[Caracterization of HLA allo-immunization and clinical impact in transfusion and organ transplantation]. / Caractérisation de l'allo-immunisation anti-HLA et impact clinique en transfusion et en transplantation d'organe.

Allo-immunizations against HLA antigens are known to be deleterious in transfusion and organ transplantation. The development of new tests based on solid phase assays for screening and identification of HLA antibodies in particular those using Luminex® bead based technology has completely changed the way of allo-immunization monitoring because of their extreme sensitivity. They allow a better characterization of these antibodies, identification of acceptable antigens and the use of virtual cross-matches. All these new possibilities improve the managing of patients before and after platelets transfusion or organ transplantation. However, this technology displays some limits that should be known in order to interpret correctly the results. Beside these bead based assays, cellular cross-matches based on Complement Dependent Cytotoxicity (CDC) and flow cytometry are still used and useful in organ transplantation since beads are produced in vitro and do not reflected exactly what happens physiologically. Moreover, differences of sensitivity between these methods make results interpretation and decision making difficult in some cases.

A novel allele HLA-C*07:445 identified in a French hematopoietic stem cell donor.

The novel allele HLA-C*07:445 has 1 nucleotide change from HLA-C*07:01 at nucleotide 277 C>A in exon 2.

Risk factors and outcome of graft failure after HLA matched and mismatched unrelated donor hematopoietic stem cell transplantation: a study on behalf of SFGM-TC and SFHI.

Graft failure remains a severe complication of hematopoietic stem cell transplantation (HSCT). Several risk factors have already been published. In this study, we re-evaluated them in a large cohort who had the benefit of the recent experience in HSCT (2006-2012). Data from 4684 unrelated donor HSCT from 2006 to 2012 were retrospectively collected from centers belonging to the French Society for Stem Cell Transplantation. Among the 2716 patients for whom HLA typing was available, 103 did not engraft leading to a low rate of no engraftment at 3.8%. In univariate analysis, only type of disease and status of disease at transplant for malignant diseases remained significant risk factors (P=0.04 and P<0.0001, respectively). In multivariate analysis, only status of disease was a significant risk factor (P<0.0001). Among the 61 patients who did not engraft and who were mismatched for 1 HLA class I and/or HLA-DP, 5 donor-specific antibodies (DSAs) were detected but only 1 was clearly involved in graft failure, for the others their role was more questionable. Second HSCT exhibited a protective although not statistically significant effect on OS (hazard ratio=0.57 [0.32-1.02]). In conclusion, only one parameter (disease status before graft) remains risk factor for graft failure in this recent cohort.

Is there any impact of HLA-DPB1 disparity in 10/10 HLA-matched unrelated hematopoietic SCT? Results of a French multicentric retrospective study.

We retrospectively analyzed the impact of HLA-DPB1 mismatches in a large cohort of 1342 French patients who underwent 10/10 HLA-matched unrelated HSCT. A significant impact of HLA-DPB1 allelic mismatches (2 vs 0) was observed in severe acute GVHD (aGVHDIII-IV) (risk ratio (RR)=1.73, confidence interval (CI) 95% 1.09-2.73, P=0.019) without impact on OS, TRM, relapse and chronic GVHD (cGVHD). According to the T-cell epitope 3 (TCE3)/TCE4 HLA-DPB1 disparity algorithm, 37.6% and 58.4% pairs had nonpermissive HLA-DPB1, respectively. TCE3 and TCE4 disparities had no statistical impact on OS, TRM, relapse, aGVHD and cGVHD. When TCE3/TCE4 disparities were analyzed in the graft-vs-host or host-vs-graft (HVG) direction, only a significant impact of TCE4 nonpermissive disparities in the HVG direction was observed on relapse (RR=1.34, CI 95% 1.00-1.80, P=0.048). In conclusion, this French retrospective study shows an adverse prognosis of HLA-DPB1 mismatches (2 vs 0) on severe aGVHD and of nonpermissive TCE4 HVG disparities on relapse after HLA-matched 10/10 unrelated HSCT.

[Impact of anti-HLA antibodies on outcomes of allogeneic stem cell transplantation: a report by the SFGM-TC]. / Impact des anticorps anti-HLA sur le devenir de l'allogreffe de cellules souches hématopoïétiques : un rapport par la SFGM-TC.

The role of anti-HLA antibodies in allogeneic stem cell transplantation setting is still unclear. In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) set up its fourth annual series of workshops which brought together practitioners from all of its member centers. These workshops took place in September 2013 in Lille. This article offers the recommendations of the group that considered the impact that have anti-HLA antibodies on outcomes in allogeneic stem cell transplantation.

Transfusion-related acute lung injury after intravenous immunoglobulin treatment in a lung transplant recipient.

Three weeks after single-lung transplantation for pulmonary fibrosis, a patient with high serum levels of de novo donor-specific antibodies received high-dose intravenous immunoglobulin (IVIG) infusion (scheduled dose: 2 g/kg on 2 days) to prevent antibody-mediated rejection. Within the first hours after completion of infusions, he experienced acute lung injury involving the transplanted lung. Given the clinical evolution and the absence of an alternative diagnosis, transfusion-related acute lung injury (TRALI) was diagnosed. The IVIG administered on each day was from the same batch. At day 110, because of an increase in the serum titers of donor-specific antibodies, IVIG therapy was reintroduced but from a different batch, with excellent clinical tolerance. The lung injury was explored biologically, but no mechanism was revealed. Given the increasing use of IVIG in solid-organ recipients, clinicians should be aware of possible TRALI after IVIG infusion.

Transcription, beta-like DNA polymerases and hypermutation.

This paper discusses two aspects of immunoglobulin (Ig) gene hypermutation. In the first approach, a transcription termination signal is introduced in an Ig light chain transgene acting as a mutation substrate, and transgenic lines are generated with control and mutant transgenes integrated in tandem. Analysis of transcription levels and mutation frequencies between mutant and control transgenes clearly dissociates transcription elongation and mutation, and therefore argues against models whereby specific pausing of the RNA polymerase during V gene transcription would trigger an error-prone repair process. The second part reports the identification of two novel beta-like DNA polymerases named Pol lambda and Pol mu, one of which (Pol mu) represents a good candidate for the Ig mutase due to its higher lymphoid expression and its similarity with the lymphoid enzyme terminal deoxynucleotidyl transferase. Peculiar features of the expression of this gene, including an unusual splicing variability and a splicing inhibition in response to DNA-damaging agents, are discussed.

Two novel human and mouse DNA polymerases of the polX family.

We describe here two novel mouse and human DNA polymerases: one (pol lambda) has homology with DNA polymerase beta while the other one (pol mu) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix-loop-helix DNA-binding motifs and polymerase X domain. mRNA expression of pol lambda is highest in testis and fetal liver, while expression of pol mu is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol mu gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, gamma-rays or H(2)O(2)). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.

Probing immunoglobulin gene hypermutation with microsatellites suggests a nonreplicative short patch DNA synthesis process.

As the rate of Ig gene hypermutation approximates the level of nucleotide discrimination of DNA polymerases (10(-3) to 10(-4)), a local inhibition of proofreading and mismatch repair during semiconservative replication could generate the mutations introduced by the process. To address this question, we have constructed transgenic mice that carry a hypermutation substrate containing a "polymerase slippage trap": an Ig gene with a mono or dinucleotide tract inserted in its V region. The low amount of slippage events as compared to the number of mutations, the absence of transient misalignment mutations at the border of the repeats, and the dissociation between the amount of frameshifts and mutations when the transgene is put on mismatch repair-deficient genetic backgrounds, suggest that Ig gene hypermutation occurs by an error-prone short patch DNA synthesis taking place outside global DNA replication.

Mismatch repair deficiency interferes with the accumulation of mutations in chronically stimulated B cells and not with the hypermutation process.

Primary responses to the hapten phenyloxazolone and chronic responses to environmental antigens occurring in Peyer's patches were analyzed in two different mismatch repair-deficient backgrounds. Paradoxically, whereas primary responses were found normal in MSH2- and only slightly diminished in PMS2-deficient mice, mutations in Peyer's patch B cells from both k.o. animals were reduced three times, the subset of Peyer's patch B cells with highly mutated sequences being specifically missing in the mismatch repair-deficient context. Strikingly, germinal center B cells from Peyer's patches of k.o. animals showed microsatellite instability at an unprecedented level. We thus propose that the amount of DNA damages generated prevents these cells from recycling in germinal centers and that mismatch repair deficiency is only the indirect cause of the lower mutation incidence observed.

Molecular typing of Enterococcus faecalis strains resistant to high levels of gentamicin and isolated in Romania.

Sixteen Enterococcus faecalis strains resistant to high levels of gentamicin, 15 of which were isolated in the same year in a Romanian hospital, harboured conjugative gentamicin resistance (Gm(r)) plasmids ranging from 55 to 85 kilobases. On the basis of restriction enzyme and DNA-DNA hybridization profiles of these plasmids, as well as of chromosomal SmaI macrorestriction and Tn916 hybridization patterns, clonal relationship was established for seven strains whereas the other strains were considered to be independent. Nine and seven of the Gm(r) plasmids carried Tn4001-like and Tn4001-truncated structures, respectively; the latter structures were truncated in the right-hand flanking extremity of the element.

Study of heterogeneity of chloramphenicol acetyltransferase (CAT) genes in streptococci and enterococci by polymerase chain reaction: characterization of a new CAT determinant.

An assay based on the utilization of degenerate primers that enable enzymatic amplification of an internal fragment of cat genes known to be present in gram-positive cocci was developed to identify the genes encoding chloramphenicol resistance in streptococci and enterococci. The functionality of this system was illustrated by the detection of cat genes belonging to four different hydridization classes represented by the staphylococcal genes catpC221, catpC194, catpSCS7, and the clostridial gene catP, and by the characterization of a new streptococcal cat gene designated catS. A sequence related to the clostridial catQ gene, which was present in one streptococcal strain, was not detected by this assay. These results reveal that these six cat genes account for chromosomal-borne chloramphenicol resistance in 12 group A, B, and G streptococci tested. By contrast, only three of these six cat genes (catpC221, catpC194, and catpSCS7) were detected on the 10 enterococcal plasmids studied here that encode resistance to chloramphenicol.

Diversity of chromosomal genetic elements and gene identification in antibiotic-resistant strains of Streptococcus pneumoniae and Streptococcus bovis.

Antibiotic-resistant Streptococcus pneumoniae (26 strains) and Streptococcus bovis (28 strains), devoid of R plasmids, were examined for DNA-DNA homology to Tn916 and Tn3701. Tn916-like structures were found in 17 S. pneumoniae and 21 S. bovis strains. Tn916-modified structures were present in 6 S. pneumoniae and 2 S. bovis strains. Two strains of each species carried elements having a Tn3701-like composite structure. All these elements were chromosome-borne. No chromosomal elements were detected in 1 S. pneumoniae and 3 S. bovis strains.