Clinicopathologic and molecular analysis of a choroidal pigmented schwannoma in the context of a PTEN hamartoma tumor syndrome.

PURPOSE: To report the first case of choroidal schwannoma in a patient affected by PTEN hamartoma tumor syndrome (PHTS) and investigate the molecular involvement of the phosphatase and tensin homolog (PTEN) and neurofibromin 2 (NF2) genes in this rare intraocular tumor. DESIGN: Observational case report. PARTICIPANT: A 10-year-old girl diagnosed with PHTS. METHODS: The enucleated specimen underwent histologic, immunohistochemical, and transmission electronic microscopy. The expression of PTEN and NF2 and their protein products were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. Somatic mutations of PTEN and NF2, as well as allelic loss, were investigated by direct sequencing of DNA extracted from the tumor. PTEN epigenetic silencing was investigated by pyrosequencing. MAIN OUTCOME MEASURES: Histopathologic and molecular characterization of a choroidal pigmented schwannoma. RESULTS: Histopathologic, immunohistochemical, and electron microscopic analysis demonstrated features consistent with a pigmented cellular schwannoma of the choroid. We found no loss of heterozygosity at the genomic level for the PTEN germline mutation and no promoter hypermethylation or other somatic intragenic mutations. However, we observed an approximate 40% reduction of PTEN expression at both the mRNA and the protein level, indicating that the tumor was nonetheless functionally deficient for PTEN. Although DNA sequencing of NF2 failed to identify any pathologic variants, its expression was abolished within the tumor. CONCLUSIONS: We report the first description of a pigmented choroidal schwannoma in the context of a PHTS. This rare tumor showed a unique combination of reduction of PTEN and absence of NF2 expression.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

Evaluation of peanut genotypes for resistance to Tomato spotted wilt virus by mechanical and thrips inoculation

The objective of this work was to evaluate the reactions of three peanut breeding lines (IC-10, IC-34, and ICGV 86388) to Tomato spotted wilt virus (TSWV) by mechanical and thrips inoculation, under greenhouse conditions, and compare them to the reactions of cultivars SunOleic, Georgia Green, and the breeding line C11-2-39. TSWV infection by mechanical inoculation was visually assessed using an index ranging from 0 (no symptoms) to 4 (apical death). Enzyme-linked immunosorbent assay was used to confirm TSWV infection from both mechanical and thrips inoculations. IC-10, IC-34, ICGV 86388, and C11-2-39 were more resistant than the cultivars SunOleic and Georgia Green based on mechanical inoculation. Upon thrips inoculation only IC-34 and ICGV-86388 were infected by TSWV, as demonstrated by reverse transcription polymerase chain reaction (RT-PCR), although no symptoms of infection were observed. The peanut breeding lines IC-10, IC-34, and ICGV 86388 show higher level of resistance to TSWV than cultivar Georgia Green considered a standard for TSWV resistance.

Respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults.

BACKGROUND: The epidemiology of respiratory viruses and their potential clinical impact when recovered in lower respiratory specimens has not been established in the hospital setting. A study was performed to investigate the association between positive viral detection and respiratory infection in an at-risk population. METHODS: 299 adult patients who underwent bronchoalveolar lavage (BAL) procedures were enrolled in a hospital-based prospective cohort study. Descriptive epidemiology is presented of 17 different respiratory viruses detected by reverse transcription-polymerase chain reaction assays in BAL fluid specimens. Multivariate analysis was conducted to identify the clinical characteristics independently associated with the presence of virus. RESULTS: Of 522 BAL fluid specimens analysed, 81% were collected in adult transplant recipients or other immunocompromised patients. Overall, PCR assays identified viral nucleic acid in 91 BAL fluid samples (17.4%). Similar rates of virus-positive BAL fluid were found in the different subpopulations studied (p = 0.113). Coronaviruses were the most frequent (32.3%), followed by rhinovirus (22.6%), parainfluenza (19.5%), influenza (9.7%), respiratory synctial virus (8.6%), human metapneumovirus (4.2%) and bocavirus (3.1%). Multivariate analysis using mixed models showed that respiratory viral infections were associated with a lack of antibiotic treatment response (OR 2.2, 95% CI 1.2 to 4.1) and the absence of radiological infiltrate (OR 0.3, 95% CI 0.2 to 0.8). In lung transplant recipients in whom a respiratory infection was suspected, the respiratory viral detection rate was 24.4% compared with 13.8% overall in other patients (p = 0.02). CONCLUSIONS: In this cohort of hospitalised adults, respiratory viruses detected in BAL fluid specimens were associated with respiratory symptoms, absence of radiological infiltrates and a poor response to antibiotic therapy.

Wound-healing gene family expression differences between fetal and foreskin cells used for bioengineered skin substitutes.

For tissue engineering, several cell types and tissues have been proposed as starting material. Allogenic skin products available for therapeutic usage are mostly developed with cell culture and with foreskin tissue of young individuals. Fetal skin cells offer a valuable solution for effective and safe tissue engineering for wounds due to their rapid growth and simple cell culture. By selecting families of genes that have been reported to be implicated in wound repair and particularly for scarless fetal wound healing including transforming growth factor-beta (TGF-beta) superfamily, extracellular matrix, and nerve/angiogenesis growth factors, we have analyzed differences in their expression between fetal skin and foreskin cells, and the same passages. Of the five TGF-beta superfamily genes analyzed by real-time reverse transcription-polymerase chain reaction, three were found to be significantly different with sixfold up-regulated for TGF-beta2, and 3.8-fold for BMP-6 in fetal cells, whereas GDF-10 was 11.8-fold down-regulated. For nerve growth factors, midkine was 36-fold down-regulated in fetal cells, and pleiotrophin was 4.76-fold up-regulated. We propose that fetal cells present technical and therapeutic advantages compared to foreskin cells for effective cell-based therapy for wound management, and overall differences in gene expression could contribute to the degree of efficiency seen in clinical use with these cells.

Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

Enhanced vascular contractility in alpha1-adrenergic receptor-deficient mice.

AIM: Alpha1-adrenergic receptors (alpha1-ARs) are classified into three subtypes: alpha1A-AR, alpha1B-AR, and alpha1D-AR. Triple disruption of alpha1A-AR, alpha1B-AR, and alpha1D-AR genes results in hypotension and produces no contractile response of the thoracic aorta to noradrenalin. Presently, we characterized vascular contractility against other vasoconstrictors, such as potassium, prostaglandin F2alpha (PGF(2alpha)) and 5-hydroxytryptamine (5-HT), in alpha1A-AR, alpha1B-AR, and alpha1D-AR triple knockout (alpha1-AR triple KO) mice. MAIN METHODS: The contractile responses to the stimulation with vasoconstrictors were studied using isolated thoracic aorta. KEY FINDINGS: As a result, the phasic and tonic contraction induced by a high concentration of potassium (20 mM) was enhanced in the isolated thoracic aorta of alpha1-AR triple KO mice compared with that of wild-type (WT) mice. In addition, vascular responses to PGF(2alpha) and 5-HT were also enhanced in the isolated thoracic aorta of alpha1-AR triple KO mice compared with WT mice. Similar to in vitro findings with isolated thoracic aorta, in vivo pressor responses to PGF(2alpha) were enhanced in alpha1-AR triple KO mice. Real-time reverse transcription-polymerase chain reaction analysis and western blot analysis indicate that gene expression of the 5-hydroxytryptamine 2A (5-HT(2A)) receptor was up-regulated in the thoracic aorta of alpha1-AR triple KO mice while the prostaglandin F2alpha receptor (FP) was unchanged. SIGNIFICANCE: These results suggest that loss of alpha1-ARs can lead to enhancement of vascular responsiveness to the vasoconstrictors and may imply that alpha1-ARs and the subsequent signaling regulate the vascular responsiveness to other stimulations such as depolarization, 5-HT and PGF(2alpha).

Role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients.

BACKGROUND: Rhinovirus is the most common cause of respiratory viral infections and leads to frequent respiratory symptoms in lung transplant recipients. However, it remains unknown whether the rhinovirus load correlates with the severity of symptoms. OBJECTIVES: This study aimed to better characterize the pathogenesis of rhinoviral infection and the way in which viral load correlates with symptoms. STUDY DESIGN: We assessed rhinovirus load in positive upper respiratory specimens of patients enrolled prospectively in a cohort of 116 lung transplant recipients. Rhinovirus load was quantified according to a validated in-house, real-time, reverse transcription polymerase chain reaction in pooled nasopharyngeal and pharyngeal swabs. Symptoms were recorded in a standardised case report form completed at each screening/routine follow-up visit, or during any emergency visit occurring during the 3-year study. RESULTS: Rhinovirus infections were very frequent, including in asymptomatic patients not seeking a specific medical consultation. Rhinovirus load ranged between 4.1 and 8.3 log copies/ml according to the type of visit and clinical presentation. Patients with highest symptom scores tended to have higher viral loads, particularly those presenting systemic symptoms. When considering symptoms individually, rhinovirus load was significantly higher in the presence of symptoms such as sore throat, fever, sputum production, cough, and fatigue. There was no association between tacrolimus levels and rhinovirus load. CONCLUSIONS: Rhinovirus infections are very frequent in lung transplant recipients and rhinoviral load in the upper respiratory tract is relatively high even in asymptomatic patients. Patients with the highest symptom scores tend to have a higher rhinovirus load.

COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen) did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR), and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells.

Inducible nitric oxide synthase (iNOS) production of nitric oxide (NO) has been mostly associated with so-called nitrosative stress or interaction with superoxide anion. However, recent investigations have indicated that, as for the other isoenzymes producing NO, guanylyl cyclase (GC) is a very sensitive target of iNOS activity. To further investigate this less explored signaling, the NO-cyclic guanosine 3'-5'-monophosphate (NO-cGMP)-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation on serine 239 was investigated in human embryonic kidney 293 cells (HEK cells). First, the expression and activity of alpha2 and beta1 NO-sensitive GC subunits was determined by Western blot analysis, reverse transcription-polymerase chain reaction and NO donors administration. Then, the expression of a functional cGMP-dependent protein kinase I (PKGI) was verified by addition of 8-Br-cGMP followed by determination of phosphorylation of VASP on serine 239. Finally, iNOS activation of this signaling pathway was characterized after transfection of HEK cells with human iNOS cDNA. Altogether our data show that iNOS-derived NO activates endogenous NO-sensitive GC and leads to VASP phosphorylation in HEK cells.

Pandemic influença A (H1N1) infection in pregnant and nonpregnant women in Spain (2009-2010)

The present study aimed to compare the main features of infection with pandemic influenza A virus in pregnant and nonpregnant women admitted to hospitals in Spain during the first waves of the 2009-2010 influenza pandemic. This was a prospective (November 2009 to June 2010), multicenter observational study. All cases were women of reproductive age who had not been vaccinated against seasonal or pandemic influenza A. Influenza infection was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The sociodemographic and clinical data of all cases were reviewed. A total of 219 inpatients, including 49 pregnant women and 170 nonpregnant women, were enrolled in the study upon admission to participating hospitals. The most substantially different symptoms between the groups were respiratory distress and unilobar consolidation, both of which were more frequent among nonpregnant women. Antibiotics and systemic corticosteroids were more frequently used in nonpregnant women; however, there were no differences in the rates of treatment with antivirals. Our findings indicated that the compared with nonpregnant women, pregnant women in this study did not have significantly different symptoms and were not at increased risk of complications from pandemic influenza virus infection.

Serological and virological surveys of the influenza A viruses in Antarctic and sub-Antarctic penguins

To evaluate the avian influenza virus (AIV) circulation in Antarctic and sub-Antarctic penguins we carried out a serosurvey on six species from Livingston, Marion and Gough islands. Seropositivity against AIV was performed on serum samples using a competitive enzyme-linked immunosorbent assay and haemagglutination and neuraminidase inhibition assays. Some oropharyngeal and cloacal swabs were also assayed to detect influenza virus genomes by real time reverse transcription-polymerase chain reaction. Overall, 12.1% (n = 140) penguins were seropositive to AIV. By species, we detected 5% (n = 19) and 11% (n = 18) seroprevalence in sub-Antarctic rockhopper penguins (Eudyptes spp.) from Gough and Marion islands, respectively, 42% (n = 33) seroprevalence in macaroni penguins (Eudyptes chysolophus Brandt), but no positives in the three other species, gentoo (Pygoscelis papua Forster; n = 25) and chinstrap penguins (P. antarctica Forster; n = 16), from Livingston Island and king penguins (Aptenodytes patagonicus Miller; n = 27) from Marion Island. While seropositivity reflected previous exposure to the AIV, the influenza genome was not detected. Our results indicate that AIV strains have circulated in penguin species in the sub-Antarctic region, but further studies are necessary to determine the precise role that such penguin species play in AIV epidemiology and if this circulation is species (or genus) specific.

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.