Sequence analysis of the germ-line VH gene corresponding to a nephritogenic antibody in MRL/lpr lupus mice.

In order to investigate the genetic origin of nephritogenic antibodies in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice, we isolated the germ-line heavy chain variable region (VH) gene corresponding to the nephritogenic antibody, B1, derived from an unmanipulated MRL/lpr mouse. Injection of this antibody into C.B-17/Icr-scid/scid mice resulted in the generation of wire loop-like glomerular lesions resembling those of lupus nephritis. Nucleotide sequences of this germ-line VH gene showed no replacement mutation in the VH region of the B1 antibody. Furthermore, this gene was identical to that found in the C3H/HeJ-lpr/lpr strain of mice. Our results suggest that germ-line VH genes can encode nephritogenic antibodies without somatic mutation, even in a mouse strain not prone to lupus.     

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis

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Primary cutaneous B‐cell lymphoma is associated with somatically hypermutated immunoglobulin variable genes and frequent use of VH1‐69 and VH4‐59 segments

Background Accurate assessment of the somatic mutational status of clonal immunoglobulin variable region (IgV) genes is relevant in elucidating tumour cell origin in B‐cell lymphoma; virgin B cells bear unmutated IgV genes, while germinal centre and postfollicular B cells carry mutated IgV genes. Furthermore, biases in the IgV repertoire and distribution pattern of somatic mutations indicate a possible antigen role in the pathogenesis of B‐cell malignancies. Objectives This work investigates the cellular origin and antigenic selection in primary cutaneous B‐cell lymphoma (PCBCL). Methods We analysed the nucleotide sequence of clonal IgV heavy‐chain gene (IgVH) rearrangements in 51 cases of PCBCL (25 follicle centre, 19 marginal zone and seven diffuse large B‐cell lymphoma, leg‐type) and compared IgVH sequences with their closest germline segment in the GenBank database. Molecular data were then correlated with histopathological features. Results We showed that all but one of the 51 IgVH sequences analysed exhibited extensive somatic hypermutations. The detected mutation rate ranged from 1·6% to 21%, with a median rate of 9·8% and was independent of PCBCL histotype. Calculation of antigen‐selection pressure showed that 39% of the mutated IgVH genes displayed a number of replacement mutations and silent mutations in a pattern consistent with antigenic selection. Furthermore, two segments, VH1‐69 (12%) and VH4‐59 (14%), were preferentially used in our case series. Conclusions Data indicate that neoplastic B cells of PBCBL have experienced germinal centre reaction and also suggest that the involvement of IgVH genes is not entirely random in PCBCL and that common antigen epitopes could be pathologically relevant in cutaneous lymphomagenesis.     

VH gene analysis of Burkitt's lymphoma in children from north-western Iran

Cases of Burkitt's lymphoma (BL) from north-western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional V_H genes were amplified from 6/12 of the tumour masses and all of these were derived from the V_H3 family, with 4/6 being derived from the most commonly used V_H3 family member, V_3-23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated V_H sequences showed a significant clustering of replacement mutations in the complementarity-determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in V_H sequences of EBV+ve endemic BL (eBL) and EBV−ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, V_H gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL.     

Assessing Bioresorbable Coronary Devices: Methods and Parameters

Bioresorbable vascular scaffolds (BRS) represent a novel approach to provide transient vessel support to drug-delivery capability without the long-term limitations of metallic drug-eluting stents (DES). The technology has the potential to overcome many of the safety concerns associated with metallic DES and possibly even convey further clinical benefit. In particular, the BRS are designed to provide short-term lumen support, and after being completely bioresorbed, eliminate the permanent caging typical of metallic DES. However, this technology has required new imaging modalities and methodologies for its assessment because the design, degradation rate, loss of mechanical property, and drug deliverability may affect its safety and efficacy. We provide an overview of all existing methods for assessing bioresorbable devices, from noninvasive to invasive, from light to sound based, and from morphological to functional parameters.     

The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors

A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74–92% and 90–96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3–IGKV1, IGHV3–IGVL1, and IGHV3–IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.     

The Anti-Non-Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells Is Encoded by a Restricted Number of Germline Progenitors

Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into nonhuman primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgV_H genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3–21 germline progenitor encode xenoantibodies directed at non-gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine α1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of 'anti-non-gal' xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti-non-gal xenoantibodies cross react with carbohydrate xenoantigens. The galactosyltransferase two gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti-non-gal xenoantibodies in primates are encoded by IgV_H genes that are restricted to IGHV3–21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.     

Incidence of novel N-glycosylation sites in the B-cell receptor of lymphomas associated with immunodeficiency

Novel N-glycosylation sites are introduced by somatic mutation into the V genes of the majority of follicular lymphomas. Sites are positively selected and rare in normal memory B cells, indicating a potential role in tumour survival in the germinal centre (GC). The incidence of c. 40% in diffuse large B-cell lymphomas (DLBCL) parallels the known heterogeneity of the disease. Immunodeficiency-related non-Hodgkin's lymphomas (NHL) include post-transplant lymphoproliferative disorders (PTLD) and acquired immunodeficiency syndrome-related NHL (AIDS-NHL). Most PTLD derive from B cells that carry mutated VH genes and that have completed the GC reaction. All AIDS-NHL carry mutated VH genes and variable features of GC or post-GC cells. To determine if N-glycosylation is a feature of immunodeficiency-related lymphomas, we analysed the VH genes of 19 PTLD and 36 AIDS-NHL. Novel sites were rare in PTLD (4/19), similar to memory B cells (P = 0.15). AIDS-NHL, including DLBCL and Burkitt's lymphomas (BL), showed heterogeneity with 16 of 36 (44%) having novel sites. The findings indicate no selection of N-glycosylation sites in PTLD, consistent with post-GC features. The variable incidence of N-glycosylation sites in AIDS-NHL mirrors that in DLBCL and sporadic BL of immunocompetent hosts, supporting the known heterogeneity of these disorders, and possibly pointing to distinct routes of tumour development.