Short-term CD4 cell response after highly active antiretroviral therapy initiated at different times from seroconversion in 1,500 seroconverters

The effect of HIV infection duration and CD4 cell count on short-term CD4 response was evaluated in treatment-naive seroconverters using logistic regression adjusted for CD4 count before highly active antiretroviral therapy (HAART) as well as for exposure category, age, sex, acute infection, and cohort. This association was also investigated in pretreated seroconverters, further adjusting for prior therapy. CD4 response (increase of >100 cells/microL at 6 months) was more likely if HAART was initiated in the first year following seroconversion (OR = 1.50 [95% CI: 1.07-2.10] compared with 2-5 years). There was no improvement in response from initiating HAART with CD4 count >350 cells/microL compared with 201 to 350 cells/microL. Below 200 cells/microL, however, the chance of a CD4 response appeared to be reduced (OR = 0.72 [95% CI: 0.40-1.28] for 0-200 cells/microL compared with 201-350 cells/microL, P = 0.26). Results were similar for pretreated individuals. Further, in pretreated individuals, a CD4 response was less likely if the CD4 nadir was lower than the pre-HAART CD4 count (OR = 0.18 [95% CI: 0.10-0.36] for >150 cells/microL difference between nadir and pre-HAART CD4 count vs. no difference, P < 0.001). Given the limitations of observational studies, particularly the inability to control for unmeasured confounders, these findings suggest that the initiation of HAART within the first year following seroconversion appears to improve short-term immunologic response. After that time, there is little to be gained in terms of short-term response from initiating HAART before reaching a CD4 count of 200 cells/microL.     

Improved Identification of Epidemiologically Related Strains of Salmonella enterica by use of a Fusion Algorithm Based on Pulsed-Field Gel Electrophoresis and Multiple-Locus Variable-Number Tandem-Repeat Analysis

Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) are used to assess genetic similarity between bacterial strains. There are cases, however, when neither of these methods quantifies genetic variation at a level of resolution that is well suited for studying the molecular epidemiology of bacterial pathogens. To improve estimates based on these methods, we propose a fusion algorithm that combines the information obtained from both PFGE and MLVA assays to assess epidemiological relationships. This involves generating distance matrices for PFGE data (Dice coefficients) and MLVA data (single-step stepwise-mutation model) and modifying the relative distances using the two different data types. We applied the algorithm to a set of Salmonella enterica serovar Typhimurium isolates collected from a wide range of sampling dates, locations, and host species. All three classification methods (PFGE only, MLVA only, and fusion) produced a similar pattern of clustering relative to groupings of common phage types, with the fusion results being slightly better. We then examined a group of serovar Newport isolates collected over a limited geographic and temporal scale and showed that the fusion of PFGE and MLVA data produced the best discrimination of isolates relative to a collection site (farm). Our analysis shows that the fusion of PFGE and MLVA data provides an improved ability to discriminate epidemiologically related isolates but provides only minor improvement in the discrimination of less related isolates.Salmonellosis is one of the most common food-borne diseases in the United States (5). Consequently, it is important to understand how Salmonella strains disseminate within and between reservoirs and environments. Many molecular typing tools have been used for this purpose (11). Of these methods, pulsed-field gel electrophoresis (PFGE) is considered by many to be the gold standard for strain typing, and variable-number tandem-repeat (VNTR) assays are powerful alternative or complementary typing tools (3, 22). Both methods offer a high degree of genetic resolution for strain typing, depending on several factors.PFGE involves separating chromosomal DNA macro-restriction fragments by size, and strains are discriminated based on the resulting band pattern observed after electrophoresis has been completed. It is one of the most reproducible and highly discriminatory typing techniques and has been widely and successfully used for a variety of Salmonella enterica serovars (12, 15); for many situations, PFGE is capable of discriminating between closely related strains. In addition, the use of the assay to analyze different serovars does not require a great deal of modification, as might be required with procedures that are dependent on PCR. Difficulties arise when strains are very closely related (i.e., poor discrimination [18, 27]) or when bands comigrate in the gel or identically sized bands represent completely different fragments of chromosomal DNA and thereby produce spurious matches (6). These complications are more pronounced when a large number of bands are generated by the restriction digest (4). In addition, while band patterns convey a crude degree of genetic relatedness, a large number of independent restriction digests would be needed to infer an accurate phylogenetic relationship (6).Multiple-locus variable-number tandem-repeat analysis (MLVA) is a PCR-based technique that relies on the amplification of chromosomal or plasmid DNA that encompasses short tandem repeats of a DNA sequence. The tandem repeats are prone to higher-than-background mutation rates due to DNA strand slippage during replication (23), and thus, the amplified fragments will vary in length depending on the number of repeats harbored at a given locus. Different fragment lengths are tallied either as the total length (base pairs) or the estimated number of repeat units, and each discretely sized fragment is considered a unique “allele” for the locus under investigation. Because of the relatively high mutation rate, strains can accumulate distinctive allele patterns within a relatively short period of time (5). Furthermore, the technique can be multiplexed and automated and is conducive to rapid and relatively high-throughput strain typing. MLVA assays are relatively robust (5, 15-17) and, while not perfect, they can provide phylogenetic information even with a limited number of loci (13, 18). While access to a sequenced genome dramatically speeds the ability to establish new assays (3), it is not a requisite to assay development. The primary limitations of the technique include the potential need for a new set of loci for every species or serovar under investigation and the fact that some loci are very “unstable” and can “disappear” from some strains or lineages; this produces the equivalent of an uninformative “null” allele. Mutation rates can also vary between loci (5, 24, 25); if ignored, this factor can introduce bias into comparative analyses.Clearly, PFGE and MLVA offer different technical and interpretive advantages and disadvantages, but it is important to emphasize that the nature of the methodological and interpretive errors is independent between the techniques. For example, errors due to comigration of bands for PFGE are independent of band size estimation errors for MLVA because differences in MLVA band size are not detectable using PFGE and macro-restriction fragments are generally independent of tandem-repeat sequences. Provided that most of the experimental variation from these two methods is uncorrelated (i.e., independent), it is possible to combine results from the two methods to produce improved estimates (8), and this premise underlies the current study.Our objective was to determine whether combining the information obtained from both PFGE and MLVA assays produces more rigorous and discriminatory analyses of bacterial pathogens, such as Salmonella. Two sets of Salmonella enterica isolates were used in this study; one set included serovar Typhimurium isolates from a wide range of sampling dates, locations, and host species while the other set included a group of serovar Newport isolates collected over a limited geographic and temporal scale for a single host species. The results of the different typing methods were assessed by comparison with those of phage-typing assays (serovar Typhimurium) and with known epidemiological relationships (serovar Newport). To interpret the MLVA data, we employed a metric that incorporates a stepwise-mutation model, and to interpret the PFGE data, we employed Dice coefficients to construct distance matrices. Our analysis shows that the fusion of the two typing methods provides an improved ability to discriminate between isolates when PFGE and MLVA separately provide partial but incomplete discrimination between strains with a high degree of probable genetic similarity.     

Screening for coeliac disease among Egyptian children

Introduction

To screen for coeliac disease in Egyptian children with non-endocrinal short stature, refractory iron deficiency anaemia and type 1 diabetes. Also, to evaluate the sensitivity and specificity of different serological tests for diagnosis of coeliac disease (CD).

Material and methods

The study included 292 patients with clinical risk of CD. Testing for coeliac antibodies was performed, together with upper gastrointestinal endoscopy and small intestinal biopsy.

Results

Eleven patients (44%) among 25 patients with refractory iron deficiency anaemia, 23 patients (34.3%) among 67 patients with non-endocrinal short stature, and 6 patients (3%) among 200 patients with type I diabetes mellitus were diagnosed by jejunal biopsy as having coeliac disease. AGA (IgG) had the highest sensitivity for diagnosing CD (80.0%) followed by the TTG (72.7%) antibody, while ARA had the highest specificity (95.9%) followed by anti-EMA (94.7%).

Conclusions

Coeliac disease is more common in Egyptian children with refractory iron deficiency anaemia, non-endocrinal short stature and type 1 diabetes than was previously thought; therefore it is mandatory to screen such patients for CD. Serological tests showed fairly good sensitivity and specificity for the diagnosis; however, intestinal biopsy remains the cornerstone for definitive diagnosis of patients with immunological reaction to gluten.     

Potential Influence of Interleukin-1 Receptor Antagonist Gene Polymorphism on Knee Osteoarthritis Risk

Objectives: Genes encoding for cytokines have been associated with susceptibility for joint osteoarthritis (OA) and interleukin (IL)-1 gene is supposed to be involved in the cartilage destruction process. In this regard, interleukin-1 receptor antagonist (IL-1RA) competing with IL-1 for binding to its receptor may act as an inhibitor of cartilage breakdown. We assessed the association of primary knee OA with IL-1RA region as a putative factor of susceptibility to knee OA in Egyptian patients. Design and methods: Eighty patients with primary knee OA and 40 aged-matched healthy controls were included into the study. DNA samples were used to study genotypes of IL-1RN gene by polymerase chain reaction (PCR) in both groups. Results: An increased frequency of the IL-1RN*1 and IL-1RN*2 alleles was found in OA patients relative to controls (60.5% vs. 39.5%, P = 0.039, 85.4% vs. 14.6%, P = 0.002, respectively) however, only the carriage rate of IL-1RN*2 allele was found to be significant when OA patients were compared to the controls. Significant higher frequencies of IL-1RN*1/*2 and IL-1RN*2/*2 genotypes in OA patients were observed as compared with controls. Both visual analogue scale (VAS) and radiographic score revealed significant correlation with both the allelic frequency and the carriage rate of IL-1RN*2 allele. Moreover, absolute frequency of IL-1RN*1/*2 genotype OA patients revealed severe VAS and high radiographic score. Conclusion: These results suggest that IL-1RN*2 allele represent a significant factor influencing the severity and course of knee OA; thereby supporting the potential role of IL-1 in the pathogenesis of this disease.     

Evaluation of an experimental dental porcelain

The aim of this study was to evaluate fracture toughness, hardness, ceramic/metal bond strength and microstructure of experimental dental porcelain and compare it with commercial type. Specimens of specific dimensions were prepared. Fracture toughness was assessed by a three-point bending test. The Vickers hardness was measured using a microhardness tester. The ceramometal bond strength was measured using a universal testing machine. The load was applied at the porcelain/metal interface via a chisel edged blade with a crosshead speed of 2.0 mm/min until fracture. The polished specimens of dental porcelain were chemically etched and the microstructure was analyzed with a scanning electron microscope. The results showed comparable fracture toughness and bond strength for both materials, while the experimental porcelain exhibited higher hardness. The experimental porcelain showed uniform cohesive failure while the commercial type showed mixed mode of failure. The microstructure of the experimental porcelain was tetragonal leucite crystals dispersed randomly in a glass matrix. The leucite crystals exist in two forms, acicular and rod like structures. It was concluded that the experimental porcelain has adequate fracture toughness and ceramic/metal bond strength that can resist the rapid crack propagation and its consequent catastrophic failure, which indicates a material serviceability in the oral cavity.     

Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis

The pathogenic protozoan parasite Theileria uilenbergi is one of the causative agents of theileriosis in small ruminants in China. The infection results in great economical losses in the northwest part of China. Efforts are underway to establish an enzyme-linked immunosorbent assay (ELISA) based on a T. uilenbergi immunodominant recombinantly expressed protein using different approaches in order to perform epidemiological studies in the area. In this study, we describe the possible use of the clone-9 protein for this purpose, which was identified as a potential immunogenic piroplasm protein by random sequencing of cDNA library clones followed by bioinformatic analyses. The clone-9 gene was partially recombinantly expressed and used for the development of an indirect ELISA for the detection of circulating antibodies in sera of T. uilenbergi-infected sheep. No cross-reactivity was observed in serum from animals infected with Theileria lestoquardi. The cut-off was calculated at 48.6% positivity using 25 serum samples from uninfected animals. A total of 101 field samples collected from an endemic area in China were used to evaluate the clone-9 ELISA for its use in the field.     

Hybrid Mice as Genetic Models of High Alcohol Consumption

We showed that F1 hybrid genotypes may provide a broader variety of ethanol drinking phenotypes than the inbred progenitor strains used to create the hybrids (Blednov et al. in Alcohol Clin Exp Res 29:1949–1958, 2005). To extend this work, we characterized alcohol consumption as well as intake of other tastants (saccharin, quinine and sodium chloride) in five inbred strains of mice (FVB, SJL, B6, BUB, NZB) and in their reciprocal F1 hybrids with B6 (FVBxB6; B6xFVB; NZBxB6; B6xNZB; BUBxB6; B6xBUB; SJLxB6; B6xSJL). We also compared ethanol intake in these mice for several concentrations before and after two periods of abstinence. F1 hybrid mice derived from the crosses of B6 and FVB and also B6 and SJL drank higher levels of ethanol than their progenitor strains, demonstrating overdominance for two-bottle choice drinking test. The B6 and NZB hybrid showed additivity in two-bottle choice drinking, whereas the hybrid of B6 and BUB demonstrated full or complete dominance. Genealogical origin, as well as non-alcohol taste preferences (sodium chloride), predicted ethanol consumption. Mice derived from the crosses of B6 and FVB showed high sustained alcohol preference and the B6 and NZB hybrids showed reduced alcohol preference after periods of abstinence. These new genetic models offer some advantages over inbred strains because they provide high, sustained, alcohol intake, and should allow mapping of loci important for the genetic architecture of these traits.     

Schizonts of Theileria annulata interact with the microtubuli network of their host cell via the membrane protein TaSP

Intracellular leukoproliferative Theileria are unique as eukaryotic organisms that transform the immune cells of their ruminant host. Theileria utilize the uncontrolled proliferation for rapid multiplication and distribution into host daughter cells. The parasite distribution into the daughter cells is accompanied by a tight association with the host cell mitotic apparatus. Since the molecular basis for this interaction is largely unknown, we investigated the possible involvement of the immunodominant Theileria annulata surface protein, TaSP, in the attachment of the parasite to host cell microtubule network. Confocal microscopic analyses showed co-localization of the TaSP protein with alpha-tubulin and reciprocal immuno-co-precipitation experiments demonstrated an association of TaSP with alpha-tubulin in vivo. In addition, the partially expressed predicted extracellular domain of TaSP co-localized with the mitotic spindle of dividing cells and was co-immunoprecipitated with alpha-tubulin in transiently transfected Cos-7 cells devoid of other T. annulata expressed proteins. Pull-down studies showed that there is a direct interaction between TaSP and polymerized microtubules. Analysis of the interaction of TaSP and host microtubulin during host cell mitosis indicated that TaSP co-localizes and interacts with the spindle poles, the mitotic spindle apparatus and the mid-body. Moreover, TaSP was demonstrated to be localized to the microtubule organizing center and to physically interact with gamma-tubulin. These data support the notion that the TaSP—microtubule interaction may be playing a potential role in parasite distribution into daughter host cells and give rise to the speculation that TaSP may be involved in regulation of microtubule assembly in the host cell.     

DFNB79: reincarnation of a nonsyndromic deafness locus on chromosome 9q34.3

Genetic analysis of an inbred Pakistani family PKDF280, segregating prelingual severe to profound sensorineural hearing loss, provided evidence for a DFNB locus on human chromosome 9q34.3. Co-segregation of the deafness trait with marker D9SH159 was determined by a two-point linkage analysis (LOD score 9.43 at θ=0). Two additional large families, PKDF517 and PKDF741, co-segregate recessive deafness with markers linked to the same interval. Haplotype analyses of these three families refined the interval to 3.84 Mb defined by D9S1818 (centromeric) and D9SH6 (telomeric). This interval overlaps with the previously reported DFNB33 locus whose chromosomal map position has been recently revised and assigned to a new position on chromosome 10p11.23–q21.1. The nonsyndromic deafness locus on chromosome 9q segregating in family PKDF280 was designated DFNB79. We are currently screening the 113 candidate DFNB79 genes for mutations and have excluded CACNA1B, EDF1, PTGDS, EHMT1, QSOX2, NOTCH1, MIR126 and MIR602.