 24 hr later with virulent T. pallidum (10(8) organisms). The degree of protection depended on the number of infused T cells and was associated with an accelerated production of IgM rheumatoid factor (RF). Fully protected animals (4 x 10(8) TPI-T) did not produce treponemal antibodies or circulating immune complexes (CIC) but produced IgM RF detectable 10 days after infection. Partially protected animals (< or = 2 x 10(8) TPI-T) produced, 30 days post-infection, relatively low levels of treponemal antibodies but high levels of CIC and RF. Control animals infused with 2 x 10(8) TPI-T lymphocytes but not infected with T. pallidum, when monitored for a period of 6 weeks, did not produce treponemal antibodies, CIC, or RF, excluding the possibility that IgM RF could be generated by the donor's B cells contaminating (circa 3%) the TPI-T lymphocytes. Moreover, unprotected syngeneic control animals infused, prior to infection, with T. phagedenis biotype Reiter-immune T cells or with T. pallidum-free testicular inflammatory fluid-immune T cells responded with increasing levels of treponemal antibodies; only a few animals produced RF and CIC 5 months after infection similarly to control guinea-pigs infected only. The production of RF in partially protected animals responding to infection with treponemal antibodies and CIC was apparently associated with the presence of the CIC; but the mechanism of RF production in fully protected animals in which no antibodies or CIC were detected is currently unknown.     

Non-specific protection against pulmonary Legionella pneumophila infection in guinea-pigs immunized and challenged with mycobacteria

Experiments were designed to test the ability of the non-specific efferent limb of cell mediated immunity (CMI) to protect guinea-pigs against a lethal L. pneumophila challenge. A secondary CMI response was generated in the lungs of guinea-pigs using an established protocol which consisted of intraperitoneal infection with Mycobacterium bovis BCG followed by intravenous infection with Mycobacterium tuberculosis H37Ra. The animals were challenged with L. pneumophila (100 LD50) by the aerosol route 3, 6 or 10 days after the H37Ra infection, and pyrexia and survival were monitored. Lungs were taken from animals killed at intervals for histology and enumeration of viable L. pneumophila. Normal guinea-pigs and others infected with either BCG or H37Ra alone were challenged with L. pneumophila as controls. Of the animals which received both BCG and H37Ra, all those challenged 3 days after H37Ra survived but this level of protection fell progressively in groups challenged 6 or 10 days after H37Ra. None of the control animals survived. Mycobacterial lung lesions were granulomatous and were readily distinguished from the acute exudative Legionella lesions. The protected animals showed evidence of a more substantial anti-mycobacterial CMI response and a delay in the development of Legionella lesions. The numbers of L. pneumophila present in the lungs indicated that protection did not result from early elimination of the Legionella challenge. The bacterial counts together with the histopathology suggest that the L. pneumophila was more effectively contained in the protected animals so that exudative damage was reduced.     

Experimental infection of cattle of different ages with infectious bovine rhinotracheitis virus (Strichen strain)

The clinical signs and pathological lesions which developed in various ages of cattle experimentally infected intranasally with the "Strichen" strain of IBR virus were similar to, but generally milder than, those of the field disease. The clinical signs were most severe 4 days after infection and had almost wholly regressed after 12 days. Serum neutralizing antibodies were detected in every animal. Virus was isolated from nasal and ocular swabs for up to 13 days and 10 days, respectively, after infection. The clinical signs and the pathological lesions were more severe in the younger animals.     

Dissociation of erythema and basophil accumulation in Jones-Mote type hypersensitivity in the guinea-pig.

Relationship between delayed-in-onset erythema and infiltrating leucocytes in the skin reaction site of Jones-Mote type hypersensitivity was observed in guinea-pigs. Guinea-pigs were immunized with formalin-fixed (F-SRBC) or native form (N-SRBC) of sheep red blood cells in incomplete Freund''s adjuvant (IFA). Elicitation was carried out with intradermal injection of F-SRBC or N-SRBC in saline 1 or 3 weeks after immunization. One week after immunization with F-SRBC or N-SRBC in IFA, erythema accompanied by basophil infiltration was detected, but antibody production was negative. Erythema reached a peak at 24 h, but basophil infiltration reached a peak at 48 h or later. In the skin reaction site elicited with N-SRBC, high levels of basophil infiltration were detected persistently even after erythema had disappeared. Three weeks after immunization, low titres of PCA (passive cutaneous anaphylaxis) were detected in guinea-pigs immunized with F-SRBC in IFA. At that time erythematous skin reaction was detected, but the level of basophil infiltration was lower than that of 1 week after immunization. In guinea-pigs immunized with N-SRBC in IFA, skin reaction of the Arthus type accompanied by haemorrhage was observed at the site elicited with N-SRBC.     

Non-specific protection against pulmonary Legionella pneumophila infection in guinea-pigs immunized and challenged with mycobacteria.

Experiments were designed to test the ability of the non-specific efferent limb of cell mediated immunity (CMI) to protect guinea-pigs against a lethal L. pneumophila challenge. A secondary CMI response was generated in the lungs of guinea-pigs using an established protocol which consisted of intraperitoneal infection with Mycobacterium bovis BCG followed by intravenous infection with Mycobacterium tuberculosis H37Ra. The animals were challenged with L. pneumophila (100 LD50) by the aerosol route 3, 6 or 10 days after the H37Ra infection, and pyrexia and survival were monitored. Lungs were taken from animals killed at intervals for histology and enumeration of viable L. pneumophila. Normal guinea-pigs and others infected with either BCG or H37Ra alone were challenged with L. pneumophila as controls. Of the animals which received both BCG and H37Ra, all those challenged 3 days after H37Ra survived but this level of protection fell progressively in groups challenged 6 or 10 days after H37Ra. None of the control animals survived. Mycobacterial lung lesions were granulomatous and were readily distinguished from the acute exudative Legionella lesions. The protected animals showed evidence of a more substantial anti-mycobacterial CMI response and a delay in the development of Legionella lesions. The numbers of L. pneumophila present in the lungs indicated that protection did not result from early elimination of the Legionella challenge. The bacterial counts together with the histopathology suggest that the L. pneumophila was more effectively contained in the protected animals so that exudative damage was reduced.     

THE SPECIFICITY OF THE SABIN-FELDMAN DYE TEST WITH REFERENCE TO PROTOZOAL INFECTIONS

Infections of Eimeria stiedae in normal and splenectomized rabbits and of Leishmania enriettii in normal rabbits and guinea-pigs and in splenectomized rabbits did not produce dye test antibodies. Guinea-pigs injected with Atoxoplasma sp. and rabbits immunized with cultures of Crithidia fasciculata were found to be negative for dye test antibodies. Mice which had been infected with Trypanosoma cruzi did not show dye test antibodies. All these animals, except one guinea-pig infected with Leishmania enriettii, were negative in the complement-fixation test for toxoplasmosis using the egg antigen. These findings are discussed in the light of previous reports.