Differential stimulation of murine lymphoma growth in vitro by normal and BCG-activated macrophages

Peritoneal macrophages from mice infected with Bacille Calmette-Guérin (BCG) and from normal mice were examined for their effects in vitro on thymidine uptake by 10 murine lymphomas, a murine fibroblast line, and a guinea pig hepatoma. Only the murine fibroblast line showed growth inhibition in the presence of BCG macrophages. For the majority of tumors, normal macrophages were profoundly stimulatory to tumor cell DNA synthesis, while BCG macrophages were much less stimulatory, without being frankly inhibitory. The effect of 2-mercaptoethanol on tumor cell growth was also studied. All lymphomas stimulated to grow more rapidly in vitro by normal macrophages were stimulated to a similar degree by 2-mercaptoethanol.     

Effect of varying nitric oxide release to prevent platelet consumption and preserve platelet function in an in vivo model of extracorporeal circulation

The gold standard for anticoagulation during extracorporeal circulation (ECC) remains systemic heparinization and the concomitant risk of bleeding in an already critically ill patient could lead to death. Normal endothelium is a unique surface that prevents thrombosis by the release of antiplatelet and antithrombin agents. Nitric oxide (NO) is one of the most potent, reversible antiplatelet agents released from the endothelium. Nitric oxide released from within a polymer matrix has been proven effective for preventing platelet activation and adhesion onto extracorporeal circuits. However, the critical NO release (NO flux) threshold for thrombus prevention during ECC has not yet been determined. Using a 4-hour arteriovenous (AV) rabbit model of ECC, we sought to find this threshold value for ECC circuits, using an improved NO-releasing coating (Norel-b). Four groups of animals were tested at variable NO flux levels. Hourly blood samples were obtained for measurement of arterial blood gases, platelet counts, fibrinogen levels and platelet function (via aggregometry). A custom-built AV circuit was constructed with 36 cm of poly(vinyl)chloride (PVC) tubing, a 14 gauge (GA) angiocatheter for arterial access and a modified 10 French (Fr) thoracic catheter for venous access. The Norel-b coating reduced platelet activation and thrombus formation, and preserved platelet function - in all circuits that exhibited an NO flux of 13.65 x 10(10) mol x cm(-2) x min(-1). These results were significant when compared with the controls. With the Norel-b coating, the NO flux from the extracorporeal circuit surface can be precisely controlled by the composition of the polymer coating used, and such coatings are shown to prevent platelet consumption and thrombus formation while preserving platelet function in the animal.     

Circulating immune complexes in patients with gram negative septic shock

Summary In order to explain complement components abnormalities observed during septic shock, circulating immune complexes (C.I.C.) were searched for in sera from 34 patients with gram negative sepsis by two different methods: polyethylene glycol precipitation test based on physical properties of C.I.C. and C1q deviation test based on the property of radiolabelled C1q to react with C.I.C. Serum immunoglobulins (IgG, IgA, IgM) and complement components (C1q, C3, C4) levels were simultaneously determined. Seventeen patients with minimal haemodynamic abnormalities had normal or increased levels (except C4 at 62% of normal) and in eleven cases both tests for C.I.C. were simultaneously positive. Seventeen patients with severe septic shock had a decrease in IgG, IgM, C1q, C3 and C4 and none had both tests for C.I.C. simultaneously positive (P<10^–4). The disappearence of C.I.C. in patients with severe septic shock associated with evidence of complement activation suggests their involvement in the pathogenesis of septic shock in man.This work was supported by grants from Conseil Scientifique de l'Université Paris Val de Marne and Délégation Générale de la Recherche Scientifique et Technique, Contracts N° 7510954 and 7771396     

Ca v 1.3 is preferentially coupled to glucose-stimulated insulin secretion in the pancreatic beta-cell line INS-1

L-Type Ca(2+) channel blockers inhibit glucose and KCl-stimulated insulin secretion by pancreatic beta cells. However, the role of the two distinct L-type channels expressed by beta cells, Ca(v)1.2 and Ca(v)1.3, in this process is not clear. Therefore, we stably transfected INS-1 cells with two mutant channel constructs, Ca(v)1.2DHPi or Ca(v)1.3 DHPi. Whole-cell patch-clamp recordings demonstrated that both mutant channels are insensitive to dihydropyridines (DHPs), but are blocked by diltiazem. INS-1 cells expressing Ca(v)1.3/DHPi maintained glucose- and KCl-stimulated insulin secretion in the presence of DHPs, whereas cells expressing Ca(v)1.2/DHPi demonstrated DHP resistance to only KCl-induced secretion. INS-1 cells were also stably transfected with cDNAs encoding the intracellular loop between domains II and III of either Ca(v)1.2 or Ca(v)1.3 (Ca(v)1.2/II-III or Ca(v)1.3/II-III). Glucose- and KCl-stimulated insulin secretion in Ca(v)1.2/II-III cells were not different from untransfected INS-1 cells. However, glucose-stimulated insulin secretion was completely inhibited and KCl-stimulated secretion was substantially resistant to inhibition by DHPs, but sensitive to omega-agatoxin IVA in Ca(v)1.3/II-III cells. Moreover, the L-type channel agonist FPL 64176 markedly enhanced KCl-stimulated secretion by Ca(v)1.3/II-III cells. Together, our results suggest that Ca(2+) influx via Ca(v)1.3 is preferentially coupled to glucose-stimulated insulin secretion in INS-1 cells.     

Hyperscanning: simultaneous fMRI during linked social interactions

"Plain question and plain answer make the shortest road out of most perplexities." Mark Twain-Life on the Mississippi. A new methodology for the measurement of the neural substrates of human social interaction is described. This technology, termed "Hyperscan," embodies both the hardware and the software necessary to link magnetic resonance scanners through the internet. Hyperscanning allows for the performance of human behavioral experiments in which participants can interact with each other while functional MRI is acquired in synchrony with the behavioral interactions. Data are presented from a simple game of deception between pairs of subjects. Because people may interact both asymmetrically and asynchronously, both the design and the analysis must accommodate this added complexity. Several potential approaches are described.     

Induction of protective therapy for autoimmune diseases by targeted DNA vaccines encoding pro-inflammatory cytokines and chemokines

T-cell-mediated autoimmune diseases such as multiple sclerosis, rheumatoid arthritis or type 1 diabetes result from an aggressive attack of self-components by autoimmune T-cells. Pro-inflammatory mediators, particularly cytokines and chemokines, direct the homing and effectorfunction of these cells. It has recently been demonstrated that the immune system, which can attack self-components, also generates 'beneficial' autoimmunity against pro-inflammatory mediators. During the course of an autoimmune condition, and to a much lesser extent in response to microbial inflammation, the immune system produces auto-antibodies to pro-inflammatory mediators. This reduces the harm from these diseases. We also discovered that targeted DNA vaccines could effectively amplify these responses to provide protective immunity. The underlying mechanism is partially understood. At the site of immunization, the relevant gene product is produced and then presented by dendritic cells/macrophages, which undergo activation due to an interaction of plasmid CpG with toll-like receptor 9 on the dendritic cell. This then activates CD4+ T-cells, which help the production of T-cell-dependent antibodies against the gene product of the vaccines. These antibodies neutralize their target product and suppress inflammation. This review explores this interesting concept and its therapeutic implications.     

Validation of the 2001 American Thoracic Society criteria for severe community-acquired pneumonia

STUDY OBJECTIVE: Ewig et al. proposed a new definition of severe community-acquired pneumonia in 1999, which was adopted by the American Thoracic Society in 2001. We evaluated this definition in an independent population of emergency department patients. DESIGN: We compared the 2001 American Thoracic Society definition of severe community-acquired pneumonia using emergency department data to intensive care unit (ICU) admission, use of mechanical ventilation, and administration of vasopressors. SETTING: LDS Hospital, a tertiary care, university-affiliated hospital with 520 total beds and 68 ICU beds in Salt Lake City, UT. PATIENTS: We studied 980 consecutive emergency department patients with a radiographically confirmed diagnosis of pneumonia between June 1995 and June 1999. Of these patients, 498 were admitted to the hospital, immunocompetent, and without a "do-not-resuscitate" order within 24 hrs of admission. MEASUREMENTS AND MAIN RESULTS: Forty-seven patients met the criteria for severe community-acquired pneumonia in the emergency department and were admitted to the ICU. Three hundred eighty patients did not meet the criteria and were admitted to a hospital unit. Nineteen patients met the definition but were admitted to a hospital unit; only one required subsequent ICU admission. Two of the 19 died after a do-not-resuscitate order was entered >24 hrs after admission; the remainder recovered. Fifty-two patients were triaged to the ICU but did not initially meet the definition of severe pneumonia. Sixteen of these 52 patients required mechanical ventilation, 13 of the 16 within 24 hrs of admission to the ICU. The sensitivity for the 2001 American Thoracic Society definition in our population was 44%, specificity was 95%, positive predictive value was 71%, and negative predictive value was 88%. CONCLUSION: The 2001 American Thoracic Society definition of severe community-acquired pneumonia had high specificity but lower sensitivity in our population compared with the derivation population. Additional factors not reflected in the definition may contribute to ICU admission and the need for mechanical ventilation.     

The Challenge of Predicting Demand for Emergency Department Services

Objectives:  The objective was to develop methodology for predicting demand for emergency department (ED) services by characterizing ED arrivals.
Methods:  One year of ED arrival data from an academic ED were merged with local climate data. ED arrival patterns were described; Poisson regression was selected to represent the count of hourly ED arrivals as a function of temporal, climatic, and patient factors. The authors evaluated the appropriateness of prediction models by whether the data met key Poisson assumptions, including variance proportional to the mean, positive skewness, and absence of autocorrelation among hours. Model accuracy was assessed by comparing predicted and observed histograms of arrival counts and by how frequently the observed hourly count fell within the 50 and 90% prediction intervals.
Results:  Hourly ED arrivals were obtained for 8,760 study hours. Separate models were fit for high- versus low-acuity patients because of significant arrival pattern differences. The variance was approximately equal to the mean in the high- and low-acuity models. There was no residual autocorrelation ( r  = 0) present after controlling for temporal, climatic, and patient factors that influenced the arrival rate. The observed hourly count fell within the 50 and 90% prediction intervals 50 and 90% of the time, respectively. The observed histogram of arrival counts was nearly identical to the histogram predicted by a Poisson process.
Conclusions:  At this facility, demand for ED services was well approximated by a Poisson regression model. The expected arrival rate is characterized by a small number of factors and does not depend on recent numbers of arrivals.     

Gender-specific and developmental influences on the expression of rat organic anion transporters

Rat organic anion transporter 1 (Oat1), Oat2, and Oat3, members of the organic anion transporter family, transport some organic anions across cellular membranes. Previously, highest Oat1 and Oat3 mRNA expression was reported in kidney and Oat2 in liver. However, gender and developmental differences in Oat expression remain unknown. This study describes gender- and age-specific patterns of rat organic anion transporter expression in various tissues. Oat mRNA expression was evaluated in adult male and female Sprague-Dawley rat tissues, and developmental expression was also determined in kidneys of Sprague-Dawley rats ranging in age from days 0 through 45. Expression was quantified using branched-DNA signal amplification. Oat1 mRNA expression was primarily observed in kidney. Surprisingly, Oat2 mRNA expression was also highest in kidney rather than in liver. Moreover, considerably higher Oat2 levels were seen in female kidney as compared with male. Finally, Oat3 mRNA expression was highest in kidney of both genders, whereas a male-predominant pattern was observed in liver. At birth, all kidney Oat mRNA levels were low. Renal Oat1 expression gradually increased throughout development, approaching adult levels at 30 days of age, where at days 40 and 45 Oat1 levels were greater in males than females. Oat2 expression in kidney was minimal through day 30 but increased dramatically at day 35 in females only. Lastly, Oat3 mRNA expression in kidney matured earliest, rapidly increasing from birth through day 10. These data indicate that Oat mRNA expression is primarily localized to the kidney, and observed expression patterns may explain some previously recognized age- and gender-dependent toxicities associated with chemical exposure.