The TL MHC class Ib molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes

Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8alphaalpha homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in beta2m-/- mice but unaffected in TCR-/- mice or CD8alpha chain-/- mice. We demonstrate that the interaction between TL and CD8alphaalpha is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of alpha-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-gamma in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-gamma production suggest that the functional consequences of TL interaction with CD8alphaalpha as well as the more general biological role of TL in mucosal immunity remains to be discovered.     

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

Pneumonia due to Bordetella bronchiseptica in a cystic fibrosis patient: 16S rRNA sequencing for diagnosis confirmation

Bordetella bronchiseptica was identified as an unusual etiologic agent of pulmonary recurrent exacerbations and pneumonia in a cystic fibrosis (CF) patient by utilizing a 16S rRNA molecular kit in our hospital's clinical laboratory. This method appears to be a useful approach for identifying new emerging CF pathogens when discrepancies exist between phenotypical tests.     

Hepatitis C virus (HCV) genotypes in the Caribbean island of Martinique: evidence for a large radiation of HCV-2 and for a recent introduction from Europe of HCV-4

Molecular epidemiological studies of hepatitis C virus (HCV) in the Caribbean may help to specify the origin and spread of HCV infection. Indeed, the Caribbean population is intermixed from European and African origins and geographically close to the American continent. We characterized HCV genotypes in the Caribbean island of Martinique. HCV genotypes were analyzed by sequencing or reverse hybridization in the 5' noncoding region (5'NC) in 250 HCV-monoinfected and 85 HCV-human immunodeficiency virus (HIV)-coinfected patients. In addition, sequencing in the nonstructural 5B (NS5B) gene was required to determine the subtype or to perform phylogenetic analysis in selected samples. Genotypes 1 to 6 were found, respectively, in 84.4, 6.8, 5.2, 2.8, 0.4, and 0.4% of 250 HCV-monoinfected patients and in 71.7, 7.1, 15.3, 5.9, 0, and 0% of 85 HCV-HIV-coinfected patients. HCV-1b was found in 66.4% of the HCV-monoinfected patients and was associated with blood transfusion, whereas HCV-1a was detected in 41.2% of the HCV-HIV-coinfected patients and was associated with intravenous drug use (IVDU). The HCV-3 strains belonged to subtype 3a and were linked to IVDU. Phylogenetic analyses were focused on HCV-2 and HCV-4, which are common in Africa. Two opposite patterns were evidenced. NS5B sequences from 19 HCV-2 isolates were affiliated with many different subtypes described either in Europe or in West Africa, suggesting an ancient radiation. In contrast, seven of the nine HCV-4 NS5B sequences ranged within HCV-4a and HCV-4d clusters spreading in continental France by the IVDU route. Epidemiological data demonstrate the recent introduction of HCV-4a and -4d subtypes into the Caribbean.     

Genetic association between the phospholipase A2 gene and unipolar affective disorder: a multicentre case-control study

The co-segregation in one pedigree of bipolar affective disorder with Darier's disease whose gene is on chromosome 12q23-q24.1, and findings from linkage and association studies with the neighbouring gene of phospholipase A2 (PLA2) indicate that PLA2 may be considered as a candidate gene for affective disorders. All relevant genetic association studies, however, were conducted on bipolar patients. In the present study, the possible association between the PLA2 gene and unipolar affective disorder was examined on 321 unipolar patients and 604 controls (all personally interviewed), recruited from six countries (Belgium, Bulgaria, Croatia, Germany, Greece, and Italy) participating in the European Collaborative Project on Affective Disorders. After controlling for population group and gender, one of the eight alleles of the investigated marker (allele 7) was found to be more frequent among unipolar patients with more than three major depressive episodes than among controls (P<0.01); genotypic association was also observed, under the dominant model of genetic transmission (P<0.02). In addition, presence of allele 7 was correlated with a higher frequency of depressive episodes (P<0.02). These findings suggest that structural variations at the PLA2 gene or the chromosomal region around it may confer susceptibility for unipolar affective disorder.     

DFNB40, a recessive form of sensorineural hearing loss, maps to chromosome 22q11.21-12.1

We report on a novel localization for a recessive form of deafness (DFNB), by linkage analysis in an Iranian consanguineous family. Affected individuals suffer from prelingual profound sensorineural hearing loss. Genome-wide analysis led to the characterization of a new locus, DFNB40, which maps to an approximately 9 Mb interval between markers D22S427 and D22S1144 at chromosome 22q11.21-12.1. Maximum lod score of 3.09 was obtained with D22S1174. Since the Bronx waltzer (bv) mouse mutant, characterized by waltzing behavior, deafness, and degeneration of cochlear inner hair cells, has been mapped to the syntenic region on murine chromosome 5, we suggest that DFNB40 and bv may result from orthologous gene defects.     

Ovarian stimulation for in-vitro fertilization combining administration of gonadotrophins and blockade of the pituitary with D-Trp6-LHRH microcapsules: pilot studies with two protocols

In women undergoing in-vitro fertilization and embryo transfer(TVF-ET), a total of 408IVF cycles were stimulated using humanmenopausal gonadotrophin (HMG) or pure follicle stimulatinghormone (FSH) plus HMG in combination with a single injectionof D-Trp⁶-LHRH microcapsules in order to enhance the ovarianresponse to gonadotrophins and to avoid spontaneous LH surges.Sixty-seven pregnancies were achieved. Two protocols were employed.In protocol 1 (‘blocking protocol’, n = 268), thepituitary was first inhibited with a full dose (3.75 mg) ofD-Trp⁶-LHRH in microcapsules and ovarian stimulation was startedafter the hypogonadotrophic hypogonadal state was ascertained(Ej >50 pg/ml). In protocol 2 (‘flareup protocol’,n = 140), the treatment with D–Trp⁶LHRH microcapsules(half-dose = 1.80 mg) and the ovarian stimulation with gonadotrophinswere started at the same time. Higher doses of gonadotrophinswere needed (39.5 11.2 ampoules FSH and/or HMG) in protocol1, in which the pituitary was blocked prior to and during thestimulation, than in protocol 2 (209 ampoules) where the exogenousgonadotrophin stimulation appeared to be augmented by the initialagonistic effect of the injection of D-Trp⁶LHRH microcapsules.In patients with purely tubal infertility, under 38 years oldand no male factor, the results obtained with protocols 1 and2 were similar in terms of pregnancy rate per cycle or per embryotransfer: 22.6 versus 20.5% and 28.3 versus 27.4%, respectively.However, considering the cost benefit, ‘flare-up’protocols appeared to be a better choice and could be recommended.     

Effect of prostaglandin E2 on agonist-stimulated cAMP accumulation in the distal convoluted tubule isolated from the rabbit kidney

The effects of calcitonin, vasoactive intestinal peptide (VIP), parathyroid hormone (PTH) and isoprenaline on intracellular cAMP accumulation were determined in the distal tubule (DCT) microdissected from collagenase-treated rabbit kidney. In DCTb (the initial bright portion) calcitonin (10 ng/ml) elicited a highly reproducible response 203.7±19.1 fmol cAMP mm^–1 4 min^–1 (SE, N=13) whereas VIP-induced cAMP accumulation was less and more variable from one experiment to another (1 M, 97.2±17.8 fmol mm^–1 4 min^–1, SE, N=12). When used in combination, these two agonists were non-additive, indicating stimulation of a single pool of cAMP in DCTb. In DCTg, (granular) which consists of at least two cell types, PTH (100 nM) elicited a marked, reproducible accumulation of cAMP (154.3±27.0 fmol mm^–1 4 min^–1; SE, N=5). Isoprenaline (1 M) and VIP (1 M) induced much smaller increases in cAMP levels 20.9±2.7 and 29.4±4.1 fmol mm^–1 4 min^–1 (SE, N=5) respectively, and, when used in combination, were non-additive, demonstrating that VIP and isoprenaline are active on the same cell type. In DCTb, prostaglandin E₂ (PGE₂) inhibited both calcitoninand VIP-stimulated cAMP accumulation (calcitonin 57.8±2.7% inhibition, SE, N=16; VIP, 80.6±2.1% inhibition, SE, N=5). The EC₅₀ values for calcitonin were 1.21±0.33 ng/ml and 1.83±0.25 ng/ ml (SD, N=3) in the absence and presence of PGE₂ (300 nM) respectively with an IC₅₀ for PGE₂ of 26.3±6.3 nM (SE, N=4). In contrast, no effects of PGE₂ were seen in DCTg vis à vis PTH, isoprenaline or VIP. The percentage inhibition of calcitonin-stimulated cAMP accumulation by PGE₂ was of the same order in the presence of isobutylmethylxanthine (an inhibitor of all types of phosphodiesterase), Ro 20-1724 (inhibitor of low-K _m cAMP-specific phosphodiesterase) or in the absence of inhibitor. Preincubation of DCTb with pertussis toxin for up to 8 h in different experimental conditions did not relieve the inhibition by PGE₂. Protein kinase C activation by phorbol ester did not attenuate calcitonin responses. These data demonstrate that the inhibition by PGE₂ of cAMP production is restricted to the initial portion (DCTb) of the distal convoluted tubule and is effective on both calcitonin and VIP responses. When tested in the presence of Ro 20-1724, ionomycin, A₁-adenosine, ₂-adrenergic and muscarinic agonists were without effect on calcitoninand PTH-stimulated cAMP accumulation in DCTb and DCTg respectively.