Cross-reactivity of the IDEXX Legiolert method with other Gram-negative bacteria and waterborne pathogens leads to false-positive assay results.

Legionella species are the causative agent of Legionnaires' disease, a potentially fatal bacterial pneumonia. New regulations and standards have prioritized the development of water safety plans to minimize the growth and spread of Legionella species in buildings. To determine the presence and type of Legionella in a water system, microbiological culturing is the gold standard method. However, recently new methodologies have been developed that claim to be sensitive and specific for Legionella at the genus or L. pneumophila at the species level. Published and anecdotal reports suggest that one of these newer culture-based, enzyme-substrate methods, the IDEXX Legiolert test, may exhibit false positivity with other microbes common to water sources. We experimentally evaluated the IDEXX Legiolert method using these other waterborne bacteria including Elizabethkingia meningoseptica, Pseudomonas aeruginosa, Proteus mirabilis and Serratia marcescens at real-world environmental concentrations. We saw false-positive results for the Legiolert test with several of these organisms, at sample concentrations as low as 60 CFU per ml. False-positive Legionella results can trigger costly remediation and water-use restrictions, that may be implemented while waiting for additional, confirmatory microbiological testing that could, in this case, yield no L. pneumophila.

12S-Lipoxygenase is necessary for human vascular smooth muscle cell survival.

Considerable evidence has been published demonstrating the importance of lipoxygenase enzymes for vascular smooth muscle cell (VSMC) growth. The current study sets out to determine whether or not 12-lipoxygenase (12LO) is also important for human placental VSMC survival. Both a pharmacological and two 12LO antisense knockdown approaches were applied. The 12LO inhibitor baicalien induced a 2-2.5-fold increase in cell death, which appeared to result from apoptosis, as indicated by DNA fragmentation, activation of procaspase 3 to caspase 3 and cytochrome C release from the mitochondria to the cytosol. This apoptosis could be prevented by treatment with the 12LO product, 12 hydroxyeicosatetraenoic acid (12HETE). Human platelet-type 12LO-antisense knockdown, by either plasmid transfection or adeno-associated virus (AAV) infection also induced substantial VSMC death over controls, which could also be prevented by treatment with 12HETE, but not 5HETE. Hence, biochemical 12LO inhibition or 12LO-antisense knockdown in VSMC can induce programmed cell death. These observations suggest a previously unrecognized association between human VSMC survivability and 12LO.

Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts.

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli, polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of P(i) and ADP. As expected of a phosphorylase, P(i) enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the complete Arabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

Phase I study of interleukin-2 and interferon alfa-2a as outpatient therapy for patients with advanced malignancy.

Twenty-six patients were treated in this phase I study with the combination of interleukin-2 (IL2) administered as a continuous infusion and interferon alfa-2a (IFN alpha-2a) administered intramuscularly to patients in an outpatient setting. The maximum-tolerated dose of both agents given as outpatient therapy was 2 x 10(6) U/m2 days 1 to 5 of IL2 and 9 x 10(6) U/m2 days 1, 3, and 5 of IFN alpha-2a for 4 consecutive weeks. A 2- to 4-week rest period was permitted after each 4 weeks of treatment. Fatigue was the treatment-limiting toxicity, and serious clinical or laboratory abnormalities occurred infrequently during this study. Patients with colon cancer metastatic to the liver tolerated treatment worse than patients with other tumors. Twelve of the 15 patients with renal cell cancer were assessable for response determinations. Of these 12 patients, three exhibited complete tumor regression, three have had partial objective regression, and three patients experienced stabilization of rapidly progressive disease. This therapy appears to be well tolerated in an outpatient treatment setting and shows significant activity against advanced renal cell cancer.

Macrophage-released proteoglycans enhance LDL aggregation: studies in aorta from apolipoprotein E-deficient mice.

Aggregated low-density lipoprotein (LDL) was shown to be present in the atherosclerotic lesion, but the mechanism responsible for its formation in vivo is not known yet. To find out whether LDL aggregation occurs in the arterial wall during atherogenesis, LDLs were extracted from the aortas of apolipoprotein E-deficient (E(0)) mice during their aging (and the development of atherosclerosis), and were analyzed for their aggregation states, in comparison to LDLs isolated from aortas of control mice. LDL isolated from aortas of E(0) mice was already aggregated at 1 month of age and its aggregation state substantially increased with age, with 3-fold elevation at 6 months of age compared to younger, 1-month-old, mice. Only minimal aggregation could be detected in LDL derived from control mice. Electron microscopy examination revealed that LDL particles from aortas of the E(0) mice were heterogeneous in their size, ranging between 20 and 300 nm. The mouse aortic LDL contained proteoglycans (PGs) and their content increased with the age of the mice, with about 2-fold higher levels than those found in LDLs derived from aortas of control mice. Macrophage-released PGs were previously demonstrated to enhance LDL aggregation in vitro. However, their involvement in LDL aggregation in vivo has not been studied yet. Thus, we next studied the effect of arterial macrophage-released PGs on the susceptibility of plasma LDL to aggregation by Bacillus cereus sphingomyelinase (SMase). Foam cell macrophages were isolated from aortas of the atherosclerotic E(0) mice at 6 months of age and were found to be loaded with cholesterol and to contain oxidized lipids. To analyze the effect of macrophage-released PGs on LDL aggregation, PGs were prelabeled by cell incubation with [35S]sulfate, followed by incubation of macrophage-released PGs with E(0) mouse plasma LDL (200 microg protein/ml) for 1 h at 37 degrees C. [35S]Sulfated PGs were found to be LDL-associated and the susceptibility of PG-associated LDL to aggregation by SMase was increased by up to 45% in comparison to control LDL. Similar results demonstrating the involvement of PGs in LDL aggregation were obtained upon incubation of LDL with increasing concentrations of PGs that were isolated from the entire aorta of E(o) mice (rather than the isolated macrophages). The stimulatory effect of macrophage-released PGs on LDL aggregation was markedly reduced when the PGs were pretreated with the glycosaminoglycan-hydrolyzing enzymes, chondroitinase ABC or chondroitinase AC, and to a much lesser extent with heparinase. We thus conclude that macrophage-released chondroitin sulfate PG can contribute to the formation of atherogenic aggregated LDL in the arterial wall.

Crystalloid and colloid resuscitation of uncontrolled hemorrhagic shock following massive splenic injury.

Using a standardized massive splenic injury (MSI) model of uncontrolled hemorrhagic shock we studied the effect of vigorous crystalloid or colloid fluid resuscitation on the hemodynamic response, and survival in rats. The value of massive fluid infusion in uncontrolled hemorrhagic shock following intra-abdominal solid organ injury is still controversial. The effect of crystalloid and colloid infusion was studied following massive splenic injury. The animals were randomized into six groups: group 1 (n = 8) sham-operated, group 2 (n = 12) MSI untreated, group 3 (n = 10) MSI treated with 41.5 mL/kg Ringer's lactate (large-volume Ringer's lactate, LVRL), group 4 (n = 14) MSI treated with 5 mL/kg 7.5% NaCl (hypertonic saline, HTS), group 5 (n = 10) MSI treated with 7.5 mL/kg hydroxyethyl starch (HES-7.5), and group 6 (n = 11) MSI treated with 15 mL/kg hydroxyethyl starch (HES-15). Following MSI mean arterial pressure (MAP) in untreated group 2 decreased from 109.1 +/- 4.5 to 49.8 +/- 9.6 mmHg (P < 0.001) in 60 min. Mean survival time was 132.1 +/- 18.7 min, and total blood loss was 30.2 +/- 4.1% of blood volume. LVRL infusion resulted in an early rise in MAP from 59.7 +/- 7.3 to 90.0 +/- 11.3 mmHg (P < 0.01), which then rapidly dropped to 11.7 +/- 4.5 mmHg (P < 0.001) after 60 min. The mean survival time was 82.5 +/- 18.2 min (P < 0.01), and total blood loss was 53.7 +/- 2.9% (P < 0.01). Total blood loss following HTS infusion was 32.2 +/- 4.0% and survival time was 127.9 +/- 19.7 min. HES-7.5 infusion only moderately increased bleeding to 44.2 +/- 3.9% (P < 0.05), but mortality remained unchanged. HES-15 infusion resulted in an increase in blood loss to 47.8 +/- 7.1% (0.01), survival time dropped to 100.7 +/- 12.3 min (P < 0.05). Vigorous large volume infusion of Ringer's lactate or HES following MSI resulted in a significant increase in intra-abdominal bleeding and shortened survival time compared to untreated, small volume HTS, or HES-7.5-treated animals. The hemodynamic response to crystalloid or colloid infusion in blunt abdominal trauma is primarily dependent on the severity of injury and the rate of fluid resuscitation.

Crystalloid or colloid resuscitation of uncontrolled hemorrhagic shock after moderate splenic injury.

Using a standardized moderate splenic injury (MSI) model of uncontrolled hemorrhagic shock, we studied the effect of vigorous crystalloid or colloid fluid resuscitation on the hemodynamic response and survival time in rats. The animals were randomized into 6 groups: group 1 (n = 8) sham-operated, group 2 (n = 10) MSI untreated, group 3 (n = 10) MSI treated with 41.5 mL/kg Ringer's lactate (large-volume Ringer's lactate [LVRL]), group 4 (n = 10) MSI treated with 5 mL/kg 7.5% NaCl (hypertonic saline [HTS]), group 5 (n = 10) MSI treated with 7.5 mL/kg hydroxyethyl starch (HES-7.5), group 6 (n = 10) MSI treated with 15 mL/kg hydroxyethyl starch (HES-15). After MSI, mean arterial pressure (MAP) in group 2 decreased from 105.0+/-5.6 to 64.0+/-12.7 mmHg (P < 0.001) after 60 min. Mean survival time was 157.4+/-28.9 min, and total blood loss was 24.0+/-5.4% of blood volume. LVRL infusion resulted in an early rise in MAP from 75.2+/-8.7 to 96.7+/-9.0 mmHg (P < 0.01), which then rapidly dropped to 43.0+/-9.7 mmHg (P < 0.001) after 60 min. The mean survival time was 140.7+/-22.3 min, and total blood loss was 41.4+/-4.8% (P < 0.05). Total blood loss following HTS infusion was 24.7+/-3.7%, and mean survival time was 177.5+/-18.9 min. HES-7.5 infusion was followed by bleeding of 25.6+/-5.1%, and mean survival time was 181+/-16.1. HES-15 infusion resulted in an increase in blood loss to 48.2+/-7.3% (P < 0.05), and mean survival time of 133.0+/-27.7 min. Large-volume Ringer's lactate (LVRL) or hydroxyethyl starch (HES-15) infusion in uncontrolled hemorrhagic shock after moderate splenic injury resulted in a significant increase in intra-abdominal bleeding, but survival time remained unchanged compared with untreated, small-volume HTS-, or HES-7.5-treated animals. The hemodynamic response to large-volume crystalloid or colloid infusion was similar to moderate large-vessel injury.

Immune potential of lymph node-derived lymphocytes in uncontrolled hemorrhagic shock.

The state of T cell immunity was evaluated in rats in early (1-4 h) hemorrhagic shock induced by a massive splenic injury. T cell subpopulations from treated and untreated shocked animals were tested by flow cytometry and the results were compared with healthy controls. A fall in CD4+ T lymphocyte and natural killer (NKR-P1+) cell number, marked decline in the T helper (CD4+) to T suppressor (CD8+) ratio, and decrease of interleukin-2 receptor (IL-2R) bearing cells in peripheral blood, mesenteric, and popliteal lymph nodes of rats was found in the early stages of hemorrhagic shock. The same phenotype profile was also revealed in lymphocytes of rats in hemorrhagic shock following massive splenic injury treated with Ringer's lactate. The number of TCRalpha beta and TCR-gamma delta positive cells, as well as the percentage of CD4 and CD8 positive cells in the thymus, was similar in all groups of rats. Culture of lymph node cells taken from rats following hemorrhage in the presence of 100 U/mL hrIL-2 resulted in a marked increase in the number of NKR-PI+ positive cells from 4.2% to 30.5% (P < 0.001). Magnet separated NKR-P1+ fractions lysed the allogeneic fibroblasts in the same manner as IL-2-activated NKR-P1 cells from the control rats. Popliteal lymph node (PLNi) CD8b+ lymphocytes from rats in hemorrhagic shock preinfected into the footpad with cytomegalovirus (CMV) 6 days prior to injury lost their ability to lyse the CMV-infected fibroblasts and protect the monolayer from CMV infection when compared with PLNi cells from control infected rats. The possible mechanisms for the observed cellular dysfunction following hemorrhage are discussed.

Overexpression of the high-affinity Fcgamma receptor (CD64) is associated with leukocyte dysfunction in sepsis.

The morbidity and mortality from sepsis and multiple organ dysfunction syndrome (MODS) continues to be high. An increase in FcgammaRI+ (CD64+) monocytes was demonstrated in septic patients, and an association between cell number, their secretory activity, and poor outcome has been described. In the present investigation further characterization of CD64+ leukocytes has been attempted. The study was aimed at examining the phagocytic activity (PA) and reactive oxygen species (ROS) production by monocytes (Mo) and neutrophils (Neu) in sepsis and sepsis-induced acute respiratory distress syndrome (ARDS) related to the pattern of CD64 expression. Twenty-three post-traumatic or post-operative male and female patients with sepsis were enrolled. The control group consisted of 10 healthy volunteers. Arterial blood samples were taken during the septic episode for flow cytometric analysis of surface leukocyte antigens, phagocytosis, and ROS production. CD64 expression on Mo and Neu was markedly increased in septic patients (P = 0.029 and P = 0.0005), and even more in sepsis with ARDS (P = 0.011). In healthy individuals, PA of CD64+ Neu was higher, than of CD64- cells (P = 0.021). In septic patients, decreased PA was detected in CD64+ Mo and Neu (P = 0.013 and P = 0.040, respectively). CD64+ Neu of patients in ARDS exhibited the most prominent PA depression (P = 0.048). ROS production in non-separated Mo and Neu was increased in sepsis (P = 0.026 and P = 0.004, respectively). In healthy individuals CD64+ Neu and stimulated CD64+ Mo demonstrated increased ROS synthesis compared to matched CD64- cells (P = 0.001 and P = 0.042, respectively). Although ROS production by CD64+ leukocytes in sepsis was also increased compared to CD64- cells, significantly less ROS was generated compared to healthy subjects (P = 0.021). In conclusion, overexpression of CD64 on blood Mo and Neu from patients with sepsis and ARDS is associated with depressed PA and decreased oxidative response.

The effects of enteral stimulation on gallbladder bile during total parenteral nutrition in the neonatal piglet.

The neonatal piglet is a satisfactory model for the human neonate requiring total parenteral nutrition (TPN). Bile status and subsequent liver and gallbladder dysfunction have long been documented as serious complications of long-term TPN. The purpose of this study was to determine whether small amounts of enteral formula during TPN will maintain normal bile appearance and composition. Thirty-one Hanford miniswine, 3 to 6 days old underwent surgery for the placement of central venous catheters. Two days postoperatively, the animals were separated into three groups, according to dietary regimens. Group 1 (n = 10), the control group, received pig formula (SPF-lac) orally (200 cal/kg/d); group 2 (n = 11), was maintained on TPN (180 cal/kg/d) with an enteral supplement of SPF-lac (20 cal/kg/d); group 3 (n = 10), was maintained on TPN only (200 cal/kg/d). The TPN formula consisted of 35 g/kg/d of glucose, 10 g/kg/d of protein, and 3 g/kg/d of lipid. The animals were maintained on these diets for 6 weeks. At necropsy, gallbladder with bile was weighed and bile volume and appearance was recorded. Chemical analyses was performed on 26 bile samples. Gallbladder weight was significantly decreased in groups 2 and 3 compared with group 1 (P less than .0003, P less than .033, respectively, using Students t test with Bonferoni adjustment). Volume was significantly decreased only in group 2 (P less than .003). Group differentiation in relation to bile appearance was determined by the presence or absence of either bile sludge or crystals.(ABSTRACT TRUNCATED AT 250 WORDS)

Necrotizing enterocolitis in a neonatal piglet model.

The aim of this study was to develop an animal model for necrotizing enterocolitis (NEC). Twenty-five neonatal Hanford minipigs had carotid artery and external jugular vein catheters and rectal Clinical Tonomitors placed under anesthesia. Experimental animals were subjected to a hypoxic insult (50% reduction in baseline PaO2 for 30 minutes) and hypothermic stress (core temperature reduced to 35 degrees C for 30 minutes). Regular oral diet was resumed and the survivors were euthanized 3 to 4 days later. All animals underwent necropsy with gross and histopathological evaluation of the entire bowel. Of 22 experimental animals, 14 survived (64%) and 8 (36%) died of pulmonary hemorrhage. Of the 14 survivors, 8 (57%) had gross and microscopic evidence of NEC. Six of the total 25 animals (24%) sustained rectal perforations from the tonometer. Of 3 control animals, one died of pulmonary hemorrhage and the two survivors had normal intestine. This model successfully produced gross and histological evidence of NEC. The tonometer shows promise as a predictor of NEC provided technical modifications can reduce the complication rate.

Transcutaneous electrical nerve stimulation (TENS) as a relief for dysmenorrhea.

Sixty-one women who suffered from primary dysmenorrhea, were treated with Transcutaneous Nerve Stimulation (TENS) for two menstrual cycles, and reported the effect of the treatment on their pain. Thirty percent of the patients reported marked pain relief, 60% reported moderate pain relief and 10% reported that TENS had no influence on their pain. No side effects were reported. We conclude that TENS is an effective and safe non-pharmacological means for the treatment of primary dysmenorrhea. It could serve as a main treatment modality for women who suffer from primary dysmenorrhea and do not wish to or cannot use the conservative pharmacological agents. In addition TENS can serve as an adjuvant therapy to the conventional pharmacological agent in severe cases of primary dysmenorrhea.

Activated CD8+ T-lymphocytes in obstructive sleep apnoea.

T-lymphocytes are implicated in the development of atherosclerosis. The aim of this study was to assess whether the CD8+ T-lymphocytes of obstructive sleep apnoea (OSA) patients undergo phenotypic and functional changes that may exaggerate atherogenic sequelae in OSA. A total of 36 OSA patients, 17 controls and 15 single-night-treated OSA patients were studied. Phenotype and cytotoxicity against K562 target cells were analysed by flow cytometry. Cytotoxicity against human umbilical vein endothelial cells (HUVECs) was assessed by 51Cr release assay. The cytotoxicity of the CD8+ T-lymphocytes of OSA patients against K562 and HUVECs was significantly greater than controls. This increased cytotoxicity directly depended on the presence of perforin and natural killer receptors (CD56, CD16), which were significantly increased in OSA CD8+ T-lymphocytes. Also the percentage of the CD56bright subset, which mediates initial interactions with vascular endothelium, significantly increased in OSA. Nasal continuous positive airway pressure treatment significantly decreased CD8+ T-cell cytotoxicity and CD56 expression, and was positively correlated with natural killer inhibitory NKB1 receptor expression either after a single-night treatment or after a prolonged treatment. In conclusion, the CD8+ T-lymphocytes of obstructive sleep apnoea patients undergo phenotypic and functional changes, rendering them cytotoxic to target cells via increased CD56+/perforin+ expression, which can be ameliorated by nasal continuous positive airway pressure treatment. These results are compatible with the current authors' hypothesis of atherogenic sequelae in obstructive sleep apnoea.

The stable prostacyclin analog, iloprost, and prostaglandin E1 inhibit monocyte procoagulant activity in vitro.

Exposure of human peripheral blood to 100 ng/mL of bacterial endotoxin for 2 hours resulted in a 20-fold increase in monocyte procoagulant activity. The activity was functionally identified as tissue factor, because it was not expressed in plasma deficient in factor VII and was specifically inhibited by a monoclonal antibody directed against human tissue factor. When the stable prostacyclin analog, iloprost, was added to blood 30 minutes before endotoxin, a dose-dependent inhibition of monocyte procoagulant activity occurred, with an I50 of 20 nmol/L. Prostaglandin E1 (PGE1) produced similar effects, with an I50 of 150 nmol/L. Exposure of THP-1 monocytic cells to 100 ng/mL endotoxin resulted in a threefold increase in procoagulant activity after 2 hours and a 20-fold increase after 6 hours. A 30-minute pretreatment with iloprost or PGE1 again inhibited development of procoagulant activity, with I50 values of 5 nmol/L and 150 nmol/L, respectively. Treatment of THP-1 cells with iloprost 2 hours after exposure to endotoxin significantly inhibited further increases in procoagulant activity. Iloprost was less potent under these conditions, 30% inhibition being obtained at 100 nmol/L and 70% at 1 mumol/L. These results suggest that prostacyclin may be a physiologic modulator of monocyte tissue factor expression; in addition, its stable analog, iloprost, may have clinical potential for the treatment of thrombotic disorders in which elevated monocyte procoagulant activity plays a role.

Family Medicine in Rural Communities: The McGill experience.

Recruitment of physicians for rural communities is a continuing problem in Canada. Medical schools can be involved through preferential admission policies. Departments of family medicine across the country are including on-site training in rural communities and are seeking to improve their rural program curriculum. The McGill rural program is described from its origins to its present state. A rural coordinator oversees 12 sites at which both residents and students are trained. One site at Shawville, Que, is described from a rural physician's point of view.

Survival following traumatic rupture of the heart in a child.

Blunt force trauma to the chest can result in rupture of the heart. We report the youngest survivor of this injury, followed by a literature review, description of the epidemiology, and mechanism and guidelines for diagnosis and aggressive management.