Cytogenetic effects of hexavalent chromium in Bulgarian chromium platers

The aim of the present study was to evaluate the genotoxic effects of hexavalent chromium (Cr(VI)) in vivo in exposed Bulgarian chromium platers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes and exfoliated buccal cells. No significant difference was observed between the exposed workers and the controls with regard to the frequency of cells with chromosome aberrations (CAs) using conventional Giemsa staining and in the frequency of sister chromatid exchanges (SCEs). However, there was a significant increase in the number of cells with micronuclei (MN) in peripheral lymphocytes from chromium exposed workers as compared to the controls. In the buccal cells from these workers, this increase was even more pronounced. Cytosine arabinoside (AraC), an inhibitor of DNA synthesis and repair, was found to significantly increase the levels of MN in vitro in the lymphocytes of both groups. The increase was more expressed in the lymphocytes of chromium exposed workers. Both centromere positive (C(+)) as well as centromere negative (C(-)) MN were observed by the fluorescence in situ hybridization (FISH) technique in both of the cell types studied. No difference between C(+) and C(-) MN frequencies was found in the lymphocytes as well as in the buccal cells. Thus, Cr(VI) appears to have both clastogenic as well as aneugenic effects in humans.     

Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide

Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.     

CD83 modulates B cell function in vitro: increased IL-10 and reduced Ig secretion by CD83Tg B cells

The murine transmembrane glycoprotein CD83 is an important regulator for both thymic T cell maturation and peripheral T cell responses. Recently, we reported that CD83 also has a function on B cells: Ubiquitous transgenic (Tg) expression of CD83 interfered with the immunoglobulin (Ig) response to infectious agents and to T cell dependent as well as T cell independent model antigen immunization. Here we compare the function of CD83Tg B cells that overexpress CD83 and CD83 mutant (CD83mu) B cells that display a drastically reduced CD83 expression. Correlating with CD83 expression, the basic as well as the lipopolysaccharide (LPS) induced expression of the activation markers CD86 and MHC-II are significantly increased in CD83Tg B cells and reciprocally decreased in CD83mu B cells. Wild-type B cells rapidly upregulate CD83 within three hours post BCR or TLR engagement by de novo protein synthesis. The forced premature overexpression of CD83 on the CD83Tg B cells results in reduced calcium signaling, reduced Ig secretion and a reciprocally increased IL-10 production upon in vitro activation. This altered phenotype is mediated by CD83 expressed on the B cells themselves, since it is observed in the absence of accessory cells. In line with this finding, purified CD83mu B cells displayed a reduced IL-10 production and slightly increased Ig secretion upon LPS stimulation in vitro. Taken together, our data strongly suggest that CD83 is expressed by B cells upon activation and contributes to the regulation of B cell function.     

APOE ε4 moderates abnormal CSF-abeta-42 levels,while neurocognitive impairment is associated with abnormal CSF tau levels in HIV+ individuals – a cross-sectional observational study

Background

Cerebrospinal fluid (CSF) biomarkers Aβ1-42, t-tau and p-tau have a characteristic pattern in Alzheimer’s Disease (AD). Their roles in HIV-associated neurocognitive disorder (HAND) remains unclear.

Methods

Adults with chronic treated HIV disease were recruited (n = 43, aged 56.7 ± 7.9; 32% aged 60+; median HIV duration 20 years, >95% plasma and CSF HIV RNA <50 cp/mL, on cART for a median 24 months). All underwent standard neuropsychological testing (61% had HAND), APOE genotyping (30.9% carried APOE ε4 and 7.1% were ε4 homozygotes) and a lumbar puncture. Concentrations of Aβ1-42, t-tau and p-tau were assessed in the CSF using commercial ELISAs. Current neurocognitive status was defined using the continuous Global Deficit Score, which grades impairment in clinically relevant categories. History of HAND was recorded. Univariate correlations informed multivariate models, which were corrected for nadir CD4-T cell counts and HIV duration.

Results

Carriage of APOE ε4 predicted markedly lower levels of CSF Aβ1-42 in univariate (r = -.50; p = .001) and multivariate analyses (R² = .25; p < .0003). Greater levels of neurocognitive impairment were associated with higher CSF levels of p-tau in univariate analyses (r = .32; p = .03) and multivariate analyses (R² = .10; p = .03). AD risk prediction cut-offs incorporating all three CSF biomarkers suggested that 12.5% of participants had a high risk for AD. Having a CSF-AD like profile was more frequent in those with current (p = .05) and past HIV-associated dementia (p = .03).

Conclusions

Similarly to larger studies, APOE ε4 genotype was not directly associated with HAND, but moderated CSF levels of Aβ1-42 in a minority of participants. In the majority of participants, increased CSF p-tau levels were associated with current neurocognitive impairment. Combined CSF biomarker risk for AD in the current HIV+ sample is more than 10 times greater than in the Australian population of the same age. Larger prospective studies are warranted.

     

Pathogenic nematodes suppress humoral responses to third-party antigens in vivo by IL-10-mediated interference with Th cell function

One third of the human population is infected with helminth parasites. To promote their longevity and to limit pathology, helminths have developed several strategies to suppress the immune response of their host. As this immune suppression also acts on unrelated third-party Ags, a preexisting helminth infection may interfere with vaccination efficacy. In this study, we show that natural infection with Litomosoides sigmodontis suppressed the humoral response to thymus-dependent but not to thymus-independent model Ags in C57BL/6 mice. Thereby, we provide evidence that reduced humoral responses were mediated by interference with Th cell function rather than by direct suppression of B cells in L. sigmodontis-infected mice. We directly demonstrate suppression of Ag-specific proliferation in OVA-specific Th cells after adoptive transfer into L. sigmodontis-infected mice that led to equally reduced production of OVA-specific IgG. Transferred Th cells displayed increased frequencies of Foxp3(+) after in vivo stimulation within infected but not within naive mice. Helminth-mediated suppression was induced by established L. sigmodontis infections but was completely independent of the individual worm burden. Using DEREG mice, we rule out a central role for host-derived regulatory T cells in the suppression of transferred Th cell proliferation. In contrast, we show that L. sigmodontis-induced, host-derived IL-10 mediated Foxp3 induction in transferred Th cells and significantly contributed to the observed Th cell hypoproliferation within infected mice.     

Strongyloides ratti infection modulates B and T cell responses to third party antigens

It is estimated that over one third of the world population is infected with helminths, Strongyloides ssp. accounting for approximately 30-100 million cases. As helminth infections often result in a modulation of the host's immune system, infected people may display impaired responses to concurrent infections and to third party antigens. Here, we employ the experimental system of murine Strongyloides ratti infection to investigate the impact of helminth infections on experimental vaccinations. We demonstrate that concurrent infection with S. ratti strongly affected the humoral response to a thymus dependent model antigen, whereby predominantly Th1 associated IgG2b production was suppressed. We provide evidence that this suppression was due to modulation of T helper cell and not B cell function as the responses to a thymus independent model antigen remained unchanged in S. ratti infected mice. Moreover, using an adoptive transfer system, we show that infection with S. ratti directly interfered with antigen-specific proliferation of T cell receptor transgenic CD4(+) T helper cells in vivo. Finally, using IL-10 deficient mice and mice that selectively lack T helper cell derived IL-10 we rule out a role for host-derived IL-10 in mediating the suppression of thymus dependent model antigen response in S. ratti infected mice.     

Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes

Background

Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved ~350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships.

Results

We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages.

Conclusion

The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the functional diversification of kinesins. Results from our gene-tree analyses indicate that there was tremendous lineage-specific duplication and diversification of kinesins in eukaryotes. Since the functions of only a few plant kinesins are reported in the literature, this comprehensive comparative analysis will be useful in designing functional studies with photosynthetic eukaryotes.     

Daily rhythms of colonic temperature and circulating blood enzymes,urea and calcium in Japanese quail (Coturnix coturnix japonica) under natural cold-dry (harmattan) and hot-dry conditions

The influence of cold-dry (harmattan) and hot-dry seasons on daily rhythmicity of colonic temperature (CT) and blood biochemical variables were investigated in 340 male Japanese quails under natural light–dark circle. Rhythm characteristics analysed using cosinor and the application of the periodic model showed a daily rhythm of CT, creatinine phosphate kinase (CPK), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase (ALP) and urea in both seasons, while calcium did not exhibit any rhythmicity. The rhythmic parameters of mesor, amplitude and acrophase analysis showed significant (p < 0.05) effects of season. The amplitudes, except for ALP, were greater during the harmattan. The acrophase, except for CPK and urea, were all restricted to the light phase of the light-dark cycle. The results, demonstrated strong seasonal changes on the daily rhythmicity of CT and blood biochemical parameters of quails under natural light-dark circle, which could be useful for researchers, clinicians and agriculturists under different climatic conditions.     

Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes

Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.