Determination of hepatitis C virus genotype by Pyrosequencing

A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping.     

Face cooling-induced reduction of plasma prolactin response to exercise as part of an integrated response to thermal stress

Summary This study was designed to verify if the decrease in blood prolactin (PRL) induced by selective face cooling during exercise could be part of a response to specific body thermal stress. Five healthy trained male cyclists presenting a significant plasma PRL elevation to exercise were, on three occasions and at weekly interval, submitted to a submaximal exercise (approx. 65% ) on ergocycle with and without selective face cooling. In absence of face cooling a first trial served to establish reference values for workload, heart rate and plasma PRL levels, the latter increasing markedly (450% of resting values) in these conditions. On a second trial but with workload maintained at reference values (222±9 W), a significant bradycardia was observed with face cooling; furthermore, plasma PRL response to exercise was significantly reduced (to 31% of original response). On a third trial with face cooling, workload had to be significantly augmented (242±10 W) to maintain heart rate at reference level (78%HR _max); in addition, plasma PRL response to exercise was almost unchanged compared to the reference-value level. The absence of a significant face cooling-induced decrease in sympathetic tonus, as evaluated through peripheral plasma catecholamines response, does not indicate a role for the autonomic nervous system in the face cooling-induced reduction of both heart rate and PRL responses during exercise. Assay of circulating peripheral beta-endorphins could indicate that the face cooling-induced PRL blunted response does not necessarily involve an opioid mediation. It was concluded that decreased plasma PRL levels could be associated to bradycardia, hyposudation and peripheral vaso-constriction to constitute an integrated response to face cooling during thermal stress. This study was supported by grants from Natural Sciences and Engineering Research Council of Canada and from Régie de la Sécurité dans les Sports du Québec     

A randomised clinical trial evaluating the efficacy of physiotherapy after rotator cuff repair

The optimal form of rehabilitation after rotator cuff repair has yet to be determined. A randomised clinical trial was undertaken to compare outcomes for two forms of rehabilitation for this condition: individualised supervised physiotherapy treatment, and a standardised unsupervised home exercise regime. Fifty-eight volunteers with all sizes of operatively repaired rotator cuff tears were allocated randomly to one of the two treatment groups. All subjects received a standardised home exercise regime. Subjects who were randomised to the physiotherapy group received additional individualised treatment. Independent, blinded assessments of range of motion, muscle force and functional outcome measures were performed pre-operatively, and at six, 12 and 24 weeks postoperation. At six, 12 and 24 weeks post-operation, comparable outcomes were demonstrated for both rehabilitation groups. By 24 weeks post-operation, most subjects demonstrated outcomes that were consistent with a favourable recovery, regardless of rehabilitation mode. On the basis of these results, outcomes for subjects allocated to individualised physiotherapy treatment after rotator cuff repair are no better than for subjects allocated to a standardised home exercise regime.     

Low quality of dying and death in patients with septic shock as perceived by nurses and resident physicians

Septic shock is a disease with both high prevalence and mortality. Few studies have evaluated the quality of dying and death (QODD) in patients with septic shock. The authors compared the QODD of patients who died of septic shock versus other causes. They prospectively collected QODD surveys from nurses and residents caring for 196 patients who died in the medical intensive care unit (ICU) at an urban, university hospital. Patients were included in the analysis if either a nurse or resident returned a survey. Chart review established cause of death. The authors compared total QODD scores (on a scale of 0–100) and a single-item score (QODD-1; on a scale of 0–10) of patients who died of septic shock versus other causes. Survey response rates were 59% (n = 155) for residents and 49% (n = 129) for nurses. Nurses rated patients as having lower total QODD and QODD-1 scores for septic (Δ 7.5 points, p = 0.03, and 0.9 points, p = 0.05, respectively). Residents rated septic patients with lower QODD-1 scores than nonseptic patients (Δ 0.8 points, p = 0.03). This study shows that nurses rate patients with septic shock as having lower QODD than patients dying of other causes. These findings are important for clinicians who counsel families of patients dying of septic shock.     

Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity

The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.     

Developing critical analysis skills in academic writing

Students may often feel confused about what is expected of their work at different academic levels. This article aims to demystify the skill of critical analysis in academic writing.     

Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes.

Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4(+) lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4(+) populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4(+) lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69(+)). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in alpha(4)beta(7) expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation.     

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation.

Apo E, a key regulator of cholesterol-rich lipoprotein metabolism, is synthesized by numerous extrahepatic tissues. Although its synthesis in macrophages is documented, the contribution of macrophage-derived apo E to hepatic clearance of serum cholesterol is unknown. To address this issue bone marrow transplantation was performed on hypercholesterolemic apo E-deficient mice with either syngeneic apo E-deficient mouse bone marrow cells (E0-control) or wild-type mouse bone marrow cells expressing apo E (E0-treated). E0-control and E0-treated mice were fed either a regular chow diet or an atherogenic diet (designated E0-control-HF and E0-treated-HF). Serum cholesterol levels dropped dramatically in the E0-treated mice largely due to a reduction in their VLDL cholesterol. No changes were seen in the E0-control mice. After 4 wk serum cholesterol in E0-treated-HF mice was about four-fold lower compared to E0-control-HF animals. Moreover, the extent of atherosclerosis in the E0-treated-HF mice after 14-16 wk was greatly reduced. Wild-type apo E mRNA was detected in the liver, spleen, and brain of the E0-treated mice indicating that apo E gene transfer was successfully achieved through bone marrow transplantation. More importantly, the level of apo E expression was sufficient to reduce the severe hypercholesterolemia of the apo E-deficient mice fed either chow or atherogenic diets.