Synthesis and evaluation of hybrid molecules targeting the vinca domain of tubulin.

Some hybrids of vinca alkaloids and phomopsin A, linked by a glycine pattern, have been synthesized in one or two steps, by an insertion reaction and shown to inhibit microtubule assembly. These compounds have been elaborated in order to interact with both the "vinca site" and the "peptide site" of the vinca domain in tubulin. Two out of three hybrids are potent inhibitors of microtubules assembly and they present good cytotoxicity against different cell lines. Molecular modelling studies show that they could bind, within the vinca domain, in similar spatial regions as those of vinca and phomopsin thanks to the flexibility provided by the glycine linker used to elaborate these hybrids.

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues--a potential cosmetic role.

The naturally occurring tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10⁻¹¹-10⁻7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10⁻5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo-cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10⁻¹¹-10⁻7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin-1, occludin, ZO-1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two-fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10⁻¹¹-10⁻7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.

Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification.

Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.

MICA compatibility and immunization in third kidney transplantations.

Significantly lower graft survival has been observed among recipients of a third (G3) compared with a first or second kidney transplantation. Because patients awaiting G3 are largely HLA immunized, they are usually transplanted with a high HLA match. Moreover, their rate of acute rejection episodes is similar to a first or second transplantation. Since major histocompatibility complex class I related chain A (MICA) molecules have been proposed as new targets for antibody recognition, we were interested to type donors and recipients for MICA alleles and to study MICA immunization of these patients. Forty-three pairs of donors and recipients were typed for MICA alleles using Luminex technology (LABtype RSSO). MICA alleles showed strong linkage disequilibrium with the B locus: some 4-digit alleles were preferentially associated with a given MICA allele. A greater frequency of patients with 2 MICA mismatches (MM) was observed among patients with rejection (40%), whereas all the graft losses were observed in patients with 0 or 1 MICA MM. MICA immunization was studied using sera from 52 patients collected on day 0 and after transplantation using a Luminex assay (LABScreen). MICA immunization was less frequent than HLA immunization, and MICA donor-specific antibody (DSA) was equally present in functional and failed grafts. These observations confirmed the potential role of MICA immunization in rejection, whereas the poor graft survival among third transplantations could not be explained by MICA incompatibility or immunization.

[Post-transfusion acute lung injury (Trali) after plasma infusion in a patient having a constitutional thrombotic microangiopathy]. / Syndrome d'oedème pulmonaire lésionnel aigu post-transfusionnel (Trali) après perfusion de plasma frais congelé au cours d'une microangiopathie thrombotique congénitale.

INTRODUCTION: Transfusion-related acute lung injury is a post-transfusion interstitial lung injury. CASE REPORT: We reported a post-transfusion acute lung injury in a 23-years old woman having a chronic thrombotic microangiopathy related to an ADAMTS 13 constitutional deficiency receiving monthly plasma infusion for six years. The temporal relationship between the lung injury and the infusion of fresh frozen plasma led to the diagnosis of transfusion-related acute lung injury. The finding in the donor of the transfused plasma of an anti-HLA class II antibody recognizing HLA-DR52 present on leucocytes of the recipient suggests a causal relationship between this antigen-antibody conflict and the triggering of the TRALI. This chronic pathologic state requiring monthly plasma transfusions for thrombotic accident prevention raises the question of the selection of plasma obtained from non-immunized donors. CONCLUSION: The occurrence of a post transfusion pulmonary edema without cardio-vascular overload, must lead to consider a TRALI especially in predisposing clinical situations. In the case reported the role of constitutional ADAMTS 13 deficiency in genesis of TRALI is considered.

Preliminary analysis of a KIR haplotype estimation algorithm: a simulation study.

The analysis of Killer cell immunoglobulin-like receptors (KIRs) in terms of haplotypes have only been done through genotyping numerous and selected families. Consequently and schematically, KIR haplotypes have been roughly described by two groups (A and B) based on their gene contents. No further KIR adapted methods have been applied to the estimation of haplotype frequencies using unrelated data. We propose here a maximum likelihood (ML) estimation of KIR haplotype frequencies. ML estimation was developed as an extension of those successfully applied to human leukocyte antigen (HLA) data including the handling of missing values and HLA nomenclature. It has been implemented using an adapted Expectation Masimisation algorithm. KIR types on 11 loci in more than 40 Irish families were used to validate the method in a simulation study. Estimated haplotype frequencies are compared to the phase known. Various allele or gene frequency estimation methods were also compared. We demonstrated the interest and reliability of the haplotype method and underline the effect of the sample size on the quality of the estimation. The ML haplotype method also provides by collapsing more accurate estimation of allele or gene frequencies in population. Such an algorithm opens new perspectives in the analysis of KIR genotypes. Large sample size studies are required using phase-known data and/or simulations. It would allow a genotype-based approach to explore the KIR gene haplotype diversity. The haplotype frequencies may be used to compare populations.

Relevance of KIR gene matching in unrelated hematopoietic stem cell transplantations.

The aim of this collaborative study was to evaluate the impact of killer cell immunoglobulin-like receptor (KIR) gene disparities on unrelated hematopoietic stem cell transplantations (HSCT) outcome. To address this question, we have determined the presence or absence of 14 functional KIR genes in HLA-matched (n= 164) or HLA-mismatched (n= 100) donor/recipient pairs and investigated whether KIR gene disparities had an impact on both the occurrence of acute graft-vs-host-disease incidence and overall survival. In a univariate analysis, our preliminary results suggest a detrimental effect of a few KIR gene disparities on patient survival that should be avoided in unrelated HSCT.

Genetic diversity of KIR natural killer cell markers in populations from France, Guadeloupe, Finland, Senegal and Réunion.

Killer cell immunoglobulin-like receptors (KIRs) belong to a diverse family of natural killer (NK) cell receptors recognizing human leukocyte antigen (HLA) class I molecules. Due to this functional link, KIR molecules are expected to display a high polymorphism, such as their HLA ligands. Moreover, many studies conducted in mouse and human models have shown that NK-KIR receptors play an important role in haematopoietic stem cell transplantation (HSCT). A beneficial impact of peculiar KIR ligand (HLA) mismatching has been reported suggesting a role to this combinatory HLA-KIR polymorphism. It is thus important to investigate KIR diversity in various human populations. To this end, we used polymerase chain reaction-sequence-specific primers to evaluate KIR gene in five selected populations (France, Guadeloupe, Senegal, Finland and Réunion). Genotypic and haplotypic frequencies were computed, as well as genetic distances and dendrogram (phylip package). These data illustrate the genetic relationship of these five populations through the KIR polymorphism. Results revealed a wide diversity in KIR gene frequencies in Guadeloupe and Réunion, and a high specificity in Senegal. The obtained dendrogram indicated small genetic distances between France, Guadeloupe and Réunion as well as between France and Finland. Senegal showed a distant genetic relationship with the other countries and, interestingly, an inverted ratio of coding/non-coding (KIR2DS4/1D) alleles compared with Caucasians. These data expose the broad diversity in KIR genes worldwide and show that KIR genes are pertinent tools in human population genetics. If the role of KIR donor-recipient incompatibilities is confirmed, KIR diversity according to ethnicity should be taken into account during the selection of HSCT donors.

[Luminex technology for HLA typing by PCR-SSO and identification of HLA antibody specificities]. / Technologie Luminex: application aux typages HLA par biologie moléculaire (PCR-SSO) et à l'identification des anticorps anti-HLA.

Luminex technology is a new flow cytometry technology enabling us to analyse numerous reactions in a unique tube or well. It is a multiplexed data acquisition and analysis platform of microsphere-based assays that performs simultaneous measurements of up to 100 different analytes. In the histo- compatibility field, individual sets of microspheres are modified with reactive components such as antigens in order to perform HLA antibodies identification, or with oligonucleotides in order to perform HLA typing after reverse PCR-SSO. Thus microspheres are the equivalent of a panel of HLA typed lymphocytes (for PRA determination and antibody identification) or equivalent to a large set of probes selected to assign HLA typing. This new tool can be very useful in HLA laboratories since it is very easy to use and the results are concordant with those obtained with reference technics.

Identification of a new HLA-DRB1 allele, DRB1*0321.

This communication reports the identification of a new HLA-DRB1*03 allele identified in three members of a Caucasian French family. This new allele has been officially named HLA-DRB1*0321 by the World Health Organization Nomenclature Committee. The complete exon 2 sequence of DRB1*0321 is identical to that of DRB1*0307 except for the first and second nucleotides of codon 37 (TT replacing AA), which lead to the substitution of a tyrosine for a phenylalanine (AAC-->TTC at position 37). The family study showed that this new allele was transmitted into the HLA-A*0101/09, -B*0801/14, -Cw*0701, -DRB1*0321, -DRB3*0101, -DQB1*0503 and -DPB1*0401 haplotype. The complete exon 2 sequence of this new allele has been previously deposited in the EMBL Sequence Database under accession number AF297266.

Estimation of the incidence of pleural mesothelioma according to death certificates in France.

BACKGROUND: The number of cases of pleural mesothelioma in France has varied substantially according to methods of assessment. MATERIALS AND METHODS: We collected information from certifying physicians about 316 subjects who died between 1 July 1992 and 30 June 1993 in three regions of France with a cause of death coded as ICD-9 category 163. The ICD codes selected as the cause of death for 178 deaths between 1 January 1987 and 31 December 1992 histologically confirmed and diagnosed as pleural mesothelioma by an expert committee were examined. Finally, we used this information to estimate the number of deaths from pleural mesothelioma in France in 1992. RESULTS: In Part I, 45% (men: 54%; women: 28%) of the cases coded as ICD-9 section 163 were definitely or probably mesothelioma; 18% (men: 16%; women: 21%) possibly mesothelioma; and 37% (men: 30%; women: 51%) other tumors, primarily adenocarcinoma metastases. In Part II, 74% of the confirmed pleural mesotheliomas were coded in category 163 (men: 75%; women: 70%). Extrapolation nationwide indicated that 902 deaths were coded as ICD-9 163 in 1992: 521 cases involved definite or probable mesothelioma and 724 definite, probable, and possible cases. CONCLUSIONS: The analysis of this sample suggests that estimating the number of mesothelioma cases from the cause-of-death statistics may overestimate their incidence, but that death certificates appeared to report the diagnosis of histologically confirmed mesothelioma accurately.

Regulation of the acetoin catabolic pathway is controlled by sigma L in Bacillus subtilis.

Bacillus subtilis grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. Acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. The acoABCL operon encodes the E1alpha, E1beta, E2, and E3 subunits of the acetoin dehydrogenase complex in B. subtilis. Expression of this operon is induced by acetoin and repressed by glucose in the growth medium. The acoR gene is located downstream from the acoABCL operon and encodes a positive regulator which stimulates the transcription of the operon. The product of acoR has similarities to transcriptional activators of sigma 54-dependent promoters. The four genes of the operon are transcribed from a -12, -24 promoter, and transcription is abolished in acoR and sigL mutants. Deletion analysis showed that DNA sequences more than 85 bp upstream from the transcriptional start site are necessary for full induction of the operon. These upstream activating sequences are probably the targets of AcoR. Analysis of an acoR'-'lacZ strain of B. subtilis showed that the expression of acoR is not induced by acetoin and is repressed by the presence of glucose in the growth medium. Transcription of acoR is also negatively controlled by CcpA, a global regulator of carbon catabolite repression. A specific interaction of CcpA in the upstream region of acoR was demonstrated by DNase I footprinting experiments, suggesting that repression of transcription of acoR is mediated by the binding of CcpA to the promoter region of acoR.

[Evaluation of occupational exposures in lung cancer]. / Evaluation des expositions professionnelles et cancer bronchopulmonaire.

Occupational lung cancers are underestimated by the number of cases compensated in the French National Insurance System. Rules of compensation of occupational diseases were recently modified in France. Therefore a study was conducted on incident cases of lung cancer in a general hospital in the Paris area. The aim was to evaluate the exposure to carcinogens using data of a detailed specific occupational questionnaire, and to determine the number of cases who could receive compensation. Two hundred and seven subjects (171 males, 36 females, mean age = 64.5 years) were eligible in 1996, and 122 had an occupational questionnaire. Definite exposure to one or more occupational carcinogens in at least one job was identified in 50 subjects, the most frequent agent was asbestos (42 subjects). Claim for compensation was done in 32 subjects, mainly for asbestos (30 subjects). This study emphasizes the frequency of occupational exposure to carcinogens, and the usefulness of systematic occupational questionnaire in subjects having lung cancer. Social and financial consequences are important for these subjects. Further studies are needed, with recruitement of control subjects to allow calculation of the attributable risk to occupational factors in lung cancer.

Interaction of anti-HLA antibodies with pig xenoantigens.

BACKGROUND: Many patients with renal failure are condemned to long-term dialysis with little prospect of transplantation because they are highly sensitized with immunoglobulin G (IgG) directed against class I human leukocyte antigens (HLA) of virtually all donors. Xenotransplantation could represent an attractive solution providing their alloantibodies (alloAb) do not recognize porcine motifs. Hitherto there has been no in vivo demonstration of any cross-reactivity and the objective of this work was to investigate this problem using a technique of extracorporeal pig kidney perfusion as a model of clinical xenografting. METHODS: Pig kidneys were perfused ex vivo with plasma from both a group of highly sensitized patients and healthy individuals. Sequential plasma samples were analyzed for the titer of anti-Galalpha1-3Gal antibody (Ab) (major natural xenoreactive Ab) by enzyme-linked immunosorbent assay and anti-HLA class I Ab against a cell panel. At the end of perfusion, kidneys were perfused with a citric acid buffer to elute bound Ab. RESULTS: Galalpha1-3Gal Ab were shown to decrease rapidly in the plasma (in less than 10 min) and then reached a plateau. A fractional decrease in anti-HLA Ab was also found in some of the perfused plasma samples. Anti-Gal Ab were readily detected in all citric acid perfusates and anti-HLA Ab in 8 of 10. The HLA specificities of eluted Ab were mainly concordant with the originally designated specificities for each patient. CONCLUSION: Anti-HLA class I Ab presumably cross-react with pig class I homologues. However, some plasma samples did not cross-react, suggesting that negatively cross-matched pig kidneys could be identified in the pig population for xenotransplantation in these patients. Further studies are required to precisely describe these cross-reactivities and to understand their functional significance in xenotransplantation.

Epidemiological evidence on the carcinogenicity of silica: factors in scientific judgement.

In view of the extended debate and differing opinions on whether crystalline silica is a human carcinogen, we have reviewed a selection of epidemiological reports, to identify the areas of uncertainty and disagreement. We have chosen to examine the papers which in a recent review were considered to provide the least confounded examinations of an association between silica exposure and cancer risk. We also refer to a study of the mortality of coalminers very recently reported by ourselves and colleagues. We find that parts of the evidence are coherent but there are contradictions. On examination this resolves mostly into differences between types of studies. The three types of epidemiological study included are: (i) exposure-response studies, the most powerful for the confirmation of a relationship between a specific exposure and a health effect; (ii) descriptive studies in which incidence of disease in an exposed population is compared with that in a reference population; and (iii) studies of incidence of disease in subjects on silicosis case-registers. Descriptive studies frequently though not invariably suggest an excess lung cancer risk in silica-exposed workers compared with the general population, but exposure-response studies consistently fail to confirm that the cause is exposure to quartz. A single exposure-response study of cristobalite suggests a positive relation. Both sets of evidence have weaknesses. There are uncertainties on whether the excess risks in the descriptive studies are related to silica exposure or to lifestyle, including smoking habits. There are doubts on whether the exposure estimates in some of the exposure-response studies were sufficiently reliable to detect a small risk or weak association, though they are unlikely to have missed a strong effect. Studies of subjects on silicosis case registers consistently show an excess of lung cancer, but it is not clear to what extent these increased risks represent a direct effect of silica exposure, a secondary effect of the silicosis, preferential inclusion of subjects suffering from the effects of smoking, or bias in diagnostic accuracy. This not unnaturally leads to differences in opinion, exacerbated by variations in the strength of proof required by different experts. The main scientific uncertainties in the evidence are: 1. Whether, in the descriptive studies, the excess lung cancer rates in silica-exposed workers are explicable in terms of smoking habits, socio-economic class differences and inappropriate comparison populations. Better smoking information and more carefully chosen comparison populations are needed; 2. Whether the exposure-response studies could have missed a real relationship between silica exposure and lung cancer, if one exists. Many of the exposure-response studies were conducted with great care, but weaknesses, in the available data on which the exposure estimations were based, could have caused a real relationship of lung cancer and silica exposure to be missed. These studies were sufficiently powerful to demonstrate relationships of silica exposure with silicosis and silico-tuberculosis, so it is unlikely that they would have missed any but a small risk, or weak relationship, for lung cancer. Our own recent study of coalminers used uniquely detailed and reliable exposure data, and failed to demonstrate convincingly an increased risk. This negative finding, though, applies only to a dust in which the proportion of quartz in the dust is usually less than 10%. Exposure-response studies are needed, with high quality exposure estimates, in populations exposed to respirable dust of which crystalline silica comprises more than 10%; 3. Whether the excess cancer risks in subjects on silicosis registers are the result of selection and diagnostic bias. Given these difficulties, case-register studies may not be capable of giving a reliable answer to the central question, though they have been useful in pointing to the possibility of a can

Highly altered V beta repertoire of T cells infiltrating long-term rejected kidney allografts.

Chronic rejection represents a major cause of long-term kidney graft loss. T cells that are predominant in long-term rejected kidney allografts (35 +/- 10% of area infiltrate) may thus be instrumental in this phenomenon, which is likely to be dependent on the indirect pathway of allorecognition only. We have analyzed the variations in T cell repertoire usage of the V beta chain at the complementary determining region 3 (CDR3) level in 18 human kidney grafts lost due to chronic rejection. We observed a strongly biased intragraft TCR V beta usage for the majority of V beta families and also a very high percentage (55%) of V beta families exhibiting common and oligoclonal V beta-C beta rearrangements in the grafts of patients with chronic rejection associated with superimposed histologically acute lesions. Furthermore, V beta 8 and V beta 23 families exhibited common and oligoclonal V beta-J beta rearrangements in 4 of 18 patients (22%). Several CDR3 amino acid sequences were found for the common and oligoclonal V beta 8-J beta 1.4 rearrangement. Quantitative PCR showed that biased V beta transcripts were also overexpressed in chronically rejected kidneys with superimposed acute lesions. In contrast, T lymphocytes infiltrating rejected allografts with chronic rejection only showed an unaltered Gaussian-type CDR3 length distribution. This pattern suggests that late graft failure associated with histological lesions restricted to Banff-defined chronic rejection does not involve T cell-mediated injury. Thus, our observation suggests that a limited number of determinants stimulates the recipient immune system in long-term allograft failure. The possibility of a local response against viral or parenchymatous cell-derived determinants is discussed.

GSTM1, smoking and lung cancer: a case-control study.

BACKGROUND: We conducted a case-control study to examine the risk of lung cancer in relation to GSTM1 polymorphism and cigarette smoking (primarily of black tobacco) in a French population. METHODS: The 611 subjects were 301 incident lung cancer cases and 310 hospital controls. We were able to constitute a DNA bank for 547 subjects (89.5%) and gather detailed information on smoking history for all of them. Results presented here concern 247 cases and 254 controls. RESULTS: Taking non- or light smokers as the reference category, we estimated odds ratios (OR) of 4.2 (95% CI: 2.6-6.7) and 5.2 (95% CI: 3.3-8.3) for the medium and heavy smokers respectively. On the other hand we estimated that the crude OR associating GSTM1 with lung cancer was 1.3 (95% CI: 0.9-1.8). Furthermore our data do not depart significantly from a multiplicative model of the combined effects of smoking and GSTM1 deficiency. CONCLUSIONS: We conclude that smoking and the GSTM1 gene are each a risk factor for lung cancer, and that their combined effect does not differ significantly from that of a multiplicative model.

Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis.

Hereditary pancreatitis (HP) is a rare inherited disorder, characterised by recurrent episodes of pancreatitis often beginning in early childhood. The mode of inheritance suggests an autosomal dominant trait with incomplete penetrance. The gene, or at least one of the genes, responsible for hereditary pancreatitis has been mapped to the long arm of chromosome 7 and a missense mutation, an arginine to histidine substitution at residue 117 in the trypsinogen cationic gene (try4) has been shown to segregate with the HP phenotype. The aim of this work was to investigate the molecular basis of hereditary pancreatitis. This study was performed on 14 HP families. The five exons of the trypsinogen cationic gene were studied using a specific gene amplification assay combined with denaturing gradient gel electrophoresis (DGGE). The present paper describes three novel mutations, namely K23R and N29I and a deletion -28delTCC in the promoter region. We also found a polymorphism in exon 4, D162D. In eight of these families we found a mutation which segregates with the disease. A segregation analysis using microsatellite markers carried out on the other families suggests genetic heterogeneity in at least one of them. Our findings confirm the implication of the cationic trypsinogen gene in HP and highlight allelic diversity associated with this phenotype. We also show that the pattern of inheritance of HP is probably complex and that other genes may be involved in this genetic disease.