IgG Subisotypes Research Articles

Passive immunity in foals born from mares vaccinated against Theileira equi and Streptococcus equi subspecies equi

ABSTRACT: The aims of this study were: 1) determine total specific IgG and subisotypes in serum and colostrum of pregnant mares vaccinated against Theileria equi and Streptococcus equi subspecies equi; 2) determine total specific IgG and subisotypes in foals born from these mares. In mares, the highest total serum IgG value for T. equi was at 335 days of gestation declining 30 days postpartum, while for S. equi was at 328 days of gestation remaining high up to 30 days postpartum. Transfer of passive immunity against both antigens was observed with specific IgG values in colostrum and foals’ serum proportional to mares’ values. The most detected specific IgG subisotypes were IgG3/5 and IgG4/7 for both antigens. Foals born from mares immunized with T. equi kept maternal IgG values until 2 months of age, while those born from mares immunized with S. equi kept maternal IgG values until 3-4 months of age. These results suggest that foals should be vaccinated after this period.

Features of immunopathogenesis of a new coronavirus infection

The main biological characteristics of viruses of the Coronaviridae family are presented. The features of the immunopathogenesis of these infections are analyzed. It was found that the structural proteins of the spine, membrane, envelope and nucleocapsid play an important role in the immunopathogenesis of COVID-19 infection. They are associated with hyperactivation of neutrophils and monocytes-macrophages, secreting large amounts of pro-inflammatory cytokines and chemokines. This contributes to the development of a cytokine storm and an unfavorable prognosis of the disease. A particularly high risk of developing pneumonia exists against the background of an increase in the production of: macrophage inflammatory protein-1 alpha, macrophage chemotactic protein, interleukin 8. At the height of infection in some patients, macrophages and dendritic cells infected with SARS-CoV-2 lose their ability to produce type I interferons and pro-inflammatory cytokines. On the part of cellular immunity, a significant decrease in the number of CD4+ and CD8+-lymphocytes was noted. Among IgG sub-isotypes, IgG3 antibodies had the highest reactivity, and IgG1 antibodies were less reactive. Antibodies to spike protein with low specificity or low titer do not neutralize the virus and contribute to the contamination of immunocompetent cells via Fc receptors. Low-affinity antibodies or their low levels can contribute to increased cell sensitivity to SARS-CoV-2 and the development of severe forms of COVID-19 disease.

Sub-isotypic differences in the immunoglobulin G response to Lawsonia intracellularis in vaccinated, seropositive, and equine proliferative enteropathy-affected horses

In the horse, Lawsonia intracellularis infection results in equine proliferative enteropathy (EPE). While upwards of 100% of weanlings on an endemic farm may seroconvert, only a small percentage (approximately 5%) will develop clinical disease. Cell-mediated immune mechanisms likely play a role in resistance to L. intracellularis and the absence of a L. intracellularis-specific IFN-γ response has been associated with the development of EPE. The goal of this study was to determine whether protection from clinical EPE is associated with the induction of a systemic IgG sub-isotypic response consistent with a Th1-type cytokine response. To describe their L. intracellularis/EPE status, horses enrolled in this study were placed into one of three categories: seropositive-only, vaccinated, and presumptive clinical EPE. An existing ELISA method was modified to detect L. intracellularis-specific IgG(a), IgG(b), and IgG(t) antibodies using the mouse anti-equine hybridomas CVS-48, CVS-39, and CVS-40, respectively. Additionally, the existing ELISA method was used to quantify total IgG antibodies specific for L. intracellularis for comparison between the groups. Total L. intracellularis-specific IgG was found to be significantly higher (p<0.05) in presumptive clinical EPE cases (n=21) when compared with seropositive (exposed but unaffected) (n=36) and vaccinated horses (n=27). Further, a similar pattern for IgG(a) was seen in that the presumptive clinical EPE horses had significantly more L. intracellularis-specific IgG(a) (p<0.05) than the seropositive or vaccinated horses. With IgG(b), however, the vaccinated horses had significantly more IgG(b) (p<0.05) than the presumptive clinical or seropositive horses. No L. intracellularis-specific IgG(t) was detected in samples from any of the groups. While the results presented here with respect to IgG(a) response in the presumptive clinical EPE group were expected, a higher concentration of IgG(a) was anticipated in the seropositive horses that failed to develop clinical disease as well as in the vaccinated horses. Future work utilizing newer reagents against a broader range of equine IgG sub-isotypes may provide additional information once these become commercially available.

Neospora caninum tachyzoites inoculated by the conjunctival route are not vertically transmitted in pregnant cattle: A descriptive study

The aim of this study was to evaluate whether Neospora caninum tachyzoites (Nc-1) inoculated by the conjunctival route in pregnant cows were able to generate infection in their fetuses. Group 1 contained 2 naturally infected cows; group 2 contained two cows inoculated intravenously with 2.5×108 tachyzoites, group 3 contained two cows inoculated with 2.5×108 tachyzoites by the conjunctival route, and group 4 contained two uninfected control cows. The four inoculated cows from groups 2 and 3 were challenged at 23 weeks of gestation. An indirect fluorescent antibody test (IFAT), recombinant NcGRA7-based ELISA, ELISA for IgG subisotypes and Western blot analysis were assessed to characterize the humoral immune response in dams. Sera from their fetuses were tested also using Western blot analysis. Routine microscopic evaluation of H&E stained fetal tissues was made and any fetal tissues and placentas with lesions compatible with Neospora-infection were processed by immunohistochemistry (IHC). DNA extraction from fresh and formalin-fixed, paraffin-embedded fetal tissues were tested by nested PCR. All dams from groups 1, 2 and 3 were seropositive by IFAT, rNcGRA7-based-ELISA and Western blot. IgG1/IgG2 ratios were ≤1 at weeks 27 and 29 of gestation. Only fetuses from groups 1 and 2 developed N. caninum specific antibodies by Western blot. Histopathological lesions compatible with those caused by N. caninum were observed in fetuses from groups 1 and 2. N. caninum cysts and tachyzoites were observed by IHC on fetal tissues from groups 1 and 2. Only fetal samples from group 2 were positive by PCR. Further work is needed not only to characterize the cellular immune response but also to clarify the consequences on the dam after conjunctival inoculation of N. caninum tachyzoites. This study shows that N. caninum tachyzoites inoculated by the conjunctival route were not vertically transmitted in pregnant cows.

Beraprost enhances production of antigen-specific IgG isotypes without modulating germinal center B cell generation and the affinity maturation

Immune regulating functions of lipid mediators are being recognized. Prostaglandins (PGs) are derived from phospholipid by cyclooxygenase-2 (COX-2) in inflammatory tissues. Based upon our previous data that imply an immune modulating activity of prostacyclin, we hypothesized that PGs promote the humoral immune responses in vivo. To test this hypothesis, we examined the effect of a prostacyclin analog beraprost and PGE2 on the antibody responses that were induced by immunizing mice with keyhole limpet hemocyanin (KLH). Beraprost was used due to the extremely unstable property of prostacyclin. Our results showed that beraprost indeed promoted the production of anti-KLH antibodies, which was dose-dependent and specific to beraprost because PGE2 did not modulate the antibody response compared with controls. The enhancing effect of beraprost was reproduced in the secondary responses, suggesting that memory B cell generation during the primary response was significantly affected by beraprost. Analysis of the isotypes of anti-KLH antibodies revealed that beraprost stimulated the production of IgA and IgG subisotypes but not IgM. However, germinal center B cell generation and the affinity of anti-KLH antibodies were not affected by beraprost administration. To confirm these results we immunized COX-2 knockout mice with KLH and analyzed whether the results with wild-type mice were reflected in the absence of PGs. The primary and secondary antibody responses were significantly impaired in the KO animals. The levels of anti-KLH IgG subisotypes and IgA were severely reduced in KO mice whereas those of IgM were comparable to controls. These results reveal an unrecognized function of PG in the humoral immune responses.

INOS potentiates mouse Ig isotype switching through AID expression

The IgA antibody plays an important role in protecting mucosal surfaces against pathogens. It has recently been shown that nitric oxide (NO) plays a critical role in mouse IgA synthesis. In the present study, we further characterized inducible-nitric oxide synthase-deficient (iNOS −/−) mice in the context of Ig expression. The amount of IgA in fecal pellets was substantially diminished in iNOS −/− mice and was paralleled by a decrease in IgA production by Peyer’s patch cells. Interestingly, the amount of all IgG subisotypes, as well as IgA, was substantially diminished in sera and in cultured spleen B cells from iNOS −/− mice. Moreover, the synthesis of TGF-β1-inducible IgA and IgG2b in iNOS −/− mice was also lower than that in WT mice. However, levels of Ig germ-line transcripts, and expression of TGF-β receptor type II (TβRII) and BAFF/APRIL, were comparable between iNOS −/− and WT mice. Expression of activation-induced cytidine deaminase (AID) was diminished in iNOS −/− B cells, but restored by a NO donor, SNAP. These results indicate that iNOS regulates Ig isotype switching events at the level of AID gene expression.

Effects of inoculum size on cell-mediated and humoral immune responses of foals experimentally infected with Rhodococcus equi: A pilot study

The objective of this pilot study was to compare the cytokine profile as well as cell-mediated and antibody responses of foals infected with a low inoculum of virulent Rhodococcus equi resulting in subclinical pneumonia to that of foals infected with a high inoculum resulting in severe clinical pneumonia. The mean (±SD) ratio of post-infection to pre-infection anti- R. equi IgG(T) concentration was significantly ( P = 0.002) higher in foals infected with the high inoculum (195 ± 145; range 62–328) compared to foals infected with the low inoculum (3.9 ± 4.5; range 0.5–11). Similarly, mean (±SD) ratio of post-infection to pre-infection IgM concentration was significantly ( P = 0.002) higher in foals infected with the high inoculum (12 ± 4.0; range 7.4–14) compared to foals infected with the low inoculum (2.5 ± 1.5; range 1.2–4.7). Proliferative responses to R. equi antigens as well as expression of mRNA for IL-2, IL-4, IL-10, and IFN-γ in BLN were not significantly different between the two groups. There was a tendency ( P = 0.073) towards a higher IFN-γ/IL-4 ratio in the low inoculum group. This study demonstrates that the size of inoculum modulates the IgG subisotype response and possibly the cytokine profile of foals.

Antibody and T‐cell responses specific for the androgen receptor in patients with prostate cancer

The androgen receptor (AR) is a steroid hormone receptor that is an essential regulator of prostate development, and the primary molecular target for the treatment of metastatic prostate cancer. In this report, we evaluated whether patients with prostate cancer have pre-existing immune responses specific for the AR as evidence that the AR also might be pursued as an immunological target antigen. The detection of auto-antibodies specific for the AR in patient sera was evaluated by ELISA and Western blotting. Peripheral blood mononuclear cells were analyzed for the presence of AR-specific T-cells, as measured by T-cell proliferation, interferon gamma (IFNgamma) and interleukin-10 secretion. We found that a significantly higher frequency of prostate cancer patients have AR LBD-specific antibody responses than do healthy male volunteers [18/105 cancer patients (17.1%) vs. 0/41 healthy volunteers, P = 0.0049], and that these responses were present regardless of the patients' disease stage [8/46 organ-confined prostate cancer patients (17.4%), 3/22 metastatic androgen-dependent patients (13.6%), and 7/37 metastatic, androgen-independent patients (18.9%)]. These antibodies were pre-dominantly of the IgG isotype, and furthermore of the IgG(2) sub-isotype. In addition, we found that patients with antibody responses also had concurrent antigen-specific CD4+ and CD8+ T-cell proliferation and IFNgamma secretion when compared to patients without antibody responses. These data demonstrate that some patients with prostate cancer have pre-existing humoral and cellular immune responses specific for the AR, suggesting that tolerance against the AR is not absolute and that the AR may be a potential immunotherapeutic target antigen.

Assessment in mice of vapA–DNA vaccination against Rhodococcus equi infection

There is a need to produce a vaccine against Rhodococcus equi pneumonia in foals in which immunity against infection is largely based on a type 1, cell-mediated, immune response. The VapA protein of the virulence plasmid of R. equi is highly immunogenic. To assess the potential of vapA-DNA to produce immunity, C57BL/6 and BALB/c mice were immunized with a DNA vaccine constructed from vapA incorporated into pcDNA3.1. The plasmid construct expressed VapA in a COS-7 cell line. Intramuscular immunization of mice resulted in enhanced clearance of R. equi from the liver of intravenously challenged mice compared to non-immunized controls. This effect was more marked when pORF-IL-12, a plasmid expressing murine IL12, was included with the vaccine. Antibody developed to VapA, with an IgG2a response being more marked in mice immunized with pcDNA-vapA than in non-immunized or in mice immunized with the mixed vapA and IL-12 plasmid constructs. In conclusion, this study has shown for the first time that DNA immunization with vapA enhances the immune responses of mice against R. equi infection, that the IgG subisotype response is consistent with a type 1-based immune response, and that this can be enhanced by injection of the IL-12 gene.

Rheumatoid factor-like IgM in Plasmodium berghei (Apicomplexa: Haemosporida) infections of BALB/c mice.

Groups of female BALB/c mice infected by intravenous injection with 50 erythrocytes containing Plasmodium berghei Vincke et Lips, 1948 were sacrificed on days 3 through 12 after infection. Rheumatoid factor-like IgM (RF-IgM) and parasite-specific IgG levels were determined by enzyme-linked immunosorbent assay in serum specimens and in culture medium removed from spleen cell cultures established at sacrifice. All four mouse IgG subisotypes were recognized by RF-IgM molecules induced by Plasmodium berghei infection, and in this regard, the parasite-induced RF-IgM response resembled that induced by lipopolysaccharide polyclonal activation. Plasmodium berghei infection resulted in a biphasic RF-IgM response, with infected animals demonstrating significantly increased levels of RF-IgM early in the infection and significantly decreased levels late in the infection, compared to uninfected control mice. The decreased levels of RF-IgM observed late in infection correlated with increasing parasitaemia levels, and were primarily due to a decrease in RF-IgM specific for mouse IgG2a. Late infection levels of RF-IgM specific for IgGI, IgG2b, and IgG3 were not significantly different from those of control animals.

Passive transfer of maternal immunoglobulin isotype antibodies against tetanus and influenza and their effect on the response of foals to vaccination.

Influenza and tetanus-specific antibodies of the IgG sub-isotypes are posively transferred to foals via colostrum and inhibit their response to inactivated influenza vaccines and tetanus toxoid. High titres of influenza antibodies of IgGa and IgGb subisotypes and tetanus antibodies of the IgGa, IgGb and IgG(T) subisotypes were detected in postsucking serum samples collected from foals born to mares that had received booster doses of multicomponent vaccines during the last 2 months of gestation. Thereafter, titres declined in an exponential manner but were still detectable in all foals at age 26 weeks, regardless of whether they had been vaccinated prior to age 26 weeks. Mean +/- s.e. half-life of decline of influenza IgGa antibodies (27.0 +/- 2.3 days) was significantly shorter than that of influenza IgGb antibodies (39.1 +/- 2.7 days; P<0.005). Tetanus IgGa and IgGb antibodies declined with half-lives of 28.8 +/- 3.0 and 34.8 +/- 5.1 days, respectively. Titres of tetanus IgG(T) antibodies were substantially higher than those of influenza IgG(T) antibodies in postsucking samples and remained so through age 26 weeks, declining with a half-life of approximately 35 days. Postsucking titres of tetanus and influenza antibodies of the IgA isotype were low and declined rapidly to undetectable levels. Yearlings showed significant increases in titre of influenza IgGa, IgGb and IgG(T) subisotype antibodies but no increase in influenza IgA antibodies in response to 2 doses of multicomponent vaccines containing tetanus toxoid and inactivated influenza A-1 and A-2 antigens. Yearlings also showed strong tetanus IgGa, IgGb and IgG(T) subisotype responses to one dose of vaccine and a substantial further rise in titre in response to administration of a second dose 3 weeks later, but failed to show an increase in titre of tetanus IgA antibodies. The influenza and tetanus IgGa, IgGb and IgG(T) subisotype responses of 6-month-old foals to vaccination followed the same pattern as those shown by yearlings but titres were generally lower. In contrast, 3-month-old foals failed to show increases in titre of either influenza or tetanus IgG subisotypes in response to 2 doses of vaccine and generally needed 1-3 additional booster doses of vaccine to achieve titres similar to those achieved by yearlings after 2 doses. Based on the finding that maternal antibodies exert a significant inhibitory effect on the response of foals to tetanus toxoid and inactivated influenza antigens, it is recommended that primary immunisation of foals born to vaccinated mares should not commence before age 6 months.

Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli.

Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice.

Monoclonal antibodies to subclass-specific antigenic determinants on equine immunoglobulin gamma chains and their characterization

This paper describes the production of a panel of monoclonal antibodies (mAbs) identifying the four recognised equine IgG subisotypes IgG, IgGa, IgGb, IgGc and IgG(T). Pure preparations of the subisotypes for use in immunisations and testing were produced using a combination of gel filtration, salt precipitation, ion exchange chromatography and protein A and Protein G affinity chromatography. The specificity of mAbs for the IgG subisotypes was confirmed using ELISA assays, by characterisation of affinity purified proteins recognised by the mAbs, and by Western blotting of equine serum proteins. The expression of equine IgG subisotypes by B cells was examined by flow cytometry using the panel of mAbs.

Report of the Second Equine Leucocyte Antigen Workshop, Squaw Valley, California, July 1995

The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaborations were fostered among the participating laboratories and observers. Overall, enormous advances have been made in the past decade since mAbs specific for equine leucocyte antigens and immunoglobulins were first reported. There remains enormous scope and need for further studies of equine leucocyte antigens and immunoglobulins, both for the purposes of comparative immunology and for the good of the horse. In the future novel techniques will be required to develop reagents for specific target antigens such as the orthologues of the CD25 or CD45 isoforms. In studies of equine immunoglobulins the functional role of the IgG isotypes must be better established, reagents for IgE must be developed, and cloning of the immunoglobulin heavy chain genes will be essential if the complexities of the IgG sub-isotypes are to be elucidated. The tasks still facing the currently small group of equine immunologists throughout the world remain formidable, and will only be tackled successfully in a spirit of collaboration.

Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated horses

Equine strangles, caused by the clonal pathogen Streptococcus equi, is a source of serious economic loss despite the widespread use of commercial vaccines. The anti-phagocytic 58 kDa M-like protein (SeM) is an important protective antigen. The objective of this study was to define differences, if any, between SeM-specific convalescent serum and mucosal IgA and IgG subisotypes and those induced by vaccination with commercial strangles vaccine. SeM-specific opsonophagocytic IgGb was the predominant serum antibody in horses intramuscularly vaccinated or recently recovered from infection. Infection also induced high levels of specific opsonophagocytic serum IgGa during and shortly after S. equi infection whereas vaccination stimulated only low levels of serum IgGa. Specific serum IgGc and opsonophagocytic IgA were present at very low levels following infection or vaccination. A strong specific mucosal antibody response occurred during the acute and convalescent phases of infection whereas vaccinated horses made no mucosal response. Specific IgGb was generally predominant in nasopharyngeal washings during the acute phase but was replaced by specific IgA during convalescence. SeM-specific mucosal IgGa and IgG(T) but not IgGc were detected only during the acute and early convalescent phase. The results therefore indicate that vaccination, although inducing SeM-specific serum isotype responses qualitatively and quantitatively similar to those seen in convalescence, did not induce mucosal responses. This suggests that mucosal immunity may be important in acquired resistance to strangles.

Characterization of Estrogen-Induced Autoantibodies to Cardiolipin in Non-Autoimmune Mice

Antibodies to cardiolipin, in humans, have been associated with a variety of autoimmune disorders including anti-phospholipid syndrome, systemic lupus erythematosus and Sjögren's syndrome. These antibodies have also been demonstrated in autoimmune-prone MRL-Mp-lpr/lpr (MRL/lpr), BXSB-Mp-+yaa(BXSB) and (NZW×BXSB)F1mice. In previous work, we had shown that gonadectomized or intact male and female non-autoimmune C57BL/6 mice, upon treatment with estrogen, express autoantibodies to cardiolipin. In this study, we extend these findings and show that the expression of these antibodies persists for months even after the exposure to exogenous estrogen has been terminated. These antibodies are of IgM and IgG, but not IgA, isotypes, and the predominant IgG subisotype is IgG2b. Estrogen-induced antibodies to cardiolipin only minimally cross-reacted with DNA, actin or ovalbumin. The binding of antibodies to cardiolipin from autoimmune human patients in general has been shown to depend upon the presence of a cofactor, β2-glycoprotein I. We found that in estrogen-treated C57BL/6 mice, as well as in SLE-prone MRL/lpr and BXSB mice, the binding of anti-cardiolipin antibodies to cardiolipin was not enhanced, but rather reduced, in the presence of human β2-glycoprotein I. Further, addition of exogenous human β2-glycoprotein I to purified immunoglobulin fractions containing anti-cardiolipin antibodies reduces, rather than enhances, the binding to cardio-lipin. Together, these data show that persistent detectable levels of IgG and IgM autoantibodies specific for cardiolipin can be induced in normal mice by estrogen treatment alone (i.e. without administration of autoantigens). Further, we characterize these antibodies regarding their kinetics, cross-reactivity, isotype distribution and cofactor (β2-glycoprotein I) requirements.

Separation of equine IgG subclasses (IgGa, IgGb and IgG(T)) using their differential binding characteristics for staphylococcal protein A and streptococcal protein G

Equine IgG possesses four well-defined subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of their increasing anodal mobility in electrophoresis. However, the preparation of IgGa and IgGb reference proteins has not previously been reported. Certain bacterial cell wall proteins, termed protein A and protein G, have been used for purification of IgG subisotypes from the serum of domestic animals which, combined with other techniques utilising differences in the physico-chemical properties of the proteins, has allowed the purification of Ig isotypes. This paper describes purification of the subisotypes of equine IgG. Purification of IgGa and IgGb was achieved by the separation of a ‘fall-through’ peak from ion-exchange chromatography consisting of IgGa and IgGb into two fractions (peaks C and D) by FPLC protein A and protein G affinity chromatography. Peak C consisted of IgGb and peak D consisted of IgGa exhibiting slightly faster cathodal migration than peak C in IEP analysis. Affinity chromatography using protein A and G columns also indicated that there may be two different components of IgG(T); one with a low affinity for protein G and the other having a greater affinity for protein G.

Differentiation of B cells in the bovine. Role of cytokines in immunoglobulin isotype expression

Cytokines such as IFN-γ, IL-4 and TGF-β, are known to regulate Ig class switching by positively or negatively regulating germline C H transcription in the mouse and human. Here we show at the protein level that similar regulatory mechanisms are in place in the bovine using various modes of B cell activation and cytokine combinations. Using antigen-receptor cross-linkage or mitogen (pokeweed mitogen) costimulation, we demonstrate that recombinant bovine IL-4 upregulates the expression of IgM, IgG 1 and IgE in vitro. Upregulation of IgG 1 and IgE production by rboIL-4 is inhibited in a dose-dependent manner by the addition or rboIFN-γ to the cultures. Using similar methodologies, we have also investigated the effects of TGB-β on IgA production in the presence or absence of various cofactors including IL-10 and IL-2. We find that TGF-β will not solely upregulate IgA production from noncommitted precursors (sIgM +) in coculture with mitogen or with formalin fixed Staphylococcus cowan Strain I (SAC), but does have positive effects on IgA production in the presence of both IL-2 and IL-10. These observations suggest that different modes of T-independent costimulation using classical B cell activators does not result in differential IgG subisotype or IgE production by bovine B cells in the presence of cytokines. Together these results suggest that individual cytokines regulate isotype expression in cattle.

Monoclonal antibodies specific for equine IgG sub-isotypes including an antibody which recognizes B lymphocytes

Equine immunoglobulin G is currently classified as consisting of five sub-isotypes: IgGa, b, and c, IgG(T), and IgG(B). The study of the role of these immunoglobulins in antigen-specific responses, and the examination of their functional properties would be greatly facilitated by the availability of monoclonal antibodies (Mabs) that distinguish between them. The production and characterization of two Mabs that recognize an IgG sub-isotype with the characteristics of IgG(ab) is described. The immunoglobulin identified by these Mabs had a heavy chain weight of 53 kDa, was of rapid cathodal electrophoretic mobility in immuno-electrophoretic analysis, and reacted only with anti-sera to IgG, and not with anti-sera to IgG(T), IgA, or IgM in radia-immunodiffusion analysis. In addition, one of these two Mabs (CVS1) also recognized the majority of peripheral blood B lymphocytes in indirect immunofluorescent staining analysis, suggesting either that equine IgD may share a common antigenic epitope with an IgG sub-isotype, or that a large proportion of equine B lymphocytes may express an IgG sub-isotype on their surface.

Immunoglobulin binding by Tritrichomonas foetus

A better method for diagnosis of bovine trichomoniasis is needed because culture is slow and somewhat lacking in sensitivity. Immunodiagnosis of Tritrichomonas foetus infection usually involves detection of antigen-antibody reactions with an anti-immunoglobulin conjugate. However, nonspecific immunoglobulin (Ig), bound to the surface of T. foetus, would also be detected by an anti-Ig conjugate and thus would interfere with the specificity of the immunoassay. The goals of this study were to define the binding of bovine immunoglobulins to T. foetus. To determine whether nonimmune binding of Ig to T. foetus occurs, we immunized rabbits with organisms that had been grown in medium containing normal bovine serum and vigorously washed three times with phosphate-buffered saline. The rabbit antiserum had similar titers to T. foetus and to normal bovine serum by enzyme-linked immunosorbent assay (ELISA). Furthermore, two bovine serum proteins were immunoprecipitated by the rabbit antiserum in an immunoelectrophoretogram. One of the serum proteins had a distribution characteristic of IgG2. The rabbit antiserum was then shown to react with purified bovine IgG and IgM by ELISA. Reactivity to IgG was greater. To identify the IgG subisotypes bound and to confirm nonimmune binding of Ig, we grew T. foetus in agammaglobulinemic fetal calf serum and reacted it with IgG1, IgG2, and IgM specific for dinitrophenol (DNP) in ELISA. The binding of IgG2 was greatest, that of IgG1 was next, and that of IgM was least. Little competitive inhibition by DNP was detected, indicating that binding of DNP-specific antibodies was predominantly nonimmune rather than antigen-specific Ig binding. We also demonstrated that T. foetus grew well in medium containing agammaglobulinemic fetal calf serum or serum made agammaglobulinemic by ammonium sulfate precipitation of Igs. This may overcome the problem of low specificity in diagnostic assays as a result of antigen with Ig bound by nonimmune mechanisms.