Clostridium difficile toxin: cytoskeletal changes and lactate dehydrogenase release in hepatocytes

BACKGROUND: We have found that Clostridium difficile toxins can evoke hepatocyte acute-phase protein synthesis, and that this effect is dependent on a functioning interleukin-1 (IL-1) receptor. The present study was undertaken to determine if C. difficile toxicity, as determined by actin rearrangement and lactate dehydrogenase (LDH) release, also requires a functioning IL-1 receptor. METHODS: Primary hepatocyte cultures were prepared from normal mice, knockout mice deficient in the IL-1-converting enzyme (ICE), and knockout mice deficient in the IL-1 p80 receptor. Hepatocytes were treated for 24 h with C. difficile culture extract, purified C. difficile toxin A, or purified C. difficile toxin B. The actin cytoskeleton was examined using confocal microscopy, and LDH release was measured by spectrophotometric analysis. RESULTS: C. difficile culture extract, toxin A, and toxin B induced collapse of the actin cytoskeleton in hepatocytes from normal mice. Hepatocytes from both the ICE-deficient mice and the IL-1 p80 receptor-deficient mice demonstrated similar responses to both toxins. These toxins also induced significant LDH release in a concentration-dependent fashion in the normal hepatocytes and the ICE-deficient hepatocytes. However, no significant increase in LDH release was observed in hepatocytes from IL-1 p80 receptor-deficient mice. CONCLUSIONS: C. difficile toxins induce actin cytoskeletal collapse independent of IL-1 or the IL-1 receptor. In contrast, toxin-stimulated LDH release was dependent on the presence of the IL-1 receptor. Thus, separate pathways appear to mediate toxic effects as manifested by actin rearrangement and LDH release.     

Risk of cardiovascular disease in relation to the use of combined postmenopausal hormone therapy: detection bias and resolution of discrepant findings in two Women's Health Initiative studies.

CONTEXT: In a recent analysis of data in the Women's Health Initiative (WHI) clinical trial and observational studies, the overall risks of coronary heart disease, stroke and venous thromboembolism, in postmenopausal women who used combined therapy with conjugated estrogens and medroxyprogesterone acetate, were lower in the observational data. However, the risks became more similar within similar duration categories of follow-up. In both studies, there was initial elevation, followed by a duration-dependent reduction, in the risks. The investigators suggest that the discrepancies in the overall risks may be due to weighting by short-duration exposure in the clinical trial, and long-duration exposure in the observational study. CRITIQUE: In the clinical trial, 44% of the hormone therapy recipients were unblinded, mainly because of persistent vaginal bleeding, and awareness of exposure status was therefore common; in the observational study, all exposed women were aware. When follow-up in the WHI studies commenced, extensive publicity was given to evidence to suggest that, among women who had already sustained a myocardial infarction, combined hormone therapy may not decrease the risk of a second myocardial infarct, as hypothesized, but instead increase it. From the commencement of follow-up onward, detection bias was therefore possible in both WHI studies. That possibility was further reinforced after about 3 years of follow-up, and again a year later, when the clinical trial participants were explicitly informed that supplemental hormones were associated with increased cardiovascular risks. That information was also given extensive publicity. CONCLUSIONS: Detection bias could not be ruled out in either WHI study, and there may have been systematic overestimation of the risks of cardiovascular outcomes associated with the use of combined hormone therapy. To a limited extent, an analysis of the clinical trial data according to blinding status might be informative in determining whether detection bias could have given rise to overestimation of the overall and duration-specific risks in both WHI studies. If numbers permit, comparisons within strata of severity might also be informative. If the risks were overestimated in the WHI studies, a duration-related protective effect of combined hormone therapy on the risk of coronary heart disease and stroke may have gone undetected.     

'Low dose' 99mTc-Sestamibi for radioguided surgery of primary hyperparathyroidism.

AIM: In this study, we evaluated the efficacy of low dose (99m)Tc-Sestamibi administration for radioguided parathyroid surgery in patients with primary hyperparathyroidism (PHPT). METHODS: Three hundred consecutive PHPT patients were studied between September, 1999 and July, 2003. Pre-operative work-up included (99m)Tc-pertechnetate/(99m)Tc-Sestamibi subtraction scintigraphy and high resolution ultrasonography (US). 37MBq of (99m)Tc-Sestamibi was injected i.v. in the operating suite approximately 10 min prior to the beginning of the surgical procedure for intraoperative radiolocalization; quick parathyroid hormone (QPTH) assays were performed. RESULTS: Two hundred and seven of the 211 patients selected for minimally-invasive radioguided parathyroidectomy (MIRP) were successfully treated for a solitary parathyroid adenoma (PA) through a 2-2.5 cm skin incision (mean operative time 35 min, mean hospital stay 1.2 days). In the 89 patients selected for traditional bilateral neck exploration (BNE), radioguided surgery was not as successful in the identification of the PA, especially in patients with (99m)Tc-Sestamibi-avid thyroid nodules. Nevertheless, the combination of probe and QPTH measurement was very helpful in patients with multigland disease. CONCLUSIONS: Low-dose (99m)Tc-Sestamibi administered few minutes before surgery is sufficient for MIRP in patients with high likelihood of a solitary PA and without concomitant (99m)Tc-Sestamibi-avid thyroid nodules. The combination of radioguided surgery and QPTH measurements is very useful in the early identification of unanticipated multigland disease.     

Respiratory failure. Conventional and high-tech support

Although significant progress has been made in the treatment of patients with acute lung failure in the critical care setting, the mortality rate from acute lung injury and ARDS is unacceptably high, given the numbers of patients treated for these syndromes each year. The improved understanding of the pathophysiology of respiratory failure from basic science and clinical research is reflected in improved survival rates over the years. Advances in the mechanical ventilator (through microprocessor technology); biosurface technology; liquid ventilation; and, in some cases, returning to so-called "antiquated" practices of patient care (e.g., prone positioning) seem to have had an impact nonetheless. As refinement continues to occur in these areas, morbidity and mortality from lung failure will have a lesser impact on patients as physicians treat the consequences of organ failure in the ICU.     

The injured elderly in the trauma intensive care unit

The injured elderly patient in the ICU presents many challenges. Demographic changes in western society will dramatically increase the patient population in question, and new, older, subsets are growing. The association of severe injury, preinjury comorbidity, and the aging process narrows the ability of the patient to respond to the stress of injury. When compared with younger patients, the elderly have greater mortality, morbidity, and higher costs. Age alone, however, does not predict outcome. Although aggressive or maximally supportive care is advocated, controlled data supporting this approach are lacking. Significant economic, sociologic, and ethical issues confront the care providers in almost every case. Continued and heightened study of all aspects of our injured elders focusing on the determinants of outcome is required. A realistic appraisal of the limitations of care and a reassessment of the financial implications of providing extended care are critical to the continuing ability to respond to this growing need.     

Lipidomic Profiling of Bronchoalveolar Lavage Fluid Extracellular Vesicles Indicates Their Involvement in Lipopolysaccharide-Induced Acute Lung Injury

Emerging data support the pivotal role of extracellular vesicles (EVs) in normal cellular physiology and disease conditions. However, despite their abundance, there is much less information about the lipid mediators carried in EVs, especially in the context of acute lung injury (ALI). Our data demonstrate that C57BL/6 mice subjected to intranasal Escherichia coli lipopolysaccharide (LPS)-induced ALI release, a higher number of EVs into the alveolar space, compared to saline-treated controls. EVs released during ALI originated from alveolar epithelial cells, macrophages, and neutrophils and carry a diverse array of lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The eicosanoids in EVs correlated with cellular levels of arachidonic acid, expression of cytosolic phospholipase A2, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome epoxygenase p450 proteins in pulmonary macrophages. Furthermore, EVs from LPS-toll-like receptor 4 knockout (TLR4^-/-) mice contained significantly lower amounts of COX and LOX catalyzed eicosanoids and ω-3 PUFA metabolites. More importantly, EVs from LPS-treated wild-type mice increased TNF-α release by macrophages and reduced alveolar epithelial monolayer barrier integrity compared to EVs from LPS-treated TLR4^−/− mice. In summary, our study demonstrates for the first time that the EV carried PUFA metabolite profile in part depends on the inflammatory status of the lung macrophages and modulates pulmonary macrophage and alveolar epithelial cell function during LPS-induced ALI.     

Impact of Stagnation on the Diversity of Cyanobacteria in Drinking Water Treatment Plant Sludge

Health-related concerns about cyanobacteria-laden sludge of drinking water treatment plants (DWTPs) have been raised in the past few years. Microscopic taxonomy, shotgun metagenomic sequencing, and microcystin (MC) measurement were applied to study the fate of cyanobacteria and cyanotoxins after controlled sludge storage (stagnation) in the dark in a full-scale drinking water treatment plant within 7 to 38 days. For four out of eight dates, cyanobacterial cell growth was observed by total taxonomic cell counts during sludge stagnation. The highest observed cell growth was 96% after 16 days of stagnation. Cell growth was dominated by potential MC producers such as Microcystis, Aphanocapsa, Chroococcus, and Dolichospermum. Shotgun metagenomic sequencing unveiled that stagnation stress shifts the cyanobacterial communities from the stress-sensitive Nostocales (e.g., Dolichospermum) order towards less compromised orders and potential MC producers such as Chroococcales (e.g., Microcystis) and Synechococcales (e.g., Synechococcus). The relative increase of cyanotoxin producers presents a health challenge when the supernatant of the stored sludge is recycled to the head of the DWTP or discharged into the source. These findings emphasize the importance of a strategy to manage cyanobacteria-laden sludge and suggest practical approaches should be adopted to control health/environmental impacts of cyanobacteria and cyanotoxins in sludge.