Differential expression of the NMDA NR2B receptor subunit in motoneuron populations susceptible and resistant to amyotrophic lateral sclerosis

We have compared the expression pattern of NMDA receptor subunits (NR1 and NR2A-D)and NRI splice variants (NR1-1a/1b,-2a/2b,-3a/3b,4a/4b) in motor neuron populations from adult Wistar rats that are vulnerable (hypoglossal, XII) or resistant (oculomotor, III) to death in amyotrophic lateral sclerosis (ALS). The major finding was higher levels of expression of the NR2B subunit in the hypoglossal nucleus. Quantitative real-time PCR showed that NR1 was expressed at a greater level than any of the NR2 subunits (> 15 fold greater, P

Characterization of tumour-infiltrating lymphocytes and apoptosis in colitis-associated neoplasia: comparison with sporadic colorectal cancer

The development of colorectal cancer is a major complication for patients with chronic idiopathic colitis. Colitis-associated tumours tend to occur at a younger age and be more aggressive than sporadic colorectal cancers. While we have previously associated the presence of tumour-infiltrating lymphocytes (TILs) and increased apoptosis in sporadic colorectal cancer with high-level microsatellite instability and improved prognosis, little is known of the relationship between these variables in colitis-associated colorectal cancer. The aim of this study was to correlate TILs and tumour cell apoptosis in colitis-associated neoplasms stratified according to microsatellite instability. Twenty tumour and 11 dysplastic samples resected from 21 patients with long-standing colitis were analysed for microsatellite instability at 10 microsatellite markers. TIL distribution (CD3, CD8) and function (granzyme B) were quantified by immunohistochemistry. Neoplastic cell apoptosis was assessed using the M30 CytoDEATH antibody. These findings were compared with 40 microsatellite stable (MSS) sporadic colorectal cancers previously evaluated for TILs and neoplastic apoptosis. Low-level microsatellite instability was found in 1/20 colitis-associated tumours. All other colitis-associated lesions were designated MSS. CD3(+) and CD8(+) TIL counts were significantly higher in colitis-associated lesions compared with NISS sporadic colorectal cancer (p < 0.0001, p = 0.001 respectively). Despite their higher TIL density, colitis-associated tumours were more likely to present late (Dukes' stage C or D) (P = 0.02). Functionally, colitis-associated TILs demonstrated significantly less granzyme B expression compared to sporadic cancers (p = 0.002). The level of tumour cell apoptosis was similar between the two groups (sporadic, 1.53%; colitis cancers, 1.45%). In conclusion, NISS colitis-associated tumours have a higher prevalence of CD3(+)/CD8(+) TILs but no associated increase in tumour cell killing by apoptosis. Unlike cytotoxic T cells in sporadic colorectal cancer, TILs do not appear to enhance the prognosis of colitis-associated colorectal cancer. This may be related to an impairment of granzyme B expression within these lesions. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Liver fluke-associated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes

Aim: To compare cell phenotypes displayed by cholangiocarcinomas and adjacent bile duct lesions in patients from an area endemic in liver-fluke infestation and those with sporadic cholangiocarcinoma. Methods: 65 fluke-associated and 47 sporadic cholangiocarcinomas and 6 normal livers were studied. Serial paraffin-wax sections were stained immunohistochemically with monoclonal antibodies characterising a Brunner or pyloric gland metaplasia cell phenotype (antigens D10 and 1F6), intestinal goblet cells (antigen 17NM), gastric foveolar apomucin (MUC5AC), a gastrointestinal epithelium cytokeratin (CK20) and the p53 protein. Results: 60% of the 112 cholangiocarcinomas expressed antigen D10, 68% MUC5AC, 33% antigen 17NM and 20% CK20; 37% showed overexpression of p53. When present together in a cholangiocarcinoma, cancer cells expressing D10 were distinct from those displaying 17NM or MUC5AC. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinomas displayed 17NM and p53 expression. Most cases of hyperplastic and dysplastic biliary epithelium expressed D10 strongly. Pyloric gland metaplasia and peribiliary glands displayed D10 and 1F6, with peribiliary gland hyperplasia more evident in the livers with fluke-associated cholangiocarcinoma; goblet cells in intestinal metaplasia stained for 17NM. No notable association of expression between any two antigens (including p53) was found in the cancers. Conclusions: Most cases of dysplastic biliary epithelium and cholangiocarcinoma display a Brunner or pyloric gland cell phenotype and a gastric foveolar cell phenotype. The expression of D10 in hyperplastic and dysplastic epithelium and in cholangiocarcinoma is consistent with a dysplasia-carcinoma sequence. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinoma display an intestinal goblet cell phenotype and overexpress p53, indicating differences in the aetiopathology of the cancers in the two groups of patients.

Topical interferon alfa-2b for the treatment of recalcitrant ocular surface squamous neoplasia

center dot PURPOSE: To evaluate topical interferon alfa-2b (IFN-alpha 2b) for the treatment of recalcitrant ocular surface squamous neoplasia (OSSN). center dot DESIGN: Prospective, noncomparative, interventional consecutive case series. center dot METHODS: Ten patients with recalcitrant OSSN were treated with topical IFN-alpha 2b (1 million IU/ml) four times a day until clinical resolution of the lesion or until the lesion appeared nonresponsive-that is, treatment failure. Progress was assessed by clinical examination and photographic records, with a minimum follow,up of six months. center dot RESULTS: Eight of 10 patients achieved clinical resolution from topical IFN-alpha 2b treatment. One patient developed invasive squamous cell carcinoma and underwent exenteration. The other patient required further mitomycin C therapy to achieve clinical resolution. The mean duration to clinical resolution for the eight patients treated with IFN-alpha 2b was 21.9 weeks (range six to 59 weeks). There have been no recurrences for any of the nine patients during follow-up (mean 55.0 weeks; range 26 to 84 weeks). center dot CONCLUSIONS: Topical IFN-alpha 2b is an important treatment modality for recalcitrant OSSN; it avoids the risks of further limbal stem cell destruction from other agents and surgical excision. If invasive disease is diagnosed at any stage, topical therapy is contraindicated, necessitating surgical excision. (Am J Ophthalmol 2006; 142:568-571. (c) 2006 by Elsevier Inc. All rights reserved.)

Population pharmacokinetics of itraconazole and its active metabolite hydroxy-itraconazole in paediatric cystic fibrosis and bone marrow transplant patients

Objective: The objective of the study was to characterise the population pharmacokinetic properties of itraconazole and its active metabolite hydroxyitraconazole in a representative paediatric population of cystic fibrosis and bone marrow transplant (BMT) patients and to identify patient characteristics influencing the pharmacokinetics of itraconazole. The ultimate goals were to determine the relative bioavailability between the two oral formulations (capsules vs oral solution) and to optimise dosing regimens in these patients. Methods: All paediatric patients with cystic fibrosis or patients undergoing BMT at The Royal Children's Hospital, Brisbane, QLD, Australia, who were prescribed oral itraconazole for the treatment of allergic bronchopulmonary aspergillosis (cystic fibrosis patients) or for prophylaxis of any fungal infection (BMT patients) were eligible for the study. Blood samples were taken from the recruited patients as per an empirical sampling design either during hospitalisation or during outpatient clinic visits. ltraconazole and hydroxy-itraconazole plasma concentrations were determined by a validated high-performance liquid chromatography assay with fluorometric detection. A nonlinear mixed-effect modelling approach using the NONMEM software to simultaneously describe the pharmacokinetics of itraconazole and its metabolite. Results: A one-compartment model with first-order absorption described the itraconazole data, and the metabolism of the parent drug to hydroxy-itraconazole was described by a first-order rate constant. The metabolite data also showed one-compartment characteristics with linear elimination. For itraconazole the apparent clearance (CLitraconazole) was 35.5 L/hour, the apparent volume of distribution (V-d(itraconazole)) was 672L, the absorption rate constant for the capsule formulation was 0.0901 h(-1) and for the oral solution formulation was 0.96 h-1. The lag time was estimated to be 19.1 minutes and the relative bioavailability between capsules and oral solution (F-rel) was 0.55. For the metabolite, volume of distribution, V-m/(F (.) f(m)), and clearance, CL/(F (.) fm), were 10.6L and 5.28 L/h, respectively. The influence of total bodyweight was significant, added as a covariate on CLitraconazoie/F and V-d(itraconazole)/F (standardised to a 70kg person) using allometric three-quarter power scaling on CLitraconazole/F, which therefore reflected adult values. The unexplained between-subject variability (coefficient of variation %) was 68.7%, 75.8%, 73.4% and 61.1% for CLitraconazoie/F, Vd(itraconazole)/F, CLm/(F (.) fm) and F-rel, respectively. The correlation between random effects of CLitraconazole and Vd((itraconazole)) was 0.69. Conclusion: The developed population pharmacokinetic model adequately described the pharmacokinetics of itraconazole and its active metabolite, hydroxy-itraconazole, in paediatric patients with either cystic fibrosis or undergoing BMT. More appropriate dosing schedules have been developed for the oral solution and the capsules to secure a minimum therapeutic trough plasma concentration of 0.5 mg/L for these patients.

Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum

Aim-Colorectal cancer has been described in association with hyperplastic polyposis but the mechanism underlying this observation is unknown. The aim of this study was to characterise foci of dysplasia developing in the polyps of subjects with hyperplastic polyposis on the basis of DNA microsatellite status and expression of the DNA mismatch repair proteins hMLH1, hMSH2, and hMSH6. Materials and methods-The material was derived from four patients with hyperplastic polyposis and between one and six synchronous colorectal cancers. Normal (four), hyperplastic (13), dysplastic (13), and malignant (11) samples were microdissected and a PCR based approach was used to identify mutations at 10 microsatellite loci, TGF beta IIR, IGF2R, BAX, MSH3, and MSH6. Microsatellite instability-high (MSI-H) was diagnosed when 40% or more of the microsatellite loci showed mutational bandshifts. Serial sections were stained for hMLH1, hMSH2, and hMSH6. Result-DNA microsatellite instability was found in 1/13 (8%) hyperplastic samples, in 7/13 (54%) dysplastic foci, and in 8/11 (73%) cancers. None of the MSI-low (MSI-L) samples (one hyperplastic, three dysplastic, two cancers) showed loss of hMLH1 expression. All four MSI-H dysplastic foci and six MSI-H cancers showed loss of hMLH1 expression. Loss of hMLH1 in MSI-H but not in MSI-L lesions showing dysplasia or cancer was significant (p< 0.001, Fisher's exact test). Loss of hMSH6 occurred in one MSI-H cancer and one MSS focus of dysplasia which also showed loss of hMLH1 staining. Conclusion-Neoplastic changes in hyperplastic polyposis may occur within a hyperplastic polyp. Neoplasia may be driven by DNA instability that is present to a low (MSI-L) or high (MSI-H) degree. MSI-H but not MSI-L dysplastic foci are associated with loss of hMLH1 expression. At least two mutator pathways drive neoplasia in hyperplastic polyposis. The role of the hyperplastic polyp in the histogenesis of sporadic DNA microsatellite unstable colorectal cancer should be examined.

A D-optimal designed population pharmacokinetic study of itraconazole capsules and solution in adults with cystic fibrosis

Background: Oral itraconazole (ITRA) is used for the treatment of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis (CF) because of its antifungal activity against Aspergillus species. ITRA has an active hydroxy-metabolite (OH-ITRA) which has similar antifungal activity. ITRA is a highly lipophilic drug which is available in two different oral formulations, a capsule and an oral solution. It is reported that the oral solution has a 60% higher relative bioavailability. The influence of altered gastric physiology associated with CF on the pharmacokinetics (PK) of ITRA and its metabolite has not been previously evaluated. Objectives: 1) To estimate the population (pop) PK parameters for ITRA and its active metabolite OH-ITRA including relative bioavailability of the parent after administration of the parent by both capsule and solution and 2) to assess the performance of the optimal design. Methods: The study was a cross-over design in which 30 patients received the capsule on the first occasion and 3 days later the solution formulation. The design was constrained to have a maximum of 4 blood samples per occasion for estimation of the popPK of both ITRA and OH-ITRA. The sampling times for the population model were optimized previously using POPT v.2.0.[1] POPT is a series of applications that run under MATLAB and provide an evaluation of the information matrix for a nonlinear mixed effects model given a particular design. In addition it can be used to optimize the design based on evaluation of the determinant of the information matrix. The model details for the design were based on prior information obtained from the literature, which suggested that ITRA may have either linear or non-linear elimination. The optimal sampling times were evaluated to provide information for both competing models for the parent and metabolite and for both capsule and solution simultaneously. Blood samples were assayed by validated HPLC.[2] PopPK modelling was performed using FOCE with interaction under NONMEM, version 5 (level 1.1; GloboMax LLC, Hanover, MD, USA). The PK of ITRA and OH‑ITRA was modelled simultaneously using ADVAN 5. Subsequently three methods were assessed for modelling concentrations less than the LOD (limit of detection). These methods (corresponding to methods 5, 6 & 4 from Beal[3], respectively) were (a) where all values less than LOD were assigned to half of LOD, (b) where the closest missing value that is less than LOD was assigned to half the LOD and all previous (if during absorption) or subsequent (if during elimination) missing samples were deleted, and (c) where the contribution of the expectation of each missing concentration to the likelihood is estimated. The LOD was 0.04 mg/L. The final model evaluation was performed via bootstrap with re-sampling and a visual predictive check. The optimal design and the sampling windows of the study were evaluated for execution errors and for agreement between the observed and predicted standard errors. Dosing regimens were simulated for the capsules and the oral solution to assess their ability to achieve ITRA target trough concentration (Cmin,ss of 0.5-2 mg/L) or a combined Cmin,ss for ITRA and OH-ITRA above 1.5mg/L. Results and Discussion: A total of 241 blood samples were collected and analysed, 94% of them were taken within the defined optimal sampling windows, of which 31% where taken within 5 min of the exact optimal times. Forty six per cent of the ITRA values and 28% of the OH-ITRA values were below LOD. The entire profile after administration of the capsule for five patients was below LOD and therefore the data from this occasion was omitted from estimation. A 2-compartment model with 1st order absorption and elimination best described ITRA PK, with 1st order metabolism of the parent to OH-ITRA. For ITRA the clearance (ClItra/F) was 31.5 L/h; apparent volumes of central and peripheral compartments were 56.7 L and 2090 L, respectively. Absorption rate constants for capsule (kacap) and solution (kasol) were 0.0315 h-1 and 0.125 h-1, respectively. Comparative bioavailability of the capsule was 0.82. There was no evidence of nonlinearity in the popPK of ITRA. No screened covariate significantly improved the fit to the data. The results of the parameter estimates from the final model were comparable between the different methods for accounting for missing data, (M4,5,6)[3] and provided similar parameter estimates. The prospective application of an optimal design was found to be successful. Due to the sampling windows, most of the samples could be collected within the daily hospital routine, but still at times that were near optimal for estimating the popPK parameters. The final model was one of the potential competing models considered in the original design. The asymptotic standard errors provided by NONMEM for the final model and empirical values from bootstrap were similar in magnitude to those predicted from the Fisher Information matrix associated with the D-optimal design. Simulations from the final model showed that the current dosing regimen of 200 mg twice daily (bd) would provide a target Cmin,ss (0.5-2 mg/L) for only 35% of patients when administered as the solution and 31% when administered as capsules. The optimal dosing schedule was 500mg bd for both formulations. The target success for this dosing regimen was 87% for the solution with an NNT=4 compared to capsules. This means, for every 4 patients treated with the solution one additional patient will achieve a target success compared to capsule but at an additional cost of AUD $220 per day. The therapeutic target however is still doubtful and potential risks of these dosing schedules need to be assessed on an individual basis. Conclusion: A model was developed which described the popPK of ITRA and its main active metabolite OH-ITRA in adult CF after administration of both capsule and solution. The relative bioavailability of ITRA from the capsule was 82% that of the solution, but considerably more variable. To incorporate missing data, using the simple Beal method 5 (using half LOD for all samples below LOD) provided comparable results to the more complex but theoretically better Beal method 4 (integration method). The optimal sparse design performed well for estimation of model parameters and provided a good fit to the data.

Paediatric population pharmacokinetics of itraconazole and its active metabolite hydroxy-itraconazole in cystic fibrosis and bone marrow transplant patients

Objectives: The aim of the study was to characterise the population pharmacokinetics (popPK) properties of itraconazole (ITRA) and its active metabolite hydroxy-ITRA in a representative paediatric population of cystic fibrosis (CF) and bone marrow transplant (BMT) patients. The goals were to determine the relative bioavailability between the two oral formulations, and to explore improved dosage regimens in these patients. Methods: All paediatric patients with CF taking oral ITRA for the treatment of allergic bronchopulmonary aspergillosis and patients undergoing BMT who were taking ITRA for prophylaxis of any fungal infection were eligible for the study. A minimum of two blood samples were drawn after the capsules and also after switching to oral solution, or vice versa. ITRA and hydroxy-ITRA plasma concentrations were measured by HPLC[1]. A nonlinear mixed-effect modelling approach (NONMEM 5.1.1) was used to describe the PK of ITRA and hydroxy-ITRA simultaneously. Simulations were used to assess dosing strategies in these patients. Results: Forty-nine patients (29CF, 20 BMT) were recruited to the study who provided 227 blood samples for the population analysis. A 1-compartment model with 1st order absorption and elimination best described ITRA kinetics, with 1st order conversion to hydroxy-ITRA. For ITRA, the apparent clearance (ClItra/F) and volume of distribution (Vitra/F) was 35.5L/h and 672L, respectively; the absorption rate constant for the capsule formulation was 0.0901 h-1 and for the oral solution formulation it was 0.959 h-1. The capsule comparative bioavailability (vs. solution) was 0.55. For hydroxy-ITRA, the apparent volume of distribution and clearance were 10.6 L and 5.28 L/h, respectively. Of several screened covariates only allometrically scaled total body weight significantly improved the fit to the data. No difference between the two populations was found. Conclusion: The developed popPK model adequately described the pharmacokinetics of ITRA and hydroxy-ITRA in paediatric patients with CF and patients undergoing BMT. High inter-patient variability confirmed previous data in CF[2], leukaemia and BMT[3] patients. From the population model, simulations showed the standard dose (5 mg/kg/day) needs to be doubled for the solution formulation and even 4 times more given of the capsules to achieve an adequate target therapeutic trough plasma concentration of 0.5 mg/L[4] in these patients.

Origin and possible roles of the Sox8 transcription factor gene during sexual development

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a critical gene involved in mammalian sex determination and differentiation. Both genes encode proteins with the ability to bind similar DNA target sequences, and to activate transcription in in vitro assays. Expression studies indicate that the two genes have largely overlapping patterns of activity during mammalian embryonic development. A knockout of Sox8 in mice has no obvious developmental phenotype, suggesting that the two genes are able to act redundantly in a variety of developmental contexts. In particular, both genes are expressed in the developing Sertoli cell lineage of the developing testes in mice, and both proteins are able to activate transcription of the gene encoding anti-Mullerian hormone (AMH), through synergistic action with steroidogenic factor I (SF1). We have hypothesized that Sox8 may substitute for Sox9 in species where Sox9 is expressed too late to be involved in sex determination or regulation of Amh expression. However, our studies involving the red-eared slider turtle indicate that Sox8 is expressed at similar levels in males and females throughout the sex-determining period, suggesting that Sox8 is neither a transcriptional regulator for Amh, nor responsible for sex determination or gonad differentiation in that species. Similarly, Sox8 is not expressed in a sexually dimorphic pattern during gonadogenesis in the chicken. Since a functional role(s) for Sox8 is implied by its conservation during evolution, the significance of Sox8 for sexual and other aspects of development will need to be uncovered through more directed lines of experimentation. Copyright (C) 2003 S. Karger AG, Basel.

Characterizations of the major coral diseases of the Philippines: Ulcerative white spot disease and novel growth anomalies of Porites

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

Prenatal environmental exposure and neurodevelopmentally important gene expression in malformed brain tissue from pediatric intractable epilepsy patients

The primary objective of this proposal was to determine whether mitochondrial oxidative stress and variation in a particular mtDNA lineage contribute to the risk of developing cortical dysplasia and are potential contributing factors in epileptogenesis in children. The occurrence of epilepsy in children is highly associated with malformations of cortical development (MCD). It appears that MCD might arise from developmental errors due to environmental exposures in combination with inherited variation in response to environmental exposures and mitochondrial function. Therefore, it is postulated that variation in a particular mtDNA lineage of children contributes to the effects of mitochondrial DNA damage on MCD phenotype. Quantitative PCR and dot blot were used to examine mitochondrial oxidative damage and single nucleotide polymorphism (SNP) in the mitochondrial genome in brain tissue from 48 pediatric intractable epilepsy patients from Miami Children’s Hospital and 11 control samples from NICHD Brain and Tissue Bank for Developmental Disorders. Epilepsy patients showed higher mtDNA copy number compared to normal health subjects (controls). Oxidative mtDNA damage was lower in non-neoplastic but higher in neoplastic epilepsy patients compared to controls. There was a trend of lower mtDNA oxidative damage in the non-neoplastic (MCD) patients compared to controls, yet, the reverse was observed in neoplastic (MCD and Non-MCD) epilepsy patients. The presence of mtDNA SNP and haplogroups did not show any statistically significant relationships with epilepsy phenotypes. However, SNPs G9804A and G9952A were found in higher frequencies in epilepsy samples. Logistic regression analysis showed no relationship between mtDNA oxidative stress, mtDNA copy number, mitochondrial haplogroups and SNP variations with epilepsy in pediatric patients. The levels of mtDNA copy number and oxidative mtDNA damage and the SNPs G9952A and T10010C predicted neoplastic epilepsy, however, this was not significant due to a small sample size of pediatric subjects. Findings of this study indicate that an increase in mtDNA content may be compensatory mechanisms for defective mitochondria in intractable epilepsy and brain tumor. Further validation of these findings related to mitochondrial genotypes and mitochondrial dysfunction in pediatric epilepsy and MCD may lay the ground for the development of new therapies and prevention strategies during embryogenesis.

Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients

Chronic bronchopulmonary bacterial infections remain the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Recent community sequencing work has now shown that the bacterial community in the CF lung is polymicrobial. Identifying bacteria in the CF lung through sequencing can be costly and is not practical for many laboratories. Molecular techniques such as terminal restriction fragment length polymorphism or amplicon length heterogeneity-polymerase chain reaction (LH-PCR) can provide many laboratories with the ability to study CF bacterial communities without costly sequencing. The aim of this study was to determine if the use of LH-PCR with multiple hypervariable regions of the 16S rRNA gene could be used to identify organisms found in sputum DNA. This work also determined if LH-PCR could be used to observe the dynamics of lung infections over a period of time. Nineteen samples were analysed with the V1 and the V1_V2 region of the 16S rRNA gene. Based on the amplicon size present in the V1_V2 region, Pseudomonas aeruginosa was confirmed to be in all 19 samples obtained from the patients. The V1 region provided a higher power of discrimination between bacterial profiles of patients. Both regions were able to identify trends in the bacterial population over a period of time. LH profiles showed that the CF lung community is dynamic and that changes in the community may in part be driven by the patient's antibiotic treatment. LH-PCR is a tool that is well suited for studying bacterial communities and their dynamics.

Characterizations of the Major Coral Diseases of the Philippines: Ulcerative White Spot Disease and Novel Growth Anomalies of Porites

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

A randomized trial comparing the diagnostic accuracy of visual inspection with acetic acid to Visual Inspection with Lugol's Iodine for cervical cancer screening in HIV-infected women.

Visual inspection with Acetic Acid (VIA) and Visual Inspection with Lugol’s Iodine (VILI) are increasingly recommended in various cervical cancer screening protocols in low-resource settings. Although VIA is more widely used, VILI has been advocated as an easier and more specific screening test. VILI has not been well-validated as a stand-alone screening test, compared to VIA or validated for use in HIV-infected women. We carried out a randomized clinical trial to compare the diagnostic accuracy of VIA and VILI among HIV-infected women. Women attending the Family AIDS Care and Education Services (FACES) clinic in western Kenya were enrolled and randomized to undergo either VIA or VILI with colposcopy. Lesions suspicious for cervical intraepithelial neoplasia 2 or greater (CIN2+) were biopsied. Between October 2011 and June 2012, 654 were randomized to undergo VIA or VILI. The test positivity rates were 26.2% for VIA and 30.6% for VILI (p = 0.22). The rate of detection of CIN2+ was 7.7% in the VIA arm and 11.5% in the VILI arm (p = 0.10). There was no significant difference in the diagnostic performance of VIA and VILI for the detection of CIN2+. Sensitivity and specificity were 84.0% and 78.6%, respectively, for VIA and 84.2% and 76.4% for VILI. The positive and negative predictive values were 24.7% and 98.3% for VIA, and 31.7% and 97.4% for VILI. Among women with CD4+ count < 350, VILI had a significantly decreased specificity (66.2%) compared to VIA in the same group (83.9%, p = 0.02) and compared to VILI performed among women with CD4+ count ≥ 350 (79.7%, p = 0.02). VIA and VILI had similar diagnostic accuracy and rates of CIN2+ detection among HIV-infected women.

Selective serotonin reuptake inhibitor antidepressant use in first trimester pregnancy and risk of specific congenital anomalies: a European register-based study

Evidence of an association between early pregnancy exposure to selective serotonin reuptake inhibitors (SSRI) and congenital heart defects (CHD) has contributed to recommendations to weigh benefits and risks carefully. The objective of this study was to determine the specificity of association between first trimester exposure to SSRIs and specific CHD and other congenital anomalies (CA) associated with SSRI exposure in the literature (signals). A population-based case-malformed control study was conducted in 12 EUROCAT CA registries covering 2.1 million births 1995-2009 including livebirths, fetal deaths from 20 weeks gestation and terminations of pregnancy for fetal anomaly. Babies/fetuses with specific CHD (n = 12,876) and non-CHD signal CA (n = 13,024), were compared with malformed controls whose diagnosed CA have not been associated with SSRI in the literature (n = 17,083). SSRI exposure in first trimester pregnancy was associated with CHD overall (OR adjusted for registry 1.41, 95% CI 1.07-1.86, fluoxetine adjOR 1.43 95% CI 0.85-2.40, paroxetine adjOR 1.53, 95% CI 0.91-2.58) and with severe CHD (adjOR 1.56, 95% CI 1.02-2.39), particularly Tetralogy of Fallot (adjOR 3.16, 95% CI 1.52-6.58) and Ebstein's anomaly (adjOR 8.23, 95% CI 2.92-23.16). Significant associations with SSRI exposure were also found for ano-rectal atresia/stenosis (adjOR 2.46, 95% CI 1.06-5.68), gastroschisis (adjOR 2.42, 95% CI 1.10-5.29), renal dysplasia (adjOR 3.01, 95% CI 1.61-5.61), and clubfoot (adjOR 2.41, 95% CI 1.59-3.65). These data support a teratogenic effect of SSRIs specific to certain anomalies, but cannot exclude confounding by indication or associated factors.