Sequence confirmation of the EWS-WT1 fusion gene transcript in the peritoneal effusion of a patient with desmoplastic small round cell tumor

Desmoplastic small round cell tumor (DSRCT) is a rare undifferentiated neoplasm. The prognosis is poor, even if therapy is instituted promptly. and thus it is important to differentiate it from other histologically and cytologically similar-looking malignancies of the young adult. We present a case of DSRCT in a 17-yr-old male with disseminated peritoneal disease and peritoneal effusion. The cytology sample showed a malignant small round cell tumor, the classical cytological features of DSRCT, and immunohistochemistry performed in the prepared cell block exhibited an antibody expression profile in keeping with DSRCT. Further material front the effusion was prepared for RNA extraction, following which a reverse-transcriptase polymerase chain reaction (RTPCR) and sequencing of the t(l l;22)(p13;q11 or q12) were carried out. The result showed the presence of the reciprocal translocation and thus confirmed the diagnosis of DSRCT. This case shows how molecular techniques (including sequencing) call be applied to cytology in clarifying and confirming certain difficult diagnosis of undifferentiated neoplasms, DSRCT in this particular case. Diagn. Cytopathol. 2003;29:341-343. (C) 2003 Wiley-Liss. Inc.

Pharmacokinetics of UC781-loaded intravaginal ring segments in rabbits: a comparison of polymer matrices

UC781 is a potent and poorly water-soluble nonnucleoside reverse transcriptase inhibitor being investi- gated as a potential microbicide for preventing sexual transmission of HIV-1. This study was designed to evaluate the in vivo release and pharmacokinetics of UC781 delivered from matrix-type intravaginal ring segments in rabbits. Three polymer matrices (polyurethane, ethylene vinyl acetate copolymer, and silicone elastomer) and two drug loadings (5 and 15 mg/segment) were evaluated in at least one of two independent studies for up to 28 days in vivo. Inter-study comparison of in vivo release, vaginal tissue, and plasma concentrations for similar formulations demonstrated good reproducibility of the animal model. Mean estimates for a 28-day in vivo release ranged from 0.35 to 3.17 mg UC781 per segment. Mean proximal vaginal tissue levels (adjacent to the IVR segment) were 8– 410 ng/g and did not change significantly with time for most formulations. Distal vaginal tissue levels of UC781 were 6- to 49-fold lower than proximal tissue levels. Mean UC781 plasma levels were low for all formulations, at 0.09–0.58 ng/mL. All formulations resulted in similar UC781 concentrations in vaginal tissue and plasma, except the low loading polyurethane group which provided significantly lower levels. Loading dependent release and pharmacokinetics were only clearly observed for the polyurethane matrix. Based on these results, intravaginal ring segments loaded with UC781 led to vaginal tissue concen- trations ranging from below to approximately two orders of magnitude higher than UC781’s EC50 under in vitro conditions (2.8 ng/mL), with little influence by polymer matrix or UC781 loading. Moreover, these findings support the use of rabbit vaginal pharmacokinetic studies in preclinical testing of microbicide intravaginal rings.

Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia.

Platinum-catalysed intravaginal rings

The present invention provides improved intravaginal drug delivery devices, i.e., intravaginal rings, useful for the prophylactic administration of an antimicrobial compound, e.g., Dapivirine, to a human. The intravaginal rings of the invention address previous stability issues by utilizing a platinum catalyst (e.g., in the form of a platinum-siloxane complex) for the cross-linking reaction. The vaginal rings surprisingly achieve relatively high and steady release rates in vivo with a matrix ring containing a relatively small loading dose. While the matrix rings of the present invention have in vivo the steady release rates associated with reservoir rings, they are easier and less expensive to manufacture. The present invention also provides methods of blocking DNA polymerization by an HIV reverse transcriptase enzyme, methods of preventing HIV infection in a female human, methods of treating HIV infection in a female human, and methods of preparing platinum-catalyzed intravaginal rings.

Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system

Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Effects of oxidized and glycated LDL on gene expression in human retinal capillary pericytes

Modified (oxidized and/or glycated) low-density lipoproteins (LDLs) have been implicated in retinal pericyte loss, one of the major pathologic features of early-stage diabetic retinopathy. To delineate underlying molecular mechanisms, the present study was designed to explore the global effects of modified LDL on pericyte gene expression.

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes:associations with lipoprotein subclasses

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P <.0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells. © The Author 2008. Published by Oxford University Press. All rights reserved.

Preparation of planar retinal specimens:verification by histology, mRNA profiling, and proteome analysis

Elucidation of the transcriptome and proteome of the normal retina will be difficult since it is comprised of at least 55 different cell types. However the characteristic layered cellular anatomy of the retina makes it amenable to planar sectioning, enabling the generation of enriched retinal cell populations. The aim of this study was to validate a reproducible method for preparing enriched retinal layers from porcine retina.

Human Parechovirus Infection in Neonatal Intensive Care

Background: Approximately 5-6% of all infective episodes in NICU are of viral origin. Previous studies suggest that human parechovirus (HPeV) infection presents most commonly in term infants, as a sepsis-like syndrome in which meningoencephalitis is prominent. Our aim was to study the infection rate and associated features of HPeV.

Methods: Blood samples were taken from NICU babies greater than 48 hours old, who were being investigated for late onset sepsis. Clinical and laboratory data were collected at the time of the suspected sepsis episode. Samples were tested using universal primers and probe directed at the 5'-untranslated region of the HPeV genome by reverse transcriptase PCR. Results were confirmed by electrophoresis and DNA sequencing.

Results: HPeV was detected in 11 of 84 samples (13%). These infants had a mean (interquartile range, IQR) gestational age of 28.9 (26.9 - 30.6) weeks and mean birth weight of 1.26 (SD = 0.72) kg. The median day of presentation was 16 (IQR: 11-27). These characteristics were similar to the infants without positive viral detection. Six infants presented with respiratory signs. One infant presented with signs of meningitis. Six of the 11 episodes of HPeV infection occurred during the winter months (December - February). No HPeV positive infants had abnormal findings on their 28-day cranial ultrasound examination.

Conclusions: We found a HPeV infection rate of 13% in infants being tested for late onset sepsis. HPeV should be considered as a possible cause of sepsis-like symptoms in preterm infants.

Narrow-band ultraviolet B exposure increases serum vitamin D levels in haemodialysis patients

Chronic kidney disease (CKD) patients are especially prone to vitamin D insufficiency. Narrow-band ultraviolet B (NB-UVB) treatment increases serum 25-hydroxyvitamin D [25(OH)D] in dermatological patients, and we studied whether it also improves vitamin D balance in CKD patients on haemodialysis.

GDF-15 contributes to proliferation and immune escape of malignant gliomas

Growth and differentiation factor (GDF)-15 is a member of the transforming growth factor (TGF)-beta family. GDF-15 is necessary for the maintenance of pregnancy but has also been linked to other physiologic and pathologic conditions.

Seroepidemiology of norovirus-associated travelers' diarrhea

Background: Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers' diarrhea (TD) in US and European travelers to Mexico, Guatemala, and India.

Methods: Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using reverse transcription-polymerase chain reaction (RT-PCR). Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLPs) to measure serum levels of immunoglobulin A (IgA) and IgG by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA); serum IgM was measured by capture enzyme-linked immunosorbent assay (ELISA), and the 50% antibody-blocking titer (BT50 ) was determined by a carbohydrate-blocking assay.

Results: NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or fourfold or more rise in antibody titer. Significantly more individuals had detectable preexisting IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p = 0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p = 0.0037), IgG (p = 0.003), and BT50 (p = <0.0001). None of the NoV-infected TD travelers had BT50  > 200, a level that has been described previously as a possible correlate of protection.

Conclusion: We found that GI-NoVs are commonly associated with TD cases identified in US adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.