IgG Plaque-forming Cells Research Articles

A New Activated Water Charged by Electrophoresis, Effect on the Experimentally Immuno-Suppressed Animal and Their Anti-Oxidative Activity

Every individuals expose to the lisk of conventional immunodeficiency in daily life with both internal and externals. In a series of investigation, we have been assessed the various health promoting menu such as hot spring hydrotherapy, acupuncture & moxibution, light excurse, evidenced Control water western medicine technique. In this report, we try to investigate the regulatory effect of hydrotherapy by activated water molecule as our representative menu under the influence of hypothalamus system. We set up both animal and human model for assessment of hydrotherapy. Before starting experimental assessment, six males and females seven-week-old ddY mice, are used for the acute oral toxicity study. There is no sign for toxicity as judged by body weight, organ inspection by veterinary doctor. Next, young ddY mice are experimentally induced diabetes and immune-deficient status by administrating cancer chemotherapeutic agent, Streptzotosin. Charged Water was evident to control the blood sugar level compared to the Control Water group together with conventional control group. Both experimental group of mice wear statistically significant for recovery from blood sugar level and antibody secreting cell both IgM and IgG plaque-forming cell. We confirm also activated water group delivered the granulocyte that slow down the oxidative stress by super oxide.

Defective neutrophil and lymphocyte function in leucocyte adhesion deficiency

We report a Chinese girl with the moderate phenotype of leucocyte adhesion deficiency (LAD), presenting with persistent omphalitis and recurrent soft tissue infections. She had subnormal adhesion-dependent neutrophil functions, such as chemotaxis and chemiluminescence response to a particulate stimulant (opsonised zymosan). Despite her adequate humoral response to documented herpes simplex virus type 1, parainfluenza type 2 and adenovirus infection in vivo, there was marked impairment in the generation of plaque-forming cells (PFC) driven by pokeweed mitogen (PWM) in vitro. IgM PFC were less severely affected than IgG and IgA PFC, probably because IgM production is less dependent on T cell help than IgA and IgG production. The patient's B cells and accessory cells had reduced function compared with the control subsets, while helper function of her CD4+ cells was virtually absent in the PWM-driven PFC assay. She also had marked defect in natural killer cell activity. The proliferation of her lymphocytes was normal to several plant lectins, including phytohaemagglutinin, concanavalin A and PWM, but markedly defective to OKT3.

Modulation of the antibody response to sheep red blood cells in normal and immunodeficient XID mice by myo-inositol.

Over the dose ranges tested, dietary vitamin A and myo-inositol induce similar changes in murine fetal growth and development. Because vitamin A is also known to affect the immune response, studies were conducted to determine if dietary myo-inositol might have an effect on antibody production. It was found that in vivo in inbred mice myo-inositol (4 mg/g of diet) accelerated the rate of appearance of plaqueforming cells (PFC) in a primary response to sheep red blood cells (SRBC). In vitro, myo-inositol accelerated the rate of appearance of colonies of anti-SRBC PFC (foci) and significantly increased the number of PFC per colony, but did not affect the number of foci per culture noted at the end of the culture period, myo-inositol had no effect on the PFC IgM:IgG ratio following a single exposure to the agent, but exposure to myo-inositol in vivo and in vitro resulted in a decrease in the number of IgM PFC per focus in a primary response and IgM and IgG PFC per focus in a secondary response. Based on studies suggesting that myo-inositol or a phosphorylated metabolite might act downstream from Bruton's tyrosine kinase (Btk) in a signal transduction pathway in B cells, immunodeficient CBA/CaHN-XID/J mice were fed a standard diet or the same diet supplemented with 0.4% myo-inositol. Mice given the supplemented diet produced significantly more IgM anti-SRBC antibody than did XID mice given the control diet (4.3 +/- 2.5 vs 1.7 +/- 2.8, 1/log2), and produced approximately the same amount as immunocompetent controls (2.9 +/- 0.9). When rechallenged with SRBC, XID mice given supplemental inositol produced significantly more IgM antibody than did the XID and immunocompetent controls (3.6 +/- 0.5 vs 1.8 +/- 1.1 and 1.5 +/- 0.7, respectively). Added dietary inositol did not have a significant effect on primary or secondary IgG responses to SRBC, which remained impaired. These results suggest that dietary myo-inositol or a derivative may be able to modulate B-cell IgM responses by interacting within the inositol second messenger system downstream from Bruton's tyrosine kinase.

Immunological activities of a Candida albicans protein which plays an important role in the survival of the microorganism in the host.

A protein with an isoelectric point of 4.3 and a relative molecular mass of 43 kDa (p43) was purified from the supernatants of the cultures of pathogenic Candida albicans but could not be detected in the supernatants of cultures of this fungus with pathogenicity previously attenuated after being repeatedly subcultured in vitro. Treatment of BALB/c and C57BL/6 mice with p43 resulted in (i) marked increases in the numbers of splenic immunoglobulin-secreting plaque-forming cells (PFC) with peak responses of immunoglobulin M (IgM) PFC preceding those of IgG PFC, with an isotype restriction pattern of IgG2a > IgG2b > IgG3 > IgG1 > IgM, and (ii) specific immunosuppression of the murine primary immune response against sheep erythrocytes. Immunosuppressive and B-cell mitogenic properties of p43 were quantitatively associated and inversely correlated with susceptibility to C. albicans infection. C57BL/6 mice treated with p43 2 days before inoculation with C. albicans were considerably more susceptible to the fungal infection than untreated mice. The immunobiological and chemical properties of p43 are compared with previously described immunosuppressive and B-cell mitogenic proteins produced by bacteria and viruses, and strategies for immunointervention are discussed.

Assessment of the immunotoxic potential of the fungicide dinocap in mice

The immunotoxic potential of dinocap was evaluated in female C57BL/6J mice following in vivo and in vitro exposure to this fungicide. In in vivo studies, groups of mice were dosed by gavage with technical grade dinocap at dosages ranging from 12.5 to 50 mg/kg per day for 7 or 12 days and selected immune functions examined. Mice dosed at 50 mg/kg per day dinocap died after 4 days of dosing. Twelve days of dosing with dinocap at 25 mg/kg per day resulted in decreased thymus weights and cellularity, and increased spleen weights. No changes were observed in body weight, absolute differential peripheral leukocyte counts, the lymphoproliferative responses to B- or T-cell mitogens, the mixed lymphocyte reaction, or natural killer (NK) cell activity of spleen cells from mice exposed to dinocap. Lymphoproliferative responses to concanavalin A (Con A) and phytohemagglutinin (PHA), however, were reduced in thymocytes from mice dosed at 25 mg/kg per day dinocap. The cytotoxic T lymphocyte (CTL) response to P815 mastocytoma cells was enhanced in mice exposed for 7 days to 25 mg/kg per day dinocap. Exposure of mice for 7 days to 25 mg/kg per day dinocap also caused a significant reduction in the IgM and IgG plaque-forming cell (PFC) response to sheep red blood cells (SRBC). A time-course study indicated that dinocap-induced suppression of the IgM PFC response was due to a delay in the peak PFC response to SRBC. In vitro studies using murine thymocytes cultured with dinocap (10 μg/ml for 72 h) resulted in suppression of the proliferative response to Con A and PHA. Exposure of thymocytes to dinocap in vitro for as little as 30 min resulted in suppression of the mitogen-stimulated response in the absence of any apparent direct cytotoxic effect. These results suggest that dinocap alters the immune system of the mouse, however, these effects are relatively modest in terms of adverse immune function and are only seen at relatively high exposure levels.

Lack of immunosuppression by ketoconazole and itraconazole

The antifungal drugs ketoconazole and itraconazole were evaluated for their effects in the following test systems: in vitro, phytohaemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells and IL-2-driven proliferation of CTLL-2 cells; in vivo, antibody response to sheep red blood cells (SRBC) and delayed-type hypersensitivity (DTH) reaction to oxazolone. At a concentration of 10 μM, ketoconazole moderately and itraconazole strongly inhibited thymidine (Thd) incorporation in human peripheral blood mononuclear cells cultured in medium supplemented with 5% human serum. Increasing the serum concentration from 5 to 20% almost completely reversed these inhibitory effects. Also, cell viability, found to be less than 15% in cultures containing 10 μM itraconazole was restored by increasing the serum concentrations in the culture medium. Similar observations were made in experiments using IL-2-stimulated CTLL-2 cells: the growth inhibition in the presense of 10 μM ketoconazole or 1 μM itraconazole could be counteracted by increased serum supplementation. In vivo, subchronic intraperitoneal dosing with 40 mg/kg ketoconazole or itraconazole to mice had no effect on the antibody response to SRBC as measured by the number of splenic IgM and IgG plaque-forming cells and did not significantly affect the DTH response to oxozalone. These data indicate that neither ketoconazole nor itraconazole exert immunosuppressive properties in vivo. Their in vitro inhibitory effects on PHA-induced lymphocyte proliferation and IL-2-dependent CTLL-2 growth are reversed by the serum supplementation to the culture medium and these activities should therefore be considered as in vitro artefacts.

Brugia pahangi: Effects of maternal filariasis on the responses of their progeny to homologous challenge infection

Granulomatous lesion formation and immune responses to Brugia pahangi infections were compared in age-matched male progeny of homologously infected and uninfected female jirds. Infections initiated in 2-week-old offspring yielded X ± SD adult worm recoveries of 6.0 ± 5.7 and 4.2 ± 5.4 in offspring from infected or uninfected mothers, respectively. Infections initiated in 4-week-old offspring resulted in an X ± SD recovery of adult worms of 11.3 ± 11.3 and 10.2 ± 5.8 in offspring from infected and uninfected mothers, respectively. The ratio of intralymphatic thrombi per intralymphatic worm was similar between infected offspring from infected or uninfected mothers within experiments. Areas of granulomas around B. pahangi antigen-coated beads embolized in the lungs were not significantly affected by maternal origin in infected or uninfected progeny. Offspring infected at 2 or 4 weeks of age from infected mothers exhibited significantly reduced titers of serum IgG antibodies to Brugia antigens at 5–8 weeks postinfection compared to infected offspring of uninfected mothers. Infected offspring from infected mothers also had significantly fewer splenic IgG plaque-forming cells to B. pahangi antigens at 5 weeks postinfection than similarly infected offspring from uninfected mothers. Western immunoblot analysis indicated qualitative and quantitative reductions in serum antibody reactivity to adult B. pahangi antigens in infected progeny of infected females compared to age-matched infected controls. Reduced homologous serum antibody responses in progeny exposed to maternal B. pahangi infection suggest that maternal immunoregulation to filarial antigens may occur. Reduced antibody responsiveness to B. pahangi antigens observed in infected offspring from infected mothers, however, had no demonstrable effect on adult worm burdens, microfilaremias, lymphatic lesion formation, or antigen-specific granulomatous inflammatory responses compared to infected progeny of uninfected mothers.

Modulation of Chicken Plaque-Forming Cells by Serotonin and Dopamine

Serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA) are endogenous components of the central nervous and endocrine systems of the chicken. To determine the effects of these monoamines antibody-mediated immunity, New Hampshire chickens of Line UNH 105 were injected intravenously with 5-HT (100 μg/kg of body weight) and DA (1 mg/kg of body weight). One milliliter of a 5% SRBC suspension was injected intravenously 30 min later. Both IgM and IgG splenic plaque-forming cells were assayed 5 after antigen injection. For in vitro studies, spleen lymphocytes from SRBC-primed chicks were incubated with DA and 5-HT followed by quantitation of IgM and IgG plaque-forming cells. The in vitro incubation of splenic lymphocytes with specific antagonists was used to ascertain the presence of monoamine receptors on lymphocytes. The 5-HT significantly enhanced IgM plaque-forming cells compared with controls following in vivo [550 ± 85 (SE) cells/106 splenic lymphocytes versus 359 ± 44] but not in vitro exposure. The IgG plaque-forming cells were not affected by 5-HT. The DA significantly suppressed IgM plaque-forming cells responses following in vivo (284 ± 46 versus 499 ± 66) and in vitro (254 ± 57 versus 451 ± 51) exposure. Significant suppression of IgG plaque-forming cells was found in vivo (287 ± 40 versus 462 ± 75) and in vitro (153 ± 36 versus 371 ± 81) following treatment Specific DA antagonists, apomorphine and metoclopramide, did not alleviate the in vitro suppressive effect of DA.

The role of monocytes and T cells in 1,25-dihydroxyvitamin D3 mediated inhibition of B cell function in vitro

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) inhibits immunoglobulin production by human mononuclear cells (MNC) in vitro. The present study was undertaken to evaluate the role of T cells and monocytes in 1,25-(OH)2D3 induced suppression of B cell functions. The synthetic vitamin D3 analogue MC 903 was examined in parallel. 1,25-(OH)2D3 and MC 903 showed a dose-related inhibition of IgM, IgG and IgA plaque-forming cells in poke-weed mitogen (PWM) activated cultures of MNC. This effect was most likely mediated through impairment of T cell and monocyte functions. First, the inhibitory effect was seen after PWM stimulation, but not after Epstein-Barr virus stimulation which activates B cells independently of T cells and monocytes. Second, 1,25-(OH)2D3 was not effective in T cell and monocyte-depleted cultures. Third, the effect of 1,25-(OH)2D3 on PWM driven MNC was reversed by addition of the recombinant monokines: interleukin (IL)-1β, tumour necrosis factor α (rTNFα), rIL-6, as well as the lymphokines: lymphotoxin (rLT) and rIL-2. This is consistent with the finding that 1,25-(OH)2D3 also inhibited IL-1α, TNFα and LT production in these cultures. The assumption that B cells are not directly affected by 1,25-(OH)2D3 was further supported by the fact that 24 h of culture with 10−8 M 1,25-(OH)2D3 failed to reduce immunoglobulin production by in vivo activated B cells.

Induction of antibody synthesis by CD4+ T cells: IL 5 is essential for induction of antigen‐specific antibody responses by TH2 but not TH1 clones

Murine B cells stimulated with lipopolysaccharide (LPS) and interleukin (IL) 4 produce IgG1 and IgE, but synthesize IgG2a when stimulated with LPS and interferon-gamma. The cytokines, however, that regulate immunoglobulin (Ig) synthesis induced in normal B cells under antigen-driven major histocompatibility complex (MHC)-restricted conditions in the absence of potent B cell mitogens have not been fully elucidated. We and others have shown that under cognate MHC-restricted conditions, CD4+ T cell clones of the TH1 subset, which produce IL 2 and interferon-gamma, and T cell clones of the TH2 subset, which produce IL 4 and IL 5, are both capable of inducing anti-trinitrophenyl IgG plaque-forming cells. In this report we have examined in further detail the cytokine requirements for the induction of Ig synthesis in B cells cultured directly with TH1 and TH2 T cell clones. Using (a) TH2 clones that varied in the amount of IL 5 secreted, (b) a neutralizing monoclonal antibody against IL 5 and (c) T cell clones pretreated with cyclosporin A to inhibit cytokine secretion, we found that IL 5 was essential for induction of IgG1 synthesis by TH2 but not TH1 T cells. Although we demonstrated that IL 2 could actually up-regulate the synthesis of IL 5 by TH2 clones, the induction of IgG synthesis by TH2 clones was entirely independent of IL 2. In contrast, induction of IgG1 synthesis by TH1 clones was absolutely dependent upon the presence of IL 2 and was not affected by the presence of IL 5. Thus, these studies demonstrate the idea that at least two independent pathways exist for the induction of IgG1 synthesis, and that one of these pathways is IL 4/IL 5 dependent and the other IL 2 dependent.

Evidence for differential induction of helper T cell subsets during Trichinella spiralis infection.

The H-2-compatible mouse strains, AKR and B10.BR, exhibit disparate responses to infection with the parasitic nematode Trichinella spiralis. The resistant AKR mice expel intestinal adult worms faster than susceptible B10.BR mice. We tested antibody and lymphokine responses in these strains. With respect to antibody responses, the B10.BR mice had 3- to 10-fold more serum IgE and T. spiralis-specific IgG1 and IgA than AKR mice. The B10.BR mice also had greater numbers of IgG and IgA plaque-forming cells than AKR mice. In contrast, AKR mice produced T. spiralis-specific IgG2a, whereas the B10.BR mice did not. The antibody response kinetics of these strains were similar. We also analyzed lymphokine secretion after restimulating lymphocytes in vitro with T. spiralis Ag. The AKR mesenteric lymph node cells produced more IFN-gamma and less IL-4 than the B10.BR mesenteric lymph node cells. The B10.BR splenocytes produced more IL-4 than the AKR splenocytes, although splenocyte IFN-gamma production was not different. The kinetics of IL-4 production also differed between the two strains. In summary, resistant AKR mice produced more IFN-gamma and T. spiralis-specific IgG2a than susceptible B10.BR mice, which produced more IL-4, IgE, and T. spiralis-specific IgG1. Our results are consistent with differential activation of Th cell subsets in T. spiralis-infected AKR and B10.BR mice.

X-linked severe combined immunodeficiency in the dog

This study documents the occurrence of a form of X-linked severe combined immunodeficiency (SCID) in the dog with clinical, immunologic, and pathologic features similar to those of X-linked SCID with B cells in man. The disease in the dog is characterized by growth retardation and increased susceptibility to bacterial and viral infections in young pups. Affected pups have all died or were euthanatized by 5 months with signs of canine distemper, infectious hepatitis, or bacterial pneumonia. Laboratory findings include normal numbers of circulating B lymphocytes and normal concentrations of serum IgM, but low to absent concentrations of serum IgG and IgA, indicating a defect in the terminal differentiation of IgG and IgA B cells into immunoglobulin-secreting plasma cells. This is supported by the failure of peripheral lymphocytes to produce IgG or IgA plaque-forming cells in response to polyclonal activation. There are low-to-normal numbers of circulating T cells, but a severely depressed blastogenic response to T cell mitogens. Postmortem findings include thymic dysplasia and hypoplasia of lymphoid tissue. Family studies and breeding experiments are consistent with an X-linked recessive mode of inheritance.

Immunomodulation in C57BI/6 mice following consumption of halogenated aromatic hydrocarbon‐contaminated coho salmon(Oncorhynchus kisutch)from lake Ontario

This report describes studies designed to assess the immunomodulatory effects associated with the consumption of coho salmon containing halogenated aromatic hydrocarbons (HAHs) and other compounds naturally bioaccumulated from Lake Ontario. Diets containing 33% coho salmon from Lake Ontario or the Pacific Ocean were fed to juvenile C57Bl/6 mice for 2-4 mo. Following 60 d, the mice that consumed Lake Ontario salmon had reduced IgM, IgG, and IgA plaque-forming cell responses to sheep erythrocytes. No changes were observed in total numbers of spleen lymphocytes, total T-lymphocytes or T-lymphocyte subsets as determined by flow cytometry. Cellular immunity, assessed by the cytotoxic T-lymphocyte response to allogeneic tumor target cells, was not altered following dietary exposure to Lake Ontario coho salmon for 4 mo. The observed humoral immunomodulation correlated with elevated PCB levels in the Lake Ontario salmon diets. The levels of pollutants such as mercury, tin compounds and other metals, PCDDs, and PCDFs were not examined.

Induction of antigen-specific antibody responses in primed and unprimed B cells. Functional heterogeneity among Th1 and Th2 T cell clones.

CD4+ T cells have been recently divided into two subsets. The functions of these subsets are thought to be distinct: one subset (Th1) is responsible for delayed type hypersensitivity responses and another (Th2) is primarily responsible for induction of antibody synthesis. To more precisely define the roles of both subsets in humoral immune responses, we examined the ability of a panel of nominal antigen specific Th1 and Th2 clones to induce anti-TNP specific antibody synthesis in TNP-primed or unprimed B cells. Four of nine Th1 clones induced little or no antibody synthesis with TNP-primed B cells. However, five other Th1 clones were very effective at inducing IgG anti-TNP plaque-forming cell (PFC) responses in primed B cells. One of these Th1 clones was analysed in detail and found to also provide helper function for unprimed B cells. Cognate B-T cell interaction was required for induction of both primary and secondary responses with this clone, indicating that a Th1 clone could function as a "classical" Th cell. The seven IL-4 producing Th2 clones examined were also heterogeneous in their ability to induce antibody secretion by TNP-primed B cells. Although four of the Th2 clones induced IgG and IgM anti-TNP PFC responses, two Th2 clones induced only IgM and no IgG antibody, and another clone failed to induce any anti-TNP PFC. All Th2 clones failed to induce any anti-TNP PFC. All Th2 clones produced high levels of IL-4, but "helper" Th2 clones produced significantly greater amounts of IL-5 than "non-helper" Th2 clones. These studies indicate that some IL-2- and some IL-4-producing T cell clones can induce TNP-specific antibody in cell clones can induce TNP-specific antibody in primed and unprimed B cells, and that Th1 and Th2 clones are heterogeneous in their ability to induce Ig synthesis. Therefore, although T cell clones can be classified as Th1 or Th2 types according to patterns of IL-2, IFN-gamma, or IL-4 synthesis, the functional capacity to induce antibody synthesis cannot be predicted solely by their ability to secrete these lymphokines.

Characterization of activation of a partially defective B cell subpopulation with immunocompetence restricted to IgM and IgA.

A B-cell subpopulation (BM-A cell) responding to an antigen with the production of IgM and IgA plaque-forming cells but not of IgG plaque-forming cells was isolated from neonatally bursectomized chickens and was examined for the mode of activation by B-cell mitogens. The BM-A cells did not elevate both glucose consumption and protein synthesis with the B-cell mitogens, in striking contrast to normal B cells. The BM-A cells were also not activated by an activator of protein kinase C, phorbol myristate acetate. Both anti-Ig and a calcium ionophore, A23187, however, primed the BM-A cells to increase intracellular free calcium ion as well as normal B cells. From these results it is conceived that the lack of protein kinase C activation may be responsible for the failure of activation of BM-A cells.

Primary response to lysozyme (HEL) and HEL-LPS in neonatal A/J mice: Presence of characteristic adult pattern of regulatory idiotype and fine specificity restriction

The adult primary murine plaque-forming cell (PFC) response to hen egg-white lysozyme (HEL) is characterized by a predominant idiotype (IdXE) (∼-50% of population) and a predominant specificity (∼-50% of population) for a determinant dependent on the 3 amino-terminal residues of HEL (TIP-dependent PFC). IdXE and TIP specificity, however, are not congruent: approximately 1 3 of each population do not overlap (1). To determine whether these characteristics result from a prolonged selection process during development, we compared the neonatal A/J response profile to HEL-CFA with the adult A/J response and found that the adult pattern was present for mice immunized as early as 5 days of age. At 5 days, A/J splenic PFC were 80% TIP dependent, 71% IdXE positive compared to the adult levels of 56% (±14) TIP dependent, 43% (±19) IdXE positive. To attempt to address the B cells directly and avoid the need for antigen-specific T-dependent processes, we studied the PFC response to HEL-LPS conjugates in adult and 12-day-old A/J mice. At the earliest age studied (12 days), the indirect splenic PFC had similar idiotypy and specificity as the adult, and the response was similar to that induced by HEL-CFA. Although the absolute IgM PFC level was equal in adult and neonate, only 11% of the adult PFC response was IgM while a large proportion of the 12-day-old HEL-LPS response was IgM. This IgM PFC response was also characterized by the same idiotypy and specificity as the IgG PFC response. These results suggest that the characteristic adult mosaic of specificity and idiotypy in the anti-HEL response may exist prior to antigenic stimulation since it occurs as early as 5 days after immunization and in the IgM PFC responses. Although IdXE predominance may merely reflect the germ line repertoire, T cells may also be involved in an idiotypebased selection, the mechanism of which has not been determined.

Biochemical Characterization of an Antigen-Specific Suppressor T Cell Factor

We describe here the biochemical properties of suppressor T cell factor (TsF2) released from an inducible anti-idiotypic T cell hybridoma (C57BL/6 T cell x BW5147) which mediates antigen (keyhole limpet hemocyanin) specific and genetically (H-2b) restricted suppression of IgG plaque-forming cell responses. We examined the suppressive activity by in vitro functional assay in fractions of chromatography and in the materials eluted from gels of sodium dodecyl sulfate polyacrylamide and isoelectric focusing and determined the molecular weight(s) (22-37 kD) and the isoelectric point(s) (pH 6.0-6.1) of this secreted factor. Messenger RNA products of the hybridoma translated in the rabbit reticulocyte lysate system were similarly examined, and the functionally active molecule was seen to migrate to almost the similar molecular weight(s) (23-40 kD), and isoelectric point(s) (pH 5.5-6.2) range as those of secreted TsF. Moreover, the TsF activity was recovered from gel slice corresponding to the similar molecular weight range analyzed in sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Thus, we speculate that this molecule is composed of a single chain, biologically active. Comparison of autoradiograms on in vitro translation products between activated and resting hybridomas by two-dimensional gel electrophoresis showed that the molecular weights and isoelectric points of two spots (28 kD, 5.7; 25 kD, 5.5) newly appearing or markedly enhanced after activation in the area with suppressor activity were concordant with the data on secreted TsF, suggesting that one of these two spots represents the functional molecule which causes antigen-specific and genetically restricted suppression of IgG responses.

Solid phase enzyme-linked immunosorbent assay (ELISA) for anti-sheep erythrocyte antibody in mouse serum

The solid phase enzyme-linked immunosorbent assay (ELISA) was developed to measure IgM and IgG anti-SRBC antibody titers in mouse serum. Sheep erythrocytes, which have a surface negative charge, were attached directly to the bottom of an aminoplate well (96-well flat bottom, Sumitomo Bakelite Co.) which is charged positively to provide a solid phase for the ELISA antigen. Serum samples titrated were obtained from mice 5 and 10 days after SRBC immunization. Alkaline phosphatase-labeled goat anti-mouse IgM and/or IgG preparations were used as second antibody. Alkaline phosphatase activity in a well was measured by the Kind and King method [Kind, P.R.N. & King, E.J. (1954). J. clin. Path., 7, 322–326]; the optical density (OD) value of quinone as a final product was measured at 492 nm. (1) A linear relationship was observed between the antiserum concentration and OD value over a wide enough dilution range to assay antibody titers of serum samples; (2) IgM and IgG fractions from antiserum showed almost the same antibody titer as did the reconstituted samples; (3) a good correlation was observed between the serum IgM titer and the number of IgM hemolytic plaque-forming cells (PFCs) in spleen 5 days after the immunization, and was also observed between serum IgG antibody titer and the number of IgG PFCs 5 and 10 days after. Therefore, the ELISA described here requires no fixative for preparation of cell-coated plates, and serum IgM and IgG anti-SRBC antibody titers can be measured without any fractionation technique.

Evaluation of the immunotoxicity of β-hexachlorocyclohexane (β-HCH)

β-HCH, an isomeric contaminant formed during the manufacture of the insecticide lindane, is a persistent environmental and food chain pollutant which has been reported to exhibit estrogenic activity in rodents and in fish. To investigate potential toxic effects on the reproductive and immune systems, β-HCH was fed to female B6C3F1 mice for 30 days. Mice exposed to 0, 100, or 300 mg of β-HCH/kg of diet were evaluated for changes in ovarian and uterine histology, body weight, lymphoid organ weight and histology, splenic cellularity, antigen-specific IgM and IgG plaque-forming cells (PFC), proliferative responses to mitogens, natural killer (NK) cell activity, and induction of cytolytic T lymphocytes. The ovaries and endometrial epithelium exhibited normal architecture. No alterations were observed in body weight, lymphoid organ weight and histology, or splenic cellularity whereas significant changes were found in several immune functions at the 200 mg/kg dose. Proliferation of splenocytes to the mitogens LPS, PHA, and Con A was decreased by 39, 43, and 57%, respectively. T-lymphocyte-mediated cytolysis of tumor targets was decreased by 25% with a concurrent reduction of 45% in NK activity. There was no significant reduction in the number of IgM or IgG PFC in exposed animals. These data indicate that β-HCH causes nonestrogenic immune function changes in the adult mouse without gross changes in lymphoid organ weight, histology, or cellularity.

Keyhole limpet hemocyanin-propagated Peyer's patch T cell clones that help IgA responses.

Four T cell clones, isolated from Peyer's patches of keyhole limpet hemocyanin (KLH)-primed BALB/c mice, were selected on the basis of their ability to help IgA responses by TNP-KLH-primed BALB/c mouse B cells. Two were KLH-dependent both in terms of their own proliferative response and in terms of their help for that of B cells. The other two were autoreactive and helped B cells proliferate independently of the presence of Ag. Both primed and unprimed B cells proliferated to some extent when helped by the KLH-reactive clones in the presence of high concentrations of either KLH or TNP-KLH. Much higher proliferation was, however, induced when primed, but not unprimed, B cells were exposed to the T cells in the presence of low concentrations of TNP-KLH but not KLH, i.e., under conditions favoring direct, cognate interaction between the T and B cells. Only modest IgM, and no IgG or IgA plaque-forming cell (PFC) responses were generated by TNP-primed B cells upon interaction with either autoreactive T cells in the absence of Ag or KLH-reactive T cells in the presence of high concentrations of KLH. For high IgM responses as well as for the appearance of IgG and IgA PFC responses, TNP-KLH was required whatever the source of the T cell help. The isotype ratios depended on the TNP-KLH concentration; IgA responses were highest and IgM responses lowest at the lowest TNP-KLH concentrations suggesting that the precursors of the IgA PFC have higher average affinity for TNP than the precursors of IgM PFC. Overall, the results are compatible with the idea that the precursors of IgA and IgG PFC and many of the precursors of IgM PFC in the long term primed B cell populations used in these experiments require engagement of their Ag-receptors before they express sufficient class II Ag and/or receptors for "switch" and differentiation factors for cognate interaction with T cells leading to PFC responses.