Effect of adenovirus-mediated RNA interference of IL-1ß expression on spinal cord injury in rats.

STUDY DESIGN: We introduced an adenoviral vector expressing interleukin-1ß (IL-1ß) small-hairpin RNA (shRNA) into the injured spinal cords to evaluate the therapeutic potential of IL-1ß downregulation in a rat model of spinal cord injury (SCI). OBJECTIVES: The purpose of this study was to investigate the possible protective effects of the IL-1ß downregulation on traumatic SCI in rats. SETTING: Department of Orthopedic Surgery, The Second Affiliated Hospital, Fujian Medical University, Quanzhou, People's Republic of China. METHODS: An adenoviral shRNA targeting IL-1ß was constructed and injected at the T12 section 7 days before SCI. The rats' motor functions were evaluated by the Basso-Beattie-Bresnahan (BBB) rating scale. Immunofluorescence, enzyme-linked immunosorbent assay, flow-cytometric analysis and western blots were also performed. RESULTS: Animals downregulating IL-1ß had significantly better recovery of locomotor function and less neuronal loss after SCI. In addition, IL-1ß downregulation significantly decreased tumor necrosis factor-alpha (TNF-α) level and Bax expression, reduced the activity of caspase-3 and increased Bcl-2 expression after SCI. CONCLUSION: This study demonstrated that the IL-1ß downregulation may have potential therapeutic benefits for both reducing secondary damages and improving the outcomes after traumatic SCI.

Salmonella Weltevreden food poisoning in a tea garden of Assam: An outbreak investigation.

BACKGROUND: Salmonella enterica serovar Weltevreden has been a rare cause of acute gastroenteritis occurring worldwide. Here, we report an outbreak of food poisoning in a tea garden. OBJECTIVES: To determine the aetiological agent and risk factors responsible for the outbreak and to take necessary steps for prevention of future outbreaks. MATERIALS AND METHODS: Affected area was visited by a team of microbiologists for collecting stool samples/rectal swabs from affected patients. Samples were processed by culture followed by confirmation of the isolates biochemically, automated bacterial identification system, conventional serotyping and molecular typing. Water samples were also processed for detection of faecal contamination. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique according to the Clinical Laboratory Standard Institute guidelines. RESULTS: The isolates were confirmed as S. enterica subspecies enterica serovar Weltevreden. They were found sensitive to ampicillin, amoxycillin-clavulanic acid, ciprofloxacin, ofloxacin, norfloxacin, cefotaxime, ceftriaxone, co-trimoxazole and doxycycline. Water samples showed high-level faecal contamination. Source of outbreak was found to be drinking water contaminated with dead livestock. House to house visit was made for early diagnosis and treatment of the cases, awareness campaigning and chlorination of drinking water. CONCLUSIONS: This report emphasises the geographical distribution of this organism in Assam. As S. Weltevreden is widely distributed in domestic animals, people should be made aware of immediate reporting of any unusual death among the livestock and their safe disposal which can significantly reduce the incidence of non-typhoidal salmonellosis in the country.

Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress.

UNLABELLED: Brain ischemia and reperfusion (I/R) injury occurs in various pathological conditions, but there is no effective treatment currently available in clinical practice. Methylene blue (MB) is a century-old drug with a newly discovered protective function in the ischemic stroke model. In the current investigation we studied the MB-induced neuroprotective mechanism focusing on stabilization and activation of hypoxia-inducible factor-1α (HIF-1α) in an in vitro oxygen and glucose deprivation (OGD)-reoxygenation model. METHODS: HT22 cells were exposed to OGD (0.1% O2, 6h) and reoxygenation (21% O2, 24h). Cell viability was determined with the calcein AM assay. The dynamic change of intracellular O2 concentration was monitored by fluorescence lifetime imaging microscopy (FLTIM). Glucose uptake was quantified using the 2-[N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino]-2-Deoxy-d-Glucose (2-NBDG) assay. ATP concentration and glycolytic enzyme activity were examined by spectrophotometry. Protein content changes were measured by immunoblot: HIF-1α, prolyl hydroxylase 2 (PHD2), erythropoietin (EPO), Akt, mTOR, and PIP5K. The contribution of HIF-1α activation in the MB-induced neuroprotective mechanism was confirmed by blocking HIF-1α activation with 2-methoxyestradiol-2 (2-MeOE2) and by transiently transfecting constitutively active HIF-1α. RESULTS: MB increases cell viability by about 50% vs. OGD control. Compared to the corresponding control, MB increases intracellular O2 concentration and glucose uptake as well as the activities of hexokinase and G-6-PDH, and ATP concentration. MB activates the EPO signaling pathway with a corresponding increase in HIF-1α. Phosphorylation of Akt was significantly increased with MB treatment followed by activation of the mTOR pathway. Importantly, we observed, MB increased nuclear translocation of HIF-1α vs. control (about three folds), which was shown by a ratio of nuclear:cytoplasmic HIF-1α protein content. CONCLUSION: We conclude that MB protects the hippocampus-derived neuronal cells against OGD-reoxygenation injury by enhancing energy metabolism and increasing HIF-1α protein content accompanied by an activation of the EPO signaling pathway.

Isolation of Shewanella algae from rectal swabs of patients with bloody diarrhoea.

Shewanella algae is an emerging bacteria rarely implicated as a human pathogen. It was infrequently recovered from clinical specimens probably because of inadequate processing of non-fermenting oxidase-positive gram-negative bacilli. We report here isolation of S. algae in pure culture and mixed with E. coli from two cases of acute gastroenteritis with bloody mucous containing diarrhea occurring at the same time. As this organism is not a normal flora of the gut, the possible source of infection may be fish contaminated with the organism. Whether this bacterium can be considered an enteric pathogen needs to be evaluated. The cases were clinically diagnosed as acute bacillary dysentery. The bacterium was identified by 16S r-RNA gene sequence analysis.

Seven-day antibiotic courses have similar efficacy to prolonged courses in severe community-acquired pneumonia--a propensity-adjusted analysis.

There are no studies to guide the optimal duration of therapy in severe community-acquired pneumonia (CAP). The aim of this study was to determine whether 7 days of antibiotic treatment is equivalent to longer-course therapy in severe CAP. In this prospective observational study, we included patients with severe CAP (CURB65 score 3-5) admitted to the hospital with signs and symptoms consistent with pneumonia. A propensity score, derived through multiple logistic regression, was used to match patients into two groups: treated for 7 days vs. treated for >7 days. Patients who died, were admitted to the intensive-care unit, developed complicated pneumonia, failed to reach clinical stability or had positive cultures for microorganisms requiring prolonged treatment within the first 7 days were excluded. Patients outside the mutual range of the propensity score were also excluded. The primary outcome of this study was 30-day mortality. Secondary outcomes were subsequent requirement for mechanical ventilation and/or inotropic support and the development of complicated pneumonia or re-admission within 30 days. Four hundred and twelve patients were suitable for derivation of the propensity score. After matching on propensity score, 164 patients treated for 7 days were compared with 164 treated for >7 days; they were well matched in terms of age, gender, comorbidities, and physiological parameters. The results showed no significant differences in the primary and the secondary outcomes between the two groups. This study therefore suggests that, in the majority of severe CAP patients who have clinically responded, antibiotics can be safely discontinued at 7 days.

Physician judgement is a crucial adjunct to pneumonia severity scores in low-risk patients.

This study investigates the reasons for hospitalisation in patients with low-risk (CURB-65 score 0-1) community-acquired pneumonia (CAP), with a view to identifying the potential for improving outpatient management. As part of a prospective observational study of CAP, we evaluated reasons for hospitalisation in these low-risk patients. 565 patients had low-risk CAP and 420 of these were admitted (for >12 h). 39.3% had additional markers of severity justifying admission, 29.5% of the admissions were required for further management that could not be provided rapidly in the community, 11.9% had unsafe social circumstances and 19.3% had no clinical reason justifying hospitalisation. 30-day mortality was increased in patients with additional severity markers (6.7%), which was significantly higher compared with 0% for patients awaiting investigations (p=0.009) and 0% without a clear indication for hospitalisation (p=0.04). In a logistic regression analysis, parameters associated with 30-day mortality were chronic cardiac comorbidity (adjusted odds ratio (aOR) 5.73, 95% CI 1.52-21.6; p=0.01), acidosis (aOR 5.14, 95% CI 1.44-18.3; p=0.01), hypoxia (aOR 9.86, 95% CI 2.39-40.7; p=0.002) and multilobar chest radiograph shadowing (aOR 4.54, 95% CI 1.21-17.1; p=0.03). This study supports recommendations from international guidelines that pneumonia severity scores should be used as an adjunct to clinical judgement, when deciding on hospitalisation.

Vascular complications are associated with poor outcome in community-acquired pneumonia.

BACKGROUND: Recognition of cardiovascular risk factors is important for primary and secondary prevention strategies. Recent evidence has linked lower respiratory tract infections with the development of acute myocardial infarction. AIM: The aim of this study was to determine the frequency of cardiovascular and cerebrovascular events and the clinical outcomes, during hospitalization for community-acquired pneumonia (CAP). DESIGN: We performed a retrospective study of 4408 patients with CAP presenting to five hospitals over a 2-year period. Clinical information, co-morbidities, cardiovascular events and 90-day mortality were collected from review of medical case notes. The relationship between cardiovascular events and outcomes were analysed using multivariable logistic regression. RESULTS: From a total of 4408 patients, 2.2% developed stroke, 5% acute coronary syndrome or myocardial infarction and 9.3% new onset atrial fibrillation. These were associated with increased 90-day mortality [odds ratio (OR), 1.49 95% CI 1.18-1.87, P=0.0006]. Vascular events were independently associated with increased length of hospital stay-median 12 days (IQR 5-22), compared to patients with no vascular events 8 days (IQR 3-17 days, P<0.0001). CONCLUSION: Cardiovascular and cerebrovascular events are common during hospitalization for CAP and are associated with increased 90-day mortality.

Novel mechanisms of protein synthesis in diabetic nephropathy--role of mRNA translation.

Ambient protein levels are affected by both synthesis and degradation. Synthesis of a protein is regulated by transcription and messenger RNA (mRNA) translation. Translation has emerged as an important site of regulation of protein expression during development and disease. It is under the control of distinct factors that regulate initiation, elongation and termination phases. Regulation of translation occurs via signaling reactions, guanosine diphosphate-guanosine triphosphate binding and by participation of non-coding RNA species such as microRNA. Recent work has revealed an important role for translation in hypertrophy, matrix protein synthesis, elaboration of growth factors in in vivo and in vitro models of diabetic nephropathy. Studies of translation dysregulation in diabetic nephropathy have enabled identification of novel therapeutic targets. Translation of mRNA is a fertile field for exploration in investigation of kidney disease.

Necessity of antibiotics prophylaxis during extracorporeal shock wave lithotripsy.

A total of 360 patients with renal and ureteral calculi who had sterile urine before extracorporeal shock wave lithotripsy (ESWL) and did not have any increased risk of infection received Tab. Ciprfloxacin (500 mg) 12 hourly for the next 5 days or no prophylaxis were included in this prospective study. Patients were followed by urinalysis and culture together with clinical evaluations. In antibiotic prophylactic group 10 (6.4%) had post ESWL urine culture positive while in without prophylaxis 13 (8.8%) had positive urine culture. The incidence of urinary tract infection after ESWL is extremely low, provided that patients have sterile urine before the procedure.

Role of Ambulatory Blood Pressure Measurement in Diagnosis and Control of Hypertension.

Office blood pressure (BP) measurements by sphygmomanometer are not necessarily representative of patient's usual blood pressure. In contrast, ambulatory blood pressure measurements (ABPM) represent a large number of readings and may reflect the actual BP status of an individual. In this study, 150 individuals were studied in 2 groups. 110 patients (group A) had stage I & II, hypertension based on casual BP readings. 26 (23.6%) of them were found to be normontensive as per existing ABPM standard. There were 40 patients with poorly controlled hypertension on multiple drugs (group B). The trough/peak ratio of > 50% was seen in 12.5% of these patients at start of study. This increased to 84.8% after modification of drugs as per the profile on ABPM, thus indicating usefulness in achieving a smoother control.

Effect of atenolol on cadmium-induced testicular toxicity in male rats.

Cadmium-induced stress adversely affects testicular activity and causes sympathetic stimulation. To investigate the effect of atenolol, a beta-adrenergic receptor blocker, on testicular androgen synthesis after cadmium treatment, adult Sprague-Dawley strain male rats were given a single sc dose of cadmium chloride 0.45 mg/kg BW. Animals were killed on day 3 after treatment. Adrenal weight, adrenal delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) activity, serum corticosterone, and brain noradrenaline were increased significantly while testicular delta 5-3 beta-HSD and 17 beta-HSD activities, serum testosterone, and accessory sex organs weight were decreased. Oral coadministration of atenolol at a dose of 2.0 mg/kg body weight for 3 days resulted in complete protection of adrenal delta 5-3 beta-HSD, testicular delta 5-3 beta-HSD, and 17 beta-HSD activities, adrenal weight, serum corticosterone, and serum testosterone when compared with cadmium-only group and there were no significant differences in these parameters from the vehicle control values. Simultaneous administration of cadmium and atenolol also protected brain noradrenaline content and reduced the rise of testicular cadmium concentration. All the parameters were similar to control levels in rats treated with atenolol alone. We conclude that atenolol may protect testicular androgen synthesis by inhibiting the action of noradrenaline on testicular Leydig cells and adrenocortical hyperactivity in cadmium-treated rats.

Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells.

Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1 nM) or insulin (1 nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/-2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor beta-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/-2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II.

Retrograde fixation of fractures of the neck and shaft of the humerus with the 'Halder humeral nail'.

The Halder humeral nail is a new intramedullary device designed for displaced two part humeral neck and shaft fractures. It is inserted retrograde from the olecranon fossa and a unique trio wire is inserted through the nail to provide good proximal fixation. This avoids the use of proximal screws and damage to the rotator cuff thereby ensuring good rotator cuff function. The results of 100 cases are presented. At 6 weeks, 95 patients could perform the majority of daily tasks. There were four non-unions, one following a deep infection.

Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase.

Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5-10 min) but delayed activation of phosphoinositide 3-kinase (PI3-K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3-K inhibitors or by the introduction of a dominant negative PI3-K, indicating that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase-, lipoxyogenase-, and cytochrome P450-dependent metabolism did not influence AA-induced Akt/PKB activation. However, the antioxidants N-acetylcysteine and diphenylene iodonium inhibited both AA- and Ang II-induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3-K, inhibited Ang II-induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox-dependent pathway and Akt/PKB activation independent of PI3-K.

Ceramide blocks PDGF-induced DNA synthesis in mesangial cells via inhibition of Akt kinase in the absence of apoptosis.

The mechanism of action of ceramide in glomerular mesangial cells has not been studied. We investigated the effect of C2 ceramide on the mitogenic signal transduction pathways induced by PDGF in mesangial cells. Increasing concentrations of C2 ceramide inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 15 microM. This inhibition of DNA synthesis was associated with attenuation of PDGF-induced early response gene c-fos transcription. PDGF receptor beta immunecomplex kinase assay showed no inhibitory effect of C2 ceramide on PDGF receptor tyrosine kinase activity. We have recently shown that the mitogenic effect of PDGF is mediated by the enzyme phosphatidylinositol (PI) 3 kinase in mesangial cells. C2 ceramide had no effect on PDGF-induced PDGFR-associated PI 3 kinase activity. These data indicate that inhibitory effect of C2 on PDGF-induced DNA synthesis is likely due to post-receptor and post-PI 3 kinase events. To address the mechanism of C2-mediated inhibition of DNA synthesis, we investigated the downstream target of PI 3 kinase, Akt. PDGF time-dependently increased Akt kinase activity in a PI 3 kinase-dependent manner. Incubation of mesangial cells with C2 ceramide inhibited PDGF-induced Akt activity. Akt kinase inhibits apoptosis of cells via phosphorylation of multiple proapoptotic proteins. However, inhibition of Akt activity by C2 ceramide did not induce apoptosis in mesangial cells. These data provide the first evidence that in mesangial cells, ceramide cross-talks with PI 3 kinase-dependent Akt kinase to inhibit PDGF-induced DNA synthesis without inducing apoptosis.

Autoregulation of mouse BMP-2 gene transcription is directed by the proximal promoter element.

Bone morphogenetic protein-2 (BMP-2) stimulates the commitment and differentiation of precursor mesenchymal cells to mature bone. We have isolated and sequenced 2712 base pairs (bp) of the 5' flanking region of mouse BMP-2 gene. Using RNase protection assay we identified two transcription initiation sites within this 2712 bp region of the BMP-2 gene. The distal start site was mapped to -736 bp in relation to the proximal start site (+1). Recombinant BMP-2 preferentially stimulated transcription initiation from the proximal start site. To investigate the mechanism of transcription initiation from these two start sites, we identified two promoter elements upstream of the proximal and distal transcription initiation sites. Transfection of promoter-luciferase reporter constructs into cells of different organs demonstrated differential transcriptional activity of proximal and distal promoters, with highest activity in the osteoblast cell lineage. In osteoblasts, BMP-2 stimulated transcription from the proximal promoter only. Together our data provide the first evidence for the presence of two transcription initiation sites with two upstream promoter elements in mouse BMP-2 gene. Furthermore, we demonstrate for the first time that BMP-2 autoregulates its expression in osteoblasts through the proximal promoter-dependent transcriptional mechanism.