Molecular characterization of antigen B2 subunit in two genotypes of Echinococcus granulosus from Indian bubaline isolates, its stage specific expression and serological evaluation.

Echinococcus granulosus is a parasitic helminth which affects both man and animals. During infection with larval stage of the organism secretory and membrane-bound (S/M) proteins play a meaningful role for evasion of immune system. Antigen B (AgB) is one of them. Present investigation has defined sequence diversity of AgB2 subunit of cattle and buffalo isolates of the organism. A total of 55 isolates were screened by polymerase chain reaction based single stranded conformation polymorphism (PCR-SSCP). Subsequently, six conformers could be detected. Based on predicted amino acid sequences of 90 amino acid residues, three clusters could be deduced. Sequence information of two buffalo isolates was homologous to AgB4 indicating gene switching phenomenon in between closely related isoforms. Numerical value of Tajima's D test proved negative selection pressure. Using artificial neural network (ANN), B cell linear epitope and stretches of agretope were predicted. Three clusters could be defined on the basis of B cell linear epitope. Out of three clusters, two showed more than 50% binding propensity with same MHCII alleles whereas, cluster 3 exhibited binding propensity with other MHCII alleles (DRB1_1501, DRB1_1502). Relative expression of AgB2 was more in active cysts (1.636 ± 0.092) followed by degenerating (0.449 ± 0.037) and calcified (0.255 ± 0.008). This result suggested that relative expression of AgB2 declines with progression of the disease. Using recombinant AgB2 sensitivity, specificity and accuracy of the ELISA test was 96.7, 94.7 and 95.9%, respectively. No cross reactivity was found with common cestode and trematode infected cattle and buffalo because cross reactive antigen was expressed intracellularly. Finally, this was concluded that AgB2 is the suitable immunological marker for detection, diagnosis and progression of the disease.

Relative expression of the 14-3-3 gene in different morphotypes of cysts of Echinococcus granulosus isolated from the Indian buffalo.

The metacestode stage of Echinococcus granulosus is of zoonotic importance. In general, the 14-3-3 protein is involved in multiplication and survival of eukaryotes. Therefore, this communication presents succinct information on relative expression of the 14-3-3 protein in six different morphotypes of cysts of E. granulosus. All isolated E. granulosus belonged to the common sheep strain (G1). Relative expression of the 14-3-3 protein was higher in fertile cysts when compared to sterile cysts. The predicted amino acid sequence of the 14-3-3 protein was closely clustered with zeta-type isoforms 1 and 2 of the 14-3-3 protein. In addition, the present study demonstrates the presence of the 14-3-3 protein which until now had not been detected in the germinal layer. Our findings indicate that the expression of this biomolecule in the germinal layer of sterile organisms may contribute to the development and survival of the parasite in the host. The uniform expression of actin II conclusively proves the survivability of the harvested organisms.

Relative expression of antigen B coding gene of bubaline isolates of Echinococcus granulosus in fertile and sterile cysts.

This article communicates the relative quantification of five isoforms of antigen B (AgB) of Echinococcus granulosus. Relative expression of the AgB was quantified in active and inactive cysts. Cysts with germinal membrane, clear cyst fluid and protoscoleces showed uniform expression of the five isoforms and were utilized as control. Relative expression of AgB1 was the highest in cysts, where calcification has initiated. AgB2 and AgB4 were expressed more in fertile cysts irrespective of the condition of germinal membrane. The lowest expression of AgB3 was seen in calcified cysts. The relative expression of AgB5 could not be correlated with respect to the condition of the cyst because AgB5 is typically expressed by the adult stage of the parasite.

Mechanism of lead induced effects on human spermatozoa after occupational exposure.

OBJECTIVES: Occupational lead exposure caused several types of male reproductive impairments in different working populations. In the present study we examined the paint factory workers of active reproductive age and compared the data with the non-occupationally exposed desk job holders taken as control from Bangalore, India. MATERIALS AND METHODS: In the above perspective, sperm cell morphology, morphometery and motile activity were assessed. Routine seminal biochemistry, cell cycle phase analysis of sperm head DNA, estimation of serum reproductive hormones and metal levels in blood and semen were also taken into account. RESULT: Low sperm velocity, ATPase activity, gross and forward progressive motility with high stationary motile spermatozoa revealed lowering of cellular activity after lead exposure (p<0.001), which was supported by high seminal plasma fructose level (p<0.001). Lowering of seminal plasma total protein with concomitant rise in free amino acid level was prevalent as the exposure increased (p<0.001), suggesting disturbance in cellular nutritional support essential for cellular motility. Prolonged liquefaction time, reduced semen volume and viscosity as well as altered seminal plasma protein, fructose and cholesterol level among the workers indicated dysfunction of accessory sex glands viz. prostate and seminal vesicle after occupational lead exposure (p<0.001). Deterioration of sperm count, structural abnormality of spermatozoa and sperm head DNA hyploidy was also associated with high blood and semen lead levels in the paint factory workers (p<0.001) without interfering serum FSH, LH and testosterone level (non-significant at p<0.05). CONCLUSION: Therefore, the present study suggested that at the present exposure level lead might cross blood-testis-barrier and increased its value in semen of the occupationally exposed paint factory workers in Bangalore, India, thereby producing detrimental effects on semen quality and sperm characteristics.

Risk factors of acute respiratory infections in underfives of urban slum community.

To ascertain the risk factors of Acute Respiratory Infections (ARI) in children, a prospective study was conducted for a period of one year among 112 underfives in urban slum community of Calcutta. Incidence of ARI was found significantly higher in undernourished children of poor socio-economic class. Parental smoking habit and solid fuel use for cooking were recognised as important risk factors of ARI.

Prognostic significance of plasma markers of immune activation, HIV viral load and CD4 T-cell measurements.

OBJECTIVE: To evaluate the prognostic significance for AIDS occurrence of plasma levels of immune activation markers in comparison with and in conjunction with HIV viral load and CD4 T-cell measurements. DESIGN: A retrospective analysis was conducted of three plasma activation markers, the soluble tumor necrosis factor (TNF) receptor II (TNF-RII), neopterin and soluble interleukin-2 receptor levels, and of CD4 T-cell levels and plasma HIV viral load. SUBJECTS: The participants were 659 men taking part in the University of California Los Angeles Multicenter AIDS Cohort Study who were HIV-seropositive but AIDS-free in 1985. MAIN OUTCOME MEASURE: Clinically defined AIDS within 3 years. Failure time statistical regression models for the time to development of AIDS were used to assess prognostic capacity of the parameters alone and in combination. RESULTS: All the markers had prognostic capability. The levels of the three plasma activation markers correlated well with each other (median r = 0.61). They related less well with HIV RNA plasma levels (median r = 0.50) and least well with CD4 cell levels (median r = 0.36). Furthermore, plasma marker levels were shown to be able to stratify patients for prognosis within all the major categories of CD4 T-cell and HIV RNA levels. CONCLUSIONS: Plasma levels of soluble TNF-RII and other soluble markers of immune activation have prognostic capabilities which are different from HIV and CD4 T-cell levels. Combination of a single plasma activation marker measurement (such as soluble TNF-RII) with CD4 T-cell levels improved the prognostic capability of each. A new graphic technique for presenting prognostic capability indicated that plasma soluble TNF-RII and CD4 cell levels are better prognostic factors than HIV plasma level with CD4 cells < 200 x 10(6)/l. Inexpensive tests for one of the plasma activation markers, such as soluble TNF-RII or neopterin, can be useful for evaluations of HIV disease course, especially when expensive equipment, technical expertise and funding required for flow cytometry and for HIV load measurements are not readily available.

Smoke-induced inhalation injury: effects of retinoic acid and antisense oligodeoxynucleotide on stability and differentiated state of the mucociliary epithelium.

Rabbit tracheal explants, exposed to burning pine wood smoke, were cultured in a chemically defined medium with and without retinoic acid (+/- RA). Exposures of 15-20 minute led to RA-independent degeneration of the mucociliary epithelial sheath. In 10 minute exposures tissue integrity was retained, but epithelial morphology changed from normal pseudostratified columnar to the flattened appearance typical of the squamous phenotype. Despite the dramatic shift in morphology, explants exhibited normal RA-dependent mucin gene expression characteristic of the mucociliary phenotype. Furthermore, electron micrographs showed continued presence of both secretory granules and cilia. RA(+) cultures also showed a normal pattern of adherent epithelial cells. In RA(-) cultures, however, there were prominent intercellular spaces indicating an RA dependence for maintaining adhesive contacts following smoke exposure. An 18-mer mucin antisense oligomer that suppressed mucin gene expression also unexpectedly blocked the smoke induced metaplasia in RA(+) cultures, but the sense oligomer had no effect.

Sexual risk factors for cervical cancer among rural Indian women: a case-control study.

BACKGROUND: The association between sexual behaviour and cervical cancer is well established. Despite a high incidence of cervical cancer in India, its role has not been widely investigated in Indian women among whom the rate of sexual promiscuity is known to be very low. A hospital-based case-control study was carried out to investigate the role of sexual risk factors in cervical cancer among rural Indian women. METHODS: A case-control design was used in which a total of 268 subjects, comprising 134 women with invasive cervical cancer as cases and 134 control women were studied. A multiple logistic regression model was used to analyse the data. RESULTS: The risk factors found to be associated with cervical cancer were early age at first coitus, extramarital sex partners of women and the time interval since first exposure. In a multiple logistic regression model, independent effects were observed for early age at first coitus, showing maximum risk in women who reported their first intercourse at < 12 years of age, compared to that of women at > or = 18 years (odds ratio [OR] = 3.5. 95% confidence interval [CI]: 1.1-10.9). Increased risk was also seen for women who had extramarital sex relationships (OR = 5.5, 95% CI: 1.5-19.5). The significant effect of early age at first coitus persisted after adjustment for latency period which also showed its independent risk association with cervical cancer in the multivariate analysis. CONCLUSION: These findings confirm the association between early age at first coitus and cervical cancer in women with a low rate of sexual promiscuity and define the role of these risk factors in cervical carcinogenesis among rural Indian women.

PIP: The association between sex behavior and cervical cancer was investigated among rural Indian women known to have very low levels of promiscuity. 134 women with invasive cervical cancer were matched with 134 controls and analysis performed using a multiple logistic regression model. Risk factors associated with cervical cancer were early age at first coitus, extramarital sex partners of women, and the time interval since first exposure. Independent effects were observed for early age at first coitus, with maximum risk among women who reported their first intercourse at younger than age 12 years compared to that of women at age 18 years or older. Increased risk was also seen for women who had extramarital sex relationships. The significant effect of early age at first coitus persisted after adjustment for a latency period which also showed its independent risk association with cervical cancer in the multivariate analysis.

Differentiation of respiratory epithelium: the effects of retinoic acid and carcinogens on the expression of mucociliary vs. squamous phenotype.

Changes in ultrastructural characteristics and mucin gene expression were examined in rat tracheal explants cultured in a synthetic medium +/- retinoic acid (RA), benzo[a]pyrene (B[a]P) and N-methyl-N-nitrosourea (NMNU). In the RA(+) cultures, no changes in either ultrastructural features or mucin gene expression were detected after 48 h incubation. After 96 h incubation, however, the ultrastructural features associated with the squamous phenotype were characteristics of cultures containing the two carcinogens and the mucin gene expression was slightly reduced. Thus, in the presence of retinoic acid, the carcinogen induced changes in cytology to the squamous phenotypes were not matched by a marked loss of mucin gene expression. Explants cultured for 48 h without RA and +/- carcinogens showed none of the cytological changes associated with onset of the squamous phenotype. While mucin mRNA was still detected, it was clearly reduced compared to 48 h cultures in RA(+) medium. However, 48 h later, all explants exhibited pronounced squamous metaplasia and the mucin message decreased to trace levels. Thus, the results of these experiments with B[a]P and NMNU in RA(+) and RA(-) media indicates that at least the early carcinogen induced changes may be distinct from those associated with the retinoid pathway controlling expression of the mucin component of the mucociliary epithelium.

Comparative evaluation of somatic & excretory-secretory antigens of Entamoeba histolytica in serodiagnosis of human amoebiasis by ELISA.

The excretory-secretory antigens collected from the axenic culture medium and conventional somatic antigen prepared from the whole trophozoites of E. histolytica were compared for their efficacy in serodiagnosis of amoebiasis. A total of 280 sera collected from different clinically proven cases of amoebiasis and healthy subjects were analysed against both the antigens in ELISA. Both antigens showed a 100 per cent correlation in amoebic liver abscess cases, patients infected with enteropathogens other than amoeba and healthy subjects. However, excretory-secretory antigens showed slightly higher detection rate in patients suffering from acute amoebic dysentery and asymptomatic cyst passers groups. These results clearly suggested the use of excretory-secretory antigens by ELISA for serodiagnosis of amoebiasis due to its better or equal sensitivity, specificity and easier preparation compare to conventional antigen. The use of excretory-secretory antigen in serodiagnosis will not only help in performing more tests utilizing the same chemicals, but also save the cost, time and troubles for importing the foreign chemicals required for cultivation of E. histolytica.

Retinoic acid-regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal-epithelial cells in culture.

Rabbit tracheal epithelial cells were cultured in a serum-free and hormone-supplemented medium with and without retinoic acid. The cells showed time-dependent mucin gene expression when cultured in the medium with retinoic acid. In the absence of retinoic acid, however, mucin mRNA was barely detectable in the cells. When retinoic acid was added back to the medium, the mucin message was prominent again. Actinomycin D and cycloheximide did not inhibit mucin gene expression. The mucin message was slightly elevated by cAMP agonists. A mucin antisense oligomer inhibited the retinoic acid-induced mucin mRNA expression and secretion, thus offering an alternate approach in the management of mucus hypersecretion in upper airway respiratory diseases such as chronic bronchitis, asthma, and cystic fibrosis.

Retinoic acid modulation of mucin mRNA in rat tracheal explants: response to actinomycin D, cycloheximide, signal transduction effectors and antisense oligodeoxynucleotide.

Factors affecting the retinoic acid modulated expression of mucin mRNA in rat tracheal cultures were studied. Actinomycin D had no effect on mucin mRNA in cultures grown with retinoic acid (RA+). The usual precipitous drop in mucin mRNA in cultures lacking retinoic acid (RA-) was prevented by actinomycin D. Cycloheximide also had no effect on mucin mRNA in RA+ cultures, but, like actinomycin D, it prevented the precipitous drop in mucin mRNA in RA- cultures. cAMP agonists had some marginal effects on the mucin mRNA, but none as dramatic as those noted by actinomycin D and cycloheximide in the RA- cultures. An antisense oligomer (18 bases) to rat mucin cDNA inhibited the mucin mRNA expression in RA+ cultures.

In vitro effects of drugs on production of mucins in rabbit tracheal epithelial cells expressing mucin gene: a model system for studying upper airway respiratory diseases.

Rabbit tracheal epithelial cells, cultured on collagen-coated dishes in serum-free and hormone-supplemented medium, were found to incorporate [3H]glucosamine into high-molecular-weight components that were secreted in the medium. The chemical analysis of the secreted products resulted in a profile that resembled that of mucous glycoproteins (mucins). When examined by dot blot analysis, the total RNA isolated from these cells hybridized to an antisense 30-mer oligonucleotide corresponding to a rat intestine mucin peptide sequence, indicating that mucin gene was expressed in these cell lines. Lung and liver tissues of rabbit did not express this gene. Transmission electron microscopy exhibited secretory granules in these cells. The incorporation of [3H]glucosamine into mucins was inhibited by three aryl-N-acetyl-galactosaminides and a chemical carcinogen, N-nitroso-N-ethyl urea, whereas 5-azacytidine enhanced the proliferation of cells as well as the radiolabeling of mucins. Parasympathetic agent (pilocarpine), cholinergic antagonist (atropine), and beta-adrenergic agonist (isoproterenol) alone have little effect on the secretion of mucins. The cholinergic agonist, methacholine, was found to increase the production of mucins and addition of atropine to the medium before methacholine blocked this stimulation. Histamine was found to stimulate mucin production in these cells.

Neutral and acidic human tracheobronchial mucin. Isolation and characterization of core protein.

Human bronchial mucin from a patient suffering from chronic bronchitis was solubilized in aqueous solution containing sodium azide and protease inhibitors and purified by Sepharose 4B and 2B column chromatography. The mucin was further purified by cesium bromide density gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis of this material showed high-molecular-weight mucin component(s) at the top of the gel. Chemical analysis of this preparation indicated a typical mucin profile of amino acids and carbohydrates. Ion-exchange chromatography resulted in resolution of the purified mucin into neutral and acidic fractions. Comparison of the chemical composition of these two fractions showed higher mole percentage of threonine, serine, sialic acid, and sulfate in the acidic fraction. Chemical deglycosylation of the purified mucin preparation with trifluoromethane sulfonic acid was carried out at 20 degrees C for 3 1/2 h. Sialic acid, fucose, galactose, and N-acetylglucosamine were completely removed, whereas traces of N-acetylgalactosamine were still detected. High-pressure liquid chromatography of the deglycosylated products from native, neutral, and acidic mucin preparations resulted in a principal peptide, P1, with identical amino acid composition. Cyanogen bromide (CNBr) treatment of the peptide P1 from neutral and acidic mucins and subsequent fractionation of the fragments by high-pressure liquid chromatography resulted in similar peptide profiles. The P1 peptide fraction was further subjected to high-pressure liquid chromatography in a second solvent system, which resulted in two peaks, P1a and P1b. Gel filtration of both peptides in 6 M guanidine hydrochloride indicated a single peak with molecular weight of approximately 97 kDa. The amino acid profile of the two peptides was dominated by high levels of threonine, serine, and proline, which combined accounted for nearly 39% of the total residues, and in most respects, the profile resembled that of native mucin. End-group analysis of the peptide P1a indicated a blocked N-terminus, whereas serine was found to be the N-terminal amino acid in the peptide P1b. Rabbit antibodies prepared against the peptide P1 from native tracheal mucin reacted strongly with neutral and acidic mucin as well as the mucin from human colon. Both neutral and acidic human tracheal mucins were immunologically reactive with mouse monoclonal antibody HMPFG-2, which was prepared against human mammary mucin. However, the response of this antibody to human colonic mucin was rather weak.

Deglycosylation of neutral and acidic human colonic mucin.

Human colonic mucin has been isolated from normal colonic mucosa by a phenol-water extraction procedure and purified by Sepharose 2B column chromatography. The mucin was further purified by cesium bromide density gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel (5%) electrophoresis of this material showed high-molecular-weight mucin component(s) at the top of the gel. Chemical analyses of this preparation indicated a typical mucin profile of amino acids and carbohydrates. Ion-exchange chromatography resulted in the separation of two major fractions, one being more acidic than the other. Chemical deglycosylation of the purified preparation at 20 degrees C for 3 1/2 showed loss of sialic acid, fucose, galactose, and N-acetylglucosamine, whereas traces of N-acetylgalactosamine were still detected. High-pressure liquid chromatography of the deglycosylated material resulted in the purification of a major peptide, P1, with high levels of threonine, serine, and proline, resembling, in most respects, the profile of native mucin. The molecular weight of the peptide was determined to be approximately 97 kDa and serine was the single NH2 terminus.

Gas-liquid chromatographic determination of mono- and diiodotyrosines in serum.

A sensitivie, reliable gas-chromatographic assay for monoiodotyrosine and diiodotyrosine in human serum is reported. The oxazolidinone-heptafluorobutyric anhydride derivatives allow the quantitation of both compounds in the linear range of 0.2 to 7.6 mg/L of serum. Analytical recovery averaged 88%, and mean accuracy and within-run precision were 98 and 2%, respectively. Concentrations of monoiodotyrosine in serum as low as 20 microgram/L and of diiodotyrosine as low as 100 microgram/L can be detected. Normal serum contains no detectable concentration of either compound, but the method is applicable as a diagnostic tool in the early prediction of thyroid disease. Both compounds were detected in the serum of a hypothyroid subject whose normal thyroid hormone concentrations were being maintained by therapy with desiccated thyroid extract.