[Evaluation of latex agglutination test for diagnosis of leptospirosis using native strains]

Leptospirosis is a common zoonosis throughout the world and common in the flat area of Guilan, Iran, with seasonal incidence, especially in rice farmers. Clinical diagnosis of leptospirosis is difficult, because its symptoms are similar to several acute infective diseases. Serological assays are important in diagnosis of the disease and microscopic agglutination test [MAT] is a gold standard, however, it is not a routine test in diagnostic laboratories. Thus, a simple and reliable test is a necessity. In this study, we evaluated a latex agglutination test using native strains of leptospires. A number of 98 positive cases and 54 negative cases which were screened by MAT, along with 30 sera of other diseases as control samples, were examined by latex agglutination test, using an antigenic suspension [whole antigen], which was extracted from 4 common native strains. False positive and false negative rate were 15 and 12 consequently. Sensivity, specificity, positive predictive value, negative predictive value, and accuracy were 89.0%, 84.5%, 86.7%, 87.2%, and 87.0% respectively. Regarding the considerable rate of sensivity and specificity of the test which is compatible to other performed studies, in addition to the simple performance test, does not need a complex laboratory facility, which may also be carried out in rural regions, therefore, this test is valuable for primary screening

[Seroprevalence of Chlamydia pneumonia in patients with myocardial infarction]

Several predisposing and risk factors are introduced as main causes of coronary atherosclerosis which is the main cause of myocardial infarction [MI]. In recent years, chronic and persistent infections are considered as such factors. This study is basically on determination of seropositivity to Chlamydia pneumonia to reveal the role of acute and chronic infections due to these bacteria as a risk factor for MI. 273 serum samples from MI patients and 109 samples from control group were examined by using commercial quantitative ELISA kits to measure specific anti Chlamydia pneumonia antibodies [IgM and IgG]. Two groups were matched for age and sex. IgM titers [ELISA] were negative in all patients and control cases, indicating lack of acute Chlamydial infection, but IgG titers were positive in 273 patients [94.4%], [mean average titer: 108 RU/ml] and in 109 control cases [84.4%] [mean average of titer: 61.9 RU/ml]. We found no significant relationship between seropositivity to Chlamydia pneumonia antibodies [lgG] with MI [P=0. 104]. In this study, no significant relationship was observed between serpositivity to Chlamydia pneumonia and subsequent incidence of MI. It seems that a large scale serological study with a diagnostic molecular methods for detection of genome in biopsy tissue of atherosclerotic coronary artery will be more helpful to reveal the expected relationship

[Evaluation an in-house IgM-ELISA for the diagnosis of human leptospirosis]

Leptospirosisis a very common zoonosis in the world. Culture is low sensitive with high rate false negative. So, serological assays are best alternative way for its diagnosis. Microscopic agglutination test [MAT] is gold standard but performing it requires a panel of some standard strains and need periodic subculturing of them, and also requires double sera with at least two weeks interval to investigate seroconversion. Furthermore, other serological methods should be investigated. The aim of this study was to evaluate an in-house IgM-ELISA developed by using antigen extracted from endemic isolates. 14 endemic isolates belonged to the serogroups: Icterohaemorrahgia, Pomona, Hardjo, and Gripotyphosa, were inoculated in EMJH to take well grown cultures. Whole antigen was extracted from each culture by Freezing-Thawing method in distilled water. Same amount of extraction of each culture with same OD number in 550nm were mixed together and were used for coating Elisa plates. Antihuman IgM conjugated with alkaline phosphatase were used in this assay. We used a commercial quantitative IgM-ELISA [SERION ELISA classic] for cut off determination. MAT was used for confirmation positive and negative cases. Sera with titer >/= 1:100 in MAT and positive criteria in commercial quantitative IgM-ELISA were considered as positive cases. 98 positive cases and 54 negative cases were chosen by screening 200 sera of patients suspected to leptospirosis by using MAT and commercial quantitative IgM-ELISA. We also used 30 sera of patients affected by hepatitis B, salmonelosis, and brucellosis as control cases. 88 of 98 positive cases were positive [false negative=10], 1 of 54 negative and all control case were negative [false positive =1] in the test. Sensitivity, specificity, PPV, NPV, and accuracy of the test were evaluated :99.0%, 89.1%, 90.75, 98.8%, and 94.25, respectively. ELISA for measuring specific IgM to leptospires antigen[s] could be a good alternative to MAT, which is not a routine diagnostic assay to perform in clinical diagnostic laboratories and only is reliable when there is paired sera. Sensitivity and specificity of the assay is dependent to several factors, especially to the type of antigen coated on plates, quality of assay materials, and also to the time of sampling. Sera of days >/= 6 of the disease has enough antibodies to measure and a common antigen extracted from several common pathogenic leptospires, especially from endemic isolates, could be more helpful to increase accuracy of the assay

Transfection of 6-23 a rat thyroid carcinoma cell line, with the neuropeptide Y cDNA

Neuropeptide Y [NPY] is a 36 amino acid peptide found throughout the central and peripheral nervous system of rat and human. NPY has been proposed to play an important role in satiety. The aim of this study was to produce cell lines that secrete high levels of bioactive NPY. For this purpose, the complementary DNA [cDNA] that encodes NPY was isolated by PCR. The cDNA was then cloned into pCEP4, to form pCEP4NPY. 6-23 cells were transfected with pCEP4NPY by electroporation. Transfected cells were selected by the addition of hygromycin B to the culture medium. Resistant colonies were picked and transferred to 96-well plates. The medium was tested for IR-NPY using a specific NPY radioimmunoassay [RIA]. The IR-NPY secreted by the cells was characterized by sephadex G50 chromatography and reversed phase fast protein liquid chromatography [FPLC]. It was found to co-elute with the synthetic standard in both cases. RNA was extracted from the cells and subjected to Northern blot analysis using labeled NPY cDNA as a probe. The cells were found to express high levels of NPY at mRNA levels

Effects of glucosamine on pancreatic glucokinase and hexokinase activities and their relationship to insulin secretion from isolated islets of normal and type 2 diabetic rat

Glucokinase serves as a glucose sensor in pancreatic beta-cells and plays a key role in glucose homeostasis and glucose-stimulated insulin secretion [GSIS]. In the present study we examined the effect of glucosamine, a glucokinase inhibitor, on the pancreatic glucokinase and hexokinase activities and on insulin secretion from freshly rat pancreatic islets of Langerhans. Insulin concentration was measured by rat insulin ELISA kit. The pancreatic islets from normal and type 2 diabetic [nSTZ] rats were isolated by collagenase digestion method. Glucose phosphorylation was quantitated by measuring the rate of glucose-6-phosphate formation in the fluorometric assay. Insulin secretion from hand-picked islets was evaluated in static incubation system. Insulin concentration was measured by rat insulin ELISA kit. Our findings demonstrate that glucosamine in a dose dependent manner, reduced glucokinase activity in islet extract, but had no effect on hexokinase activity. The glucose-stimulated insulin secretion was inhibited by glucosamine but it had no effect on the basal insulin secretion. In diabetic rats glucokinase was decreased while the basal insulin secretion and the activity of hexokinase were higher than normals. Based on results obtained from the present study, the assumption could be made that the decrease in the activity of glucokinase of pancreatic islets could be related to the impaired glucose stimulated insulin secretion. The increase in basal insulin secretion of diabetic rats may be due to an increase in pancreatic hexokinase activity