Novel 5-flucytosine-resistant clade of Candida dubliniensis from Saudi Arabia and Egypt identified by Cd25 fingerprinting

DNA fingerprinting of Candida dubliniensis isolates using the species-specific probe Cd25 previously showed that this species consists of two distinct groups, termed Cd25 group I and Cd25 group II. The present study investigated the population structure of 30 C. dubliniensis oral isolates from Saudi Arabia and Egypt using Cd25 fingerprinting and rRNA gene internal transcribed spacer region-based genotyping. Cd25 fingerprinting analysis of these isolates revealed two distinct populations, the first of which consisted of 10 closely related genotype 1 isolates (average similarity coefficient [S(AB)] value, 0.86). The second population of 20 isolates was much more heterogeneous (average S(AB) value, 0.35) and consisted of two distinct subpopulations, one of which consisted of genotype 3 isolates (n = 13) and the other of genotype 4 isolates (n = 7). A mixed dendrogram generated from the fingerprint data from the 30 Saudi Arabian and Egyptian isolates, 5 Israeli isolates, and 51 previously characterized international isolates (32 of Cd25 group I and 19 of Cd25 group II) revealed the presence of three distinct main clades. The first corresponded to the previously described Cd25 group I and contained all the Saudi Arabian, Egyptian, and Israeli genotype 1 isolates mixed with international isolates. The second clade corresponded to the previously described Cd25 group II and contained three Israeli isolates, one genotype 2 isolate, one genotype 3 isolate, and a genotype 4 variant isolate, which were mixed with international isolates. The third clade has not been described before and consisted solely of the 20 Saudi Arabian and Egyptian genotype 3 and 4 isolates identified in this study and a previously described genotype 4 Israeli isolate. All 20 Cd25 group III isolates exhibited high-level resistance to 5-flucytosine (MIC > or = 128 microg/ml), whereas all Cd25 group I and Cd25 group II isolates tested (10 Saudi Arabian and Egyptian, 16 Israeli, and 24 international) were susceptible to 5-flucytosine (MIC < or = 0.125 microg/ml). The results of this study show for the first time the presence of a novel 5-flucytosine-resistant clade of C. dubliniensis (Cd25 group III) that is predominant among isolates from Saudi Arabia and Egypt and absent from a previously characterized international collection of 98 isolates from 15 countries.     

Comparative analysis of immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay using virus-like particles or virus-infected mouse brain antigens to detect IgM antibody in sera from patients with evident flaviviral infections

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquito-borne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 - specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain.     

The IBD6 Crohn's disease locus demonstrates complex interactions with CARD15 and IBD5 disease-associated variants

Genetic studies in inflammatory bowel disease have identified multiple susceptibility loci, whose relevance depends critically on verification in independent cohorts. Genetic variants associated with Crohn's disease have now been identified on chromosomes 5 (IBD5/5q31 risk haplotype) and 16 (IBD1 locus, CARD15/NOD2 mutations). Stratification of genome-wide linkage analyses by disease associated variants is now possible, offering both increased power for identification of other loci and improved understanding of genetic mechanisms. We performed a genome-wide scan of 137 Crohn's disease affected relative pairs from 112 families. Multipoint non-parametric linkage analyses were performed, with further stratification of affection status by common CARD15 mutations and the IBD5 haplotype. We verified linkage of Crohn's disease to regions on chromosome 3 (P=0.0009) and X (P=0.001) in our cohort. Linkage to chromosome 16 (IBD1) was observed in Crohn's disease pairs not possessing common CARD15 mutations (P=0.0007), approximately 25 cM q telomeric of CARD15. Evidence for linkage to chromosome 19 (IBD6) was observed in Crohn's disease pairs not possessing CARD15 mutations (P=0.0001), and in pairs possessing one or two copies of the IBD5 risk haplotype (P=0.0005), with significant evidence for genetic heterogeneity and epistasis, respectively. These analyses demonstrate the complex genetic basis to Crohn's disease, and show that the discovery of disease-causing variants may be used to aid identification of further susceptibility loci in complex disease.     

Bacterial superantigen-treated intestinal epithelial cells upregulate heat shock proteins 25 and 72 and are resistant to oxidant cytotoxicity

While the pathological events evoked by infection are commonly described, effective host responses to bacteria and their products should primarily be protective. Heat shock protein (Hsp) expression is upregulated by many stimuli and serves to maintain intracellular protein integrity. The ability of the prototypic superantigen, Staphylococcus aureus enterotoxin B (SEB) to induce Hsps was investigated with BALB/c mice and by in vitro addition to the murine small intestinal epithelial cell line MSIE. SEB-treated (5 or 100 microg intraperitoneally) mice revealed increased Hsp25 and Hsp72, but not Hsc73, in jejunal lymphocytes and epithelial cells. A similar Hsp response to SEB occurred in MSIE cells and was preceded by activation of the ERK1/2 and p38 mitogen-activated protein kinases but not the SAPK/JNK pathway; pharmacological inhibition of ERK1/2, but not p38, significantly reduced SEB-induced Hsps. Moreover, SEB-treated MSIE cells were protected against oxidant-induced cytotoxicity (measured by 51Cr release) and F-actin depolymerization. Thus, SEB exposure results in a rapid induction of the Hsp25 and Hsp72 in intestinal epithelial cells, both directly and through lymphocyte activation, and we suggest that this event is important in protecting the gut from damage by Staphylococcus infection or in the reparatory process and may be a generalized response to lumen-derived bacterial toxins.     

Function and genetics of dystrophin and dystrophin-related proteins in muscle

The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and alpha-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.     

Frequency of cytokine polymorphisms in populations from western Europe,Africa, Asia,the Middle East and South America

PCR-SSOP identification procedures for IL-2, IL-6, IL-10, TNF-alpha and TNF-beta cytokine polymorphisms have been developed. Application of the procedures to a range of diverse geographically distributed populations has identified ethnic differences within the groups studied. Five populations were investigated, Northern Ireland, South African Zulu, Omani, Singapore Chinese and Mexican Mestizos.     

Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn's disease

The inflammatory bowel diseases (IBDs), Crohn's disease (CD) and ulcerative colitis, are chronic inflammatory disorders of the digestive tract. The pathogenesis of IBD is complicated, and it is widely accepted that immunologic, environmental and genetic components contribute to its etiology. To identify genetic susceptibility factors in CD, we performed a genome-wide association study in Japanese patients and controls using nearly 80,000 gene-based single nucleotide polymorphism (SNP) markers and investigated the haplotype structure of the candidate locus in Japanese and European patients. We identified highly significant associations (P = 1.71 x 10(-14) with odds ratio of 2.17) of SNPs and haplotypes within the TNFSF15 (the gene encoding tumor necrosis factor superfamily, member 15) genes in Japanese CD patients. The association was confirmed in the study of two European IBD cohorts. Interestingly, a core TNFSF15 haplotype showing association with increased risk to the disease was common in the two ethnic groups. Our results suggest that the genetic variations in the TNFSF15 gene contribute to the susceptibility to IBD in the Japanese and European populations.     

Adenoviral transfer of the murine oncostatin M gene suppresses dextran-sodium sulfate-induced colitis.

The use of biologics has promising potential in the treatment of inflammation. Studies with cultured cells and mouse models of disease have ascribed proinflammatory and anti-inflammatory functions to oncostatin M (OSM) and the related cytokine, interleukin-6 (IL-6). Here, we examined the effect of systemic administration of adenoviral (Ad) vectors encoding either murine OSM (AdMuOSM) or murine IL-6 (AdMuIL-6) in a mouse model of colitis. BALB/c mice were treated with a 5-day course of 4% dextran-sodium sulfate (DSS) water with or without administration of adenoviral vectors (i.p. or i.m. at 10(7) plaque-forming units [pfu]) given as a cotreatment or therapy. The deletion variant of the adenovirus served as a control for adenoviral infection. Colitis was assessed by (1) morphology (damage score, macrophage infiltration, apoptosis) and (2) function (myeloperoxidase activity and Ussing chamber analysis of epithelial ion transport). Infection with adenovirus alone did not affect colonic form or function. AdMuOSM (either i.p. or i.m.) significantly reduced the severity of the DSS-induced colitis. There was less damage, reduced macrophage infiltration, fewer apoptotic bodies, and a significant improvement in stimulated ion transport in colonic tissues from the treated mice. No benefit of AdMuIL-6 treatment was observed in this model system. Thus, systemic administration of AdMuOSM given as a cotreatment and to a lesser extent as a therapy was found to be of benefit in DSS-induced colitis, a murine model of inflammatory bowel disease (IBD).     

Association of the dihydrolipoamide dehydrogenase gene with Alzheimer's disease in an Ashkenazi Jewish population.

Abundant biochemical evidence links deficient activity of mitochondrial alpha-ketoglutarate dehydrogenase with neuropathologically confirmed Alzheimer's disease (AD). Reduced alpha-ketoglutarate dehydrogenase activity has also been associated with anti-mortem measures of clinical disability. One of the genes encoding this complex, namely, DLD, lies within a chromosome 7 region that is in linkage disequilibrium with AD. We therefore examined the hypothesis that variation in DLD is associated with AD risk. Denaturing HPLC was used to search for sequence variations in the coding and flanking regions of all exons of DLD, but no abundant variants that alter protein sequence were found. However, four common SNPs were identified and genotyped in a case-control series of 297 Caucasians from New York City, including 229 residents of a Jewish nursing home. Logistic regression analysis was performed for the four-locus DLD genotype, sex, and ApoE4 status to determine the association of these independent variables with AD. Significant associations with AD were observed for ApoE4 (P < 10(-6)) and sex combined with DLD genotype (P = 0.013). The association with the DLD genotypes appears only in the male population in both the Caucasian series (P = 0.0009, n = 83) and the Ashkenazi Jewish subseries (P = 0.017, n = 49). The DLD genotype appears to operate independently of APOE in conferring AD risk.