First description of Mycobacterium tuberculosis and M. canettii concomitant infection: report of two cases.

We report the first two cases of tuberculous coinfection with Mycobacterium tuberculosis and M. canettii. Both patients were young Djiboutian females with pulmonary tuberculosis (TB). One had a miliary pattern with concomitant human immunodeficiency virus infection. Both recovered completely with a standard four-drug anti-tuberculosis treatment regimen. Due to the different natural reservoirs and routes of infection of these two strains, our study supports the common belief that multiple strains of infection in TB are related to superinfection rather than concomitant infection.

Genotyping of Pseudomonas aeruginosa reveals high diversity, stability over time and good outcome of eradication.

BACKGROUND: Genotyping of Pseudomonas aeruginosa (P.a) is used for surveillance at our CF clinic. METHODS: P.a from 1999 to 2012 were analysed, using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable number of tandem repeats analysis (MLVA). RESULTS: Among 232 isolates from 104 patients, we identified 78 unique strains, of which 56 were isolated from individual patients. The B-clone was isolated from 13 patients and the camp transmission clone J-strains from 8 patients at the start of the study. There was no indication of transmission within the clinic. PFGE and MLVA clone identification was in 91% agreement. For patients who provided more than 2 P.a isolates, similar strains were identified over time for 45/49 chronically- and for 6/16 intermittently-colonized patients despite, periods of no detectable P.a after eradication therapy. CONCLUSIONS: Analyses revealed high genotypic diversity, acceptable outcome of eradication therapy and no indication of cross-infection at the CF centre.

A new highly discriminatory multiplex capillary-based MLVA assay as a tool for the epidemiological survey of Pseudomonas aeruginosa in cystic fibrosis patients.

Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) has been shown to provide a high level of information for epidemiological investigations and the follow-up of Pseudomonas aeruginosa chronic infection. In the present study, an automatized MLVA assay has been developed for the analysis of 16 VNTRs in two multiplex polymerase chain reactions (PCRs), followed by capillary electrophoresis. The result in the form of a code is directly usable for clustering analyses. This MLVA-16(Orsay) scheme was applied to the genotyping of 83 isolates from eight cystic fibrosis patients, demonstrating that the same genotype persisted during eight years of chronic infection in the majority of cases. Comparison with pulsed-field gel electrophoresis (PFGE) analysis showed that both methods were congruent, MLVA providing, in some cases, additional informativity. The evolution of strains during long-term infection was revealed by the presence of VNTR variants.

High-throughput typing method to identify a non-outbreak-involved Legionella pneumophila strain colonizing the entire water supply system in the town of Rennes, France.

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of Legionella pneumophila in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.

Molecular epidemiology of human and animal tuberculosis in Ibadan, Southwestern Nigeria.

From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1-18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19-48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.

Clinical characteristics of the smooth tubercle bacilli 'Mycobacterium canettii' infection suggest the existence of an environmental reservoir.

Over a 3-year follow-up, 30 out of the 318 unique Mycobacterium tuberculosis complex isolates recovered in the Republic of Djibouti had a smooth-type morphology and were Niacine-negative, the characteristics of 'Mycobacterium canettii' strains. Unlike M. tuberculosis, 'M. canettii' grew on nutrient-poor media at 30°C, and possessed characteristic lipids. They were isolated from respiratory and extra-respiratory sites from patients with typical forms of tuberculosis. Most cases resolved with antibiotic therapy but in two human immunodeficiency virus-positive patients 'M. canettii' infection led to septicaemia and death. No cases of human-to-human transmission were observed. The proportion of tuberculosis cases caused by 'M. canettii' was higher among French patients than among Djiboutian patients. Patients with 'M. canettii' were significantly younger than those with tuberculosis caused by other M. tuberculosis complex strains. Smooth tubercle bacilli could be misidentified as non-tuberculous mycobacteria and appear to be limited to the Horn of Africa. Their characteristics are consistent with the existence of non-human sources of infection.

[Performances of the assay MTBDRplus(®) in the surveillance of rifampicin resistance in Mycobacterium tuberculosis]. / Performances du kit MTBDRplus(®) dans le cadre de la surveillance de la résistance à la rifampicine chez Mycobacterium tuberculosis.

The purpose of the survey was the routine assessment of the MTBDRplus(®) kit performance in the determination and characterization of Mycobacterium tuberculosis resistance to rifampicin. The survey was carried out on a collection of 144 strains (126 of which were resistant to rifampicin) isolated on patients from 15 countries. Sensitivity to antituberculosis drugs was determined by a liquid culture system and the reference method was the amplification and sequencing of a target region of the rpoB gene whose mutations are responsible for rifampicin resistance (codons 507 to 533). The assessed kit was based on a reverse hybridization technique using eight overlapping probes covering the target region and four probes representing the most-frequently observed mutations. The assay performance was found excellent, specificity: 100%, sensitivity: 99.2%; 17 mutations affecting 10 codons were reported, two of which were newly identified.

[Phenotypic and genotypic (randomly amplified polymorphic DNA analysis and multiple-locus variable-number tandem-repeat analysis) charaterization of 96 clinical isolates of Pseudomonas aeruginosa in the F. Bourguiba hospital (Monastir, Tunisia)]. / Caractérisation phénotypique et génotypique (randomly amplified polymorphic DNA analysis et multiple-locus variable-number tandem-repeat analysis) de 96 isolats cliniques de Pseudomonas aeruginosa à l'hôpital F. Bourguiba (Monastir, Tunisie).

AIM OF THE STUDY: Phenotypic and genotypic characterization of 96 clinical isolates of Pseudomonas aeruginosa recovered in a Tunisian teaching hospital during a 16-month period. MATERIALS AND METHODS: All the isolates were characterized by serotyping, antimicrobial susceptibility typing and genotyping with randomly amplified polymorphic DNA (RAPD) analysis and multiple-locus variable-number tandem-repeat analysis (MLVA). RESULTS: Forty-one isolates out of 96 (43%) were recovered from two intensive care units (medical and chirurgical). Most of the isolates (48%) belonged to serotype O:11. Among the 13 antibiotypes, three multidrug resistant ones were mostly observed within the two intensive care units. Genotyping showed 83 RAPD types and 52 MLVA types. Isolates showing the same serotype could show different genotypes. A limited number of clusters was highlighted with MLVA typing, of which an outbreak of nine cases within the surgical intensive care unit. CONCLUSION: Except this outbreak of nine cases, the heterogeneity observed for most of the P. aeruginosa isolates showed that outbreak situations were rare in the F. Bourguiba hospital during the study period. MLVA genotyping is a good tool for genotyping P. aeruginosa clinical isolates.

Cell-mediated and not humoral immune responseis responsible for partial protection against toxoplasmosis in SCID mice reconstituted with human PBMC

El objetivo de este estudio fue profundizar en el conocimiento de la respuesta inmunologica especifica contra Toxoplasma en humanos. Usamos ratones SCID reconstituidos con PBMC de donantes sanos con y (..)(CNS) (AU)

The aim of this study was to further our understanding of the human Toxoplasma-specific immune response. We used severe combined immune deficiency (SCID) mice that had been reconstituted with peripheral blood mononuclear cells (PBMC) from healthy donors with and without serum Toxoplasma antigens(PBMC Toxo+ and PBMC Toxo-, respectively) and subsequently infected with parasite cysts. The specific lymphocyte proliferation rate (LPR) was higher for PBMC from Toxoplasma-immune donors and these PBMC secreted more Th1 cytokines than those obtained from Toxoplasma-non-immune donors. The engraftmentof human parasite-immune cells significantly increased the survival rate following infection compared to in unreconstituted animals,where as the engraftment of human non-parasite-immune cells did not alter the survival rate. Specific human anti-Toxoplasma antibodies were found in both groups of humanised animals, suggesting that the humoral immune response does not play a major role in this protection. Ten days after infection, cytometry revealed that there were more human CD45+ cells in the spleen and peritoneum of mice from the PBMC Toxo+ group than from the PBMC Toxo- group. Furthermore, plasma levels of nitric oxide(NO) peaked at an early stage of infection in resistant animals.Finally, no human CD4 mRNA or IFN-¦Ã mRNA was found in the brain during infection. All together, our results suggest that the human cell-mediated immune response provides partial protection against toxoplasmic encephalitis (TE) in this model. However,the effector mechanisms involved may be located outside of the central nervous system (CNS) (AU)

Cell-mediated and not humoral immune response is responsible for partial protection against toxoplasmosis in SCID mice reconstituted with human PBMC

El objetivo de este estudio fue profundizar en el conocimientode la respuesta inmunológica específica contra Toxoplasma enhumanos. Usamos ratones SCID reconstituidos con PBMC dedonantes sanos con y sin antígenos séricos de Toxoplasma (PBMCToxo+ and PBMC Toxo-, respectivamente) y subsecuentementeinfectados con cistos de parásito. La proliferación linfocitaria especifica(LPR) fue mayor en los PBMC de los donantes immunes aToxoplasma y éstos PBMC secretaron más citocinas Th1 que losobtenidos de donantes no immunes a Toxoplasma. La implantaciónde células inmunológicas de humanos infectados augmentósignificativamente la supervivencia después de la infección silo comparamos con la de los animales no reconstituidos. Sinembargo, la implantación de células inmunológicas de humanosno infectados no alteró la supervivencia. Se encontraron anticuerposespecíficos humanos anti- Toxoplasma en los dos gruposde animales humanizados, lo que sugiere que la respuesta inmunológicahumoral no juega un papel determinante en ésta protección.Diez días después de la infección, los estudios de citometríarevelaron que había más células humanas CD45+ en el bazoy peritoneo de los ratones del grupo PBMC Toxo+ que en los delgrupo Toxo-. Además, los niveles plasmáticos de óxido nítrico(NO) alcanzaron un máximo en un estadío más temprano de lainfección en animales resistentes. Finalmente, no se encontróRNAm de CD4 o IFN-gama humanos en el cerebro durante la infección.Nuestros resultados sugieren que la respuesta humana inmunológicacellular aporta una protección parcial contra la encephalitistoxoplásmica (TE) en este modelo. Sin embargo, los mecanismosefectores involucrados podrían estar localizados fuera delsistema nervioso central (CNS)

The aim of this study was to further our understanding of thehuman Toxoplasma-specific immune response. We used severecombined immune deficiency (SCID) mice that had been reconstitutedwith peripheral blood mononuclear cells (PBMC) fromhealthy donors with and without serum Toxoplasma antigens(PBMC Toxo+ and PBMC Toxo-, respectively) and subsequentlyinfected with parasite cysts. The specific lymphocyte proliferationrate (LPR) was higher for PBMC from Toxoplasma-immunedonors and these PBMC secreted more Th1 cytokines than thoseobtained from Toxoplasma-non-immune donors. The engraftmentof human parasite-immune cells significantly increased the survivalrate following infection compared to in unreconstituted animals,whereas the engraftment of human non-parasite-immunecells did not alter the survival rate. Specific human anti-Toxoplasmaantibodies were found in both groups of humanised animals, suggestingthat the humoral immune response does not play a majorrole in this protection. Ten days after infection, cytometry revealedthat there were more human CD45+ cells in the spleen andperitoneum of mice from the PBMC Toxo+ group than from thePBMC Toxo- group. Furthermore, plasma levels of nitric oxide(NO) peaked at an early stage of infection in resistant animals.Finally, no human CD4 mRNA or IFN-gamma mRNA was found in thebrain during infection. All together, our results suggest that thehuman cell-mediated immune response provides partial protectionagainst toxoplasmic encephalitis (TE) in this model. However,the effector mechanisms involved may be located outsideof the central nervous system (CNS)

CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.

The remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or 'spacers'. CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair. In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic. The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate. In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y. pestis isolates. In Y. pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event. This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized. Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage. This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained. The CRISPR structure provides a new and robust identification tool.

Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto.

Leptospira interrogans sensu stricto is responsible for the most frequent and severe cases of human leptospirosis. The epidemiology and clinical features of leptospirosis are usually associated with the serovars and serogroups of Leptospira. Because of the difficulties associated with serological identification of Leptospira strains, we evaluated a novel PCR-based method for typing L. interrogans serovars. Based upon the genome sequence of L. interrogans serovar Lai type strain 5660, 44 loci were analyzed by PCR for their variability in size due to the presence of variable-number tandem repeats (VNTR). Seven VNTR loci were found to be powerful markers for serovar identification, epidemiology, and phylogenetic studies of L. interrogans. This rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.

A submicroscopic unbalanced subtelomeric translocation t(2p;10q) identified by fluorescence in situ hybridization: fetus with increased nuchal translucency and normal standard karyotype with later growth and developmental delay, rhombencephalosynapsis (RES).

Reaching an accurate diagnosis in children with mental retardation associated or not with dysmorphic signs is important to make precise diagnosis of a syndrome and for genetic counseling. A female case with severe growth and development delay, dysmorphic features and feeding disorder is presented. Antenataly, the fetus was observed to have increased nuchal translucency and a slight hypoplastic cerebellum. A standard karyotype was normal. RES and a submicroscopic unbalanced subtelomeric translocation t(2p; 10q) were demonstrated after birth. We show that within the framework of a collaborative approach, a concerted research of submicroscopic subtelomeric rearrangements should be performed in case of mental retardation associated with facial dysmorphic features, and when other etiologies or non-genetic factors (iatrogenic, toxic, infectious, metabolic...) have been ruled out.

Tandem repeats analysis for the high resolution phylogenetic analysis of Yersinia pestis.

BACKGROUND: Yersinia pestis, the agent of plague, is a young and highly monomorphic species. Three biovars, each one thought to be associated with the last three Y. pestis pandemics, have been defined based on biochemical assays. More recently, DNA based assays, including DNA sequencing, IS typing, DNA arrays, have significantly improved current knowledge on the origin and phylogenetic evolution of Y. pestis. However, these methods suffer either from a lack of resolution or from the difficulty to compare data. Variable number of tandem repeats (VNTRs) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses in a growing number of pathogens and have given promising results for Y. pestis as well. RESULTS: In this study we have genotyped 180 Y. pestis isolates by multiple locus VNTR analysis (MLVA) using 25 markers. Sixty-one different genotypes were observed. The three biovars were distributed into three main branches, with some exceptions. In particular, the Medievalis phenotype is clearly heterogeneous, resulting from different mutation events in the napA gene. Antiqua strains from Asia appear to hold a central position compared to Antiqua strains from Africa. A subset of 7 markers is proposed for the quick comparison of a new strain with the collection typed here. This can be easily achieved using a Web-based facility, specifically set-up for running such identifications. CONCLUSION: Tandem-repeat typing may prove to be a powerful complement to the existing phylogenetic tools for Y. pestis. Typing can be achieved quickly at a low cost in terms of consumables, technical expertise and equipment. The resulting data can be easily compared between different laboratories. The number and selection of markers will eventually depend upon the type and aim of investigations.

Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping.

We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome of Legionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of other Legionella species or other bacteria used as controls. A polymorphism was observed for seven markers among the L. pneumophila strains tested. To check whether these markers could be used to compare strains of L. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies of L. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of the L. pneumophila colonies and gave a first hint as to the presence of several strains in a single sample.

A dominant modifier of transgene methylation is mapped by QTL analysis to mouse chromosome 13.

The single-copy hepatitis B virus transgene in the E36 transgenic mouse strain undergoes methylation changes in a parent-of-origin, tissue, and strain-specific fashion. In a C57BL/6 background, the paternally transmitted transgene is methylated in 30% of cells, whereas it is methylated in more than 80% of cells in (BALB/c x C57BL/6) F1 mice. We established previously that several genetic factors were likely to contribute to the transgene methylation profile, some with demethylating and some with de novo methylating activities. Using quantitative trait loci (QTL) mapping, we have now localized one major modifier locus on chromosome 13 (Mod13), which explains a 30% increase in the methylation level of this transgene with no effect on the flanking endogenous sequences. No other QTL could be identified, except for a demethylating activity of low significance located on chromosome 12. Recombinant inbred mice containing a BALB/c allele of Mod13 were then used to show that the presence of Mod13 is sufficient to induce de novo methylation. A segregation between de novo methylation and repression of transgene expression was uncovered, suggesting that this genetic system is also useful for the identification of factors that interpret methylation patterns in the genome.

Negative regulation of autoreactive B cells in transgenic mice expressing a human pathogenic cold agglutinin.

Cold agglutinins (CA) are autoantibodies that bind to erythrocyte carbohydrates at low temperatures and induce complement-mediated cell lysis, thus causing hemolytic anemia. Tolerance mechanisms towards CA-expressing B cells and the factors inducing pathogenic CA production are unknown. In order to develop an animal model for CA disease, we have produced transgenic mice expressing the heavy or the light chain of a human CA, previously shown to be pathogenic to the mouse. Expression of the human H chain alone resulted in a B cell maturation block at the pro-B stage, and did not induce allelic exclusion. In double-transgenic mice, co-expression of the human H and L chains restored B cell development but the majority of bone marrow cells expressing the human IgM were eliminated by deletion. In the periphery, B cells were depleted, and a large proportion of the remaining cells co-expressed a human and a murine H chain, secreting "mixed" IgM. A few autoreactive cells, predominating in the peritoneal cavity, escaped tolerance mechanisms and secreted transgenic IgM. The autoreactive B cells are amenable to polyclonal stimulation, making these transgenic mice a suitable model for a human autoimmune disease.

A new family of genes and pseudogenes potentially expressing testis- and brain-specific leucine zipper proteins in man and mouse.

We have characterized a new mouse gene highly transcribed in the testis, and a derived intronless gene expressed in the embryo. The latter gene is present in Mus musculus domesticus and in Mus musculus castaneus but is absent in Mus spretus. The sequencing of different clones from a testis cDNA library reveals a complex transcriptional regulation for the intron-containing gene. The use of several promoters, alternative splicing and trans-splicing, and of two different polyadenylation sites account for the diversity. The different cDNAs encode proteins with features of basic helix-loop-helix leucine zipper (bHLH-ZIP) DNA-binding factors with homology to a new brain-specific factor. The presence of multiple CK2 and PKC phosphorylation sites suggests that their activity may be regulated by phosphorylation. In man, a pseudogene, apparently derived from the same transcript as in mouse and showing 90% homology in the coding region, is present within an intron of another gene. Interestingly, although the human pseudogene is highly mutated in human, in the mouse it has only four nucleotide changes compared with the cDNA of origin, and is still capable of encoding a protein.

Association of glucocorticoids and cyclosporin A or rapamycin prevents E-selectin and IL-8 expression during LPS- and TNFalpha-mediated endothelial cell activation.

BACKGROUND: Endothelial cell (EC) activation plays an important role in inflammation, hemostasis, and organ rejection of allogeneic and xenogeneic transplantation. These processes leads to rapid and transient up-regulation of proinflammatory molecules, such as the adhesion molecule E-selectin and the chemotactic cytokine IL-8. The purpose of this study was to investigate the specific effects of several major and potentially synergistic immunosuppressive drugs-cyclosporin A (CsA), rapamycin (Rap), and glucocorticoids (GC)-on lipopolysaccharide (LPS)- or tumor necrosis factor (TNF)alpha-induced EC activation METHODS: The ability of immunosuppressive drugs, used alone or in combination, to prevent in vitro TNFalpha- and LPS-induced expression of E-selectin and interleukin 8 on porcine ECs, as well as their effect on leukocyte-EC interaction, were investigated. In addition, we studied the in vivo effect of these drugs after i.v. administration of recombinant TNFalpha to rats. RESULTS: At high concentrations, which correspond to the acceptable experimental levels in primate xenograft recipients, CsA, Rap, and GC individually inhibited E-selectin protein induction in a dose-dependent manner in cultured porcine ECs treated with LPS with an additive effect when the drugs were associated. The pattern of drug-mediated inhibition was related to the stimulus used to activate ECs (i.e., LPS vs. TNFalpha). Reduced expression of E-selectin on ECs activated in the presence of the tested immunosuppressive drugs correlated with a weaker adhesion of human U937 cells to ECs. Messenger RNA analysis demonstrated that the presence of CsA, Rap, and GC during EC activation inhibited E-selectin and interleukin 8 at the gene expression level. LPS-mediated induction of IbetaBalpha expression was not observed in ECs treated with CsA, whereas GC reduced its transcripts by approximately 50%. It is interesting that in vivo studies confirmed that CsA and GC inhibited EC activation at therapeutic doses (1 mg/kg and 10 mg/kg for GC and CsA, respectively) and showed that the combination of CsA and GC efficiently prevents TNFalpha-mediated induction of E-selectin on cardiac ECs. CONCLUSION: Our data show that, besides their specific immunosuppressive effects on T cells, CsA, Rap, and GC can efficiently contribute to the attenuation of EC activation in vivo and the resulting inhibition is enhanced by the association of CsA with GC.

Mutated cytochrome b as a determinant of a new monoclonal antibody (H8.98) on renal carcinoma cell lines recognized by a Vgamma3Vdelta1(+) T-cell clone.

We generated a monoclonal antibody (MAb), H8.98, that recognizes an antigen shared by 50% of examined renal carcinoma cell (RCC) lines and is susceptible to lysis by a Vgamma3Vdelta1(+) T-cell clone derived from RCC tumor-infiltrating lymphocytes. H8.98 inhibited Vgamma3Vdelta1(+ )T-cell clone-mediated lysis of RCC lines. It did not stain normal kidney lines, melanomas, fibroblasts, Burkitt's lymphoma or Epstein-Barr virus-transformed B-cell lines but it did stain 2 of 4 tested breast cancer lines. Through screening of a renal carcinoma cDNA library using H8.98, we isolated a cDNA clone which, upon sequencing, was found to be cytochrome b with 2 point mutations.