Molecular cloning, expression and purification of protein TB10.4 secreted by Mycobacterium tuberculosis

Tuberculosis [TB] is the leading cause of mortality among the infectious diseases, especially in developing countries. One of the main goals in tuberculosis research is to identify antigens which have the ability of inducing cellular and/or humoral immunity in order to use them in diagnostic reagents or vaccine design. The aim of this study was to clone and express the TB10.4 protein in Escherichia coli expression system. DNA was extracted from Mycobacerium tuberculosis H37Rv. Gene specific primers were designed using Gene Runner software according to sanger sequence database. Gene tb 10.4 fragment was amplified by PCR method and purified tb 10.4 gene was cloned into pET 102/D vector. Plasmid containing pET 102/D-10.4 was transformed into competence E. coli TOP10. A positive transformant was chosen and plasmids DNA was isolated and subsequently transformed into competence E. coli BL21 [DE3]. The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. Purified recombinant protein was achieved using metal affinity chromatography [Ni-nitrilotriacetic acid]. TB10.4 molecule was successfully cloned, expressed, and purified. An approximately 26.4 kDa exogenous protein was observed on the SDS-PAGE. The recombinant protein was confirmed by DNA sequencing of correct insert. The success of expressing the TB10.4 protein could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of submit vaccine and diagnostic method

nasal smear for eosinophils, its value, and its relation to nasal mucosal eosinophilia in allergic rhinitis

There is no single test as a gold standard for the diagnosis of allergic rhinitis [AR]. This study was to assess the usefulness and validity of nasal smear as a quick, easy and inexpensive diagnostic method for diagnosis of allergic rhinitis. This study was conducted in a university hospital setting. Nasal smears were taken from 39 patients with a clinical history of nasal allergy and a positive skin prick test to at least one aeroallergen as well as 26 controls without any history and negative test. Biopsy specimens from the inferior turbinate as well as nasal smears of 19 cases including 9 patients and 10 controls with the same criteria were taken. Nasal smears and biopsy slides were stained with Giemsa and Hematoxilin-Eosin and were examined blindly by two separate pathologists. Fifty one percents of the patients and 11.5% of the controls showed eosinophilia in their nasal smear [>/=10% eosinophils, P=0.001]. The sensitivity of nasal eosinophil count as a diagnostic test for AR was 51.3% with a specificity of 88.5%, a positive predictive value of 87% and a negative predictive value of 54%. Eosinophilia in nasal biopsies was found in 44% and 30% of allergic patients and controls respectively. There was no significant correlation between symptoms or positive skin tests with either smear eosinophilia or tissue eosinophilia. Evaluation of eosinophils in nasal smear is an insensitive but fairly specific test for the diagnosis of allergic rhinitis. It seems that the nasal secretions and nasal tissue represent two distinct cellular compartments

Metabolic factors associated with urinary calculi in children

We aimed to identify metabolic and anatomical abnormalities present in children with urinary calculi. Metabolic evaluation was done in 142 pediatric calculus formers. Evaluation included serum biochemistry; measurement of daily excretion of urinary calcium, uric acid, oxalate, citrate, and magnesium [in older children]; and measurement of calcium, uric acid, oxalate, and creatinine in random urine samples in nontoilet-trained patients. Urinary tests for cystinuria were also performed. All of the patients underwent renal ultrasonography. Sixty-one patients [42.7%] had metabolic abnormalities. Anatomical abnormalities were found in 12 patients [8.4%]. Three children [2.1%] had infectious calculi, and 3[2.1%] had a combination of metabolic and anatomic abnormalities. In 66 children [46.2%] we did not find any reasons for calculus formation [idiopathic]. Urinalysis revealed hypercalciuria in 25 [17.6%], hyperuricosuria in 23 [16.1%], hyperoxaluria in 17 [11.9%], cystinuria in 9 [6.3%], hypocitraturia in 3 [2.1%], and low urinary magnesium level in 1 [0.7%] patients. Sixteen patients [11.2%] had mixed metabolic abnormalities. Metabolic abnormalities are common in pediatric patients with urinary calculi. In our study, calcium and uric acid abnormalities were the most common, and vesicoureteral reflux seemed to be the most common urological abnormality which led to urinary stasis and calculus formation

Correlation between serum 25 hydroxy vitamin D3 and laboratory risk markers of cardiovascular diseases in type 2 diabetic patients

To determine the association between vitamin D deficiency and cardiovascular risk markers among diabetic patients. This was a cross-sectional study conducted in Ghaem Hospital, Mashhad, Iran, from December 2007 to March 2008 in 119 type 2 diabetic patients. Coronary, cerebrovascular, and peripheral vascular diseases were confirmed. Blood biochemical parameters including laboratory risk markers of cardiovascular disease were determined. Serum 25 hydoxy [OH] D was measured during winter. The correlation between vitamin D deficiency and cardiovascular prevalence, and also laboratory variables was determined. The mean age of patients was 55.3 +/- 11.2 years. The mean 25[OH] D concentration was 32.4 +/- 21.6ng/ml. The prevalence of hypovitaminous D was 26.1% among the diabetic patients. The difference with the control group was not significant [p=0.12]. Overall, 36 [30.3%] patients were positive for coronary vascular disease [CVD]. The correlation between hypovitaminous D and CVD was not significant [p=0.11]. Patients with vitamin D deficiency had significant differences in body mass index [p=0.003], metabolic syndrome [p=0.05], high sensitive C-reactive protein [p=0.009], microalbuminuria [p=0.04], and glumerular filtration rate [p=0.02], compared to patients with sufficient vitamin D. The fasting blood sugar, glycosylated hemoglobin, lipid profiles, homocysteine, uric acid, and insulin resistance were not related to vitamin D deficiency. There is an association between hypovitaminous D and inflammatory markers that contributed to CVD, so vitamin D may be important in maintaining cardiovascular health

Optimization of Anti-Rh D immunoglobulin stability in the lyophilization processes

Anti-Rh D IgG is used for the prevention of anti-D antibody production in Rh- individuals who have been exposed to Rh+ red blood cells. The stability of IgG preparations as a solution is low, with a shelf life of a year or more. Formulation of anti-Rh D IgG as a lyophilized preparation would decrease its degradation rate and increases its shelf life. The objective of this study was to formulate the anti-Rh D as a lyophilized preparation using different formulations and optimize the lyophilization processes. The effect of various formulations on the stability of anti-Rh D was evaluated using accelerated stability test. In this method the amount of transmittance [T%] at 585 nm for the lyophilized preparations had inverse relationship with aggregation of anti-Rh D. To improve stability, the most stable formulation was selected and different concentrations of sucrose in the presence of sodium-potassium phosphate buffer 25 mM pH 7.5. Then, the bioactivity was determined, using the ELAT test and also, the amount of moisture measured in this formulation. Among different formulations, the one with anti-Rh D 5 mg/ml, tween 80 0.1%, glycine 0.15 M, manitol 7% and sucrose 60 mM in sodium-potassium phosphate buffer 25 mM pH 7.5 was the most stable formulation [P < 0.05]. The result of biological test of ELAT showed that bioactivity of more than 93% meets the requirement set by British Pharmacopoeia. The amount of moisture measured in this formulation was less than 3%. It was concluded that this formulation could be introduced as a candidate for the formulation of anti-Rh D in a lyophilized dosage form

Specific IgG antibodies [total and subclasses] against saffron pollen: a study of their correlation with specific IgE and immediate skin reactions

Saffron [Zaaferan], botanical name Crocus sativus, is the most expensive spice in the world. It is derived from the dried stigma and pistil of the purple saffron crocus flowers. Iran is the largest saffron producer accounting for more than 80% of the world's production. Saffron contains an aeroallergen that causes reactive respiratory allergic reactions in atopic subjects. IgG antibody to allergens in the serum of allergic patients is not routinely measured. In this study in order to find out more about mechanism of allergy against saffron pollen, specific antibodies [IgE and IgG, total and subclasses] in atopic subjects were assayed. We used an ELISA assay for measuring specific IgE and IgG against saffron pollen extract in the sera of 38 atopic subjects [test group] and 20 non allergic subjects [control group]. The optical densities were compared between allergic subjects and non-allergic individuals. The prick test with saffron pollen extract was used to evaluate the cutaneous and specific antibody responses in the allergic subjects. The correlation was determined by statistical analysis. Specific saffron pollen IgE and IgG subclasses were found significantly higher in the allergic subjects than the control group. The immediate skin reaction was found positive in 70% of the test group. We report here, the existence of a positive correlation between specific IgE and skin reaction by prick test in atopic subjects [R=0.433]. A negative correlation between specific IgE and IgG4 subclass was also found [R=-0.576]. These data may be useful to understand the mechanism of allergy to saffron and may help in clarifying clinical manifestations and to prevent IgE production as well as therapeutic application