Regulation of immune responses by T suppressor cells and by serum in chronic paracoccidioidomycosis

Regulation of cellular responses was studied during the course of chronic murine disseminated paracoccidioidomycosis. Regulation of peripheral blood lymphocyte (PBL) proliferative responses to concanavalin A (Con A) was studied in vitro by mixing PBL from infected and noninfected mice. PBL from mice infected for 18 weeks had depressed responses to Con A and they depressed the Con A responses of PBL from noninfected mice by 95% when they were mixed in a 1:1 ratio. After treatment of PBL from infected mice with anti-Lyt-2.2 antibody plus complement, the responses to Con A were increased to normal values. The percentage of T-cell subpopulations in PBL from infected mice did not differ significantly from those of normal mice. Immunoregulation of delayed-type hypersensitivity (DTH) responses to antigen by serum from infected animals was studied in mice 1 week after intranasal (i.n.) infection, a time when DTH responses were maximal. DTH responses to antigen 7 days after i.n. infection (10(7) CFU Paracoccidioides brasiliensis) were significantly reduced when 0.5 ml of immune mouse serum (ELISA antibody titer to P. brasiliensis antigens 1:10,240) was given i.v. 1 day before infection (P less than 0.01) or 1 day before skin testing (P less than 0.001). Normal mouse serum did not have this effect. The results indicate that progression of chronic disseminated paracoccidioidomycosis was associated with the development of T-cell suppressor activity for Con A responses of PBL, and that DTH responses to antigen were depressed by the administration of serum with specific high titer antibodies.     

The antitheilerial effects of Theileria parva parva reaction and recovery sera in vitro

Peripheral blood leucocytes (PBL) of cattle were infected in vitro with the sporozoites of Theileria parva spp. The transformed cell lines were adapted to grow in sera from the PBL donors. The cattle were then infected with T. p. parva stabilate and either treated with parvaquone or the disease allowed to run its course. Sera harvested during severe disease reaction or early recovery were substituted for pre infection sera and caused the intracellular degeneration of the Theileria macroschizonts. Cell lines passaged in these sera died out as the parasites were eliminated. The antiparasitic effects of sera were short lived and were neither host nor parasite isolate restricted.     

Heat lability of rabbit anti-Mycoplasma pneumoniae serum

The metabolic-inhibition test utilizing glucose as a substrate was used to follow the immune response of New Zealand white rabbits injected via the footpad with a suspension ofMycoplasma pneumoniae grownin vitro. Sera were collected 7 and 11 days after initial inoculation and at intervals following several booster injections. The early sera were shown to be dependent upon a heat-labile accessory factor for demonstration of the inhibitory activity. This accessory factor could be supplied by unheated normal rabbit or normal guinea pig serum. The sera collected subsequent to multiple booster injections did not require such a heat-labile factor and inhibition was not enhanced by the addition of normal rabbit sera. A preliminary TEAE cellulose fractionation of early immune serum gave evidence for two species of antibody activities, one heat-stable dependent upon a heat-labile accessory factor for activity, and the other heat-labile dependent upon a heat-labile accessory factor for activity. Junior Research Fellow supported by USPH Training Grant 5T01 A100232 This study was supported by Public Health Service Research Grant 5 R01 A109002 and National Science Foundation Grant GB 4778. The results of this study were presented, in part, at the 1970 Annual Meetings of the American Society for Microbiology in Boston, Massachusetts.     

Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens

Lymphoid cells from spleens and lymph nodes of rabbits infected with T. pallidum respond by proliferation to concanavalin A (Con A) and T. pallidum antigens. Spleen cell responsiveness to treponemal antigens appears 6 days after infection, is 100 to 600 fold higher than the response of uninfected control rabbits, and is maintained throughout the 31-day observation period. Specifically responding cells in the inguinal and popliteal lymph nodes of infected animals are demonstrable on day 10, and the magnitude of the response increases throughout the observation period. Specific responsiveness to T. pallidum antigens in vitro is enhanced in purified T cell populations and is abolished by treatment with goat anti-rabbit thymocyte serum and complement. The response of spleen and lymph node cells to Con A is unaffected during syphilitic infection. These results are consistent with a role for T cell-mediated specific immunity to treponemal antigens early after infection and do not support a hypothesis of depressed cellular immunity during syphilitic infection.     

Trichinella spiralis: inhibition of sheep hemagglutinins in mice

One hundred and twenty-four mice were injected intraperitoneally with sheep red blood cells. The mice had been previously either orally inoculated with T. spiralis (16 mice), or injected intraperitoneally during 7 consecutive days with normal saline (12 mice), normal mouse serum (6 mice), or infected mouse serum (6 mice), normal rabbit serum (6 mice), sera from lightly (36 mice) or heavily infected rabbits (36 mice), and rabbit anti-lymphocyte serum (6 mice). The homologous serum clearly demonstrated an immunosuppressive effect on the production of sheep hemagglutinins; however, it was impossible to conclude that heterologous serum has such an activity since the normal rabbit serum used as control demonstrated the same activity. The inhibition of hemagglutinin production has also been observed in mice infected with T. spiralis. The presence of a suppressive agent released by the parasite or antigenic competition is discussed as the possible mediator of immunological unresponsiveness.     

Characterization with crossed immunoelectrophoresis of some antigens differentiating a virulent Candida albicans from its derived, avirulent strain

Antigenic differences between a wild-type virulent Candida albicans 4918 (wt) and its spontaneous avirulent mutant (m-10) were found with crossed immunoelectrophoresis. Yeast cell extracts as well as soluble protein and mannoprotein fractions obtained by affinity chromatography on concanavalin A (Con A) were analyzed. Sera from patients with candidiasis and antisera from rabbits infected with live wt cells and boosted with wt extracts or rabbits immunized with purified wt cell wall preparation were used as counter reactants. Qualitative differences in serum precipitins formed by patients with suspected or culture-proven candidiasis to polysaccharide antigens of wt and m-10 origin were observed. In comparison, except for a spike-formed precipitate detected only with the wt extract, the serum from infected rabbits precipitated the wt and m-10 cell wall polysaccharide antigens about equally. The same type of precipitate was also found with the Con A wt mannoprotein fractions but was again lacking with the m-10 mannoproteins. This precipitate, with extremely slow electromobility in the first dimension, may be related to some special immunodeterminant of the wt mannan molecule. No substantial differences in the precipitation patterns of the Con A wt and m-10 proteins were found when analyzed with patients' sera or rabbit anti-cell sera. However, using these protein fractions with anti-cell wall sera revealed a larger number of precipitates for the wt as opposed to the m-10 strain. The observed antigenic differences between the virulent- and the avirulent-derived strains seem to be mainly associated with cell wall determinants (components) and might be related to the greater adherence and infectivity of the wild strain.     

Development of cellular immunity to individual soluble antigens of Treponema pallidum during experimental syphilis

The contribution of individual specific molecules of Treponema pallidum subspecies pallidum to cellular immunity in experimental syphilis was evaluated by combining the techniques of Ag identification and purification with the lymphocyte proliferation assay. Proliferative responses of splenic lymphocytes from syphilitic rabbits to complex treponemal Ag and Con A were vigorous throughout the course of intratesticular infection (6, 10, 17, 30, and 210 days). Normal rabbits did not respond to any treponemal preparations and all rabbits failed to respond to normal rabbit testicular Ag (NRT). Seven defined treponemal Ag (47 kDa, 37 kDa, 35, 33-kDa, 30-kDa, 14 kDa, and 12 kDa) stimulated lymphocytes from infected rabbits. Cellular responses to the 37-kDa and 30-kDa fractions were evident by day 6 of infection and responses to the 35, 33-kDa and 14-kDa Ag were first detected on day 10; responsiveness to these Ag continued throughout the observation period. Cellular responses to the 47-kDa molecule were detectable but lower when compared with other individual Ag. Responsiveness to the 12-kDa Ag was not evident until 7 mo postinfection. Specific immunoblot reactivity of serum from rabbits used in this study generally correlated with the development of cellular reactivity to individual Ag of T. pallidum.     

Suppression of Con A induced lymphocyte transformation by sera from filarial patients with clinical manifestations

Reduced lymphocyte transformation to Wuchereria bancrofti microfilariae excretory-secretory antigen and Con A were observed in clinical filarial patients. Pre-incubation of normal human peripheral blood mononuclear cells with sera from filarial patients with clinical manifestations such as hydrocele and elephantiasis suppressed Con A induced responses. Effect of fractionated clinical filarial serum on Con A induced lymphocyte transformation showed that the inhibitory activity was associated with high molecular weight serum fraction.     

Plasmodium chabaudi: Humoral and cell-mediated responses of immunized mice

Antigen preparations of Plasmodium chabaudi parasites enriched in merozoites and schizonts, obtained from in vitro culture, and combined with saponin protected C57BL/6J mice from P. chabaudi infection as judged by reduced primary parasitemias and recrudescences. Sera passively transferred from immunized and untreated mice after a challenge infection were more protective in recipients than serum from normal mice. Mice treated with antilymphocyte serum during immunization did not develop as strong an immunity to infection as did controls treated with normal serum. Immunized mice had depressed delayed-type hypersensitivity reactions to malarial antigen but increased serum titers of malarial antibody (measured by imniunofluorescence) after challenge with P. chabaudi when compared to immunized mice which remained unchallenged. The protective activity of sera from various groups of mice did not necessarily correlate with the serum antibody titers.     

Experimental Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Background

A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.

Methods

Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.

Findings

Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.

Conclusions

These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Effects of concanavalin A on the migration of radioactively labeled lymphoid cells

The effects of the jackbean globulin Concanaalin A (Con A) on the distribution of radioactive ⁵¹Cr-labeled lymph node cells was studied in CBA mice. Lymph node cells treated in vitro with Con A in subagglutinating noncytotoxic doses were unable to “home” to the lymph nodes of syngeneic recipients after intraenous injection. The effect was almost immediate and seemed unrelated to mitogenesis. The inhibitory effect of Con A on lymphocyte migration could be partially reersed by alpha-methyl mannoside; the degree of migratory impairment was related to the amount of Con A bound to the lymphocyte surface at the time of transfer. The membrane site at which Con A binds to the lymphocytes is similar to that which is bound by heterologous antilymphocyte serum but is probably distinct from the theta antigenic site. These data lend support to the hypothesis that surface lymphocyte carbohydrate determinants are involved in the specific lymphocyte “homing” receptor.     

Human monocyte activation by supernatants from concanavalin A (con A) stimulated lymphocytes

Human peripheral lymphocytes were stimulated with Concanavalin A (Con A) in the absence of serum. Supernatants were collected from control and mitogen stimulated lymphocyte cultures and fractions pooled according to the elution before, together with or after human serum albumin which was added as a marker. Only one fraction derived from Con A stimulated lymphocyte culture Supernatants which eluted immediately after human serum albumin had a significant effect on the metabolism and structure of human monocytes in vitro. Monocytes separated by human serum albumin and incubated with this fraction for 20 hr had an increase in nuclear RNA synthesis. Monocytes attached to cover slips in Leighton tubes showed an increase in the percentage of phagocytizing cells and phagocytic activity. Electron microscopy demonstrated highly phagocytic cells containing numerous Golgi associated granules and strands of nondilated rough surfaced endoplasmic reticulum in presence of the active fraction.     

Immunologic dysfunction during viral oncogenesis: II. Inhibition of cellular immunity to viral antigens by malignant rabbit fibroma virus

The ability of two related viruses—Shope fibroma virus (SFV) and malignant rabbit fibroma virus (MV)—to induce virus-specific immune responses in lymphocytes of recipient animals was studied. SFV produces a benign local tumor which regresses in 12–14 days. Using an assay for virus-induced lymphocyte blastogenesis lymphocytes reactive to SFV were detected, both in rabbits bearing SFV-induced tumors and in rabbits whose SFV-induced tumor had regressed. These virus-reactive cells were detected in peripheral blood and spleen, and in lymph nodes draining the primary tumor. In contrast, MV produces a disseminated tumor and eventual death. MV does not induce detectable blastogenic responses in lymphocyte populations. SFV and MV are antigenically cross reactive: rabbits immune to SFV do not develop MV-induced tumors, and antisera to each virus neutralize both equally. Lymphocytes from SFV-infected rabbits proliferate in vitro in response to MV that has been inactivated by ultraviolet light (uv/MV) but not to infectious MV. In contrast, lymphocytes from rabbits infected with MV do not respond to uv-inactivated MV or to SFV. Thus, infectious MV inhibits the development of normal blastogenic responses in vivo and prevents the expression of those responses in lymphocytes from MV-resistant, SFV-immune rabbits in vitro. The relevance of this impairment to the differences in the clinical courses of SFV- and MV-induced tumors is discussed.     

Trichinella spiralis: correlates in vitro of altered immune responsiveness in mice.

Mixed lymphocyte reactions and in vitro antibody responses to dinitrophenol (DNP) after immunization with DNP-Ficoll were measured in spleen cells from mice following infection with 200 Trichinella spiralis larvae. A depression of the mixed lymphocyte reaction was observed at 14 through 84 days after infection. A reduced response to concanavalin A stimulation was demonstrated over a similar time period, 7 through 63 days of infection. The addition of mitomycin C-treated spleen cells from mice infected with T. spiralis to cultures of normal splenocytes suppressed the mixed lymphocyte reaction by 28% to 65%. The antibody response to DNP-Ficoll immunization was enhanced 20 days after infection, a time when the T-dependent antibody response to sheep erythrocytes was depressed.     

Evidence for active synthesis of two allelic b locus markers by single lymphocytes from heterozygous rabbits.

The double expression on single cells of both b4 and b6 allotypic markers by the mixed antiglobulin technique was observed on single peripheral blood lymphocytes (PBL) from heterozygous rabbits which were treated with pronase and incubated overnight to allow for regrowth of surface immunoglobulin (Ig). The percent of double staining rosettes observed after lymphocyte processing prior to pronase stripping ranged from 10–30%. Following pronase treatment at concentrations allowing for maximum stripping but assuring high cellular viability, the total rosetting population was reduced to 1–3% in the majority of cases. Occasionally, pronase stripping was less effective. Overnight incubation in serum free medium resulted in the reappearance of surface Ig with double staining rosettes ranging from 8–30%. Exposure of b6 or b4 normal rabbit serum to b4 or b6 cells, respectively, resulted in uptake of b6 in which most of the passively acquired Ig eluted off during overnight incubation, while negligible uptake of b4 was seen. Coincubation of normal b4, 4 PBL which presumably are capable of donating Ig with pronase treated b6, 6 PBL and vice versa also resulted in the absence of mixed rosettes. That simultaneous active synthesis of both allelic markers results in double allotype expression was demonstrated by kinetics data. Supporting this was the observation that only after washing of cycloheximide inhibited cells did allotype reexpression resume.     

Human T lymphocytes become glucocorticoid-sensitive upon immune activation

A murine model for Transfer Factor (TF) was used in an attempt to identify the nature of its antigen-specific component. TF was prepared from lymph node cells of CBA/Ca/T6 mice sensitized 30 days previously with 2,4-dinitrofluorobenzene (DNFB). To assay for the specific component of TF, 2 × 10⁷ lymphocyte equivalents were injected intravenously into normal syngeneic recipients. Lymph node cells obtained 18–24 hr later gave a positive response in the macrophage migration inhibition (MMI) test in the presence of the soluble analog of DNFB (sodium 2,4-dinitrobenzenesulfonate). The activity of TF was abrogated by absorption with anti-Ia sera including both an Ia alloantiserum (A.TH anti-A.TL) and a xenogeneic rabbit anti-serum which exclusively recognizes carbohydrate-defined Ia antigens. Analysis by paper chromatography using the technique for purification of carbohydrate-defined Ia antigens revealed that MIF production was obtained exclusively with those fractions known to contain Ia antigenic activity. In addition, pretreatment of TF with insoluble conconavalin A (Con A) which has an affinity for carbohydrate-defined Ia antigens resulted in removal of its activity. Taken together these findings pointed to the presence in TF of I-region gene products. Absorption with antibody directed against the dinitrophenyl determinant abolished the capacity of TF to stimulate macrophage inhibition factor production suggesting that it might also contain antigen fragments possibly in association with Ia. No evidence was, however, obtained for H-2 restriction of the action of TF in vivo since it was found to exert an effect in a variety of strain combinations including A.TH and Balb/c which share no known common I-region specificities. Parallel experiments were carried out with the lymphocyte transformation assay since this is known to be a measure of the nonspecific components in TF. Pretreatment with mouse allo-anti-Ia^k serum directed against both protein-and carbohydrate-defined Ia antigens caused a partial reduction in the proliferative response. In contrast no change in response was observed when the TF was absorbed with insoluble Con A or anti-DNP serum. Furthermore, lymphocyte transformation was obtained with only one of the three paper chromatography fractions positive in the MMI assay as well as two other different fractions. Taken together, these findings permitted a distinction to be made between specific and nonspecific components of TF and indicated that the specificity of TF could be explained in terms of the presence of I-region gene coded products possibly in association with antigen fragments.     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Depression of lymphocyte reactivity to phytohemagglutinin by serum from patients with liver disease

The influence of serum from patients with a variety of liver abnormalities on the in vitro response of normal human peripheral blood lymphocytes to stimulation with phytohemagglutinin was studied. These experiments demonstrate that serum from patients with acute viral hepatitis, chronic Australia antigenemia, common bile duct obstruction, primary biliary cirrhosis, hepatic necrosis secondary to halothane, and alcoholic cirrhosis suppresses DNA synthesis by stimulated normal lymphocytes. Sera obtained from two patients 1 week after surgical correction of their common duct obstruction no longer demonstrated lymphocyte suppression. Dilution of normal serum with serum from either of three patients resulted in more rapid decrease of thymidine uptake by stimulated lymphocytes than when normal serum was diluted with culture medium. This indicates the presence of an inhibitory factor (s) in the patients' sera. No correlation was shown between the extent of thymidine uptake by stimulated lymphocytes and the bilirubin, transaminase, or alkaline phosphatase levels in the serum in which they were cultured. The addition of bile salts to stimulated lymphocyte cultures did result in suppression of the response but only at concentrations much higher than would be expected in serum of the patients studied. Cell death after 72-hr incubation was 42% in normal serum and only 50% in serum from a patient who had demonstrated prominent suppression of DNA synthesis.