B cell selection and allelic exclusion of an anti-DNA Ig transgene in MRL-lpr/lpr mice

We have used an Ig transgene (VH3H9) that increases the frequency of anti-DNA autoantibodies to address whether the production of antinuclear Abs in systemic lupus erythematosus is the consequence of a breakdown of B cell tolerance. We have shown that nonautoimmune mice regulate anti-DNA B cells, and that lupus-prone MRL-lpr/lpr mice are defective in this regulation. Here we show that a subset of anti-DNA B cells, namely those that stain nuclei in a homogeneous fashion, not only fail to be deleted in MRL-lpr/lpr mice, but undergo preferential clonal expansion. In addition, we describe a surprising finding: the VH3H9 transgene is less efficient at inhibiting endogenous heavy chain gene rearrangement on the autoimmune-prone MRL-lpr/lpr genetic background than on the nonautoimmune BALB/c background.     

Incidence of autoantibodies in the infertile population

The diagnosis of histoplasmosis was made by isolation of Histoplasma capsulatum from cerebrospinal fluid (CSF), blood and bone marrow or by histopathologic (biopsy or post mortem) examination. The mean age of the patients was 35.8 years; 13 patients were male (72.2%). The disease was disseminated, with the following distribution:skin (38.8%), bone marrow (27.7%), nasopharyngeal mucosa (22.2%), lungs (22.2%), colon (11.1%), central nervous system (5.5%) and esophagus (5.5%). Adenomegaly (50%), hepatomegaly (77.7%) and splenomegaly (61.1%) were frequently seen. The most common hematologic abnormality was pancytopenia (33.3%) of the patients. Eleven patients were treated, 9 with amphotericin B and 2 with itraconazole. Eight had good clinical improvement and all of them were given amphotericin B or a triazolic as maintenance therapy. This study emphasize the importance of this mycosis in immunodepressed patients, specially AIDS patients, in whom the infection tends to invade the macrophagic-lymphoid system and preferentially the cutaneous tegument.     

Development of the early B cell population in Xenopus

Like mammals, the amphibian Xenopus uses combinatorial joining of the immunoglobulin V, D and J elements and multiple rearrangements to generate its B cell repertoire. Xenopus larvae hatch 2 days after fertilization and individuals are under pressure to develop an immune repertoire when the number of available cells is small (approximately 5 and 200 IgM-positive cells on days 5 and 11 after fertilization, respectively). In the liver, in a first phase of differentiation spanning days 5-12 after fertilization before immunological competence, the heavy (H) chain locus starts rearranging followed by the light (L) chain locus 3 days later. By immunohistology the first B cells expressing H and L chain are detectable on day 10. Despite the small number of cells available and the lack of external antigen selection at these early stages, the repertoire is heterogeneous. The VH families are used stepwise, although their genes are interspersed in the genome. The earliest family used (VH1) is homologous to the VH3 family of human and to the VH7183 of the mouse which are also overrepresented in early mammalian development. In the second phase, from day 12-13 onwards, the spleen differentiates and the animal becomes immunologically competent. The V, D and J usage is similar to that of adults although VDJ junctions lack N nucleotides until metamorphosis. A preferential reading frame for D and one specific DJ junction are overrepresented during this second phase. The visible bias toward homology-based junction results in fact from selection after rearrangement.     

Repression of the c-myb gene by WT1 protein in T and B cell lines

The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.     

In vitro role of IL-1 in heightened IgG, anti-DNA, and nephritogenic idiotype production by B cells derived from the murine MRL/lpr lupus model

The MRL/lpr murine model resembles human lupus both in its serologic and immunopathologic features, and is characterized by high-level IgG and autoantibody production. The precise mechanisms for this B cell hyperactivity are poorly understood. This study explored the role of IL-1 in determining high-level IgG and autoantibody production in the MRL/lpr murine lupus model by blocking IL-1 activity with a recombinant IL-1 receptor antagonist (IL-1Ra). IgG and autoantibody production (anti-DNA ab and Id-H130 activity) by B cells derived from MRL/lpr mice was significantly suppressed by treating B cell cultures with IL-1Ra. In contrast, IgG and autoantibody production by B cells derived from young MRL/lpr, MLR/++, or normal C3H/HeJ mice showed virtually no suppression with IL-1Ra. Collectively, these findings indicate that IL-1 may be an important factor in determining the heightened production of IgG, anti-DNA, and id-H130 antibody production in lupus-prone MRL/lpr mice. Furthermore, heightened IL-1 activity appears to be influenced by both age and the presence of the lpr mutation.     

Retroviral transfer of the human MDR1 gene confers resistance to bisantrene-specific hematotoxicity

In this work, we demonstrate a protective effect conferred by the human multidrug resistance gene (MDR1) to populations of the murine hematopoietic system against the toxic effects of bisantrene, a novel intercalating cytotoxic agent under investigation as an anticancer agent. In vitro, MDR1-expressing cell lines are highly cross-resistant to bisantrene, and low levels of P-glycoprotein (the MDR1 gene product cell surface protein) confer resistance to the drug. MDR1-positive mice were generated after transplantation of bone marrow cells (BMC) transduced in vitro with a MDR1 retrovirus. Control mice were transplanted with BMC transduced with the neomycin resistance gene. Administration of a single i.v. dose of 50 mg/kg of bisantrene resulted in a decrease of the total WBC count of approximately 40%. In contrast, a decrease of the total WBC count of only 17% was observed in mice transplanted with MDR1-transduced BMC. Immunofluorescence studies with cell lineage-specific monoclonal antibodies showed that bisantrene was specifically toxic for B lymphocytes and macrophages. Double-staining with MRK16 (a monoclonal antibody specific for P-glycoprotein) demonstrated that a single dose of bisantrene increased the relative number of MDR1-transduced positive B cells, macrophages, and (to some extent) granulocytes when compared to the number found in MDR1-untreated mice or the bisantrene-treated neomycin-transduced control mice. These results provide in vivo evidence that bisantrene is a hematotoxic drug capable of selecting for MDR1-transduced hematopoietic cells. Bisantrene might be useful for gene therapy as an in vivo selective agent for cells transduced with MDR1 vectors that also coexpress therapeutic genes of interest.     

Evidence that an OX-2-positive cell can inhibit the stimulation of type 1 cytokine production by bone marrow-derived B7-1 (and B7-2)-positive dendritic cells

The present study was designed to compare the effects of typical and atypical antipsychotic drugs on extracellular dopamine (DA) levels in the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAC), using in vivo microdialysis with dual probe implantation in awake, freely moving rats. Amperozide (2 and 10 mg/kg), clozapine (5 and 20 mg/kg), and olanzapine (10 mg/kg), all of which are atypical antipsychotics, produced greater increases in extracellular DA levels in the mPFC than in the NAC. Olanzapine (1 mg/kg), risperidone (0.1 and 1 mg/kg), also an atypical antipsychotic, and S-(-)-sulpiride (25 mg/kg), a typical antipsychotic, produced comparable increases in extracellular DA levels in the mPFC and the NAC. S-(-)-sulpiride (10 mg/kg) and haloperidol (0.1 and 1 mg/kg), another typical antipsychotic, significantly increased extracellular DA levels in the NAC but not in the mPFC. The effects of the six antipsychotic drugs to increase extracellular DA levels in the mPFC relative to those in the NAC was positively correlated with the difference between their pKi values for serotonin (5-hydroxytryptamine, 5-HT2A) and DA-D2 receptors and was inversely correlated to their pKi values for D2 or D3 receptors, but was not for 5-HT2A receptors alone. These results are consistent with the hypothesis that the ability of antipsychotic drugs to produce a greater increase in prefrontal compared with NAC extracellular DA levels may be related, in part, to weak D2 and D3 receptor affinity relative to 5-HT2A receptor antagonism.     

Association of the FcepsilonRIbeta gene with bronchial hyper-responsiveness in an Italian population

A study of two DNA polymorphisms (i2 RsaI, E237G) in the gene for the beta subunit of the IgE high affinity receptor (FcepsilonRIbeta) was performed in 168 Italian families with atopic asthmatic children. The prevalence of the E237G allele in the Italian population was 4%, so this polymorphism was unsuitable for this study. The i2 RsaI polymorphism minor allele frequency was 44%, and it had a PIC value of 0.37. Linkage analysis indicated a significant allele sharing in affected sib pairs for bronchial hyper-responsiveness (BHR, p=0.048), but not for allergic asthma. These data indicate an association of bronchial hyper-responsiveness with the FcepsilonRIbeta gene.     

Promotion of antigen-specific antibody production in murine B cells by a moderate increase in histone acetylation

By employing the specific histone deacetylase inhibitor trichostatin A (TSA), we investigated whether histone acetylation modulates the production of antigen-specific antibodies in murine splenocytes in vitro. TSA caused a marked increase in both anti-sheep red blood cell (SRBC) and anti-trinitrophenyl (TNP) plaque-forming cell (PFC) responses in splenocytes at much lower concentrations than sodium butyrate. It also dose dependently augmented the production of anti-trinitrophenyl antibodies in splenic B cells with a concomitant, moderate increase in the level of histone H4 acetylation. Its optimal concentration for promoting the production of these antibodies was 10 nM. However, to gain such an effect on antibody production, TSA had to be added to cells before Day 2 in culture. Trichostatin C, an analog of TSA and a less potent inducer of Friend leukemia cell differentiation, also increased both the anti-trinitrophenyl PFC response and histone H4 acetylation in B cells, but at higher concentrations than TSA. TSA did not stimulate the production of lipopolysaccharide-induced polyclonal immunoglobulin M in B cells. These results suggest that a moderate increase in histone acetylation may play a significant role in promoting antigen-specific antibody production in B cells.     

Monocyte cytokine production in an elderly population: effect of age and inflammation

OBJECTIVE: To determine the association among aging, inflammation, and cytokine production by peripheral blood mononuclear cells. POPULATION AND METHODS: We examined production of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-1 receptor antagonist (IL-1Ra), and IL-6 in 711 elderly participants in the Framingham Heart Study (mean age, 79 y) and 21 young healthy volunteers (mean age, 39 y). The elderly subjects were categorized by serum C-reactive protein (CRP) concentration, a marker of systemic inflammation. RESULTS: Production of IL-6 (p < .00001) and IL-1Ra (p < .00001) was higher in the elderly subjects than in the control group. IL-6 production increased with increasing CRP, whereas IL-1RA was uniformly elevated in elderly subjects regardless of CRP. However, we found no difference in the production of IL-1 beta or TNF-alpha between the young and elderly groups, regardless of CRP status. IL-6 population correlated with IL-1 beta (r = .36, p < .0001) and TNF-alpha production (r = .25, p < .0001), but IL-1Ra production did not. CONCLUSION: Production of IL-6 and IL-1Ra--but not IL-1 beta or TNF-alpha--was increased in the elderly compared to healthy, young subjects. The increase in IL-6 also correlated with increased production of CRP, a marker of inflammation. However, IL-1Ra was increased in the elderly independently of CRP production. Although limited by the small control group, these data suggest that dysregulation of some inflammatory cytokines occurs with age, but the role of inflammation in aging remains unclear.     

B cell population and its autoimmune activity in the thymus of patients associated with myasthenia gravis

Immunohistochemical studies using antibodies to B lymphocyte and to immunoglobulin G bearing cell were carried out for resected thymus-specimens of 10 patients associated with myasthenia gravis. In each case, abundant B cells (L26 positive cell) resided in the follicles and the medulla of thymus, especially, were congregated in the follicles developing germinal center and around Hassall's corpuscles. Amount of B cell population was various among each case regardless of type of myasthenia gravis or age. B cells were greatly increased in the thymus of patients with values of anti-acetylcholine receptor antibody titers over 100 nmol/l. Although numerous B cells were present in the thymus of these patients, IgG bearing cells were extremely rare. In the most cases, B cells lacked IgG expression. From the results, numerous B cells pre-activating autoimmune antibody production were accumulated in the thymus of patients associated with myasthenia gravis.     

Analysis of B cell autoepitopes and mechanisms for autoantibody production in autoimmune diseases

One of the representative immunological disorders in systemic autoimmune diseases is production of autoantibodies. Recent studies were concentrated on B cell epitopes of intranuclear autoantigens using recombinant proteins. They revealed autoepitopes homologous to those on exogenous agents and multiple epitopes, which respectively indicated molecular mimicry and antigen-driven immune responses as a mechanism for autoantibody production. Further, some autoantigens are thought to be catalyzed in a disease-specific manner. These antigen-specific factors would contribute to the autoantibody production, cooperating with antigen-nonspecific factors such as polyclonal B cell activation, overexpression of cytokines and dysfunction of T cells and antigen presenting cells.