High prevalence of hepatitis C virus type 5 in central France evidenced by a prospective study from 1996 to 2002

From 1996 to 2002, hepatitis C virus (HCV) typing was prospectively performed for 1,281 unselected HCV-infected and viremic patients, irrespective of their clinical status. Eighty-three patients (6.5%) were coinfected with human immunodeficiency virus (HIV) and HCV. A total of 1,195 strains were identified by a serotype screening (Murex HCV Serotyping 1-6 assay) and/or genotyping (Inno-LiPA HCV II) test. The distribution of HCV types showed an unusually high rate of type 5 (14.2%) that was stable over time and was the third most frequent type, after type 1 (59.1%) and type 3 (15.1%). HCV type 5 was more frequent in patients who were older than 50 (P = 10(-6)), but its frequency did not differ significantly by gender (P = 0.21). Serotyping was performed for 1,160 strains but failed for 30.2% of them. The efficiency depended on HIV status (for HCV-HIV-coinfected patients, half of the strains were untypeable) and HCV type. Genotyping was performed for 428 samples, with an overall efficiency of 99.3%. It failed in three cases, which were subsequently identified as HCV type 2. Serotyping and genotyping results for 39 patients showed discrepancies between the two methods for 4 patients, who had HCV type 2, type 6, or mixed infections. Thus, HCV type 5 may also be encountered frequently in Western countries. Its apparent confinement to a restricted area raises the question of how it emerged and underscores the need for further studies of HCV type prevalence, routes of transmission, pathogenicity, and responses to treatment.     

HLA haplotype study of 53 juvenile insulin-dependent diabetic (I.D.D.) families

Fifty-three families with at least one IDD patient were genotyped for 5 markers of the HLA complex including Bf and DR. In 8 families one of the parents was also affected and in 12 families more than two children were diseased. In total, 76 patients were genotyped. Their haplotypes were compared with those of 106 unrelated controls (the parents of 53 genotyped families).

• 1) 

  Three haplotypes or segments of them (A2, Cw3, B15, BfS, DR4; Aw30, Cw5, B18, BfF I, DR3; and Al, Cw7, B8, BfS, DR3) were found more frequently in IDD patients.
• 2) 

  Measured by the 6 formula, the association of the postulated IDD susceptibility gene was very strong with the D-end of two of these haplotypes: BfF1, DR3 and BfS, DR4. However, the association was weak with the DR3 of the haplotype Al, Cw7, B8, BfS, DR3.
• 3) 

  An excess of HLA-identical affected siblings was found.
• 4) 

  An excess of DR3/DR4 heterozygotes was observed. By contrast, the observed frequency of patients homozygous for DR3 or DR4 was not increased, but even slightly decreased.

The data support a model of inheritance comprising at least two closely linked specifically "diabetic" loci (most of the time marked by B18, BfFl, DR3 and B15, BfS, DR4) and a non-specifically "diabetic" haplotype favouring auto-immunisation (most of the time marked by B8, BfS, DR3). This model is discussed in the light of the presented data and of those of the literature.     

USH2A mutation analysis in 70 Dutch families with Usher syndrome type II

Usher syndrome type II (USH2) is characterised by moderate to severe high-frequency hearing impairment, progressive visual loss due to retinitis pigmentosa and intact vestibular responses. Three loci are known for USH2, however, only the gene for USH2a (USH2A) has been identified. Mutation analysis of USH2A was performed in 70 Dutch USH2 families. Ten mutations in USH2A were detected, of which three are novel, c.949C>A, c.2242C>T (p.Gln748X) and c.4405C>T (p.Gln1468X). Including 9 previously published Dutch USH2a families, estimates of the prevalence of USH2a in the Dutch USH2 population were made. Mutations were identified in 62% of the families. In 28% both mutated alleles were identified, whereas in 34% the mutation in only one allele was found. It is estimated that about 28% of the Dutch USH2 families have a different causative gene. Analysis of deduced haplotypes suggests that c.1256G>T (p.Cys419Phe) is a Dutch ancestral mutation, occurring in 16% of the alleles.     

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.     

Inhibition of platelet adherence to brain microvasculature protects against severe Plasmodium berghei malaria

Some patients with Plasmodium falciparum infections develop cerebral malaria, acute respiratory distress, and shock and ultimately die even though drug therapy has eliminated the parasite from the blood, suggesting that a systemic inflammatory response contributes to malarial pathogenesis. Plasmodium berghei-infected mice are a well-recognized model of severe malaria (experimental severe malaria [ESM]), and infected mice exhibit a systemic inflammatory response. Because platelets are proposed to contribute to ESM and other systemic inflammatory responses, we determined whether platelet adherence contributes to experimental malarial pathogenesis. Indeed, a significant (P < 0.005) increase in the number of rolling and adherent platelets was observed by intravital microscopy in brain venules of P. berghei-infected mice compared with the number in uninfected controls. P-selectin- or ICAM-1-deficient mice exhibit increased survival after P. berghei infection. We observed a significant (P < 0.0001) reduction in the morbidity of mice injected with anti-CD41 (alpha(IIb) or gpIIb) monoclonal antibody on day 1 of P. berghei infection compared with the morbidity of infected controls injected with rat immunoglobulin G. Additionally, platelet rolling and adhesion in brain venules were reduced in P. berghei mice lacking either P-selectin or ICAM-1 or when the platelets were coated with anti-CD41 monoclonal antibody. Unlike other inflammatory conditions, we did not detect platelet-leukocyte interactions during P. berghei malaria. Because (i). leukocyte adhesion is not markedly altered in the absence of P-selectin or ICAM-1 and (ii). CD41 is not an adhesion molecule for parasitized erythrocytes, these findings support the hypothesis that inhibition of platelet adhesion to the brain microvasculature protects against development of malarial pathogenesis.     

CD4+ T cells determine the ability of spleen cells from F1 hybrid mice to induce neonatal tolerance to alloantigens and autoimmunity in parental mice

Spleen cells from F₁ hybrid mice injected into newborn parental mice induce a state of cytolytic unresponsiveness to the corresponding alloantigens. However, these mice develop a transient autoimmune syndrome characterized by the production of multiple autoantibodies and glomerulonephritis. Previous reports indicated that the depletion of F₁ donor T cells, shortly prior the injection into parental mice, does not interfere with any of these events. Here, we have explored whether the continuous absence of T cells in F₁ mice influences the ability of their spleen cells to induce neonatal tolerance to alloantigens and the associated autoimmune manifestations. Our results revealed that spleen cells from athymic (BALB/c × C57BL/6) F₁ hybrid (CB6F₁) nulnu mice or from euthymic CB6F₁ mice depleted from birth of CD4⁺ T cells, but not of CD8⁺ T cells, are unable to induce neonatal tolerance to alloantigens and autoimmune manifestations. By contrast, the partial reconstitution of T cells in CB6F¹ nulnu mice, after the neonatal graft of a syngeneic thymus, restored the capacity of spleen cells from these mice to induce tolerance and autoimmunity when injected into newborn BALB/c mice. These results demonstrate that the functional defect of spleen cells from athymic CB6F₁ nulnu mice to induce neonatal tolerance to alloantigens is directly related to the long-term absence of mature CD4⁺ T cells. Interestingly, a new increase in the titers of anti-DNA Ab was observed when spleen cells from athymic CB6F₁ nulnu mice were injected into adult BALB/c mice that had been tolerized at birth with normal CB6F₁ spleen cells. This finding indicates that B cells from CB6F₁ nulnu mice recover their capacity to interact with alloreactive Th2 cells when they are placed into mice having functional CD4⁺ T cells. These data indicate that the continuous absence of CD4⁺ T cells causes a reversible functional defect in F₁ spleen cells that determines their inability to induce neonatal tolerance and autoimmunity.     

Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam,the Vitek 2 system,and the MRSA-screen latex agglutination test

Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the "gold standard." We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-microg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter(-1) for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-microg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-microg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.     

Binding of nucleosomes to a cell surface receptor: Redistribution and endocytosis in the presence of lupus antibodies

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds)DNA and/or anti-histone autoantibodies could recognized and influence the fate of cell surface-bound nucleosomes. ¹²⁵I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of ～ 7nM and a low-affinity site (Kd ～ 400 nM) with a high capacity of 9 × 10⁷ sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived dsDNA (180–200 bp) was found to compete for binding of ¹²⁵I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from ¹²⁵-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa dsDNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-dsDNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5–10 min which moved toward the perinuclear region by 20–30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the micro-filament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-dsDNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.     

Dendritic cell populations in Leishmania major-infected skin and draining lymph nodes

Using a metacyclic promastigote ear infection model of cutaneous leishmaniasis, we examined the phenotype, parasite load, and cytokine production of dendritic cells in the skin and draining lymph nodes of resistant C57BL/6J and susceptible BALB/c mice. Five dendritic cell populations were isolated from the skin and lymph nodes, and the main difference between the groups of mice was an increased number of plasmacytoid dendritic cells in the lymph nodes of the susceptible mice. Although similar cell types were present in the skin emigrants of both strains, there was a 10-fold larger number of cells in BALB/c mouse skin early in infection than in C57BL/6J mouse skin. None of the dendritic cells in the lymph nodes harbored parasites until 3 weeks after infection, with the Langerhans cells having the largest load and the plasmacytoid dendritic cells having the smallest load but the longest lasting infection. Although parasites could be detected in the lymph nodes a few hours after infection, none of the skin emigrants harbored parasites, indicating that they are not the vehicle that ferries the parasites from the skin to the lymph nodes. The presence of larger numbers of plasmacytoid cells in infected BALB/c mice, the more protracted infection of these cells, and their production of alpha interferon point to a complex and important role for the plasmacytoid cells in leishmaniasis.