V region diversity in human anti-insulin antibodies. Preferential use of a VHIII gene subset

Data on structures used by human antibody repertoires are derived principally from lymphoid malignancies and from autoantibodies that often express VH genes from the developmentally regulated fetal repertoire. To determine whether human immune responses generated by exogenous Ag use a pool of VH genes distinct from the fetal repertoire, nucleotide and predicted amino acid sequences were determined for five anti-insulin B cell clones from a type I diabetic patient treated with human insulin. The data show that a set of VHIII genes is preferentially used by human anti-insulin B cells. Structural features indicate that these expressed VH are derived from germ-line genes that are not frequent in fetal repertoires and these genes have undergone Ag-driven somatic mutation. The preferential use of related VH segments contrasts with the BALB/c anti-insulin response, which uses multiple V genes elements largely unmutated from germ-line sequences. In addition, long CDRH3 structures in human anti-insulin mAb are generated by complex gene interaction mechanisms that are not seen in murine anti-insulin mAb. Interestingly, similar potential insulin-binding structures are used by antibodies from both species. These findings suggest that human responses to exogenous insulin may express a limited number of VH genes and depend upon somatic mutation and complex D gene interactions in CDRH3 to expand the repertoire. Although these antibodies bind autologous insulin, VH gene usage and structural features that predominate in the response are not characteristic of the fetal repertoire.     

Early anti-nucleosome autoantibodies from a single MRL+/+ mouse: fine specificity, V gene structure and pathogenicity

In systemic lupus erythematosus, the nucleosome assumes a central role in the autoimmune response to self antigens. To gain insight into the etiology and pathogenesis of anti-nucleosome antibodies (Ab), we analyzed a panel of six IgG-secreting hybridomas derived from a single young MRL +/+ mouse at the onset of the autoimmune response. All monoclonal antibodies (mAb) bound exclusively the native nucleosome, and represented five different clonotypes that recognized diverse nucleosomal epitopes, typical of a polyclonal response. The VH-complementarity-determining region (CDR)3 regions exhibited unique stretches of charged amino acids with different polarity that may be important for the interaction with the nucleosome. These early anti-nucleosome mAb displayed striking structural differences with not only anti-DNA, but also with anti-nucleosome Ab, that appear later in disease. Two of the mAb deposited in kidney glomeruli after in vivo administration to RAG-1-deficient mice, suggesting that diverse B cell clones, possibly selected by the nucleosome itself, may play a role in the initiation of kidney damage.     

Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid

To aid treatment choice in early stage of Hodgkin's disease, we analysed patients registered in the IDHD Database with clinical stages I or II Hodgkin's disease who were not staged with laparotomy and whose initial treatment was with radiotherapy alone. The factors analysed for outcome after first relapse included initial stage, age, sex, histology, number of involved areas, mediastinal involvement, E-lesions, B-symptoms, erythrocyte sedimentation rate, alkaline phosphatase, serum albumin and haemoglobin. As well as presentation variables, we analysed the disease-free interval after initial radiotherapy and the extent of disease at relapse. A total of 1364 patients with clinical stage I or II Hodgkin's disease were treated with initial radiotherapy, of whom 473 relapsed. The probability of survival 10 years after relapse was 63%. For cause-specific survival (CSS), both multivariate and univariate analysis identified the importance of age at presentation and histological subtypes. When all causes of death were considered, the multivariate analysis identified age as the only significant factor. The length of initial disease-free interval had no influence on prognosis after relapse, but the 169 patients with nodal relapse had a higher cause-specific survival than those with an extranodal component of relapse (74% versus 51% at 10 years, P < 0.005). Thus, the important factors for outcome after initial treatment with radiotherapy are those factors predicting the risk of relapse after initial treatment together with those predicting outcome after relapse, namely age, histologic subtype and extent of disease at relapse.     

CRF2-4: isolation of cDNA clones encoding the human and mouse proteins

A microbial population that biodegraded N-phosphonomethyliminodiacetic acid (PIA), a key component of glyphosate (N-phosphonomethylglycine) process waste, was established. The stoichiometric conversion of PIA to aminomethylphosphonic acid (AMPA) was observed in a laboratory sequencing batch reactor (SBR) containing activated sludge from a glyphosate-manufacturing facility and PIA as sole source of carbon. PIA degradation was determined by high-performance liquid chromatography and confirmed by radiolabeled studies. Greater than 90% of the [carboxymethyl-2-14C]-label of PIA was released as 14CO2 in 7 days using samples of sludge from the SBR. The cycle time required to biodegrade up to 7.5 mM PIA in SBRs was reduced from 21 to < 3 days. PIA biodegradation was also established in an immobilized bacteria column inoculated with mixed liquor from a SBR; > 99% PIA removal was achieved at an influent concentration of 2.2 mM and a hydraulic retention time of < 10 h. A pure bacterial culture was isolated from a SBR by streaking samples of sludge on solid media with PIA as sole carbon source. The isolate was identified as Xanthomonas maltophilia. In liquid culture, X. maltophilia degraded up to 4.4 mM PIA within 10 days and produced stoichiometric amounts of AMPA. The results demonstrate the biodegradation of PIA and suggest the potential for its treatment in industrial biological treatment systems.     

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene: a possible link between infection and lupus

The VH4-21 (V4-34) gene segment, a member of the VH4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of VH4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the I/i antigen, and also capable of encoding anti-DNA antibodies. Recognition of I/i antigen or DNA appears to be via two distinct sites on VH, with I/i binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with I/i, rendering a single sequence monospecific for I/i or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-I/i antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity.     

大截面4Cr13V耐蚀塑料模具钢的组织和性能

 大截面4Cr13V耐蚀塑料模具钢模块在生产中质量波动大,往往存在比较严重的疏松、一次碳化物残留、偏析和夹杂等缺陷,对后续使用性能有不利影响。利用金相显微镜、扫描电镜、硬度测试及冲击韧性试验等试验分析手段,研究了国内某钢厂改善锻造工艺后生产的高品质大截面4Cr13V耐蚀塑料模具钢模块不同位置的夹杂物、退火组织、碳化物尺寸、热处理组织及力学性能的差异,并分析形成差异的可能因素。结果表明：大截面4Cr13V耐蚀塑料模具钢模块的纯净度较高,边部碳化物的球化程度最好,越靠近心部大尺寸碳化物越多,这些大尺寸碳化物的存在对力学性能有一定的不利影响,是造成边心部性能差异的主要因素。     

Cytotoxicity of murine B lymphocytes induced by human VH4-34 (VH4.21) gene-encoded monoclonal antibodies

We previously found that a ventricular isovolumic pressure-time curve could be well fitted by the difference between two S-shaped logistic curves for the pressure rising and falling components, and called it "hybrid logistic" function: P(t)=A/[1+exp[-(4B/A)(t-C)]]-D/[1+exp[-(4E/D)(t-F)]]+G. We reported that the parameters of this hybrid logistic function are useful to characterize left ventricular contraction and relaxation comprehensively. In this study, we investigated how well this hybrid logistic function could fit the isometric twitch force-time curves of cross-circulated right ventricular papillary muscles of 7 dogs. This function precisely fitted the isometric force curves with correlation coefficients above 0.9996, much better than another fitting function (F(t)=C(t/A)(B)exp[1-(t/A)(B)]) proposed by Nwasokwa. The present results indicate that our hybrid logistic function can also reasonably express the canine right ventricular papillary muscle isometric twitch force-time curve. We suggest the possibility that the parameters of this hybrid logistic function are also useful to comprehensively characterize right ventricular papillary muscle twitch contraction and relaxation.     

The mouse mutation sarcosinemia (sar) maps to chromosome 2 in a region homologous to human 9q33-q34

The autosomal recessive mouse mutation sarcosinemia (sar), which was discovered segregating in the progeny of a male whose premeiotic germ cells had been treated with the mutagen ethylnitrosourea, is characterized by a deficiency in sarcosine dehydrogenase activity. Using an intersubspecific cross, we mapped the sar locus to mouse chromosome 2, approximately 15-18 cM from the centromere. The genetic localization of this locus in the mouse allows the identification of a candidate region in human (9q33-q34) where the homologous disease should map.     

Infrared brazing of Ti-6Al-4V and 17-4 PH stainless steel with a nickel barrier layer

Infrared brazing of Ti-6Al-4V and 17-4 PH stainless steel using the BAg-8 filler metal was performed in this study. A nickel barrier layer 10 μm thick was introduced on the 17-4 PH stainless steel before infrared brazing. For the specimen that was infrared brazed at 800 °C and 850 °C for less than 300 seconds, the Ni layer served as an effective barrier layer to prevent the formation of Ti-Fe intermetallics. Experimental results show that the average shear strength of the joint can be greatly improved for the specimen by Ni plating. Comparing the specimens with and without electroless-plated Ni film, the former has no Ti-Fe intermetallic compound, but interfacial CuNiTi and NiPTi phases are observed in the latter. The fractured location of the joint after the shear test is changed from the interfacial TiFe (without Ni plating) into the TiCu reaction layer (with Ni plating). The plated Ni layer is consumed for the specimen that was infrared brazed at 880 °C for 300 seconds, and its bonding strength is impaired. Consequently, a lower brazing temperature and/or time are still preferred even though a plated Ni barrier layer is applied.     

The role of protein kinase-C in signal transduction through vasopressin and acetylcholine receptors in pancreatic B-cells from normal mouse

The role of protein kinase-C (PKC) in the potentiation of insulin release by arginine vasopressin (AVP) and acetylcholine (ACh) was investigated with normal mouse islets. The islets were submitted to a short term (30-min) or long term (22-h) treatment with phorbol 12-myristate 13-acetate (PMA) to stimulate acutely or down-regulate PKC before being stimulated by AVP or ACh. Control islets were treated with the inactive 4 alpha-phorbol 12,13-didecanoate. In the presence of 15 mM glucose and 2.5 mM Ca2+, AVP and ACh stimulated inositol phosphate (IP) formation, increased cytoplasmic Ca2+ (Cai2+), and potentiated insulin release. These effects were greater with ACh than with AVP, in particular on Cai2+, which was scarcely affected by AVP. In the absence of extracellular Ca2+, only ACh induced a short-lived increase in Cai2+ and insulin. Acute treatment with PMA in the presence of extracellular Ca2+ strongly increased insulin release in spite of a marked lowering of Cai2+. Under these conditions, the effects of AVP and ACh on IP production and Cai2+ were practically abolished, and only ACh transiently increased insulin release. In the absence of Ca2+, the small mobilization of Cai2+ by ACh triggered a peak of insulin, whereas a similar mobilization of Cai2+ by AVP was ineffective on insulin. After long term treatment of the islets with PMA, AVP normally increased IP formation, but did not affect insulin release. The effect of ACh on IP was still inhibited. However, ACh produced a marked transient increase in Cai2+, with a small transient release of insulin. The releasing effect of ACh was also inhibited in the absence of Ca2+. In conclusion, PKC plays a dual role in the B-cell responses to ACh and AVP. Its activation is necessary for the sustained potentiation of insulin release that both agents produce. This effect probably results from a sensitization of the secretory machinery to Cai2+, the triggering signal. PKC also exerts a negative feedback control on the signal transduction mechanisms involving phospholipase-C, but the ACh and AVP responses are not equally affected by this feedback.     

Expression of a mouse immunoglobulin V gene in homozygote and heterozygotes

Our earlier work had demonstrated a fine-specificity idiotype in anti-NP antibodies of C57Bl/6 mice. It was controlled by a VH gene (VH-NPb). The idiotype could be demonstrated in all anti-NP of the C50Bl/6 mice (IgG, IgA, IgM including natural anti-NP). The VH-NPb gene is condominant with CBA allele F1 hybrid mice in the production of natural antibodies but dominant over the CBA allele in immune antibody formation (92% of F1 mice were like C57Bl/6). Probably the product of this gene has a higher affinity for NP than the average affinity of idiotype-negative antibody in the F1 hybrids. Both the codominance in natural antibodies and the dominance in immune antibodies had the peculiar characteristic that genotypically identical individuals varied a great deal. The share of idiotype-positive antibody in the total natural anti-NP titre of (C57Bl/6 x CBA)F1 mice varied from less than 33% to more than 67%. In immune antibodies individual differences were still greater, in ca 92% of mice only idiotype-positive anti-NP could be detected and in ca 8% only idiotype-negative antibody was detectable. Only few F1 hybrid mice had detectable amounts of both types. Homozygous mice of the LP strain behaved in all aspects like the F1 mice. Of their natural anti-NP titres 50% were due to idiotype-positive antibody and in the immune antibodies the figure was 86%. Individual non-immune sera again contained varying mixtures of idiotype-positive and idiotype-negative antibody, but immune sera contained only one or the other type. The third allotype b strain tested, C57Bl/Ka, took an intermediary position between LP and C57Bl/6. Ca 70% of natural anti-NP titres were due to idiotype-positive antibody but all immune anti-NP was idiotype-positive.     

Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+ progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38- cells. Comparable extents of human engraftment and lineage development were obtained from 5 x 10(5) CD34+ cells and 2,000 CD34+CD38- cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38- cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38- cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.     

MAdCAM-1 dependent colonization of developing lymph nodes involves a unique subset of CD4+CD3- hematolymphoid cells

During fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules briefly express the Peyer's patch addressin MAdCAM-1. This allows initial seeding by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta 7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. It was found that the CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1. They can differentiate into natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. In addition to LN, CD4+CD3- cells can also be found in fetal spleen starting at 13.5 dpc, while absent from fetal liver. In view of the necessity of lymphotoxin in lymphoid organ development, it is thought that the novel subset of CD4+CD3- LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.     

低碳含钒钢组织变化及V(C,N)析出规律

通过热模拟试验,研究了0．058％C-0．92％Mn-0．51％V钢在变形后等温过程中的组织变化规律及显微硬度变化规律,并用透射电镜研究了V(C,N)的沉淀析出规律。结果表明,变形后在650、700、750℃等温1130s时可获得单相铁素体组织,从而基本消除珠光体组织。等温转变过程中,铁素体先是主要沿奥氏体晶界形核,随后逐渐变为晶内形核。V(C,N)的沉淀析出存在两种形式：相间析出和随机析出。V(C,N)的相间析出消除了C元素在奥氏体中的富集,是获得单相铁素体组织的重要条件。