A novel class of anti-DNA antibodies identified in BALB/c mice

We have characterized four IgG monoclonal antibodies (mAbs) derived from BALB/c mice that bind double-stranded DNA (dsDNA) with high affinity. The hydridomas were selected for expression of a member of the VHS107 family. Three of the four cell lines use the VH11 gene and one uses the VH1 gene. These antibodies exhibit many characteristics of pathogenic anti-DNA antibodies. They are high affinity and not broadly crossreactive. Unlike the anti-DNA antibodies in autoimmune mice, they exhibit no somatic mutation in their VH genes. These results demonstrate that somatic mutation of VHS107 genes is not necessary for generating high affinity dsDNA binding. The fact that such antibodies have not previously been reported suggests that they are rare and that their expression may be downregulated in both nonautoimmune and autoimmune individuals.     

Protective role of fenofibrate in sepsis-induced acute kidney injury in BALB/c mice

Acute kidney injury (AKI) is a severe complication of sepsis, which largely contributes to the associated high mortality rate. Fenofibrate, a peroxisome proliferator activated receptor α (PPARα) agonist, has received considerable attention because of its effects related to renal damage-related energy metabolism and inflammation. The present study investigated the effects of fenofibrate on sepsis-associated AKI in BALB/c mice subjected to caecal ligation and puncture (CLP). Eight-week-old male BALB/c mice were divided into four groups: control group, fenofibrate group, caecal ligation and puncture (CLP) group, and fenofibrate + CLP group. CLP was performed after mice were gavaged with fenofibrate for 2 weeks. After 48 hours, we measured the histopathological alterations of the kidney tissue and plasma levels of serum creatinine (CRE), neutrophil gelatinase-associated lipocalin (NGAL), reactive oxygen species (ROS), ATP, and ADP. We evaluated PPARα and P53 protein levels as well as interleukin (IL)-1β, IL-6, and tumour necrosis factor-α mRNA levels. Our results showed that administering fenofibrate significantly reduced kidney histological alterations caused by CLP. Fenofibrate inhibited the plasma levels of ROS, CRE, NGAL, and increased the ATP/ADP ratio. Fenofibrate significantly inhibited elevations in P53, IL-1β, IL-6, and tumour necrosis factor-α expression. The results suggest that fenofibrate administration effectively modulates energy metabolism and may be a novel approach to treat sepsis-induced renal damage.

Acute kidney injury (AKI) is a severe complication of sepsis, which largely contributes to the associated high mortality rate.     

Experimental autoimmune peripheral neuritis induced in BALB/c mice by myelin basic protein-specific T cell clones

In vivo adoptive transfer of CD4+ T helper cell type 1 clones reactive with autologous myelin basic protein (MBP) may initiate an inflammatory demyelinating disease of the central nervous system called experimental autoimmune encephalomyelitis. Although MBP is also a component of peripheral nervous system (PNS) myelin, previous studies have failed to demonstrate inflammation in the PNS induced by MBP-reactive T cells. Here, we report on two MBP-specific T cell clones that preferentially initiate inflammatory and demyelinating peripheral neuritis when adoptively transferred to syngeneic recipients. The MBP epitope recognized by these clones spans the junction of exons 6 and 7 and, therefore, is present in the 21- and 18.5-kD but not the 14- and 17-kD MBP isoforms, in which exon 5 is spliced to exon 7. The data suggest that MBP may be processed and presented differently in the central nervous system and PNS, and they provide evidence for MBP as a potential target for autoimmune reactions in the PNS.     

Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice

We have previously shown that continuous administration of anti- interleukin 10 (anti-IL-10) antibodies (Abs) to BALB/c mice modifies endogenous levels of autoantibodies, tumor necrosis factor alpha (TNF- alpha), and interferon gamma, three immune mediators known to affect the development of autoimmunity in "lupus-prone" New Zealand black/white (NZB/W)F1 mice. To explore the consequences of IL-10 neutralization in NZB/W F1 mice, animals were injected two to three times per week from birth until 8-10 mo of age with anti-IL-10 Abs or with isotype control Abs. Anti-IL-10 treatment substantially delayed onset of autoimmunity in NZB/W F1 mice as monitored either by overall survival, or by development of proteinuria, glomerulonephritis, or autoantibodies. Survival at 9 mo was increased from 10 to 80% in anti- IL-10-treated mice relative to Ig isotype-treated controls. This protection against autoimmunity appeared to be due to an anti-IL-10- induced upregulation of endogenous TNF-alpha, since anti-IL-10- protected NZB/W F1 mice rapidly developed autoimmunity when neutralizing anti-TNF-alpha Abs were introduced at 30 wk along with the anti-IL-10 treatment. Consistent with the protective role of anti-IL-10 treatment in these experiments, continuous administration of IL-10 from 4 until 38 wk of age accelerated the onset of autoimmunity in NZB/W F1 mice. The same period of continuous IL-10 administration did not appear to be toxic to, or cause development of lupus-like autoimmunity in normal BALB/c mice. These data suggest that IL-10 antagonists may be beneficial in the treatment of human systemic lupus erythematosus.     

Molecular basis of Escherichia coli colonization of the upper urinary tract in BALB/c mice. Gal-Gal pili immunization prevents Escherichia coli pyelonephritis in the BALB/c mouse model of human pyelonephritis.

Most human pyelonephritis Escherichia coli isolates express both mannose (MS)- and globoside (Gal-Gal)-binding pili. An ascending E. coli urinary tract infection model was established in the 16-wk-old female BALB/c mouse to compare the pathogenic significance of MS and Gal-Gal pili and their efficacy as vaccines for the prevention of pyelonephritis. The distribution and density of pilus receptor compounds in urogenital tissues and as soluble compounds in urine were determined with antibodies to the synthetic receptor analogues, alpha D-Gal(1----4) beta D-Gal and alpha D-Man(1----2) alpha D-Man. Both carbohydrates were detected in vagina, bladder, ureter, and renal pelvis epithelium and in collecting duct and tubular cells. A pilus receptor compound also was detected in urine. It competitively inhibited the binding capacity of MS pili and was found to be physically, chemically, and immunologically related to Tamm-Horsfall uromucoid. Infectivity and invasiveness were quantitatively and histologically characterized for four E. coli strains: J96, a human pyelonephritis strain that expresses both MS and Gal-Gal pili; two recombinant strains prepared from J96 chromosomal DNA encoding MS pili or Gal-Gal pili; and the nonpiliated K12 recipient. Intravesicular administration of J96 (10(6) colony-forming units [CFU]) resulted in renal colonization and invasion in each of nine mice. The Gal-Gal clone (10(6) CFU) colonized the kidneys in each of 10 mice but did not invade. In contrast, the MS clone (10(6) CFU) did not colonize renal epithelium or invade. This effect was superceded when larger doses (greater than or equal to 10(10) CFU) of the MS clone were administered in volumes that cause acute vesicoureteric reflux. The efficacy was determined of vaccines composed of pure MS or Gal-Gal pili or the lipopolysaccharide containing O somatic antigen of the challenge strain, J96. The Gal-Gal pilus vaccine blocked renal colonization in 19 of 22 mice and renal invasion in 10 of 11 mice. Gal-Gal pili may be useful immunogens for the prevention of pyelonephritis in anatomically normal urinary tracts.     

Safety and efficacy of various combinations of injectable anesthetics in BALB/c mice.

Four combinations of drugs--ketamine-xylazine, ketamine-xylazine-acepromazine (KXA), ketamine-xylazine-buprenorphine, and ketamine-xylazine-carprofen--were compared for their ability to produce anesthesia in BALB/c mice. Induction time, anesthetic duration, blood pressure, pulse rate, and time to recovery were recorded. The anesthesia induced by each anesthetic combination was assessed by using reflex responses to standardized stimuli. The KXA combination produced stable physiologic parameters and was associated with the longest duration of anesthesia (40 +/- 8 min); immobility was produced in all other groups (38 +/- 5 min), but a surgical plane of anesthesia could not be confirmed. All anesthetic protocols produced significant hypotension. No deaths occurred. We recommend KXA as a safe and reliable anesthetic for mice requiring a surgical plane of anesthesia.     

Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice.

Immunization with cardiac myosin induces T cell-mediated myocarditis in genetically predisposed mice and serves as a model for autoimmune heart disease. This study was undertaken to identify pathogenic epitopes on the myosin molecule. Our approach was based on the comparison of the pathogenicity between cardiac (alpha-)myosin and soleus muscle (beta-)myosin. We show that alpha-myosin is the immunodominant isoform and induces myocarditis at high severity and prevalence whereas beta-myosin induces little disease. Therefore the immunodominant epitopes of alpha-myosin must reside in regions of different amino acid sequence between alpha- and beta-myosin isoforms. Cardiac myosin peptides corresponding to these regions of difference were synthesized and tested for their ability to induce inflammatory heart disease. Three pathogenic peptides were identified. One peptide that is located in the head portion of the molecule induced severe myocarditis, whereas two others that reside in the rod portion possessed only minor pathogenicity. The identification of pathogenic epitopes on the cardiac myosin molecule will allow detailed studies on the recognition of this antigen by the immune system and might be used to downmodulate ongoing heart disease.     

Enhanced induction of SARS-CoV nucleocapsid protein-specific immune response using DNA vaccination followed by adenovirus boosting in BALB/c mice

OBJECTIVE: To investigate immunogenicity in the induction of humoral and cellular immune responses to genetic vaccines of the recombinant severe acute respiratory syndrome-associated coronavirus (SARS-CoV)-N gene expressing the same protein plasmid, pcDNA3.1-N, and replication-defective adenoviral vector, rAd-N, in a pcDNA3.1-N prime-rAd-N boost regimen and the reverse sequence in a rAd-N prime-pcDNA3.1-N boost regimen. METHOD: After the mice had been immunized intramuscularly and/or intraperitoneally with pcDNA3.1-N and rAd-N in prime-triple boost immunization, humoral and cellular immune responses were detected. RESULTS: After detection, different levels of anti-N humoral and cellular responses are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen and the reverse sequence of heterogeneous combinations. There is a significant difference between heterogeneous and homologous vaccinations. However, the cytotoxic T lymphocyte (CTL) response was not significantly altered by the different prime-boost immunizations or the recombinant adenovirus of pcDNA3.1-N prime-rAd-N boost regimen alone, but lymphoproliferation and interferon-gamma (IFN-gamma) secretion were all enhanced by heterologous combination immunizations compared to homologous combinations. For the reverse sequence immunization regimen, lymphoproliferation, IFN-gamma and CTL responses were all significantly weaker compared with pcDNA3.1-N prime-rAd-N boost regimen. CONCLUSION: Taken together, of all the combinations, the prime-triple boost immunization of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N can effectively induce SARS-CoV-N-specific and strong humoral and cellular immune responses in mice. The present results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine in the induction of an enhanced humoral and cellular immune response.     

Values of the U.S. National Reference Serum for human antibodies to native DNA obtained with commercial immunoassays for anti-DNA in systemic lupus erythematosus

The Arthritis Foundation and the Centers for Disease Control have recently prepared a "U.S. National Reference Serum" for human antibodies to native DNA. We tested this serum with 13 commercial assays for antibodies to native DNA, to permit comparisons of the values obtained in each test. Titers ranged from 10 to 2560 in Crithidia luciliae immunofluorescence assays. The serum produced 794 int. units/mL in the Cordis ELISA assay, 136 Amersham units/mL in a radioimmunoassay, and 88 FIAX units in a fluorometric immunoassay. These results can be used for interlaboratory comparisons of differing methodologies for measuring anti-DNA.     

(NZW x BXSB)F1 hybrid. A model of acute lupus and coronary vascular disease with myocardial infarction

Both sexes of the (NZW x BXSB)F1 mice developed an early systemic lupus erythematosus-like disease. In males, the disease resembled that in the BXSB male parent and was not affected by sex hormonal manipulation. In females, the disease duplicated that of (NZB x NZW)F1 females by virtue of a delayed onset and estrogen dependence. Autoantibody production, circulating Ig-bound gp 70 immune complexes, and deposition of Ig and gp 70 in the affected glomeruli were demonstrated in both males and females. The abnormally high incidence of degenerative coronary vascular disease with myocardial infarction, particularly in these F1 males, provides a useful model for the investigation of a possible immunologic component in coronary vascular disease.     

Efficacy of ciprofloxacin and moxifloxacin against Nocardia brasiliensis in vitro and in an experimental model of actinomycetoma in BALB/c mice

The efficacy of ciprofloxacin and moxifloxacin against Nocardia brasiliensis was evaluated by applying 25 mg of each drug/kg subcutaneously every 8 h in BALB/c mice infected with N. brasiliensis. A statistically significant difference was observed only with moxifloxacin. A moxifloxacin-trimethoprim-sulfamethoxazole combination was as active as when each compound was used alone.     

Regulation of azophenylarsonate-specific repertoire expression. 1. Frequency of cross-reactive idiotype-positive B cells in A/J and BALB/c mice

A large proportion of p-azophenylarsonate (ARS)-specific antibodies from A/J mice share a cross-reactive idiotype (CRIA) that comprises a family of closely related but nonidentical clonotypes. I determined that only 2.6 % (7 out of 267) A/J ARS-specific monoclonal antibodies generated in the splenic focus system possess the predominant CRIA. Because ARS-specific B cells are present at a frequency of 1/68,000 B cells, the frequency of the entire idiotype family is 1 per 2.8 X 10(6) splenic B cells. Thus, there is a striking discrepancy between the representation of this idiotype at the clonal precursor cell level and the serum antibody response. In addition, BALB/c mice have the potential to generate CRIA-positive precursor cells within their nonimmune repertoire. When A/J mice are immunized with ARS-protein conjugates, the serum antibody response and precursor cell population are both dominated by CRIA. The frequency of CRIA-positive B cells increases over 100-fold after immunization, whereas CRIA-negative precursor cells may initially decrease, followed by a later rise in frequency. Finally, although ARS-specific precursor cells are present in high frequency at birth, CRIA-positive monoclonal anti-ARS antibodies are not observed during the early neonatal period. These data provide evidence to suggest that complex regulatory networks influence precursor cell and serum antibody expression.     

Suppression of NZB/NZW murine nephritis by administration of a syngeneic monoclonal antibody to DNA. Possible role of anti-idiotypic antibodies.

Suppression of circulating antibodies to double-stranded DNA was achieved in NZB/NZW f1 female mice by repeated administration of an IgG2a monoclonal antibody to DNA. Deaths from nephritis were delayed; glomerular deposition of IgG and of the cationic IgG DNA antibodies characteristic of murine lupus nephritis were diminished. Quantities of circulating antibodies to single-stranded DNA were not reduced compared with untreated or IgG myeloma-treated control mice. Antibodies directed against the monoclonal anti-DNA appeared in the circulation of treated mice after three inoculations of the idiotype. Those antibodies did not react with another monoclonal anti-DNA of the same allotype. One monoclonal anti-idiotypic antibody was obtained in hybridoma cultures derived from a spleen of a treated mouse. Cross-reactive or common idiotypes were found in 30-50% of NZB/NZW f1 sera and monoclonal DNA antibodies. Deletions of portions of the spectrotype of antibodies to DNA were found in sera containing anti-idiotypic antibodies, suggesting suppression of clones producing antibodies with isoelectric points similar to that of the immunizing idiotype. Deletions of some of the anti-idiotypic antibodies also occurred as the mice aged. Rheumatoid factors were not detectable in any sera. Therefore, administration of an antibody to DNA bearing an idiotype occurring with high frequency in NZB/NZW f1 females resulted in relatively specific suppression of the antibody response to double-stranded DNA, as well as suppression of nephritis. Reduction of anti-DNA synthesis by anti-idiotypic antibodies may have been an important suppressive mechanism. Experiments are in progress to test this hypothesis.     

Structural characterization of a novel Lactarius volemus Fr. polysaccharide and its immunity activity in BALB/c mice

Lactarius volemus Fr. has been regarded as a great edible medicinal fungal resource in China. In this study, L. volemus Fr. polysaccharide (LVP) with an average molecular weight of 16.842 kDa was obtained by water extraction. The structure of LVP was characterized to be mannan, and the linkages in the mannan were found to comprise the Manp, (1→4)-α-Man and (1→4,6)-α-Man. Furthermore, intraperitoneal administration of LVP increased the thymus, spleen and liver indices, dose-dependently. Additionally, LVP enhanced the immune response and the phagocytic activities. Pathological evaluations showed that LVP in mice increased the proliferation of red medullary lymphocytes (60–70%). Collectively, these results indicated that LVP might be a potential resource of raw material for further investigations of functional foods.

Lactarius volemus Fr. has been regarded as a great edible medicinal fungal resource in China.     

Alteration of clonal profile. I. Effect of sublethal irradiation on the responses to phosphorylcholine in BALB/c mice

BALB/c mice exhibit greater than 90% H8 clonal dominance in the immune response to phosphorylcholine. Adult mice exposed to 500 rads were initally unable to produce a humoral immune response to both phosphorylcholine and trinitrophenol antigens, and the direct plaque- forming cell response was slowly regained over several weeks. Clonotypic analysis wity antisera directed against the H8 idiotype showed that the H8 clone initially dominated the recovery of the response to phosphorycholine but that 60 days after the irradiation significant numbers of non-H8 clones could be detected. This same pattern could be seen in mice irradiated with 100 rads, a dose that does not completely abrogate the H8 response to phosphorylcholine. Sublethal irradiation of neonates before they had acquired responsiveness to phosphorylcholine could also eventually lead to the emergence of non-H8 idiotypes. Thus, a radiosensitive element regulates the expression of clonal dominance in anti-phosphorylcholine responses of BALB/c mice.     

Repeated oral administration of chitosan/DNA nanoparticles delivers functional FVIII with the absence of antibodies in hemophilia A mice

Summary. Background: Current treatment of hemophilia A is expensive and involves regular infusions of factor (F)VIII concentrates. The supply of functional FVIII is further compromised by the generation of neutralizing antibodies. Thus, the development of an alternative safe, cost effective, non‐invasive treatment that circumvents immune response induction is desirable. Objectives: To evaluate the feasibility of oral administration of chitosan nanoparticles containing FVIII DNA to provide sustainable FVIII activity in hemophilia A mice. Methods: Nanoparticles were characterized for morphology, DNA protection and transfection efficiency. Oral administration of nanoparticles containing canine FVIII in C57Bl/6 FVIII^?/? hemophilia A mice was evaluated for biodistribution, plasma FVIII activity and phenotypic correction. Sustainable FVIII expression was elucidated after repeated nanoparticle administration. Immune responses to repeated oral nanoparticle administration were also investigated. Results: Chitosan nanoparticles had a particle size range of 200–400 nm and protected DNA from endonuclease and pH degradation. In addition, nanoparticles transfected HEK 293 cells resulted in expression of eGFP, luciferase and FVIII. Hemophilia A mice that ingested chitosan nanoparticles demonstrated transient canine FVIII expression reaching > 100 mU 1 day after treatment, together with partial phenotypic correction. The delivered FVIII plasmid DNA was detected in the intestine and, to a lesser extent, in the liver. Importantly, repeated weekly administrations restored FVIII activity. Furthermore, inhibitors and non‐neutralizing FVIII antibodies were not detectable. Conclusions: Repeat oral administration of FVIII DNA formulated in chitosan nanoparticles resulted in sustained FVIII activity in hemophilic mice, and thus may provide a non‐invasive alternative treatment for hemophilia A.     

Idiotype-antiidiotype regulation. V. The requirement for immunization with antigen or monoclonal antiidiotypic antibodies for the activation of beta 2 leads to 6 and beta 2 leads to 1 polyfructosan-reactive clones in BALB/c mice treated at birth with minute amounts of anti-A48 idiotype antibodies

The anti-beta 2 leads to 6 fructosan antibodies sharing the idiotypes (Id) of ABPC48 (A48) monoclonal protein represent a silent fraction of the anti-beta 2 leads to 6 fructosan repertoire, since these antibodies cannot be detected during a conventional immune response elicited by bacterial levan (BL). However, the administration at birth of minute amounts of anti-A48 Id antibodies causes a long-lasting activation of A48 Id+-bearing clones. This activation is related to direct interaction of anti-A48 Id antibodies with precursors bearing the A48 Id+ immunoglobulin receptor, since an A48 Id+ response can be transferred with highly purified B cells in lethally irradiated mice. The maturation of these precursors into A48 Id+ anti-beta 2 leads to 6 fructosan antibody-secreting cells requires challenge by the antigen. Isoelectric focusing (IEF) data showed that in 1-mo-old mice an UPC10 (U10)-like spectrotype was observed, whereas in 3-mo-old mice, a new spectrotype binding BL rather than inulin (In) was identified. This spectrotype was observed only in CXBJ mice, the single strain in which an A48 Id+ response was observed. The antigenic challenge can be replaced by a monoclonal anti-A48 Id antibody (i.e., 17-38). Interestingly, in 1-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of A48 Id- anti-beta 2 leads to 6 fructosan-reactive clones with BALB/c type IEF spectrotypes, whereas in 3-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of W3082 IdX+ anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan-reactive clones. In these animals, inhibition of A48 Id+ anti-beta 2 leads to 6 fructosan clones was observed. This antibody probably represents a homobody carrying the internal image of the antigen, which through its paratope suppresses the A48 Id+ response and through its Id activates an A48 Id- anti-beta 2 leads to 6 fructosan response in 1-mo-old mice and in 3-mo-old mice leads to an anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan response dominated by the W3082 IdX.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the substantia nigra and the locus coeruleus in BALB/c mice.

The purpose of the present study was to determine whether 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), administered systemically or as a local infusion, has a direct neurotoxic action upon dopamine (DA) neurons in the substantia nigra (SN) and norepinephrine (NE) neurons in the locus coeruleus (LC) in BALB/c mice. Results indicated that both acute and repeated MPTP infusions (2 micrograms/0.3 microliter per side) significantly impaired locomotor activity, decreased stereotyped behavior and caused a disturbed pattern of locomotion in mice. The biochemical changes parallel the behavioral changes. Repeated MPTP infusions to the SN decreased DA levels markedly in the SN and the striatum; chronic MPTP infusions to the LC reduced NE levels markedly in the LC and the hippocampus. Furthermore, repeated MPTP injections for 7 days (30 mg/kg, one injection per day) have resulted in a long-lasting effect on both the nigral-striatal and the coeruleus-hippocampal systems. DA levels in the SN and the striatum were decreased at 1, 3, 7 and 28 days after the last MPTP injection. Similarly, NE levels in the LC and the hippocampus were also reduced markedly at the same time intervals examined. Behaviorally, repeated MPTP treatment also produced long-lasting motor deficits in mice at all time intervals studied. Moreover, the LC appeared to be more sensitive than the SN to the neurotoxic effects of MPTP. Immunohistochemical results have similarly revealed that repeated MPTP treatment markedly decreased tyrosine hydroxylase-positive cells and fibers in the SN and the LC. It also markedly decreased DA-beta-hydroxylase-positive cells and fibers in the LC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic control of specific immune suppression. I. Experimental conditions for the stimulation of suppressor cells by the copolymer L- glutamic acid50-L-tyrosine50 (GT) in nonresponder BALB/c mice

In the present studies we have confirmed that the random copolymer of L- glutamic acid50-L-tyrosine50 (GT) fails to induce an antibody response in a large number of inbred strains of mice. Nevertheless, GT complexed to methylated bovine serum albumin (MBSA) elicits a GT-specific IgG PFC response in vivo. Furthermore, injection of BALB/c mice with 10 to 100 mug of GT specifically decreases their ability to develop anti-GT PFC responses to a subsequent challenge with GT-MBSA. GT-specific tolerance can be transferred to normal, syngeneic recipients by spleen cells or thymocytes of GT-primed animals. These results indicate that the stimulation of suppressor cells can be observed in nonresponder mice with another synthetic polypeptide besides GAT. Various parameters of GT-specific immunosuppression in BALB/c mice are described. The application of these techniques to the study of the genetic factors controlling the stimulation of specific immune suppression is discussed.     

Genotypic analysis of B cell colonies by in situ hybridization. Stoichiometric expression of three VH families in adult C57BL/6 and BALB/c mice

The filter paper disc method for cloning inducible lymphocytes was used to census the splenic B cell population of C57BL/6 and BALB/c mice for the expression of three VH gene-families, VH X-24, -Q52, and -J558. B cell colonies, arising from single founder lymphocytes, were identified by in situ hybridization with VH family- and C mu-specific cDNA probes. Some 6.7 X 10(4) C mu+ colonies were screened. Among C57BL/6- or BALB/c-derived colonies, approximately 3% were VH X-24+, approximately 19% were VH Q52+, and approximately 54% were VH J558+. These frequencies are consistent with a process of equiprobable expression for individual VH segments, and provide direct evidence that normal splenic B lymphocytes use a process of random genetic combinatorics to generate the antibody repertoire.