Detection of nuclear protein antigens to antinuclear antibodies in serum of NOD mouse

Nuclear protein antigens to the antinuclear antibodies in serum of non-obese diabetic (NOD) mice were investigated. In the serum of diabetic NOD female mice (20 weeks old), the antinuclear antibodies were detected by indirect immunofluorescence assay using frozen sections of liver of C 57 BL/6 J or NOD mice as antigen. Nuclei were separated from the liver of C 57 BL/6 J mice and solubilized. Solubilized nuclear antigens were analyzed by SDS PAGE-Western immunoblotting techniques. Nuclear protein antigens with molecular weights of 26,000, 32,000 and 65,000 showed strongly positive reactions with the antinuclear antibodies in the serum of the NOD mouse.     

The nonobese diabetic mouse model. Independent expression of humoral and cell-mediated autoimmune features

Nonobese diabetic (NOD) mice present concomitant signs of cell-mediated and humoral autoimmunity. Whereas the involvement of the cell-mediated manifestations in the pathogenesis of diabetes has been clearly demonstrated, the origin and the relevance of the humoral manifestations is still unclear. In the present study, we have tried to determine whether the humoral manifestations observed in NOD mice were secondary to the cell-mediated antiislet reaction, or whether they resulted from an autonomous polyclonal activation of B cells, a possibility suggested by the notorious presence of antilymphocyte antibodies with thymocytotoxic properties, in the serum of old NOD females. To discriminate between the two alternatives, we have followed the titers of thymocytotoxic autoantibodies in aging males and females, as well as in F1 hybrids where the organ-specific disease is recessive, and in back-crossed mice where the susceptibility genes responsible for insulitis and diabetes have segregated. In addition to thymocytotoxic antibodies, we have also screened the sera of these animals for hyperglobulinemia, antiinsulin, and anti-DNA autoantibodies that are classically associated with polyclonal B cell activation in autoimmune strains of mice. The results indicate that these humoral anomalies are clearly disconnected from the occurrence of diabetes and even of insulitis. Lymphocytotoxic antibodies appear several weeks after the onset of insulitis in NOD mice, are not correlated with disease occurrence and have no predictive value for its onset. The humoral manifestations that include, beside thymocytotoxic antibodies, antiinsulin antibodies, hyperglobulinemia, but no anti-DNA antibodies, are found at the same frequency in F1 mice as in parental mice in spite of the fact that the former are practically free of insulitis lesions. These anomalies are also randomly distributed among back-crossed mice independently of the presence and the severity of the organ-specific lesions. Altogether, these results suggest that NOD mice, like other autoimmune strains, suffer from a genetically inherited defect of B cell regulation resulting in the hyperproduction of natural autoantibodies.     

Agonist-induced 4-1BB activation prevents the development of Sjӧgren's syndrome-like sialadenitis in non-obese diabetic mice

Activation of costimulatory receptor 4-1BB enhances T helper 1 (Th1) and CD8 T cell responses in protective immunity, and prevents or attenuates several autoimmune diseases by increasing Treg numbers and suppressing Th17 or Th2 effector response. We undertook this study to elucidate the impact of enforced 4-1BB activation on the development of Sjögren's syndrome (SS)-like sialadenitis in non-obese diabetic (NOD) model of this disease. An anti-4-1BB agnostic antibody was intraperitoneally injected to female NOD mice aged 7 weeks, prior to the disease onset that occurs around 10–11 weeks of age, 3 times weekly for 2 weeks, and the mice were analyzed for SS pathologies at age 11 weeks. The salivary flow rate was markedly higher in the anti-4-1BB-treated NOD mice compared to the IgG-treated controls. Anti-4-1BB treatment significantly reduced the leukocyte infiltration of the submandibular glands (SMGs) and the levels of serum antinuclear antibodies. Flow cytometric analysis showed that the percentages of CD4 T cells, Th17 cells and plasmacytoid dendritic cells among SMG leukocytes were markedly reduced by anti-4-1BB treatment, in conjunction with a reduction in SMG IL-23p19 mRNA levels and serum IL-17 concentrations. Although the proportion of Tregs and IL-10 mRNA levels in SMGs were not altered by 4-1BB activation, IL-10 mRNA levels in salivary gland-draining lymph nodes and serum IL-10 concentrations were both markedly increased. While anti-4-1BB treatment did not affect the amount of Th1 cells and IFNγ mRNA in the SMGs, it increased these measurables in salivary gland-draining lymph nodes. Hence, agonistic activation of 4-1BB impedes the development of SS-like sialadenitis and hyposalivation.     

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.     

Genetic analysis of diabetes in the nonobese diabetic mouse. I. MHC and T cell receptor beta gene expression

Backcross nonobese diabetic (NOD) ((NOD x SWr)F1 x NOD) mice (108 females and 105 males) were typed for MHC, TCR V beta, and monitored for 350 days for the onset of diabetes. The presence of "antipolar" antibodies in the sera and the occurrence of insulitis was examined in a proportion of these backcross mice. There was no difference in the incidence of diabetes in mice heterozygous for TCR V beta b/a vs those homozygous for TCR V beta b/b. Among the 17 diabetics (all female) detected in this backcross, 14/17 were H-2nod/nod but 3/17 were H-2nod/q. This supports a previous observation suggesting that the MHC-linked diabetogenic gene originally thought to be recessive may rather be dominant but have a low penetrance in the heterozygous state. Antipolar autoantibodies were found in both female and male backcross mice, and were similarly distributed in diabetic and nondiabetic mice. There appeared to be no correlation between the level of these auto-antibodies and development of diabetes. The incidence and severity of insulitis was linked to MHC but no influence of TCR genes on insulitis nor an association between insulitis and antipolar antibodies could be demonstrated in this study. Further analyses of H-2nod/nod intercross mice homozygous for TCR V beta a or TCR V beta b are currently underway.     

False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.     

Antibodies to extractable nuclear antigens in graft-vs-host F1 mice as determined by immunoblotting: absence of anti-Sm antibody

Antibodies to extractable nuclear antigens (ENA) were determined in autoimmune graft-vs-host F1 (GVH F1) mice by the immunoblotting method. Major populations of anti-ENA known in human sera including anti-Sm were not detected. It is thus conceived that different kinds of ENA proteins may be recognized by human autoimmune and GVH F1 sera.     

Australian Antigen and Autoantibodies in Chronic Hepatitis

The sera of 110 patients with chronic hepatitis and adequate controls were examined for antibodies to smooth muscle (S.M.), mitochondria (M.), and for antinuclear factors by the immunofluorescence method, and for Australia (Au(1)) antigen by a modified micro-Ouchterlony immunodiffusion technique. Twelve out of 13 patients with primary biliary cirrhosis had M. antibodies, two had antinuclear factor, and none had Au(1) in their sera. In chronic aggressive hepatitis 23·5% of the sera contained antinuclear factor, 13% S.M. antibodies, 10·5% M. antibodies, and 22% Au(1) antigen. Of the 12 patients with chronic persistent hepatitis, one had antinuclear factor, one S.M. antibodies, and three Au(1) antigen.The most striking finding was a mutual exclusion between Au(1) antigen and M. and S.M. antibodies. None of the 33 patients with one or the other form of chronic hepatitis and M. or S.M. antibodies had Au(1) antigen; 22 out of 77 (28%) patients without such antibodies were positive.     

Effects of sleep deprivation on the development of autoimmune disease in an experimental model of systemic lupus erythematosus

Sleep is hypothesized to play a restorative role on immune system. In addition, disturbed sleep is thought to impair host defense mechanisms. Chronic sleep deprivation is a common occurrence in modern society and has been observed in a number of chronic inflammatory conditions, such as systemic lupus erythematosus (SLE). New Zealand Black/New Zealand White (NZB/NZW) F1 mice develop an autoimmune disease that strongly resembles SLE in humans, exhibiting high titers of antinuclear antibodies associated with the development of rapidly progressive and lethal glomerulonephritis. On the basis of this evidence, the present study examined the onset and progress of lupus in as-yet healthy female mice submitted to sleep deprivation. Sleep deprivation was accomplished by two 96-h periods in the multiple-platform method when mice were 10 wk old, and they were observed until 28 wk of age. Blood samples were collected from the orbital plexus fortnightly to evaluate serum antinuclear antibodies and anti-double-stranded DNA. Proteinuria and longevity as well as body weight were also assessed. The results indicated that mice submitted to sleep deprivation exhibited an earlier onset of the disease, as reflected by the increased number of antinuclear antibodies. However, no statistical difference was found in the other parameters analyzed. According to these results, sleep deprivation could be considered as a risk factor for the onset but not for the evolution of the disease.     

Murine graft vs host disease. A model for study of mechanisms that generate autoantibodies to ribonucleoproteins

We established chronic graft vs host disease in (BALB/c x A/J) F1 mice with the injection of lymphoid cells from the parental A/J strain. These animals developed glomerulonephritis, forefoot edema, alopecia, splenomegaly, and lymphadenopathy to various degrees, and all developed antinuclear antibodies. To determine whether these antibodies were directed against the small nuclear ribonucleoprotein (snRNP) particles that are characteristic targets for autoimmune responses in human rheumatic diseases, sera were studied in the 32P immunoprecipitation and immunoblotting assays. Among 20 mice, antibodies to snRNP developed in 10. These antibodies usually reached maximal levels about 4 wk after induction of graft vs host disease and generally fell thereafter. However, two mice developed antibodies to snRNP between the 10th and 20th wk of follow-up. Sera from six mice strongly recognized the U1 snRNP and an additional serum strongly bound both the U1 and U3 particles. Several sera contained lower levels of antibodies specific for the U3 and possibly pre-U2 snRNP particles. In immunoblots, sera that immunoprecipitated the U1 snRNP bound epitopes located on its 70,000 Da, A, B'/B, and/or C polypeptides. Sera that immunoprecipitated the U3 snRNP recognized a 34,000-Da polypeptide. These polypeptides are known to bear the autoantigenic epitopes that are recognized by human sera containing anti-U1 RNP and anti-U3 RNP autoantibodies. We conclude that chronic graft vs host disease in mice provides a model for the study of the autoimmune responses that characterize human diseases such as mixed connective tissue disease, scleroderma, and SLE.     

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice

BACKGROUND: Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. METHODS: HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. RESULTS: Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. CONCLUSIONS: We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.     

Schistosoma mansoni: immunoblot analysis of adult worm proteins

Proteins of adult Schistosoma mansoni were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 31-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-rad-irradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes.     

Maternal immunization with both hemagglutinin- and neuraminidase-expressing DNAs provides an enhanced protection against a lethal influenza virus challenge in infant and adult mice

Maternal immunization is the major form of protection against many infectious diseases in early life. In this report, transmission of vaccine-specific maternal antibodies and protection of offspring against a lethal influenza virus challenge were studied. Adult female BALB/c mice were immunized intramuscularly with plasmid DNAs encoding influenza virus hemagglutinin (HA), neuraminidase (NA), or mixture of the two plasmids. The levels of specific antibodies in sera of offspring at different ages and the survival rates following the lethal viral challenge were valued. The results showed effective transmission of maternal antibodies and long-lasting protection in offspring. Along with the growth of offspring, the antibody titers in vivo decreased and the ability against virus infection decreased accordingly. The HA-specific maternal antibodies protected the offspring from a lethal influenza infection up to 2 weeks old, and the NA-specific maternal antibodies protected offspring up to 4 weeks old. Furthermore, antibodies transferred by the mother immunized with the mixture of HA and NA DNAs protected the offspring up to 6 weeks old. This suggests that maternal immunization with a mixture of HA and NA DNAs provide the most effective protection against the virus challenge for the offspring of mice.     

Transfer of diabetes from prediabetic NOD mice to NOD-SCID/SCID mice: association with pancreatic insulin content.

Splenocytes from prediabetic female NOD mice can transfer diabetes to NOD-SCID mice. Whereas the kinetics of disease transfer was shown to be a function of the age of donor splenocytes, information is scarce as to how the stage of autoimmune disease, as evaluated by pancreatic insulin content, is related to the diabetogenic potency of splenic T-cells. We therefore determined individual diabetes transfer times after an i. v. injection of splenocytes from prediabetic NOD mice of different ages into female NOD-SCID mice in relation to the diabetes incidence in NOD donor mice and their pancreatic insulin contents. Three groups (n = 8) of NOD mice aged 5, 11, and 17 weeks (wk) underwent splenectomy and hemipancreatectomy. After that, 10x10 (6) splenocytes either pooled from all donor NOD mice of the different age groups or individually from single donor mice were transferred to groups of four 6-week-old NOD-SCID mice, respectively, in two sets of experiments. Insulin was extracted from the resected hemipancreas, and the insulin content was determined by a RIA. Diabetes in the NOD-SCID cohort occurred after a mean time of 126 days after transfer of pooled splenocytes from 5-week-old NODs, after 68 days (transfer from 11-week-old NODs), and after a mean time of 43 days (transfer from 17-week-old NODs, 5 vs. 11 wk: p < 0.02, 11 vs. 17 wk: p < 0.001). Individual time to diabetes positively correlated with diabetes transfer times in NOD-SCID recipients (p < 0.0001) in the 17-week-old NOD mice, confirming previous diabetes transfer studies in hemi-pancreatectomized NOD mice. Furthermore, individual insulin concentrations in 17-week-old NOD mice also positively correlated to diabetes transfer times in recipient mice (p < 0.0001). No such correlations for these parameters were seen for the 5 and 11-week-old NOD mice (time to diabetes: 11 wk, p = 0.14, 5 wk, p = 0.75; insulin content: 11 wk, p = 0.81, 5 wk, p = 0.14). These data suggest that destructive T-cell activity increases during the course of islet autoimmunity. The immune response seems to be programmed for beta-cell destruction just before diabetes onset. This is the only time that pancreatic insulin content predicts the impending onset of diabetes.     

Immune-Mediated Fever in the Dog. Occurrence of Antinuclear Antibodies, Rheumatoid Factor, Tumor Necrosis Factor and Interleukin-6 in Serum

Contents of antinuclear antibodies (ANA), rheumatoid factor (RF), tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were measured in serum from 20 dogs with immune-mediated fever. Seven out of 20 patients were ANA positive, 1 out of 20 was positive to antibodies against extractable nuclear antigens (ENA), 1 out of 20 was positive to antibodies against deoxynucleoproteins (DNP), 2 out of 13 were RF positive and none out of 20 patients had antibodies against native DNA in the serum. TNF-α was not detected in any serum of 15 dogs with immune-mediated fever, while 10 out of 13 presented with elevated IL-6. The results varied between patients, but the IL-6 level was high in most of them. This indicate a role for IL-6 in the pathogenesis of immune-mediated fever in most cases.     

Contribution of different mechanisms to pancreatic beta-cell hyper-secretion in non-obese diabetic (NOD) mice during pre-diabetes

The development of insulin-dependent diabetes mellitus (IDDM) results from the selective destruction of pancreatic beta-cells. Both humans and spontaneous models of IDDM, such as NOD mice, have an extended pre-diabetic stage. Dynamic changes in beta-cell mass and function during pre-diabetes, such as insulin hyper-secretion, remain largely unknown. In this paper, we evaluated pre-diabetic female NOD mice at different ages (6, 10, and 14 weeks old) to illustrate alterations in beta-cell mass and function as disease progressed. We found an increase in beta-cell mass in 6-week-old NOD mice that may account for improved glucose tolerance in these mice. As NOD mice aged, beta-cell mass progressively reduced with increasing insulitis. In parallel, secretory ability of individual beta-cells was enhanced due to an increase in the size of slowly releasable pool (SRP) of vesicles. Moreover, expression of both SERCA2 and SERCA3 genes were progressively down-regulated, which facilitated depolarization-evoked secretion by prolonging Ca(2+) elevation upon glucose stimulation. In summary, we propose that different mechanisms contribute to the insulin hyper-secretion at different ages of pre-diabetic NOD mice, which may provide some new ideas concerning the progression and management of type I diabetes.     

A new autoantigen reactive with prediabetic nonobese diabetic mice sera.

The identification and characterization of new autoantigens would widen the knowledge of the pathogenic mechanism of insulin dependent diabetes mellitus. Screening of lambda gt11 mouse insulinoma (MIN6N8a) cell cDNA library with prediabetic nonobese diabetic (NOD) mice sera resulted in the isolation of a strong positive clone, named the clone 3-5, of 1579 nucleotides without a poly A region. After 5'-rapid amplification of the cDNA end (RACE), complete nucleotide sequence of the clone 3-5 gene consisting of 2231 nucleotides showed that the 3-5 gene had the theoretical open reading frame of 634 amino acids. However, the real antigenic protein of the clone 3-5 was only 21 amino acids long encoded by only 63 nucleotides. The 21 amino acids were expressed as a fusion protein in E. coli and purified by affinity chromatography. The purified 3-5 recombinant protein was examined for its reactivity with prediabetic NOD mice sera by immunoblotting. The only non-denatured form of the 3-5 protein showed a binding reactivity with NOD mice sera, demonstrating that the conformational epitope of 3-5 protein was important for antibody recognition. The prevalence of autoantibody reactive to the 3-5 protein was about 78% (14/18) and 46% (11/24) in prediabetic and acute diabetic NOD mice sera, respectively. However, the sera from other mouse strains such as BALB/c, ICR, C57BL/6, SJL/J, and NOD/SCID did not show a positive reactivity to the 3-5 protein, which indicated that immune reactivity against the 3-5 protein was autoimmune diabetic mouse-specific.     

A comparison of two antigen strains of each of mouse hepatitis virus and mouse adenovirus for detection of complement fixation antibody in mouse and rat sera

Detection rates of complement fixation antibodies in mice and rats were compared between two antigen strains of each of mouse hepatitis virus (MHV) and mouse adenovirus (MAV). Among 66 and 47 naturally infected MHV-positive sera of mice (18 facilities) and rats (16 facilities) respectively, 17 mouse and 21 rat sera reacted with both Nu-67 and MHV-2 strains, but 49 mouse and 25 rat sera were positive to Nu-67 strain alone. Only one rat serum reacted with MHV-2 strain alone. In comparison with K87 and FL strains of MAV, all 8 positive mouse sera (3 facilities) reacted with K87 strain alone whereas out of 53 positive rat sera (20 facilities), 43, 6 and 4 sera reacted with K87 strain alone, with FL strain alone and with both the two strains, respectively.     

Inactivated SARS-CoV vaccine prepared from whole virus induces a high level of neutralizing antibodies in BALB/c mice

We tested the ability of inactivated SARS-CoV vaccine to induce neutralizing antibodies in BALB/c mice. The inactivated vaccine was prepared by SARS-CoV virus propagation in Vero cells, with subsequent beta-propiolactone inactivation and Sepharose 4FF column chromatography purification. One hundred forty BALB/c female mice were divided into seven groups of 20 mice each. Of the seven groups, three groups were inoculated with 0.1, 1, and 3 microg of the vaccine without adjuvant while three other groups were inoculated at the same three dosages of vaccine with aluminum hydroxide as adjuvant, respectively. The remaining group was set up as a blank control. Each mouse was inoculated twice at an interval of 3 weeks. One week after the second immunization, mice sera were collected to detect serum neutralizing antibodies. An assay for determining neutralizing antibody titers was developed. The results can be summarized as follows: (1) higher dosages of vaccine induced higher levels of neutralizing antibody titer; (2) the level of neutralizing antibodies induced by the inoculation with aluminum hydroxide adjuvant was slightly higher than that without adjuvant, but the difference was not statistically significant.     

Anti-idiotypic T lymphocyte responsiveness in murine Schistosomiasis mansoni

Pooled sera from CBA/J mice infected for greater than or equal to 16 weeks with the blood fluke Schistosoma mansoni were immunoaffinity purified using soluble schistosome egg antigens (SEA) coupled to Sepharose 4B. The bound and then eluted fraction was shown to contain only immunoglobulins and to have anti-SEA activity. These anti-SEA antibodies stimulated proliferation of lymph node cells from mice infected with S. mansoni for 8, 12, or greater than or equal to 16 weeks but not from uninfected mice. The cells stimulated by anti-SEA antibodies were nylon wool adherent, Thy-1.2+, L3T4+, Lyt-2-lymphocytes. Immunoglobulins without anti-SEA activity isolated from the sera of syngeneic uninfected mice were not stimulatory for cells from normal or infected animals. Thus the responding T cells appear to be stimulated by the idiotypes expressed on the syngenic anti-SEA antibodies. These data present evidence for anti-idiotypic cellular reactions in murine schistosomiasis that could play important immunoregulatory roles in this disease.