Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents – transforming growth factor β₁ (TGF-β₁), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) – on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-β₁; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-β₁, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca(2+) changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K(+) solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in a subpopulation of ECs 5-11s after a [Ca(2+)](i) rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca(2+) or inositol 1,4,5-trisphosphate (IP(3)). First, phospholipase C inhibitor U-73122 was added to prevent IP(3) production in response to the [Ca(2+)](i) increase in SMCs. In high K(+) solution, all SMCs presented global and synchronous [Ca(2+)](i) increase, but no [Ca(2+)](i) variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP(3)-induced Ca(2+) release, reduced the number of flashing ECs by 75+/-3% (n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP(3).     

Pre-exercise hyperhydration delays dehydration and improves endurance capacity during 2 h of cycling in a temperate climate

Whether the use of pre-exercise hyperhydration could improve the performance of athletes who do not hydrate sufficiently during prolonged exercise is still unknown. We therefore compared the effects of pre-exercise hyperhydration and pre-exercise euhydration on endurance capacity, peak power output and selected components of the cardiovascular and thermoregulatory systems during prolonged cycling. Using a randomized, crossover experimental design, 6 endurance-trained subjects underwent a pre-exercise hyperhydration (26 ml of water x kg body mass(-1) with 1.2 g glycerol x kg body mass(-1)) or pre-exercise euhydration period of 80 min, followed by 2 h of cycling at 65% maximal oxygen consumption (VO(.)2max) (26-27 degrees C) that were interspersed by 5, 2-min intervals performed at 80% V(.)O2max. Following the 2 h cycling exercise, subjects underwent an incremental cycling test to exhaustion. Pre-exercise hyperhydration increased body water by 16.1+/-2.2 ml.kg body mass(-1). During exercise, subjects received 12.5 ml of sports drink x kg body mass(-1). With pre-exercise hyperhydration and pre-exercise euhydration, respectively, fluid ingestion during exercise replaced 31.0+/-2.9% and 37.1+/-6.8% of sweat losses (p>0.05). Body mass loss at the end of exercise reached 1.7+/-0.3% with pre-exercise hyperhydration and 3.3+/-0.4% with pre-exercise euhydration (p<0.05). During the 2 h of cycling, pre-exercise hyperhydration significantly decreased heart rate and perceived thirst, but rectal temperature, sweat rate, perceived exertion and perceived heat-stress did not differ between conditions. Pre-exercise hyperhydration significantly increased time to exhaustion and peak power output, compared with pre-exercise euhydration. We conclude that pre-exercise hyperhydration improves endurance capacity and peak power output and decreases heart rate and thirst sensation, but does not reduce rectal temperature during 2 h of moderate to intense cycling in a moderate environment when fluid consumption is 33% of sweat losses.     

Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.