Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity

Pseudomonas aeruginosa is a persistent pathogen in the airways of patients with cystic fibrosis or bronchiectasis from other causes and appears to have evolved strategies to survive the inflammatory response of the host. We hypothesized that the secreted hemolytic phospholipase C (PLC) of P. aeruginosa (PlcHR) would decrease neutrophil respiratory burst activity. We found that while intact wild-type P. aeruginosa cells stimulated moderate respiratory burst activity from human neutrophils, an isogenic mutant pseudomonas (DeltaHR strain) containing a targeted deletion of the plcHR operon induced a much more robust oxidative burst from neutrophils. In contrast, a second pseudomonas mutant (DeltaN) containing a disruption in the gene encoding the nonhemolytic PLC (PlcN) was not different from the wild type in stimulating neutrophil O2.- production. Readdition of purified PlcHR to the DeltaHR strain suppressed neutrophil O2.- production to levels stimulated by wild-type bacteria. Interestingly, purified PlcHR decreased phorbol myristate acetate (PMA)- but not formyl methionyl-leucyl-proline (fMLP)-induced respiratory burst activity, suggesting interference by PlcHR with a protein kinase C (PKC)-specific signaling pathway. Accordingly, the PKC inhibitor bisindolylmaleimide inhibited the oxidative burst induced by either PMA or intact pseudomonas, but not by fMLP, whereas the p38 kinase inhibitor SB-203580 fully inhibited the respiratory burst induced by fMLP or the PlcHR-replete wild-type bacteria, but not PMA or the PlcHR-deficient DeltaHR bacterial mutant. We conclude that expression of PlcHR by P. aeruginosa suppresses bacterium-induced neutrophil respiratory burst by interfering with a PKC-dependent, non-p38 kinase-dependent pathway.     

Carcinoembryonic antigen like antigen in granular cell myoblastomas

Summary A series of granular cell myoblastomas (GCM) and other benign and malignant tumours of soft tissue were examined for cytoplasmic content of carcinoembryonic antigen (CEA) by the two-layer conjugated immunoperoxidase technique. Using a commercial rabbit anti-CEA serum only granular cell myoblastomas showed positive cytoplasmic reaction. Pretreatment with periodic acid made this reaction less intense, but when the commercial rabbit anti-CEA serum was absorbed with tissue powder from normal human spleen the positive reaction was totally abolished. It is concluded that the positivity of GCM for CEA using commercial rabbit anti-CEA serum is due to the content of non-specific cross-reacting antigen (NCA) and maybe other cross-reacting glycoproteins in this tumour, and not to CEA as claimed in a previous study.     

Detection of nocturnal hypoglycemia in insulin-treated diabetics by a skin temperature--skin conductance meter

The efficacy and credibility of a skin temperature--skin conductance meter (Teledyne Sleep Sentry) for detecting hypoglycemia was studied during night-time in 22 adult insulin-treated diabetics. Capillary blood glucose concentration was measured 99 times (when the alarm sounded, in case of hypoglycemic symptoms, and at 3 a.m.). Hypoglycemia was defined as a capillary blood glucose concentration of less than or equal to 3 mmol/l. Blood glucose was measured 61 times in connection with sounding of the alarm and 38 times without the alarm sounding. At 3 a.m. the Sleep Sentry sounded the alarm 22 times, of which hypoglycemia was present 6 times giving a diagnostical specificity or diagnostical true positive rate of 0.27 (95% confidence limits 0.11-0.50). In 35 of 38 cases of no alarm the blood glucose was greater than 3 mmol/l, giving a diagnostical sensitivity of 0.92 (95% confidence limits 0.79-0.98). The Sleep Sentry sounded the alarm in 6 of 9 cases of hypoglycemia, giving a nosological sensitivity of 0.67 (95% confidence limits 0.30-0.93). The Sleep Sentry did not sound the alarm in 35 of 51 cases of non-hypoglycemia, giving a nosological specificity of 0.69 (95% confidence limits 0.54-0.81). In other words, the Sleep Sentry detects about 2/3 of blood glucose values less than or equal to 3 mmol/l, but in addition it sounds a false alarm in 2/3 of the cases.     

European seroepidemiology network 2: Standardisation of assays for seroepidemiology of varicella zoster virus.

BACKGROUND: The aim of the European Sero-Epidemiology Network (ESEN2) is to harmonise the serological surveillance of vaccine-preventable diseases in Europe. OBJECTIVE: To allow comparison of antibody prevalence in different countries by standardising results into common units. STUDY DESIGN: For varicella zoster virus (VZV), a reference laboratory established a panel of 148 samples, characterised by indirect enzyme-immunoassay (ELISA), indirect immunofluorescence, and complement fixation test. Fifty-seven samples were also studied by the fluorescence antibody to membrane antigen test. The geometric mean of the antibody activity (GMAA) obtained from four ELISA determinations was used to characterise each sample of the panel as positive (GMAA: >100 mIU/ml), equivocal (GMAA: 50-100 mIU/ml) or negative (GMAA: <50 mIU/ml) for antibody to VZV (anti-VZV). Thirteen laboratories, using five different ELISA tests, tested the panel. RESULTS: Agreement with the reference laboratory was above 85% in all cases, and the R(2) values obtained from regression analysis of the quantitative results were always higher than 0.87. Finally, the regression equations could be used to convert national values into a common unitage. CONCLUSION: This study confirmed that results for anti-VZV obtained by different ELISA methods can be converted into common units, enabling the comparison of the seroprevalence profiles obtained in the participant countries.     

Experimental immunization with Pseudomonas aeruginosa alginate induces IgA and IgG antibody responses.

We tried experimentally to induce a specific antibody response against Pseudomonas aeruginosa locally in the airways and systemically in rats by three different routes of immunization; intragastric feeding, intratracheal inoculation or subcutaneous vaccination. Three groups of rats were immunized with live mucoid P. aeruginosa PAO 579 by intragastric feeding or with killed PAO 579 intratracheally or subcutaneously. Three other groups were immunized with purified P. aeruginosa alginate either by intragastric feeding, intratracheally or subcutaneously. At weekly intervals for four weeks animals were sacrificed and serum and bronchial fluid were obtained. The specific IgA and IgG antibody response in lavage fluid and serum was measured. Only traces of antibodies could be detected in the bronchial lavage fluids. Anti-alginate IgA and IgG antibodies developed in all rats immunized with alginate but no antibodies against other P. aeruginosa antigens were detected. The highest IgA and IgG titer against alginate was induced by the subcutaneous immunization. IgA and IgG antibodies against other P. aeruginosa antigens developed in rats immunized with liver and sonicated bacteria. The highest IgA and IgG titers were obtained after intratracheal and subcutaneous immunization with sonicated bacteria. The present work has shown that IgA and IgG antibodies develop with high specificity after immunization. The different titers obtained do not necessarily reflect different degrees of protection.     

Identification of viral agents associated with diarrhea in young children during a winter season in Beijing, China.

BACKGROUND: Viral diarrhea remains a major cause of childhood morbidity and mortality worldwide. Although rotavirus was extensively studied in China, few comprehensive studies of all viral agents related to diarrhea in children have been conducted. OBJECTIVES: Our study was performed to investigate the role of enteric viruses in acute diarrhea in our country and to evaluate methods that could be used in routine diagnostics. STUDY DESIGN: One hundred stool samples were collected from children under 5 years of age seeking medical care for acute diarrhea during the winter season 2000/2001 in Beijing Children's Hospital. All specimens were initially screened microscopically for leucocytes/red blood cells. Samples with negative results were analyzed for virus presence using commercial EIAs and/or in-house RT-PCRs. RESULTS: At least one viral agent was found in 67% of the specimens. The frequency of rotavirus, astrovirus, norovirus and enteric adenovirus was 59%, 8%, 6% and 2%, respectively. Dual infections were found in 9.0% (6/67) of the positive samples. The results from rotavirus and astrovirus EIAs were concordant with those of rotavirus and astrovirus RT-PCRs. CONCLUSIONS: Enteric viruses play an important role in pediatric diarrhea during the winter season in China. A combination of microscopic examination of stool samples with specific EIA assays to detect virus antigen in stool specimens may be suitable for routine diagnostics.     

Skeletal muscle adaptations to physical training in type II (non-insulin-dependent) diabetes mellitus

Seven middle-aged men with manifest type II diabetes mellitus underwent an endurance training programme for 10-15 weeks. The maximal aerobic capacity, as well as the endurance capacity, was improved by 10% (p less than 0.05). The intramuscular glycogen store increased by more than 80% (p less than 0.05) from 350 mumol/g dw (dry weight), and the activities of citrate synthase and 3-hydroxy-acyl-CoA dehydrogenase increased by more than 50% (p less than 0.05) and 30% (p less than 0.05). The activity of glycogen synthase was decreased by approximately 20% (p less than 0.05), whereas lactate dehydrogenase remained unchanged. Capillaries/fibre and fibre area increased by more than 50% (p less than 0.05) and 30% (p less than 0.05) leaving the area of supply constant. Training did not influence fasting blood lipids and glucose, glycosylated hemoglobin, oral glucose tolerance, and insulin response to an oral glucose load measured 72 hours post-exercise. It is concluded that patients with manifest type II diabetes, as normoglycaemic individuals, adapt to physical training. However, no persistent effect on glucohomeostasis and lipaemia is produced by short-term training in the diabetic patients.     

Respiratory modulation of shivering intensity in the pigeon

Respiration and shivering were measured in unanaesthetized, cold-exposed pigeons using pneumotachography and electromyography, respectively. The instantaneous intensity of shivering in the pectoral muscle varied in phase with respiration. Power spectral analysis showed that the main frequency components of respiration and demodulated EMG coincided exactly. The intensity of shivering was highest during end-expiration and lowest at end-inspiration. This was confirmed by cross-correlation analysis of respiration and demodulated EMG. The absolute level of modulation remained constant (c. 10 microV peak-to-peak) despite changes in the general intensity of shivering. On the other hand, the relative depth of modulation was highest during incipient shivering. These facts indicate that only a part of the motor units recruited for shivering is susceptible to respiratory modulation and that this part is first recruited during incipient shivering. Inhalation of 5% CO2 did not affect the interaction between respiration and shivering although respiration frequency varied from 25 to 60 min-1. Thus, pulmonary chemoreceptors do not mediate this effect. It is suggested that the interaction between respiration and shivering occurs directly in the CNS. The question whether the interaction is adaptive for the animal or merely reflects a common evolutionary history of the underlying neural circuits is discussed.