The Vaginal Microbiota over an 8- to 10-Year Period in a Cohort of HIV-Infected and HIV-Uninfected Women

BackgroundWe identified predominant vaginal microbiota communities, changes over time, and how this varied by HIV status and other factors in a cohort of 64 women.MethodsBacterial DNA was extracted from reposited cervicovaginal lavage samples collected annually over an 8–10 year period from Chicago Women’s Interagency HIV Study participants: 22 HIV-negative, 22 HIV-positive with stable infection, 20 HIV-positive with progressive infection. The vaginal microbiota was defined by pyrosequencing of the V1/V2 region of the 16S rRNA gene. Scheduled visits included Bacterial vaginsosis (BV) screening; clinically detected cases were referred for treatment. Hierarchical clustering identified bacterial community state types (CST). Multinomial mixed effects modeling determined trends over time in CST, by HIV status and other factors.ResultsThe median follow-up time was 8.1 years (range 5.5–15.3). Six CSTs were identified. The mean relative abundance (RA) of Lactobacillus spp. by CST (with median number of bacterial taxa) was: CST-1–25.7% (10), CST-2–27.1% (11), CST-3–34.6% (9), CST-4–46.8% (9), CST-5–57.9% (4), CST-6–69.4% (2). The two CSTs representing the highest RA of Lactobacillus and lowest diversity increased with each additional year of follow-up (CST-5, adjusted odds ratio (aOR) = 1.62 [95% CI: 1.34–1.94]; CST-6, aOR = 1.57 [95 CI: 1.31–1.89]), while the two CSTs representing lowest RA of Lactobacillus and higher diversity decreased with each additional year (CST-1, aOR = 0.89 [95% CI: 0.80–1.00]; CST-2, aOR = 0.86 [95% CI: 0.75–0.99]). There was no association between HIV status and CST at baseline or over time. CSTs representing lower RA of Lactobacillus were associated with current cigarette smoking.ConclusionsThe vaginal microbial community significantly improved over time in this cohort of women with HIV and at high risk for HIV who had regular detection and treatment referral for BV.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.     

Normative Database of Retinal Oximetry in Asian Indian Eyes

Objective

To study the oxygen saturation profile in normal Asian Indian eyes.

Design

A cross sectional prospective study.

Subjects

Ninety eight consecutive patients presenting to our hospital with best corrected distance visual acuity (BCVA) of 20/20 and a normal ophthalmic examination were included in the study. Patients having any ocular or systemic disease were excluded from the study.

Materials and Methods

Oximetry was performed on all subjects with the Oxymap T1 retinal oximeter (Oxymap hf, Reykjavik, Iceland).

Main Outcome Measures

The images were analysed for oxygen saturation and diameter.

Results

The mean age was 33 years (Range: 18-63; SD: 12.4). The average arteriolar saturation was 90.3 ± 6.6% and the venous saturation was 56.9% ± 6.3. The average A-V (arterio-venous) difference was 33.2% ± 5.2. There was an increase in arteriolar (R² = 0.264; p=0.001) and venous saturation (R² = 0.151; p=0.001) with age. There was no significant change in the arterio-venous saturation difference (AVSD). The inferotemporal quadrant had the lowest saturations. Age correlated positively with ocular perfusion pressure (OPP)(R² = 0.07; p=0.007). OPP correlated positively with global arteriolar saturation (R²=0.057, p=0.018).

Conclusion

This study provides the normative database for an Indian population and is comparable to previous studies. Age, vessel diameter and OPP were the significant factors that influenced the saturation. Arteriolar and venous saturations increased with age while the AVSD did not change significantly.     

Determination of growth inhibitory action point of interferon gamma on WISH cells in cell cycle progression and the window of responsiveness of the cells to the interferon

We had earlier shown that human foetal epithelial cells (WISH), growth-inhibited by interferon gamma (IFNgamma), were reversibly detained at a point prior to DNA synthesis. In the present study, we determined the window of action of IFNgamma in the G1 phase duration and the exact point of detention of WISH cells in cell cycle progression with respect to the known points of detention by the inhibitors of DNA replication initiation (aphidicolin and carbonyl diphosphonate) and of activation of replication protein A (6-dimethylaminopurine), of which RPA activation being the earlier event compared to DNA replication initiation in cell cycle progression. WISH cells, which were released from IFNgamma-induced arrest, permeabilised and exposed independently to these inhibitors show that IFNgamma detains WISH cells prior to initiation of DNA synthesis. Further, exposure of IFNalpha-synchronized (at G0/G1) or mimosine-synchronized (at G1/S) WISH cells to IFNgamma, which was added at different time points post-release from the synchronizing agent, showed that the cells were promptly responsive to the growth inhibitory action of IFNgamma only during the first 11h in G1 phase. Taken together, these results suggest that IFNgamma inhibits growth of WISH cells by detaining them at a point prior to initiation of DNA synthesis and that the IFN acts within the first 11h in G1 phase of the cell cycle.     

Dietary Intakes and Health Risk of Toxic and Essential Heavy Metals through the Food Chain in Agricultural,Industrial, and Coal Mining Areas of Northern India

Minerals can enter the food chain through industrial and mining activities. Soil-to-vegetable transfer is higher than soil-to-cereal, but human consumption of metals is attributable to balanced diets containing both vegetables and cereals and drinking water. However, the impact of location on intakes of metals from predominantly cereal-based Indian diets is not clear. Hence, the present study was undertaken in selected Agricultural, Industrial, and Coal Mining Areas (AA, IA, CMA) around the Allahabad District in Northern India to compare transfer of toxic metals, Pb, Cd, Cr and essential metals, Fe, Zn, Cu, Co in soil and water to common crops: cereals (rice, wheat, maize) and vegetables (spinach, potato), and to assess Daily Intake of Metal (DIM) and consequent Health Risk Index (HRI). The overall content of all metals, except Cu, in water, soils, and crops followed the pattern CMA > IA > AA. Transfer factors (TFs) followed the sequence spinach > potato > cereals. Quantitatively, however, cereals contribute maximally to a balanced diet, so DIM and HRI were higher from cereals than vegetables. Even though spinach had the highest TFs, cereals contributed maximally to HRI. CMA had the highest metal content so locally grown cereals contributed significantly to intake of both toxic and essential metals.     

Integrin expression in developing human salivary glands

The development and complete differentiation of salivary glands is a complex process that involves a large number of co-ordinated events. Little is known about the molecular basis for salivary gland development. However, we have reported previously that integrins appear to play a role. Integrins are heterodimeric transmembrane receptors consisting of one α and one β subunit that play a pivotal role in the interaction of cells with the extracellular matrix. Such interactions regulate the organisation of cells of tissues and organs during development as well as cell proliferation and differentiation. Using immunohistochemistry and Western and Northern blot analysis, we mapped the localisation and expression of integrins β1, β3 and β4 in human salivary glands obtained from foetuses ranging from weeks 4–24 of gestation and compared it with adult salivary glands. Integrin β1 first appeared during the canalisation stage and during the differentiation stage. A message first appeared at week 6 of development. The expression of β4 integrin protein and message was observed only in the late stage of differentiation. Integrin β3 was not detected in the developing glands; however, integrins β1, β3 and β4 were all expressed in adult salivary gland tissues. The data suggest that integrins, particularly β1, have a role to play in salivary gland development and differentiation.     

Females with paired occurrence of cancers in the UADT and genital region have a higher frequency of either Glutathione S-transferase M1/T1 null genotype

Upper Aero digestive Tract (UADT) is the commonest site for the development of second cancer in females after primary cervical cancer. Glutathione S-transferase (GSTM1 and / or T1) null genotype modulates the risk of developing UADT cancer (primary as well as second cancer). The aim of this study was to evaluate the difference in GST null genotype frequencies in females with paired cancers in the UADT and genital region as compared to females with paired cancers in the UADT and non-genital region. Forty-nine females with a cancer in the UADT and another cancer (at all sites-genital and non-genital) were identified from a database of patients with multiple primary neoplasms and were analyzed for the GSTM1 and T1 genotype in addition to known factors such as age, tobacco habits, alcohol habits and family history of cancer. Frequencies of GSTM1 null, GSTT1 null, and either GSTM1/T1 null were higher in females with paired occurrence of cancer in the UADT and genital site (54%, 33% and 75% respectively) in comparison to females with paired occurrence of cancer in the UADT and non-genital sites (22%, 6% and 24% respectively). The significantly higher inherited frequency of either GSTM1/T1 null genotype in females with a paired occurrence of cancers in UADT and genital region (p = 0.01), suggests that these females are more susceptible to damage by carcinogens as compared to females who have UADT cancers in association with cancers at non-genital sites.     

Immune activation of type I IFNs by Listeria monocytogenes occurs independently of TLR4, TLR2, and receptor interacting protein 2 but involves TNFR-associated NF kappa B kinase-binding kinase 1

Type I IFNs are well established antiviral cytokines that have also been shown to be induced by bacteria. However, the signaling mechanisms regulating the activation of these cytokines during bacterial infections remain poorly defined. We show that although Gram-negative bacteria can activate the type I IFN pathway through TLR4, the intracellular Gram-positive bacterium Listeria monocytogenes (LM) can do so independently of TLR4 and TLR2. Furthermore, experiments using genetic mutants and chemical inhibitors suggest that LM-induced type I IFN activation occurs by an intracellular pathway involving the serine-threonine kinase TNFR-associated NF-kappaB kinase (TANK)-binding kinase 1 (TBK1). Interestingly, receptor-interacting protein 2, a component of the recently discovered nucleotide-binding oligomerization domain-dependent intracellular detection pathway, was not involved. Taken together, our data describe a novel signal transduction pathway involving TBK1 that is used by LM to activate type I IFNs. Additionally, we provide evidence that both the LM- and TLR-dependent pathways converge at TBK1 to activate type I IFNs, highlighting the central role of this molecule in modulating type I IFNs in host defense and disease.     

TIA proteins are necessary but not sufficient for the tissue-specific splicing of the myosin phosphatase targeting subunit 1

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.     

Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death

Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 anti-apoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins.