Role of CD40 antigen and interleukin-2 in T cell-dependent human B lymphocyte growth

In the present study, we examined the participation of CD40 ligand (L)-CD40 interaction in T cell-dependent B cell responses. To this end, purified B lymphocytes were cultured over irradiated CD4⁺ cloned T cells activated with immobilized anti-CD3 antibody. The anti-CD40 mAb 89 strongly blocked, in a specific fashion, both proliferation and Ig secretion of tonsil B cells. Interestingly, proliferation of surface (s)IgD⁺ B cell was significantly less inhibited by anti-CD40 than that of sIgD^? cells. Preactivated T cells induced B cells to grow and secrete immunoglobulins preferentially in response to IL-2. This contrasts with the CD40 system where B cells are essentially responsive to IL-4 and IL-10 but not to IL-2 alone. Collectively, these data indicate that CD40L-CD40 interaction plays an important role in IL-2-mediated T cell-dependent B cell responses. However, the activation of a subset of sIgD⁺ cells may be independent of this interaction.     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.     

Expression of a 32-kDa ligand for the CD40 antigen on activated human T lymphocytes

To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgGl. The CD40-Fc fusion protein was able to specifically bind to CD4⁺ and various CD8⁺ T cell clones activated with immobilized anti-CD3. The ¹²⁵I-labeled CD40-Fc fusion protein bound anti-CD3 activated CD4⁺ T cell clone (MT9) with an equilibrium dissociation constant (Ka) of 10-20 nM. The human CD40-binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of ≈ 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8⁺ blood mononuclear T cells activated by anti-CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40-Fc obtained after anti-CD3 activation.     

Frequency of Cytotoxic T Lymphocyte Precursors to Herpes Simplex Virus Type 1 as Determined by Limiting Dilution Analysis

The conditions for establishing a limiting dilution assay to measure cytotoxic T lymphocyte precursors (CTL-P) against herpes simplex virus type 1 (HSV-1) were determined. Analysis by Poisson statistics demonstrated that the estimated frequency of HSV-1-reactive cells in the spleens of normal mice was less than 1/250,000. In contrast, mice immunized previously with infectious HSV-1 demonstrated a CTL-P frequency between 1/3,500 and 1/15,670. The generation of a maximum cytotoxic T lymphocyte response required that mice be primed in vivo with infectious virus. Immunization with inactivated virus either failed to elicit detectable CTL-P frequencies or gave frequencies markedly less than those induced with infectious virus. To obtain positive cultures, the responder cell population had to be exposed to stimulator splenocytes expressing viral antigens. Normal splenocytes without virus or normal splenocytes with T cell growth factor did not result in significant cytotoxicity. Split culture analysis comparing cytotoxicity against syngeneic and allogeneic virus-infected targets provided evidence for specificity, H-2 restriction, and the T cell nature of the CTL-P. It was determined that precursors were eliminated by treatment with anti-Thy 1, Lyt 2.1, or Lyt 1.1, indicating the CTL-P were Lyt 1(+)2(+) cells. Cytotoxicity was reduced after treatment of the responders with anti-Lyt 2 plus complement, which gave further evidence of the T cell nature of the cytotoxic T lymphocytes. These experiments demonstrated the feasibility of using the limiting dilution approach as a highly sensitive and quantitative means to measure the cell-mediated immune response to HSV-1 antigens.     

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.     

The pathogenesis of molybdenum cofactor deficiency, its delay by maternal clearance, and its expression pattern in microarray analysis

Molybdenum cofactor (Moco)-deficiency is a lethal autosomal recessive disease, for which until now no effective therapy is available. The biochemical hallmark of this disorder is the inactivity of the Moco-dependent sulfite oxidase, which results in elevated sulfite and diminished sulfate levels throughout the organism. In humans, Moco-deficiency results in neurological damage, which is apparent in untreatable seizures and various brain dysmorphisms. We have recently described a murine model for Moco-deficiency, which reflects all enzyme and metabolite changes observed in the patients, and an efficient therapy using a biosynthetic precursor of Moco has been established in this animal model. We now analyzed these mice in detail and excluded morphological brain damage, while expression analysis with microarrays indicates a massive cell death program. This neuronal damage appears to be triggered by elevated sulfite levels and is ameliorated in affected embryos by maternal clearance.     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.     

Variola and camelpox virus-specific sequences are part of a single large open reading frame identified in two German cowpox virus strains

A large open reading frame (ORF) has been identified in two German cowpox virus strains. The ORFs (5676 and 5679 nt, respectively) differ in 10 nucleotides, resulting in an amino acid homology of 99.8%. In searching GenBank nucleotide sequences (>90% identity) were present in several small ORFs in variola major, variola minor and camelpox virus genomes. Alignments revealed that these small ORFs are fragments of a large ORF. However, sequences of the ORF described here are entirely absent in the two cowpox virus reference strains. Databank analysis revealed amino acid identities (ranging from 25 to 39%) with so-called B22R-like poxviral proteins with unknown function encoded by several chordopoxviruses. Further sequencing of one cowpox virus strain under study identified an ORF (5790 nt) which displays high levels of nucleotide identity to ORFs present in several orthopoxvirus species. Taken together, the two cowpox viruses analyzed here contain one large ORF which is conserved within the genus Orthopoxvirus and a unique, more distantly related ORF of similar size, which is conserved in the subfamily Chordopoxvirinae.     

应用四环素调控系统建立可调控抗恶性疟原虫DNA疫苗的初步研究

目的 :应用四环素 (tetracycline,Tet)可调控系统建立可调控的抗恶性疟原虫的DNA疫苗。方法 :首先构建恶性疟原虫顶端膜抗原 1(AMA 1)基因和转录激活因子 (tTA或rtTA)基因的真核表达质粒pTL 8/AMA 1和pTL 8/AMA 1(rtTA) ,并大量制备这两种质粒及表达转录抑制子 (tTS)的质粒pUHS6 1。以pTL 8/AMA 1、pTL 8/AMA 1(rtTA)和pTL 8/AMA 1(rtTA)加pUHS6 1/tTS免疫小鼠后 ,用四环素类似物强力霉素 (doxcycline ,dox)诱导或抑制上述DNA载体内AMA 1的表达 ,并分离小鼠血清检测针对AMA 1抗体的表达情况。结果 :①构建了真核表达质粒pTL 8/AMA 1和pTL 8/AMA 1(rtTA) ;②pTL 8/AMA 1和pTL 8/AMA 1(rtTA)在没有被诱导表达时 (仅基础表达 ) ,可诱导明显的免疫应答。在以pTL 8/AMA 1(rtTA)与含tTS的pUHS6 1共同免疫后 ,可极大地降低其免疫应答。应用dox诱导pTL 8/AMA 1(rtTA)中的AMA 1基因表达后 ,可明显引起免疫应答。结论 :pTL 8/AMA 1(rtTA)附加pUHS6 1构成了可调节的DNA免疫系统。该系统的建立将对进一步深入研究DNA疫苗的免疫机制和调控基因表达 ,减少不良免疫反应以及进行可精确调控的基因治疗打下一定的基础