A hospital-based study on knowledge, attitude and practice of pregnant women on gender preference, prenatal sex determination and female feticide.

India has witnessed a decline in sex ratio in the past few decades. A hospital-based cross-sectional study was carried out to find out the attitude toward gender preference and knowledge as well as practice toward prenatal sex determination and female feticide among pregnant women. A majority (66.0%) of the pregnant women did not show any gender preference, followed by male preference (22.2%) and female preference (11.8%). A high proportion, i.e. 84.7% and 89.7%, of the total subjects were aware that prenatal sex determination and female feticide is illegal, respectively.

Growth promotion of Mycobacterium avium-M. intracellulare complex by enterobacterial acetate.

A mycobacterial growth factor was present in the conditioned media of Escherichia coli and Enterobacter cloacae cultures, but not in Pseudomonas aeruginosa culture. This factor potentiated the growth of Mycobacterium avium-M. intracellulare complex (MAC), a patient isolate, and well-established strains of M. avium and M. intracellulare. The growth factor was not a polypeptide; it was heat-stable and possessed a molecular weight < 500 D. Acetate production by enterobacteria was responsible for the biological activities observed. Acetate promoted mycobacterial growth at concentrations up to 3 mM; higher levels were toxic. The effects of acetate on MAC growth were not influenced by the pH of the media. Our data suggest that production of acetate by enterobacteria may regulate mycobacterial growth, and therefore, intestinal acetate might be a cofactor in the pathogenicity of MAC.

Correlation between antibodies to mannophosphoinositides & detection of antigen in circulating immune complexes in patients with pulmonary tuberculosis.

Enzyme linked immunosorbent assays were used to detect the concentration of mannophosphoinositides in circulating immune complexes (CICs) and antibodies to these antigens in the sera of patients with pulmonary tuberculosis. Serum samples from 235 tuberculosis patients at various levels of disease progression were analysed. Multiple regression analysis showed a direct correlation between antigen levels in CICs and serum antibodies to mannophosphoinositides.

Protective efficacy of different cell-wall fractions of Mycobacterium tuberculosis.

Immunization with various cell-wall fractions of M. tuberculosis H37Ra, progressively depleted of lipids (cell-wall-insoluble fraction; CWIF), soluble proteins (cell-wall core; CWC), mycolic acids and arabinogalactans (cell-wall-protein-peptidoglycan complex; CW-PPC) elicited significant levels of both humoral and cell-mediated immune response. Mice immunized with these fractions, when challenged with an LD50 dose of M. tuberculosis H37Rv, exhibited significant protection as revealed by high survival rates and decreased bacterial load in lungs, liver and spleen, as compared to nonimmunized animals.

Lack of functional correlation between gamma-glutamyl transpeptidase and amino acid transport in the lactating mouse mammary gland.

Gamma-glutamyl transpeptidase (EC 2.3.2.2) in lactating mouse mammary gland was inhibited by affinity labelling of the tissue with 6-diazo-5-oxo-L-norleucine. Amino acid (L-alanine, L-methionine) uptake by the affinity labelled mammary gland tissue and the control tissue was measured in vitro. Uptake of amino acids by the affinity-labelled tissue was comparable to that of control tissue. These findings suggest that gamma-glutamyl cycle is not involved in amino acid uptake by the mammary gland.

Use of polymerase chain reaction in analysing recombinant cDNA clones encoding storage proteins of chickpea Cicer arietinum L.

A simple and reliable method was undertaken for the use of polymerase chain reaction in analyzing cDNA clones. Amplification was done of the inserts from positive legumin clones isolated from a cDNA library constructed from developing chickpea cotyledons in the expression vector, gt11. Amplification was made simple by using oligonucleotide primers which allowed convenient sizing, subcloning and sequencing of inserts by di-deoxy chain termination method. This simple method may provide opportunity to isolate large number of agronomically important genes from gene libraries.

Fetal radiation exposure and moyamoya disease.

Moyamoya disease is a progressive occlusive disease of the cerebral vasculature with particular involvement of the circle of Willis. We present the first reported patient with moyamoya disease possibly due to radiation exposure during fetal development. A 10-year-old male, whose mother had undergone radiotherapy when pregnant, presented with moyamoya disease. The relevant literature is discussed.

Susceptibility to severe Streptococcal sepsis: use of a large set of isogenic mouse lines to study genetic and environmental factors.

Variation in responses to pathogens is influenced by exposure history, environment and the host's genetic status. We recently demonstrated that human leukocyte antigen class II allelic differences are a major determinant of the severity of invasive group A streptococcal (GAS) sepsis in humans. While in-depth controlled molecular studies on populations of genetically well-characterized humans are not feasible, it is now possible to exploit genetically diverse panels of recombinant inbred BXD mice to define genetic and environmental risk factors. Our goal in this study was to standardize the model and identify genetic and nongenetic covariates influencing invasive infection outcomes. Despite having common ancestors, the various BXD strains (n strains=33, n individuals=445) showed marked differences in survival. Mice from all strains developed bacteremia but exhibited considerable differences in disease severity, bacterial dissemination and mortality rates. Bacteremia and survival showed the expected negative correlation. Among nongenetic factors, age -- but not sex or weight -- was a significant predictor of survival (P=0.0005). To minimize nongenetic variability, we limited further analyses to mice aged 40-120 days and calculated a corrected relative survival index that reflects the number of days an animal survived post-infection normalized to all significant covariates. Genetic background (strain) was the most significant factor determining susceptibility (P< or =0.0001), thus underscoring the strong effect of host genetic variation in determining susceptibility to severe GAS sepsis. This model offers powerful unbiased forward genetics to map specific quantitative trait loci and networks of pathways modulating the severity of GAS sepsis.

Detection of mannophosphoinositide antigens in sputum of tuberculosis patients by dot enzyme immunoassay.

A simple and economical dot ELISA for the detection of mannophosphoinositide antigen in sputum samples of tuberculosis patients has been developed using affinity-purified antibodies. This test is able to detect free as well as bound antigen. Sputum samples from 94 patients suffering from tuberculosis and 30 non-tuberculosis patients were screened and an overall sensitivity and specificity of 89% and 93.3%, respectively, was obtained. The sensitivity of the test among the different groups of tuberculosis patients did not vary significantly.

Change in colony morphology influences the virulence as well as the biochemical properties of the Mycobacterium avium complex.

Factors that influence colony morphology are of crucial importance for drug development as well as for understanding the virulence of Mycobacterium avium complex (MAC) strains. The MAC 101 strain used in the present study grows as smooth transparent (SmT) colonies that tend to become opaque and pigmented when incubated for long periods of time. However, when MAC was passaged in animals, two types of colonies were recovered. The new rough transparent (RgT) colony morphology appeared more flat and transparent, having a central spot, irregular edges at times, and a dry, granular appearance like that of the rough mutants. In animal studies, the RgT bacilli multiplied at a much faster rate than that of the SmT bacilli, causing 60-80% mortality compared with the 10% mortality observed in mice infected with SmT. In vitro studies indicated that the SmT MAC did not grow and multiply as well in resident peritoneal macrophages as the RgT MAC did. The two morphotypes did not differ in their growth ratesin vitro but the RgT MAC failed to reduce dimethylthiazol-diphenyltetrazolium bromide (MTT), alamar blue and neutral red, suggesting that there might be significant changes in the cell wall or elsewhere causing changes in cellular permeability. These two morphotypes could serve as models for studying the biochemical markers or the identification of factors responsible for the virulence of the MAC.

Enhancement of antibacterial activity of clofazimine against Mycobacterium avium-Mycobacterium intracellulare complex infection induced by IFN-gamma is mediated by TNF-alpha.

The effects of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) alone or in combination with free or liposomal clofazimine against Mycobacterium avium-Mycobacterium intracellulare complex (MAC) were investigated. Treatment of murine resident peritoneal macrophages with 50 U/mL IFN-gamma (pre-infection) or 30 U/mL TNF-alpha (post-infection), caused significant reduction in intracellular MAC growth; this response was suppressed by anti-TNF-alpha antibodies or pentoxifylline. Activation of macrophages with IFN-gamma or TNF-alpha enhanced the intracellular activities of free and liposomal clofazimine against MAC; the individual antimycobacterial activities of free and liposomal clofazimine, however, were comparable. In the beige mouse model, IFN-gamma was ineffective, while liposomal clofazimine significantly decreased the infection in liver and spleen; the use of N(G)-monomethyl-L-arginine, a nitric oxide inhibitor, enhanced the effect of IFN-gamma against MAC infection. In addition, treatment of infected mice with either IFN-gamma or liposomal clofazimine significantly reduced the infection in peritoneal macrophages.

Therapeutic efficacy of liposomal clofazimine against Mycobacterium avium complex in mice depends on size of initial inoculum and duration of infection.

The therapeutic efficacy of liposomal clofazimine (L-CLF) against Mycobacterium avium complex (MAC) was evaluated in the acute and chronic infection models of the beige mouse (C57BL/6J bgj bgj). The maximum tolerated dose of L-CLF was inversely proportional to the infection level. L-CLF showed higher antibacterial activity than free clofazimine. Treatment with 25 mg of L-CLF per kg of body weight (intravenously) was started at days 1, 8, 15, and 22 postinfection and was studied at three levels of MAC infection (10(4), 10(5), and 10(6) bacilli/mouse). L-CLF treatment caused a significant (P < 0.05 to 0.001) reduction in the numbers of viable bacteria in lung, liver, and spleen at all infection levels, irrespective of time of treatment. However, the best results were obtained when an already established infection was treated (day 22). The organ-related differences in response to the treatment were also affected by the level of infection. A marked reduction in the numbers of CFU was observed in the lungs of mice with lower infection levels, whereas liver and spleen were treated more efficiently at higher infection levels. These studies might help in evaluations of host responses to therapy.

Mycobacterium avium-intracellulare complex activates nuclear transcription factor-kappaB in different cell types through reactive oxygen intermediates.

Mycobacterium avium-intracellulare complex (MAC) is one of the most common opportunistic pathogens in HIV-infected patients. Their synergistic interaction leads to a rapid deterioration of the host defense. In vivo, MAC manifests as a disseminated granulomatous disease that produces a massive inflammatory tissue response perhaps through its activation of inflammatory cytokines. The intracellular signaling following interaction of the mycobacterium with host cells is incompletely understood. Because the response is dependent, in part, on the activation of NF-kappaB, we investigated the effect of MAC on this nuclear transcription factor in cells of macrophage and nonmacrophage lineage. We demonstrate that both high and low virulence strains of MAC potently and rapidly activated NF-kappaB. In supershift assays, using specific Abs against the NF-kappaB subunits, we identified a p50/p65 heterodimer that was formed within 5 min after incubation with the bacterium too rapidly for cytokines to be involved in the activation. This activation was instead mediated through the generation of reactive oxygen intermediates, inasmuch as preincubation of cells with a variety of antioxidants inhibited NF-kappaB activation. Likewise, the transfection of cells with Mn-superoxide dismutase blocked the NF-kappaB activation induced by the bacterium. These data suggest that NF-kappaB activation is a consequence of interaction of host cells with the bacterium and that the interaction may play a pivotal role in the pathogenesis of the disease.

Inverse relation between disease severity and expression of the streptococcal cysteine protease, SpeB, among clonal M1T1 isolates recovered from invasive group A streptococcal infection cases.

The streptococcal cysteine protease (SpeB) is one of the major virulence factors produced by group A streptococci (GAS). In this study we investigated if differences exist in SpeB production by clonally related M1T1 clinical isolates derived from patients with invasive infections. Twenty-nine of these isolates were from nonsevere cases and 48 were from severe cases, including streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) cases. The expression and amount of the 28-kDa SpeB protein produced were determined by quantitative Western blotting, and protease activity was measured by a fluorescent enzymatic assay. A high degree of variation in SpeB expression was seen among the isolates, and this variation seemed to correlate with the severity and/or clinical manifestation of the invasive infection. The mean amount of 28-kDa SpeB protein and cysteine protease activity produced by isolates from nonsevere cases was significantly higher than that from STSS cases (P = 0.001). This difference was partly due to the fact that 41% of STSS isolates produced little or no SpeB compared to only 14% of isolates recovered in nonsevere cases. Moreover, the cysteine protease activity among those isolates that expressed SpeB was significantly lower for STSS isolates than for isolates from nonsevere cases (P = 0.001). Increased SpeB production was also inversely correlated with intact M protein expression, and inhibition of cysteine protease activity blocked the cleavage of the surface M protein. Together, the data support the existence of both an "on-off" and a posttranslational regulatory mechanism(s) controlling SpeB production, and they suggest that isolates with the speB gene in the "off" state are more likely to spare the surface M protein and to be isolated from cases of severe rather than nonsevere invasive infection. These findings may have important implications for the role of SpeB in host-pathogen interactions via regulation of the expression of GAS virulence genes and the severity of invasive disease.

Relative neutralizing activity in polyspecific IgM, IgA, and IgG preparations against group A streptococcal superantigens.

In this study we compared the ability of different immunoglobulin (Ig) preparations containing IgG, IgM, and/or IgA to neutralize the activity of streptococcal pyrogenic exotoxin A (SpeA) or culture supernatant from a clinical group A streptococcal isolate. All Ig preparations markedly inhibited the mitogenic and cytokine-inducing activity of SpeA and culture supernatant at concentrations of 0.05-0.5 mg/mL, and at 0.5 mg/mL, most caused 95-100% inhibition of both stimuli. A significantly higher (P< or =.05) inhibition of SpeA was achieved by Pentaglobin (IgG, IgM, and IgA) and IgAbulin (IgA and IgG), as compared with pure IgG preparations. IgM- and IgA-enriched preparations had significantly higher inhibitory activity against SpeA than against culture supernatant, whereas the reverse was true for the IgG preparations (P< or =.05). The data show that IgM and IgA are potent inhibitors of specific streptococcal superantigens. These findings may have implications for the optimization of immunotherapy in invasive streptococcal infections.

Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and nonsevere invasive diseases.

The relatedness of group A streptococcal (GAS) strains isolated from 35 Canadian patients with invasive disease of different severity was investigated by a variety of molecular methods. All patients were infected with M1T1 strains and, based on clinical criteria, were classified as severe (n = 21) and nonsevere (n = 14) invasive GAS infection cases. All the M1 strains studied had the emm1.0 allele and the same streptococcal pyrogenic exotoxin (Spe) genotype, speA(+) speB(+) speC speF(+) speG(+) speH smeZ(+) ssa. All isolates had the same speA allotype, speA2. The randomly amplified polymorphic DNA banding pattern with two different primers was identical for all strains, and pulsed field gel electrophoresis analysis showed that 33 and 30 isolates had identical banding patterns after DNA digestion with SfiI or SmaI, respectively; the nonidentical isolates differed from the main pattern by only one band. A relatively high degree of polymorphism in specific regions of the sic gene was observed among isolates; however, this polymorphism was not associated with disease severity. Likewise, although the phenotypic expression of SpeA, SpeB, and SpeF proteins varied among the M1T1 isolates, there was no correlation between the amount of Spe expressed and disease severity. Importantly, mitogenic and cytokine responses induced by partially purified bacterial culture supernatants containing a mixture of expressed superantigens were very similar for isolates from severe and nonsevere cases (P > 0.1). Together, the data indicate that highly related invasive M1T1 isolates, some indistinguishable, can cause disease of varying severity in different individuals. These findings underscore the contribution of host factors to the outcome of invasive GAS infections.

Reciprocal, temporal expression of SpeA and SpeB by invasive M1T1 group a streptococcal isolates in vivo.

The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including speA, speB, speF, speG, and smeZ. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of the speA-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that speA gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of the speA gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced speA gene expression and suppressed speB gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of streptococcal virulence factors in vivo. The model described here should facilitate such studies.