GM and KM allotypes in eight tribal populations of madyha Pradesh and Orissa, India

Summary Serum samples from eight endogamous Indian tribal populations of Madhya Pradesh (Dhurwa, Halba, Bhatra, Muria, Maria) and Orissa (Deshia Khond, Binjhal, Kisan) with a total of n=731 unrelated individuals were typed for G1M (1,2,3,17), G3M (5,10,11,13,14,15,16,21,26), and KM (1). In seven of these populations five different GM haplotypes were found:GM*1,17;21,26; GM*1,17;10,11,13,15,16; GM*1,2,17;21,26; GM*1,3;5,10,11,13,14,26; andGM*3;5,10,11,13,14,26. In the Kisan sample the haplotypeGM*1,2,17; 21,26 is absent. The intergroup variability in the distribution of these haplotypes is considerable and statistically highly significant. The reasons for that can be attributed to the ethnohistory and to the genetic isolation of these eight endogamous tribal populations. The GM haplotype distribution pattern of all these groups is quite different from that of the non-tribal populations of India, whereas it is in good agreement with that of the so far tested other tribal populations from India. This can be explained by different origin and history of the Indian tribal and non-tribal populations. In the KM system, too, remarkable variability is seen in the distribution of phenotype and allele frequencies among the eight tribal populations under study.     

Virtually full-length subtype F and F/D recombinant HIV-1 from Africa and South America

For reliable classification of HIV-1 strains appropriate reference sequences are needed. The HIV-1 genetic subtype F has a wide geographic spread, causing significant epidemics in South America, Africa, and some regions of Europe. Previously only two full-length sequences of each of the HIV-1 subtype F subclusters F1 and F2 have been described. To extend the knowledge of subtype F variation on a complete genome level, three new virtually full-length F1 sequences were cloned and sequenced, two from Africa and one from South America. Comparison of the new and previously described sequences showed that monophyletic clustering of the subcluster F1 of subtype F is consistent and highly supported in all genome regions. Two additional full-length strains were shown to be mosaics of subtypes F and D. These epidemiologically unrelated F/D sequences showed similar chimeric structure, suggesting that they may represent a previously undescribed circulating recombinant form (CRF). This was supported by partial sequences from three additional unlinked F/D recombinants. Genetic distances in the phylogenetic trees suggest that the recombination event leading to the putative CRF occurred relatively long ago, close to the divergence of the F1 and F2 subclusters. Furthermore, all five F/D recombinants are linked to the Democratic Republic of Congo, suggesting that the original recombination event took place in central Africa.     

Increased in vivo frequency of IA-2 peptide-reactive IFNgamma+/IL-4- T cells in type 1 diabetic subjects

Active T cell recognition of islet antigens has been postulated as the pathogenic mechanism in human type 1 diabetes, but evidence is scarce. If T cells are engaged, they are expected to display increased clonal size and exhibit a T helper (Th)1/Th2 differentiation state. We used a peptide library that covers tyrosine phosphatase IA-2, a target antigen expressed in pancreatic beta cells, to probe 8 diabetic patients and 5 HLA-matched controls. When tested in a high resolution IFNgamma/IL-4 double color ELISPOT assay directly ex vivo, the number of IA-2-reactive IFNgamma producing cells was 17-fold higher in patients than in controls and IL-4 producing cells were not present. An average of 9 peptides was recognized in the patients vs. one in the controls. Determinant recognition primarily involved CD4+ cells and showed high variability among the patients. Furthermore, anti-CD28 antibody signal enhances quantitative assessment of effector T cells in T1D patients. In vitro expansion with peptides and IL-2 results in detection of responding cells in the controls and loss of disease specificity of the T cell response. Together these data provide strong evidence for the active targeting of IA-2 by Th1 memory effector cells in human type 1 diabetes.     

Molecular Characterization of Vancomycin-Resistant Enterococci from Hospitalized Patients and Poultry Products in The Netherlands

Vancomycin-resistant enterococci (VRE) pose an emerging health risk, but little is known about the precise epidemiology of the genes coding for vancomycin resistance. To determine whether the bacterial flora of consumer poultry serves as a gene reservoir, the level of contamination of poultry products with VRE was determined. VRE were genotyped by pulsed-field gel electrophoresis (PFGE), and transposon structure mapping was done by PCR. The vanX-vanY intergenic regions of several strains were further analyzed by sequencing. A total of 242 of 305 (79%) poultry products were found to be contaminated with VRE. Of these VRE, 142 (59%) were high-level-vancomycin-resistant Enterococcus faecium strains (VREF). PFGE revealed extensive VREF heterogeneity. Two genotypes were found nationwide on multiple occasions: type A (22 of 142 VREF [15%]) and type B (14 of 142 VREF [10%]). No PFGE-deduced genetic overlap was found when VREF from humans were compared with VREF from poultry. Two vanA transposon types were identified among poultry strains. In 59 of 142 (42%) of the poultry VREF, the size of the intergenic region between vanX and vanY was ~1,300 bp. This transposon type was not found in human VREF. In contrast, all human strains and 83 of 142 (58%) of the poultry VREF contained an intergenic region 543 bp in size. Sequencing of this 543-bp intergenic vanX-vanY region demonstrated full sequence conservation. Though preliminary, these data suggest that dissemination of the resistance genes carried on transposable elements may be of greater importance than clonal dissemination of resistant strains. This observation is important for developing strategies to control the spread of glycopeptide resistance.     

Colonization of in vitro-formed cervical human papillomavirus- associated (pre)neoplastic lesions with dendritic cells: role of granulocyte/macrophage colony-stimulating factor

The purpose of this study was to investigate the ability of CD1a+ Langerhans/dendritic cells (LCs/DCs) to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. Migration of LCs/DCs in the presence of keratinocytes derived from normal cervix and HPV-transformed cell lines was evaluated in Boyden chambers and in organotypic cultures and correlated with granulocyte/macrophage colony-stimulating factor (GM-CSF) production by the cells, as determined by ELISA. Conditioned media of HPV-transformed keratinocytes contained lower amounts of GM-CSF and induced a decreased motile response of LCs/DCs in the Boyden chamber assay compared with those of normal cervical keratinocytes. The migration of LCs/DCs in the presence of conditioned media from normal keratinocytes could be blocked by an anti-GM-CSF antibody, and the migration of LCs/DCs in the presence of conditioned media from HPV-transformed keratinocytes could be increased by supplementing the media with recombinant GM-CSF. GM-CSF was also a potent factor in enhancing the colonization of LCs/DCs into organotypic cultures of HPV-transformed keratinocytes, as the infiltration of LCs/DCs in the in vitro-formed (pre)neoplastic epithelium was minimal under basal conditions and dramatically increased after the addition of GM-CSF to the cultures. These results suggest that GM-CSF could play an important role in the recruitment of LCs/DCs into the HPV-transformed (pre)neoplastic cervical epithelium and be useful as a new immunotherapeutic approach for cervical (pre)cancers.     

In vivo monitoring of age-related changes in rat brain using quantitative diffusion magnetic resonance imaging and magnetic resonance relaxometry

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.     

Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods

Extended-spectrum beta-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were > or =2 microg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of > or =5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by > or =3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were > or =32 microg/ml, suggesting either the presence of an AmpC-type beta-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta-lactamase with a pI of > or =8.3 suggestive of an AmpC-type beta-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla(OXA) genes. The IEF profiles of the remaining 16 isolates showed a variety of beta-lactamase bands, all of which had pIs of < or =7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla(OXA), and bla(CTX-M) genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained beta-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained beta-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.     

HnRNP CBP35-CBP67 interaction during stress response and ageing

Previous studies have demonstrated the existence of nuclear carbohydrate binding proteins in a variety of mammalian cells with molecular masses of 35 000, 67 000, and 70 000 (CBP35, CBP67, and CBP70), which are associated with nuclear ribonucleoprotein (RNP) complexes. CBP35 consists of two domains, an aminoterminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear RNP complex, and a carboxyl-terminal portion homologous to β-galactoside-specific lectins. CBP35 it has been proposed, like the glucose-specific lectin, CBP67, to guide RNP complexes through the nuclear pore. Here we show that the exposure of mature rats to stress induces an increase in nuclear CBP35 bound to CBP67 and retained on immobilized glucose. Nuclear extracts from the livers of old rats displayed no detectable stress response. This CBP35·CBP67 association detected in rat liver is considered with respect to the CBP35·CBP70 association recently observed in HL60 cell nuclear extracts.