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1.  Asthma, Allergy, and Responses to Methyl Donor Supplements and Nutrients 
After a brief period of stabilization, recent data has shown that the prevalence of asthma and allergic diseases continue to increase. Atopic diseases are major public health problems resulting in significant disability and resource utilization globally. While environmental factors influence the development of atopic disease, dietary changes may partially explain the high burden of atopic disease. One mechanism by which diet is suspected to impact asthma and allergy susceptibility is through epigenetic mechanisms including DNA methylation. Dietary methyl donors are important in the one-carbon metabolism pathway that is essential for DNA methylation. Findings from both observational studies and interventional trials of dietary methyl donor supplementation on the development and treatment of asthma and allergy have produced mixed results. While issues related to the differences in study design partially explain the heterogeneous results, two other issues have been largely overlooked in these studies. Firstly, these nutrients affect one of many pathways and occur in many of the same foods. Secondly, it is now becoming clear that the human intestinal microbiome is involved in the metabolism and production of the B vitamins and other methyl donor nutrients. Future studies will need to account for both the interrelationships between these nutrients and the effects of the microbiome.
PMCID: PMC4004707  PMID: 24360248
Allergy; Asthma; Betaine; Choline; Vitamin B2; Vitamin B6; Vitamin B12; Folate; Methyl Donor; Microbiome
2.  The genomic origins of asthma 
Thorax  2014;69(5):481-487.
Lung function tracks from the earliest age that it can be reliably measured. Genome wide association studies (GWAS) suggest that most variants identified for common complex traits are both regulatory in function and active during fetal development. Fetal programming of gene expression during development is critical to the formation of a normal lung. An understanding of how fetal developmental genes related to diseases of the lungs and airways is a critical area for research. This review article will consider the developmental origins hypothesis, the stages of normal lung development and a variety of environmental exposures that might influence the developmental process: in utero cigarette smoke exposure, vitamin D and Folate. We conclude with some information on developmental genes and asthma.
PMCID: PMC4343201  PMID: 24668408
Genomics; Asthma; Fetal Lung Development
3.  Congenital Malformations among Babies Born Following Letrozole or Clomiphene for Infertility Treatment 
PLoS ONE  2014;9(10):e108219.
Clomiphene citrate (CC) is the first line drug for ovulation induction but because of its peripheral antiestrogenic effect, letrozole was introduced as the 2nd line drug. It lacks the peripheral antiestrogenic effect and is associated with similar or even higher pregnancy rates. Since letrozole is a drug for breast cancer, its use for the purpose of ovulation induction became controversial in the light of studies indicating an increased incidence of congenital malformations.
To evaluate and compare the incidence of congenital malformations among offsprings of infertile couples who conceived naturally or with clomiphene citrate or letrozole treatment.
Settings and Design
A retrospective cohort study done at a tertiary infertility centre.
Methods and Material
A total of 623 children born to infertile women who conceived naturally or following clomiphene citrate or letrozole treatment were included in this study. Subjects were sorted out from medical files of both mother and newborn and follow up study was done based on the information provided by parents through telephonic conversations. Babies with suspected anomaly were called and examined by specialists for the presence of major and minor congenital malformations. Other outcomes like multiple pregnancy rate and birth weight were also studied.
Overall, congenital malformations, chromosomal abnormalities were found in 5 out of 171 (2.9%) babies in natural conception group and 5 out of 201 babies in the letrozole group (2.5%) and in 10 of 251 babies in the CC group (3.9%).
There was no significant difference in the overall rate of congenital malformations among children born to mothers who conceived naturally or after letrozole or CC treatment.
Key Messages
Congenital malformations have been found to be comparable following natural conception, letrozole and clomiphene citrate. Thus, the undue fear against letrozole may be uncalled for.
PMCID: PMC4182712  PMID: 25272289
4.  Spatial memory is enhanced in long-living Ames dwarf mice and maintained following kainic acid induced neurodegeneration 
Age associated cognitive impairment is associated with low levels of IGF-1, oxidative stress, and neuronal loss in the hippocampus. Ames dwarf mice are long-lived animals that exhibit peripheral IGF-1 deficiency. Hippocampal-based spatial memory (a homolog of cognitive function) has not been evaluated in these long-living mice.
Materials and methods
We evaluated the hippocampal-based spatial memory in 3-, 12- and 24-month-old Ames dwarf and wild type mice using the Barnes maze and the T-maze. We also examined the effect of a hippocampal-specific toxin, kainic acid (KA), on spatial memory to determine whether Ames mice were resistant to the cognitive impairment induced by this compound.
We found that Ames dwarf mice exhibit enhanced learning, making fewer errors and using less time to solve both the Barnes and T-mazes. Dwarf mice also have significantly better short-term memory as compared to wild type mice. Both genotypes exhibited neuronal loss in the CA1 and CA3 areas of the hippocampus following KA, but Ames dwarf mice retained their spatial memory.
Our results show that Ames dwarf mice retained their spatial memory despite neurodegeneration when compared to wild type mice at an “equiseizure” dose of KA.
PMCID: PMC4104773  PMID: 20561541
Ames dwarf; Hippocampus; Spatial memory; Barnes maze; Kainic acid
5.  Disulfiram Suppresses Growth of the Malignant Pleural Mesothelioma Cells in Part by Inducing Apoptosis 
PLoS ONE  2014;9(4):e93711.
Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and β proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.
PMCID: PMC3972204  PMID: 24690739
7.  Targeted proteasome inhibition by Velcade induces apoptosis in human mesothelioma and breast cancer cell lines 
Cancer chemotherapy and pharmacology  2009;66(3):10.1007/s00280-009-1181-8.
Thoracic malignancies and human breast cancer (HBC) continue to be aggressive solid tumors that are poor responders to the existing conventional standard chemotherapeutic approaches. Malignant pleural mesothelioma (MPM) is an asbestos-related tumor of the thoracic pleura that lacks effective treatment options. Altered ubiquitin proteasome pathway is frequently encountered in many malignancies including HBC and MPM and thus serves as an important target for therapeutic intervention strategies. Although proteasome inhibitor Velcade (Bort-ezomib) has been under clinical investigation for a number of cancers, limited preclinical studies with this agent have thus far been conducted in HBC and MPM malignancies.
To study the biological and molecular responses of MPM and HBC cells to Velcade treatments, and to identify mechanisms involved in transducing growth inhibitory effects of this agent.
Flow-cytometric analyses coupled with western immunoblotting and gene-array methodologies were utilized to determine mechanisms of Velcade-dependent growth suppression of five MPM (H2595, H2373, H2452, H2461, and H2714) and two breast cancer (MDA MB-468, SKBR-3) cell lines.
Our data revealed significant reduction in cell growth properties that were dose and time dependent. Velcade treatment resulted in G2M phase arrest, increased expression of cyclin-dependent kinase inhibitor p21 and pro-apoptotic protein Bax. Pretreatment of mesothelioma cells with Velcade showed synergistic effect with cisplatin combination regimens. High-throughput gene expression profiling among Velcade treated and untreated mesothelioma cell lines resulted in identification of novel transducers of apoptosis such as CARP-1, XAF1, and Troy proteins.
Velcade targets cell cycle and apoptosis signaling to suppress MPM and HBC growth in part by activating novel transducers of apoptosis. This pilot study has paved way for further in-depth analysis of the downstream target molecules associated with presensitization of mesothelioma cells in finding effective therapeutic treatment options for both mesothelioma and recalcitrant breast cancers.
PMCID: PMC3826182  PMID: 19960346
Malignant pleural mesothelioma; Bortezomib (Velcade); Apoptosis; Gene expression
8.  Vitamin D related genes in lung development and asthma pathogenesis 
BMC Medical Genomics  2013;6:47.
Poor maternal vitamin D intake is a risk factor for subsequent childhood asthma, suggesting that in utero changes related to vitamin D responsive genes might play a crucial role in later disease susceptibility. We hypothesized that vitamin D pathway genes are developmentally active in the fetal lung and that these developmental genes would be associated with asthma susceptibility and regulation in asthma.
Vitamin D pathway genes were derived from PubMed and Gene Ontology surveys. Principal component analysis was used to identify characteristic lung development genes.
Vitamin D regulated genes were markedly over-represented in normal human (odds ratio OR 2.15, 95% confidence interval CI: 1.69-2.74) and mouse (OR 2.68, 95% CI: 2.12-3.39) developing lung transcriptomes. 38 vitamin D pathway genes were in both developing lung transcriptomes with >63% of genes more highly expressed in the later than earlier stages of development. In immortalized B-cells derived from 95 asthmatics and their unaffected siblings, 12 of the 38 (31.6%) vitamin D pathway lung development genes were significantly differentially expressed (OR 3.00, 95% CI: 1.43-6.21), whereas 11 (29%) genes were significantly differentially expressed in 43 control versus vitamin D treated immortalized B-cells from Childhood Asthma Management Program subjects (OR 2.62, 95% CI: 1.22-5.50). 4 genes, LAMP3, PIP5K1B, SCARB2 and TXNIP were identified in both groups; each displays significant biologic plausibility for a role in asthma.
Our findings demonstrate a significant association between early lung development and asthma–related phenotypes for vitamin D pathway genes, supporting a genomic mechanistic basis for the epidemiologic observations relating maternal vitamin D intake and childhood asthma susceptibility.
PMCID: PMC4228235  PMID: 24188128
Vitamin D; Cholecalciferol; Lung development; Asthma; Fetal programming
9.  Curcumin suppresses growth of mesothelioma cells in vitro and in vivo, in part, by stimulating apoptosis 
Molecular and cellular biochemistry  2011;357(0):10.1007/s11010-011-0878-2.
Malignant pleural mesothelioma (MPM) is an aggressive, asbestos-related malignancy of the thoracic pleura. Although, platinum-based agents are the first line of therapy, there is an urgent need for second-line therapies to treat the drug-resistant MPM. Cell cycle as well as apoptosis pathways are frequently altered in MPM and thus remain attractive targets for intervention strategies. Curcumin, the major component in the spice turmeric, alone or in combination with other chemotherapeutics has been under investigation for a number of cancers. In this study, we investigated the biological and molecular responses of MPM cells to curcumin treatments and the mechanisms involved. Flow-cytometric analyses coupled with western immunoblotting and gene-array analyses were conducted to determine mechanisms of curcumin-dependent growth suppression of human (H2373, H2452, H2461, and H226) and murine (AB12) MPM cells. Curcumin inhibited MPM cell growth in a dose- and time-dependent manner while pretreatment of MPM cells with curcumin enhanced cisplatin efficacy. Curcumin activated the stress-activated p38 kinase, caspases 9 and 3, caused elevated levels of proapoptotic proteins Bax, stimulated PARP cleavage, and apoptosis. In addition, curcumin treatments stimulated expression of novel transducers of cell growth suppression such as CARP-1, XAF1, and SULF1 proteins. Oral administration of curcumin inhibited growth of murine MPM cell-derived tumors in vivo in part by stimulating apoptosis. Thus, curcumin targets cell cycle and promotes apoptosis to suppress MPM growth in vitro and in vivo. Our studies provide a proof-of-principle rationale for further in-depth analysis of MPM growth suppression mechanisms and their future exploitation in effective management of resistant MPM.
PMCID: PMC3809898  PMID: 21594647
Malignant pleural mesothelioma; Curcumin; Apoptosis; Gene expression
10.  Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis 
Carbohydrate research  2012;359:44-53.
Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome. The protein was purified to homogeneity and utilized to accept a chromophore containing farnesyl diphosphate analogue as a substrate. The analogue was utilized by the enzyme and resulted in a bactoprenyl diphosphate product with an easy to monitor tag associated with it. Furthermore, the diphosphate is shown to be readily converted to monophosphate using a common molecular biology reagent. This monophosphate product allowed for the investigation of complex oligosaccharide biosynthesis, and was used to probe the activity of glycosyltransferases involved in the well characterized Campylobacter jejuni N-linked protein glycosylation. Novel reagents similar to this will provide key tools for the study of uncharacterized oligosaccharide assemblies, and open the possibility for the development of rapid screening methodology for these biosynthetic systems.
PMCID: PMC3449032  PMID: 22925763
bactoprenol; undecaprenol; undecaprenyl diphosphate synthase; cis-prenyl transferases; Bacteroides fragilis
11.  Pseudo Chediak Higashi-like inclusions in myeloblasts in AML-M2 
PMCID: PMC3889064  PMID: 24455661
12.  Aspirin and Low-Molecular Weight Heparin Combination Therapy Effectively Prevents Recurrent Miscarriage in Hyperhomocysteinemic Women 
PLoS ONE  2013;8(9):e74155.
The management of recurrent pregnancy loss (RPL) still remains a great challenge, and women with polycystic ovarian syndrome (PCOS) are at a greater risk for spontaneous abortion. Treatment with low-molecular-weight heparin (LMWH) has become an accepted treatment option for women with RPL; however, the subgroup of women, who are likely to respond to LMWH, has not been precisely identified. The present study evaluated the efficacy of LMWH with reference to PCOS and associated metabolic phenotypes including hyperhomocysteinemia (HHcy), insulin resistance (IR) and obesity. This prospective observational study was conducted at Institute of Reproductive Medicine, Kolkata, India. A total of 967 women with history of 2 or more consecutive first trimester abortions were screened and 336 were selected for the study. The selected patients were initially divided on the basis of presence or absence of PCOS, while subsequent stratification was based on HHcy, IR and/or obesity. The subjects had treatment with aspirin during one conception cycle and aspirin-LMWH combined anticoagulant therapy for the immediate next conception cycle, if the first treated cycle was unsuccessful. Pregnancy salvage was the sole outcome measure. The overall rate of pregnancy salvage following aspirin therapy was 43.15%, which was mostly represented by normohomocysteinemic women, while the salvage rate was lower in the HHcy populations irrespective of the presence or absence of PCOS, IR, or obesity. By contrast, aspirin-LMWH combined therapy could rescue 66.84% pregnancies in the aspirin-failed cases. Logistic regression analyses showed that HHcy remained a significant factor in predicting salvage rates in the PCOS, IR, and obese subpopulations controlled for other confounding factors. With regard to pregnancy salvage, combined anticoagulant therapy with aspirin and LMWH conferred added benefit to those with HHcy phenotype.
PMCID: PMC3764119  PMID: 24040195
13.  Chronic Childhood Idiopathic Myelofibrosis in Down’s Syndrome: A Case Report 
Idiopathic myelofibrosis a disease of elderly is rarely seen in children. A case of chronic idiopathic myelofibrosis in an 8 year old boy with Down’s syndrome is reported here, who presented with progressive pallor and hepatosplenomegaly. Peripheral blood examination revealed pancytopenia, macrocytic anemia and tear drop cells. No blasts were found. Bone marrow aspirate yielded a dry tap and trephine biopsy showed marrow fibrosis with osteosclerosis. Focally megakaryocytes were increased in number with atypical morphology. No blasts were seen. Review of literature revealed 47 reported cases of childhood idiopathic myelofibrosis. Six cases were associated with Down’s syndrome and only 3 of them had features of chronic idiopathic myelofibrosis without evidence of acute megakaryoblastic leukaemia.
PMCID: PMC3422385  PMID: 23997458
Childhood; Down’s syndrome; Idiopathic myelofibrosis; Osteosclerosis
14.  The Impact of Self-Identified Race on Epidemiologic Studies of Gene Expression 
Genetic epidemiology  2011;35(2):93-101.
Although population differences in gene expression have been established, the impact on differential gene expression studies in large populations is not well understood. We describe the effect of self-reported race on a gene expression study of lung function in asthma. We generated gene expression profiles for 254 young adults (205 non-Hispanic whites and 49 African Americans) with asthma on whom concurrent total RNA derived from peripheral blood CD4+ lymphocytes and lung function measurements were obtained. We identified four principal components that explained 62% of the variance in gene expression. The dominant principal component, which explained 29% of the total variance in gene expression, was strongly associated with self-identified race (P<10−16). The impact of these racial differences was observed when we performed differential gene expression analysis of lung function. Using multivariate linear models, we tested whether gene expression was associated with a quantitative measure of lung function: pre-bronchodilator forced expiratory volume in one second (FEV1). Though unadjusted linear models of FEV1 identified several genes strongly correlated with lung function, these correlations were due to racial differences in the distribution of both FEV1 and gene expression, and were no longer statistically significant following adjustment for self-identified race. These results suggest that self-identified race is a critical confounding covariate in epidemiologic studies of gene expression and that, similar to genetic studies, careful consideration of self-identified race in gene expression profiling studies is needed to avoid spurious association.
PMCID: PMC3718033  PMID: 21254216
ancestry; gene expression; population stratification; self-identified race
15.  Association of variants in innate immune genes with asthma and eczema 
The innate immune pathway is important in the pathogenesis of asthma and eczema. However, only a few variants in these genes have been associated with either disease. We investigate the association between polymorphisms of genes in the innate immune pathway with childhood asthma and eczema. In addition, we compare individual associations with those discovered using a multivariate approach.
Using a novel method, case control based association testing (C2BAT), 569 single nucleotide polymorphisms (SNPs) in 44 innate immune genes were tested for association with asthma and eczema in children from the Boston Home Allergens and Asthma Study and the Connecticut Childhood Asthma Study. The screening algorithm was used to identify the top SNPs associated with asthma and eczema. We next investigated the interaction of innate immune variants with asthma and eczema risk using Bayesian networks.
After correction for multiple comparisons, 7 SNPs in 6 genes (CARD25, TGFB1, LY96, ACAA1, DEFB1, and IFNG) were associated with asthma (adjusted p-value<0.02), while 5 SNPs in 3 different genes (CD80, STAT4, and IRAKI) were significantly associated with eczema (adjusted p-value < 0.02). None of these SNPs were associated with both asthma and eczema. Bayesian network analysis identified 4 SNPs that were predictive of asthma and 10 SNPs that predicted eczema. Of the genes identified using Bayesian networks, only CD80 was associated with eczema in the single-SNP study. Using novel methodology that allows for screening and replication in the same population, we have identified associations of innate immune genes with asthma and eczema. Bayesian network analysis suggests that additional SNPs influence disease susceptibility via SNP interactions.
Our findings suggest that innate immune genes contribute to the pathogenesis of asthma and eczema, and that these diseases likely have different genetic determinants.
PMCID: PMC3412627  PMID: 22192168
asthma; Bayesian network; genetic association; eczema; innate immunity
16.  NMDA and kainate receptor expression, long-term potentiation, and neurogenesis in the hippocampus of long-lived Ames dwarf mice 
Age  2011;34(3):609-620.
In the current study, we investigated changes in N-methyl d-aspartate (NMDA) and kainate receptor expression, long-term potentiation (LTP), and neurogenesis in response to neurotoxic stress in long-living Ames dwarf mice. We hypothesized that Ames dwarf mice have enhanced neurogenesis that enables retention of spatial learning and memory with age and promotes neurogenesis in response to injury. Levels of the NMDA receptors (NR)1, NR2A, NR2B, and the kainate receptor (KAR)2 were increased in Ames dwarf mice, relative to wild-type littermates. Quantitative assessment of the excitatory postsynaptic potential in Schaffer collaterals in hippocampal slices from Ames dwarf mice showed an increased response in high-frequency induced LTP over time compared with wild type. Kainic acid (KA) injection was used to promote neurotoxic stress-induced neurogenesis. KA mildly increased the number of doublecortin-positive neurons in wild-type mice, but the response was significantly enhanced in the Ames dwarf mice. Collectively, these data support our hypothesis that the enhanced learning and memory associated with the Ames dwarf mouse may be due to elevated levels of NMDA and KA receptors in hippocampus and their ability to continue producing new neurons in response to neuronal damage.
PMCID: PMC3337943  PMID: 21544578
Ames dwarf; Hippocampus; NMDA receptors; LTP; Neurogenesis; Life Sciences; Molecular Medicine; Geriatrics/Gerontology; Cell Biology
17.  Recurrent Pregnancy Loss in Polycystic Ovary Syndrome: Role of Hyperhomocysteinemia and Insulin Resistance 
PLoS ONE  2013;8(5):e64446.
Recurrent pregnancy loss (RPL) in polycystic ovary syndrome (PCOS), which occurs in ∼50% of total pregnancies is a frequent obstetric complication. Among the several hypotheses, insulin resistance (IR), obesity and hyperhomocysteinemia (HHcy) play significant role/s in RPL. This study was conducted to assess the link between elevated levels of homocysteine and IR in PCOS-associated women with RPL in Kolkata, India. A retrospective study was conducted of one hundred and twenty six PCOS women (<30 years) who experienced two or more spontaneous abortions during the first trimester presenting to Institute of Reproductive Medicine (IRM) in Kolkata during the period of March 2008 through February 2011. One hundred and seventeen non-PCOS subjects with matching age range were randomly chosen as controls. Incidence of HHcy and IR was 70.63% (n = 89) and 56.34% (n = 71), respectively, in RPL-affected PCOS population which was significantly higher (p<0.04; p<0.0001) when compared to the non-PCOS set (HHcy: 57.26%; IR: 6.83%). Rates of miscarriage were significantly higher (p<0.008; p<0.03) in hyperhomocysteinemia-induced miscarriage when compared to the normohomocysteinemic segment (PCOS: 70.63% vs.29.36% & non-PCOS: 57.26% vs. 42.73%) along with the insulin resistant (p<0.04; p<0.0001) population (PCOS: 70.63% vs. 56.34% & non-PCOS: 57.26% vs. 6.83%) in both groups. A probabilistic causal model evaluated HHcy as the strongest plausible factor for diagnosis of RPL. A probability percentage of 43.32% in the cases of HHcy- mediated RPL suggests its increased tendency when compared to IR mediated miscarriage (37.29%), further supported by ROC-AUC (HHcy: 0.778vs. IR: 0.601) values. Greater susceptibility towards HHcy may increase the incidence for miscarriage in women in India and highlights the need to combat the condition in RPL control programs in the subcontinent.
PMCID: PMC3660299  PMID: 23700477
18.  Durable Protection from Vaginal Simian-Human Immunodeficiency Virus Infection in Macaques by Tenofovir Gel and Its Relationship to Drug Levels in Tissue 
Journal of Virology  2012;86(2):718-725.
A vaginal gel containing 1% tenofovir (TFV) was found to be safe and effective in reducing HIV infection in women when used pericoitally. Because of the long intracellular half-life of TFV and high drug exposure in vaginal tissues, we hypothesized that a vaginal gel containing TFV may provide long-lasting protection. Here, we performed delayed-challenge experiments and showed that vaginal 1% TFV gel protected 4/6 macaques against vaginal simian-human immunodeficiency virus (SHIV) exposures occurring 3 days after gel application, demonstrating long-lasting protection. Despite continued gel dosing postinfection, neither breakthrough infection had evidence of drug resistance by ultrasensitive testing of SHIV in plasma and vaginal lavage. Analysis of the active intracellular tenofovir diphosphate (TFV-DP) in vaginal lymphocytes collected 4 h to 3 days after gel dosing persistently showed high TFV-DP levels (median, 1,810 fmol/106 cells) between 4 and 24 h that exceed the 95% inhibitory concentration (IC95), reflecting rapid accumulation and long persistence. In contrast to those in peripheral blood mononuclear cells (PBMCs) following oral dosing, TFV-DP levels in vaginal lymphocytes decreased approximately 7-fold by 3 days, exhibiting a much higher rate of decay. We observed a strong correlation between intracellular TFV-DP in vaginal lymphocytes, in vitro antiviral activity, and in vivo protection, suggesting that TFV-DP above the in vitro IC95 in vaginal lymphocytes is a good predictor of high efficacy. Data from this model reveal an extended window of protection by TFV gel that supports coitus-independent use. The identification of protective TFV-DP concentrations in vaginal lymphocytes may facilitate the evaluation of improved delivery methods of topical TFV and inform clinical studies.
PMCID: PMC3255839  PMID: 22072766
19.  Letrozole in a low-cost in vitro fertilization protocol in intracytoplasmic sperm injection cycles for male factor infertility: A randomized controlled trial 
Letrozole, a selective aromatase inhibitor, reduces the total dose of gonadotrophin required for inducing follicular maturation. We evaluated if incorporation of letrozole could be an effective alternative for low-cost in vitro fertilization (IVF) protocol particularly in intracytoplasmic sperm injection (ICSI) cycles where male factor infertility is the sole indication for IVF.
It is a randomized controlled single-blind trial. 94 women with history of severe male factor infertility were selected. 42 women (study group) received letrozole, 5 mg daily from day 3-7 and recombinant FSH (rFSH) 75IU/day from day 5 continuously till hCG injection. 52 women (control group) underwent continuous stimulation by rFSH (150-225IU/day) from day 2. GnRH-antagonist (Inj. Orgalutran 0.25 ml sub-cutaneous) was started at maximum follicle size of 14 in both groups. Ovulation was triggered by 10,000IU of hCG followed by IVF-ET. Main outcome measures were total dose of rFSH (IU/cycle), terminal E2 (pg/ml), number of mature follicles, number of oocyte retrieved, transferable embryo, endometrial thickness, pregnancy rate and mean expenditure. Statistical analysis is done by using SPSS11.
As compared to control group (1756 ± 75IU), the study group i.e., Let-rFSH received (625 ± 98IU) significantly lower (P = 0.0001) total dose of rFSH. Terminal E2 was significantly lower (P = 0.0001) in study group than control (830 ± 36 vs. 1076 ± 41 pg/ml) with significant increment in endometrial thickness (P = 0.0008) in study group, (9.1 ± 0.32 vs. 8.7 ± 0.69) which maintained an improved pregnancy rate though nonsignificant. The risk of hyperstimulation had significantly (P = 0.01) reduced in study group than control (0 vs. 7).Treatment outcome in all other aspects including pregnancy rate were statistically comparable. Per cycle mean expenditure was reduced by 34% in study group than control.
Adjunctive use of letrozole may be an effective mean of low-cost IVF therapy.
PMCID: PMC3493831  PMID: 23162355
Antagonist; ICSI; letrozole; low-cost IVF; male factor infertility
20.  Assessment of oocyte quality in polycystic ovarian syndrome and endometriosis by spindle imaging and reactive oxygen species levels in follicular fluid and its relationship with IVF-ET outcome 
The aim of this study is to examine meiotic spindle in oocytes along with reactive oxygen species (ROS) levels in follicular fluid of women undergoing IVF and to correlate these findings with embryo quality and pregnancy outcome.
167 women aged 25–35 years with endometriosis (Group A), polycystic ovarian syndrome (PCOS) (Group B) and tubal block (Group C) were included. Long protocol downregulation using recombinant follicular stimulating hormone was used for ovarian stimulation. Aspirated follicular fluid containing mature oocytes were analyzed for ROS levels and the oocytes were assessed for the presence of meiotic spindle using Cri-Oosight™ Polscope. Fertilization, embryo quality, endometrial assessment, and final pregnancy outcome were assessed.
Meiotic spindles were visualized in a higher proportion of mature oocytes retrieved from women with endometriosis (66%) as compared to those with PCOS (50.5%) and tubal block (62.3%). ROS levels were also observed to be significantly less in the follicular fluid of oocytes in women with endometriosis (Group A) as compared to the other two groups (P ≤ 0.001). However, pregnancy rates were observed to be lower in Group A (32%) than Groups B (39%) and C (44%), respectively. Within each group, oocytes with spindle visualization yielded a higher number of Grade 1 embryos (P < 0.05) as well as lower ROS levels in follicular fluid (P ≤ 0.001) as compared to those where spindle could not be visualized.
There was good correlation between spindle imaging and ROS levels as reliable predictors of oocyte assessment. Women with endometriosis had low ROS levels and good spindle imaging results suggesting a possible role of endometrial receptivity accounting for lower pregnancy rates in these women. Poor oocyte quality, as reflected by higher mean ROS levels and low number of oocytes with spindle visualization, could be the factor impeding pregnancy in women with PCOS as compared to women with tubal block.
PMCID: PMC3493834  PMID: 23162358
Endometriosis; meiotic spindle imaging; oocyte quality; PCOS; reactive oxygen species
22.  Evaluation of the clinical efficacy of potassium nitrate desensitizing mouthwash and a toothpaste in the treatment of dentinal hypersensitivity 
Potassium Nitrate has been used as a desensitizing agent to treat dentinal hypersensitivity. The effectiveness of a potassium nitrate is evaluated both in the form of a toothpaste and a mouthwash in a clinical study. Thirty patients were assessed using evaporative stimuli and thermal stimuli and response was evaluated using Visual Analogue Scale at baseline, at 2 weeks and 4 weeks. The patients were divided into. group I: fifteen patients who used toothpaste containing 5% potassium nitrate, sodium fluoride, xylitol and triclosan, group II: Fifteen patients who used mouthwash containing 3% potassium nitrate, sodium fluoride, xylitol and triclosan . The results of both the assessment methods indicated that potassium nitrate toothpaste as well as mouthwash showed statistically significant decrease in the sensitivity score on a Visual Analogue Scale. This was effective in reducing the symptoms of dentinal hypersensitivity when used either as toothpaste or as a mouthwash. But, there were no statistically significant differences between the groups, although both were effective in the treatment of hypersensitivity.
Key words:Dentinal hypersensitivity, potassium nitrate toothpaste, potassium nitrate mouthwash, desensitizing agents.
PMCID: PMC3908806  PMID: 24558521
23.  Effects of endotoxin exposure on childhood asthma risk are modified by a genetic polymorphism in ACAA1 
BMC Medical Genetics  2011;12:158.
Polymorphisms in the endotoxin-mediated TLR4 pathway genes have been associated with asthma and atopy. We aimed to examine how genetic polymorphisms in innate immunity pathways interact with endotoxin to influence asthma risk in children.
In a previous analysis of 372 children from the Boston Home Allergens and the Connecticut Childhood Asthma studies, 7 SNPs in 6 genes (CARD15, TGFB1, LY96, ACAA1, DEFB1 and IFNG) involved in innate immune pathways were associated with asthma, and 5 SNPs in 3 genes (CD80, STAT4, IRAK2) were associated with eczema. We tested these SNPs for interaction with early life endotoxin exposure (n = 291), in models for asthma and eczema by age 6.
We found a significant interaction between endotoxin and a SNP (rs156265) in ACAA1 (p = 0.0013 for interaction). Increased endotoxin exposure (by quartile) showed protective effects for asthma in individuals with at least one copy of the minor allele (OR = 0.39 per quartile increase in endotoxin, 95% CI 0.15 to 1.01). Endotoxin exposure did not reduce the risk of asthma in children homozygous for the major allele.
Our findings suggest that protective effects of endotoxin exposure on asthma may vary depending upon the presence or absence of a polymorphism in ACAA1.
PMCID: PMC3252252  PMID: 22151743
24.  Hemolytic Anemia as a Presenting Feature of Wilson’s Disease: A Case Report 
Wilson’s disease is a rare inherited disorder of copper metabolism causing severe damage to vital organs. Liver and brain disorders are the main manifestations. Severe hemolytic anemia is an unusual complication of Wilson’s disease. We present a case who developed spherocytic acute hemolytic anemia (Coomb’s negative) as the initial manifestation of Wilson’s disease. On examination Kayser- Fleischer ring was found. Laboratory data supported a diagnosis of Wilson’s disease.
PMCID: PMC3002091  PMID: 21886393
Wilson’s disease (WD); Spherocytic anemia
25.  A patient presenting with a perivascular epithelioid cell tumor in the broad ligament: a case report 
Perivascular epithelioid cell tumors are a family of rare mesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular epithelioid cells. They can originate in any visceral organ or soft tissue and include a range of lesions such as angiomyolipoma, clear cell 'sugar' tumor of the lung, lymphangioleiomyomatosis and clear cell myomelanocytic tumors of the falciparum ligament/ligament teres. Due to their rarity and varied sites and presentation, management of these tumors remains highly challenging.
Case Presentation
A 46-year-old para 2 Caucasian woman initially presented to the general surgeons at our hospital in North West London with abdominal pain. Laparoscopy revealed a right broad ligament hematoma, which was thought to be iatrogenic in origin, from insertion of the Veress needle at the time of surgery, and was managed conservatively. Upon her re-presentation two months later with severe pain, ultrasound scanning revealed the hematoma had increased in size and she underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy. Histology results from necrotic tissue from the hematoma led to a diagnosis of perivascular epithelioid cell tumor. She was then referred to a tertiary oncology center, where she underwent several further operations in an attempt to debulk the tumor for symptomatic relief of her pain, with limited success. She is now taking the immunosuppressive drug sirolimus, which has produced a modest reduction in tumor size. She is now 47 months on from initial presentation.
A literature search has revealed only six other case reports of broad ligament perivascular epithelioid cell tumors, with varied presentations and management. The longest duration of follow-up was 21 months. Only five other cases of perivascular epithelioid cell tumor managed with sirolimus have been reported. We therefore feel that this report highlights some of the difficulties in diagnosing perivascular epithelioid cell tumors, and sheds light on management strategies for a very rare gynecological tumor in addition to sharing our experience in the use of sirolimus in its treatment.
PMCID: PMC3169496  PMID: 21846376

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