Initiation of the extrinsic pathway of coagulation by human and rabbit alveolar macrophages: a kinetic study

We examined assembly and expression of the factor X activating complex on human and rabbit alveolar macrophages. Kinetic parameters of the factor X activating reaction were determined by functional titrations of factors VII and X with macrophage tissue factor (TF) added. We found rapid activation of factor X to Xa on alveolar macrophage surfaces. Detection of rapid factor Xa formation on macrophages required addition of exogenous factors VII and X. At plasma concentrations of the purified factors, factor Xa was formed on freshly isolated macrophages at approximately 5.4 pmol/min/10(6) cells. After macrophage maturation in culture for 20 hours with LPS (endotoxin) added, the factor X activation rate was increased two- to sixfold. The km' (apparent km) of TF-factor VII enzymatic complexes assembled on alveolar macrophages for factor X were (258 +/- 55 and 475 +/- 264 nmol/L for human and rabbit cells, respectively). The km' did not change during macrophage maturation in culture, but V'max (apparent Vmax) was consistently increased. The K1/2 of human factor VII (concentrations giving half maximal rates of factor X activation) for the interaction with human and rabbit alveolar macrophage TF were 0.191 +/- 0.096 and 1.7 +/- 0.7 etamol/L, respectively. The K1/2 were not significantly changed after maturation, whereas rates of Xa formation at saturation with factor VII were increased. The fast rates of factor X activation observed at physiologic concentrations of plasma-derived factors VII and X indicate that TF on alveolar macrophages is likely to provide sites for binding of factor VII and activation of factor X in vivo during clotting reactions associated with alveolar edema and inflammation.     

Studies of an antiendotoxin antibody in preventing the physiologic changes of endotoxemia in awake sheep

In sheep, endotoxin (LPS) causes pulmonary hypertension, hypoxemia, leukopenia, exudation of protein-rich lung lymph, reduced dynamic compliance (Cdyn), and increased resistance to airflow (RL), changes similar to those seen in human sepsis and sepsis-induced ARDS. We used well-described methods in the awake sheep-endotoxin model to evaluate the effectiveness of a commercially manufactured antibody to prevent the physiologic changes of endotoxemia. In awake sheep with chronic lung lymph fistulas, we used a whole-body plethysmograph to measure Cdyn, RL, and FRC. Pulmonary artery, left atrial, and systemic arterial pressures were recorded continuously. Arterial blood gases (for calculating AaPO2), leukocyte counts, and lymph samples were collected every 30 min. Animals received a 30-min (2 mg/kg) infusion of antiendotoxin antibody 4 h before LPS (0.75 micrograms/kg) challenge (n = 4), or were given a mixture of LPS (0.75 micrograms/kg) and antibody (2 mg/kg) that had been incubated in vitro at 37 degrees C for 30 min before infusion (n = 6). A control group given only 2 mg/kg of antibody (n = 4) showed no change in any measured parameter, whereas control animals receiving LPS alone (n = 6) exhibited a typical endotoxin response. In all animals receiving endotoxin, Cdyn declined by approximately 50% within 30 to 60 min, and RL increased approximately sixfold over a similar time course. Accompanying the abnormalities in lung mechanics were pulmonary hypertension, leukopenia, and widening of the AaPO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo expression of the B7 costimulatory molecule by subsets of antigen-presenting cells and the malignant cells of Hodgkin's disease

The B-lymphocyte/accessory-cell activation antigen B7 (BB1) has been shown in vitro to stimulate T-lymphocyte proliferation and cytokine production via CD28 present on the latter cells. In this study, benign lymphoid tissues, lymphomas, and extralymphoid inflammatory sites were examined immunohistochemically using anti-B7 and other relevant monoclonal antibodies. B7 was expressed by benign transformed germinal center B cells, as it was by B cells of follicular lymphomas. B7 was also expressed by a subpopulation (a mean of 31% to 65%) of macrophages and dendritic cells in a variety of lymphoid tissues. It was present in abundance on all macrophages constituting sarcoid granulomas in lymph nodes. In extralymphoid inflammation, 17% to 35% of macrophages expressed B7 only weakly. Cases of Hodgkin's disease showed expression of B7 by the majority of Reed-Sternberg cells or malignant mononuclear variants, a phenomenon that potentially contributes to the lymphocytic accumulation that is a feature of this condition. CD28+ T cells were seen in all areas where T cells were present. B7+ and CD28+ cells colocalized in, for example, lymphoid follicles, lymph node paracortex, sarcoid granulomas, and Hodgkin's disease tissue, indicating a potential for cellular interaction via these molecules at these sites.     

A Qualitative Inquiry About Pruno,an Illicit Alcoholic Beverage Linked to Botulism Outbreaks in United States Prisons

Objectives. Since 2011, 3 outbreaks of botulism in US prisons have been attributed to pruno, which is an alcoholic beverage made by inmates. Following 1 outbreak, we conducted a qualitative inquiry to understand pruno brewing and its social context to inform outbreak prevention measures.Methods. We interviewed staff, inmates, and parolees from 1 prison about pruno production methods, the social aspects of pruno, and strategies for communicating the association between botulism and pruno.Results. Twenty-seven inmates and parolees and 13 staff completed interviews. Pruno is fermented from water, fruit, sugar, and miscellaneous ingredients. Knowledge of pruno making was widespread among inmates; staff were familiar with only the most common ingredients and supplies inmates described. Staff and inmates described inconsistent consequences for pruno possession and suggested using graphic health messages from organizations external to the prison to communicate the risk of botulism from pruno.Conclusions. Pruno making was frequent in this prison. Improved staff recognition of pruno ingredients and supplies might improve detection of brewing activities in this and other prisons. Consistent consequences and clear messages about the association between pruno and botulism might prevent outbreaks.Botulism is a rare but serious illness that can lead to respiratory failure and death. Botulism patients may initially present with blurred or double vision, drooping eyelids, slurred speech, and difficulty swallowing before developing more severe signs and symptoms, such as paralysis and difficulty breathing. In the United States, an average of 145 confirmed botulism cases are reported each year, of which approximately 15% are attributable to foodborne botulism.1 Foodborne botulism is caused by ingestion of botulinum toxin, a bacterial toxin that is produced under the following rarely attained conditions: an anaerobic environment with warm temperatures and low acid, salt, and sugar concentrations.2 Alaska Native foods and home-canned vegetables are the food items most commonly associated with foodborne botulism3,4; however, in the past decade, 5 foodborne botulism outbreaks have been attributed to pruno, an illicit alcoholic beverage made in prisons.5 Pruno was initially recognized as a botulism vehicle after it was implicated in 2 outbreaks in California in 2004 and 2005; these outbreaks resulted in confirmed botulism in 5 inmates, 3 of whom were critically ill and mechanically ventilated.6 No additional outbreaks were reported until 2011, when 8 maximum security inmates in Utah developed botulism after drinking pruno. Three were mechanically ventilated, and most reported persistent symptoms, such as weakness, 11 months after the outbreak.7 The following year, 12 Arizona inmates were sickened in 2 outbreaks of botulism associated with pruno consumption; 8 were mechanically ventilated.5 In all 5 outbreaks, pruno was made with potatoes,5 an uncommon pruno ingredient according to online sources, and a food historically associated with botulism.8,9 Because of these outbreaks, pruno-related botulism accounted for 40% and 48% of foodborne botulism cases in the United States in 2011 and 2012, respectively (Centers for Disease Control and Prevention, unpublished data).Internet sources indicate that pruno is common in US prisons.10–12 Although recent trends suggest future pruno-related botulism outbreaks are likely to occur,5,7 there is a dearth of information about how pruno is made and distributed in prisons, and the social and entrepreneurial aspects of its production and use. To address these gaps and inform botulism outbreak prevention measures, 4 months after the Utah outbreak, we conducted a qualitative inquiry in the affected prison to better understand the brewing process, social context of pruno, and communication strategies for informing inmates about the risk of botulism from pruno.     

Premature cardiovascular disease in chronic renal failure

There is a remarkable lack of reliable information about the determinants of risk of cardiovascular disease (CVD) among patients with chronic renal failure. Indeed, such patients have often been deliberately excluded from randomised trials of treatments of CVD, perhaps because of concerns about drug safety. But the absolute risk of CVD among them may be large, so the potential absolute benefits of treatments may also be large, and may well exceed any increased hazards. Hence, as well as further investigation of the underlying mechanisms of cardiac disease, it would be helpful to have some large-scale randomised trials in a wide range of renal patients of interventions (such as cholesterol-lowering drugs, antihypertensives, aspirin, B-vitamins, and antioxidant vitamins) that are of proven or suspected benefit in other settings. If safe and effective treatments can be identified, and started early in the natural history of renal failure, the exceptionally high risk of CVD experienced by these patients could be decreased before and after end-stage renal failure has occurred.     

Overexpression of sarcolipin decreases myocyte contractility and calcium transient

OBJECTIVE: Sarcolipin (SLN) is a novel 31-amino-acid protein associated with the sarcoplasmic reticulum (SR) whose function in cardiac muscle is poorly defined. In this study, we tested the hypothesis that SLN is a regulator of SR Ca(2+) transport function by overexpressing SLN in adult rat ventricular myocytes which express low levels of SLN. METHODS: Expression of SLN mRNA in rat tissues was analyzed by Northern blot as well by RT-PCR analysis. To define the role of SLN in cardiac muscle contractility, we overexpressed SLN in adult rat ventricular myocytes using adenoviral gene transfer techniques. Localization of SLN in the adult rat ventricular myocytes was determined using confocal microscopy. Myocyte contractility and calcium transients were measured using edge detection and Fura 2AM. RESULTS: Our results demonstrate that overexpression of SLN decreased the cell shortening significantly when compared to control myocytes, whereas the time to peak contraction was not altered. In addition, SLN overexpression prolonged the time of 50% relaxation. Calcium transient analysis shows that time to 50% decay of [Ca(2+) ]i was markedly prolonged in SLN-overexpressing myocytes (control -245.0+/-3.78 vs. SLN -199.0+/-3.25 ms, p<0.001). However, there were no significant differences in peak amplitudes of [Ca(2+)](i) between SLN-overexpressing and control myocytes. We further demonstrate that SLN is localized within the SR membrane similar to PLB and SR Ca(2+) ATPase. Co-immunoprecipitation studies indicate that SLN can physically interact with phospholamban. CONCLUSIONS: We conclude that SLN may play an important role in regulating the SR calcium ATPase pump, possibly by interacting with phospholamban.     

Functional coronary microvascular injury evident as increased permeability due to brief ischemia and reperfusion

Although morphological studies suggest that coronary vascular injury is a result of prolonged ischemia and subsequent reperfusion, whether functional coronary microvascular injury develops during brief in vivo ischemia is unclear. In other organs, permeability is a sensitive indicator of functional vascular injury. Therefore, a new double-indicator method of assessing vascular protein permeability, a method that is both sensitive and specific for vascular injury, was used to investigate the effects of ischemia of graded duration followed by reperfusion on coronary microvascular function. To help confirm functional coronary vascular injury, endothelium-dependent vasodilation of isolated coronary vascular rings also was examined. Microvascular permeability was quantitatively assessed as a protein leak index by measuring the rate of extravascular accumulation of radiolabeled protein (indium 113m transferrin) normalized for vascular surface area (technetium 99m erythrocytes). Anesthetized dogs underwent 0 (control), 15, 30, or 60 minutes of left anterior descending coronary artery occlusion followed by 60 minutes of reperfusion. Even 15 minutes of ischemia increased the protein leak index by 50% (3.16 +/- 0.30 ischemic vs. 2.09 +/- 0.11 control). Longer periods of ischemia increased the protein leak index in proportion to the duration of ischemia. The protein leak index increased threefold (6.51 +/- 0.60) after 60 minutes of ischemia. At each duration of ischemia, there was significant regional variation in the protein leak index that correlated with the severity of ischemic blood flow to that region measured with microspheres. Endothelial injury also was evident after 15 and 30 minutes of ischemia as impaired vasodilation of isolated coronary rings in response to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Electron microscopy and in vitro direct immunofluorescence revealed evidence of vascular injury after 60 minutes but not after 15 minutes of ischemia. We conclude that even brief ischemia and reperfusion cause functional coronary vascular injury evident as increased microvascular permeability and impaired endothelium-dependent vasodilation and that regional differences in the degree of microvascular injury correlate with differences in the severity of ischemia.     

Rectal hyperplasia after jejunoileal bypass for morbid obesity.

Jejunoileal bypass (JIB) has been widely used to treat patients with morbid obesity for the past 20 years. In rats JIB causes adaptive colonic hyperplasia and enhances colorectal neoplasia. In this study crypt cell production rate (CCPR) was measured stathmokinetically in cultured rectal biopsies from nine patients with JIB and seven controls without intestinal operations or disease. Crypt cell production rate in the group with JIB was more than double that of controls (12.80 (2.67) v 6.23 (1.49) cells/crypt/h: p less than 0.001). There were no significant differences in crypt morphometry and histological examination of rectal biopsies was normal. Patients with JIB have a marked and persistent increase in cell proliferation in the large intestine and may be at increased risk of developing colonic cancer.     

Prospective study of autonomic neuropathy as a predictor of mortality in patients with diabetes

To investigate the relationships between serum concentrations of soluble adhesion molecules and hyperglycemia, insulin resistance, or other conventional risk factors in type 2 diabetes, we measured soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-selectin), insulin sensitivity, and conventional risk factors in 150 Japanese type 2 diabetic patients without apparent diabetic macroangiopathy. High serum concentrations of sVCAM-1 and sE-selectin were observed in patients with type 2 diabetes. Serum concentrations of soluble adhesion molecules were not significantly influenced by sex, hypertension, dyslipidemia, or microangiopathy. Spearman correlation showed that sVCAM-1 concentrations correlated significantly with fasting plasma glucose (FPG), fasting C-peptide, and insulin sensitivity [K index of the insulin tolerance test (K(ITT))] (rho=0.19,0.23, and -0.23, respectively). Soluble E-selectin concentrations correlated significantly with body mass index (BMI), FPG, fasting C-peptide, insulin sensitivity, and triglyceride (rho=0.33,0.42,0.26,-0.48, and 0.29, respectively). Multiple regression analysis showed that FPG, fasting C-peptide, and total cholesterol were independent factors that correlated with sVCAM-1 levels. BMI, FPG, and insulin sensitivity were independent factors that correlated with sE-selectin levels. Serum concentrations of sE-selectin significantly increased associated with clustering of conventional risk factors those obesity, hypertension, dyslipidemia, and current smoking (P<0.01). Thus, sVCAM-1 and sE-selectin levels are related to both hyperglycemia and insulin resistance. Soluble E-selectin levels may be related to obesity, hyperglycemia, and insulin resistance and may reflect the presence of a multiple risk factor clustering syndrome.