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.     

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.     

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.     

Lingual tuberculosis

Oral tuberculosis is very rare and when present they are usually secondary to pulmonary tuberculosis. Tuberculous lesions of the tongue have become so infrequent that they are virtually a forgotten disease entity and may pose a diagnostic problem. The case reported in this paper emphasizes the importance of including tuberculosis in the differential diagnosis of any chronic oral ulcer. The low number of oral infections by M. tuberculosis could be due to underreporting.     

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.     

New views of the immunological synapse: variations in assembly and function

The interaction of T cells with antigen-presenting cells results in the formation of a contact face, termed the immunological synapse. The prototypical dynamics of this process are well established and involve cessation of crawling, a highly fluid 'immature' synapse phase during which signaling is initiated, and ultimately the formation of a 'mature' synapse characterized by centralized and peripheral supramolecular activating complexes. Ongoing research is directed towards defining how these supramolecular assemblies are formed and, more importantly, to what end. With regard to the former, progress has been made in defining the order in which various molecules are recruited to signaling centers in prototypical settings. With regard to the latter, however, the issue now appears more complex, as both developmental changes in T cells and variations in the environment appear to modulate features of mature synapse development. Although many details of the immunological synapse have been established, emerging evidence suggests a great variability in the ultimate form of these contacts and their effects on T-cell functions.     

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.     

Four days of citalopram increase suppression of cortisol secretion by prednisolone in healthy volunteers

Rationale Chronic antidepressant treatment increases glucocorticoid-mediated negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis, and thus reduces HPA axis activity, in depressed patients and healthy controls. In contrast, acute antidepressant treatment induces an activation of basal HPA axis activity.Objectives We examined the effects of 4 days of treatment with the selective serotonin reuptake inhibitor, citalopram, on basal salivary cortisol and on suppression of salivary cortisol by prednisolone.Methods We used a single-blind, placebo-controlled, repeated-measure design. Salivary cortisol was measured from 0900 to 1700 hours. In the first phase of the study, basal salivary cortisol secretion was measured on 2 study days, before and after 4 days of treatment with citalopram (orally, 20 mg/day). In the second phase, salivary cortisol secretion after suppression by prednisolone (5 mg, given at 2200 hours the night before) was measured on 2 study days, again before and after 4 days of treatment with citalopram (orally, 20 mg/day). Eight volunteers participated to the study.Results Citalopram increased basal salivary cortisol in the morning (0900–1100 hours) by approximately 47% (P=0.003). Moreover, citalopram increased suppression by prednisolone in the morning (0900–1100 hours): suppression was approximately 22% before citalopram and 45% after citalopram (P=0.05).Conclusions Citalopram increases glucocorticoid-mediated negative feedback on the HPA axis after as little as 4 days of treatment. This effect could be due to an increased function of the corticosteroid receptors. Our findings further support the notion that one of the mechanisms by which antidepressants exert their therapeutic effects is by normalizing HPA axis hyperactivity in depressed patients.     

The effect of abolition of the endogenous corticosteroid rhythm on the circadian variation in methotrexate toxicity in the rat

Summary Monitoring of indices of haematological, renal and hepatic toxicity in rats after a single i.v. bolus of methotrexate has shown that they vary with the time of day at which the drug is administered. Maximum toxicity occurs after administration at 0600 h. Further experimentation has shown that the amount of corticosteroid present in the blood has a profound effect on the toxicity of methotrexate in the rat. If the endogenous production of corticosterone is suppressed by treatment with dexamethasone the toxicity of methotrexate is markedly increased at whatever clock time it is administered. However, if constantly high plasma levels are achieved by giving supplementary corticosterone methotrexate toxicity is diminished regardless of what time it is given.Since the timing of maximum methotrexate toxicity corresponds to the circadian nadir of endogenous plasma corticosterone concentration in the rat the possibility that it might be related to corticosterone production must be considered. Whether this phenomenon occurs in man and has any clinical relevance has yet to be investigated.