Analysis of HIV-1 expression level and sense of transcription by high-throughput sequencing of the infected cell.

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

Virus infection speeds: theory versus experiment

In order to explain the speed of Vesicular Stomatitis Virus VSV infections, we develop a simple model that improves previous approaches to the propagation of virus infections. For VSV infections, we find that the delay time elapsed between the adsorption of a viral particle into a cell and the release of its progeny has a veryimportant effect. Moreover, this delay time makes the adsorption rate essentially irrelevant in order to predict VSV infection speeds. Numerical simulations are in agreement with the analytical results. Our model satisfactorily explains the experimentally measured speeds of VSV infections

Progress with schistosome transgenesis

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.

A phase I clinical and pharmacological study evaluating vinflunine in combination with doxorubicin as first line treatment in metastatic breast cancer.

Vinfunine (VFL) is a novel bifluorinated tubulin-targeted agent of the vinca alkaloids class active in advanced stage breast cancer. We conducted a phase I study combining VFL with doxorubicin (DXR) to define the recommended dose (RD), safety, pharmacokinetic (PK) interaction and efficacy. Two schedules (day 1 every 3 weeks; days 1 and 8 every 3 weeks) were investigated as first line chemotherapy in metastatic breast cancer patients. Thirty-two patients received a total of 162 cycles of the VFL-DXR combination (median 6). The RDs were VFL 250 mg/m(2)/DXR 40 mg/m(2) every 3 weeks for schedule 1 and VFL 120 mg/m(2)/DXR 25 mg/m(2) days 1 and 8 every 3 weeks for schedule 2. The main dose-limiting toxicity was neutropenia. The most frequent non-hematological adverse events were nausea, fatigue, constipation, vomiting, anorexia, stomatitis and dyspnea. Objective response rate was reached in 47.1% of the patients. No PK interaction was observed. VFL-DXR combination is feasible with manageable toxicity. The antitumor activity was promising and supports further evaluation.

PFAPA syndrome is not a sporadic disease.

OBJECTIVES: To determine whether PFAPA (periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis) patients have a positive family history (FH) for recurrent fever syndromes. METHOD: For all patients with PFAPA seen in two paediatric rheumatology centres (Romandy, Switzerland and Bordeaux, France), parents were interviewed to record the FH for periodic fever. As controls, we interviewed a group of children without history of recurrent fever. RESULTS: We recruited 84 patients with PFAPA and 47 healthy children. The FH for recurrent fever (without an infectious cause and recurring for at least half a year) was positive in 38/84 (45%), and was positive for PFAPA (diagnosis confirmed by a physician) in 10/84 (12%) of the PFAPA patients. For 29 of the 38 patients with positive FH, the affected person was a sibling or a parent. None of the healthy children had a positive FH for recurrent fever or PFAPA. A positive FH for rheumatological diseases was seen in both groups of children. CONCLUSION: These data show that a significant percentage of PFAPA patients present a positive FH of recurrent fever and PFAPA. This familial susceptibility suggests a potential genetic origin for this syndrome.

Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions

The availability of induced pluripotent stem cells (iPSCs)has created extraordinary opportunities for modeling andperhaps treating human disease. However, all reprogrammingprotocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirelydevoid of xenobiotics. We first developed a xeno-free cellculture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derivedprimary cultures of human dermal fibroblasts under strictxeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free humaniPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colonymorphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiationability in vitro and in teratoma assays. Overall, the resultspresented here demonstrate that human iPSCs can be generatedand maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.

The role of antibody concentration and avidity in antiviral protection.

Neutralizing antibodies are necessary and sufficient for protection against infection with vesicular stomatitis virus (VSV). The in vitro neutralization capacities and in vivo protective capacities of a panel of immunoglobulin G monoclonal antibodies to the glycoprotein of VSV were evaluated. In vitro, neutralizing activity correlated with avidity and with neutralization rate constant, a measure of on-rate. However, in vivo, protection was independent of immunoglobulin subclass, avidity, neutralization rate constant, and in vitro neutralizing activity; above a minimal avidity threshold, protection depended simply on a minimum serum concentration. These two biologically defined thresholds of antibody specificity offer hope for the development of adoptive therapy with neutralizing antibodies.

Wear of two artificial tooth materials in vivo: a 12-month pilot study.

STATEMENT OF PROBLEM: Wear of methacrylate artificial teeth resulting in vertical loss is a problem for both dentists and patients. PURPOSE: The purpose of this study was to quantify wear of artificial teeth in vivo and to relate it to subject and tooth variables. MATERIAL AND METHODS: Twenty-eight subjects treated with complete dentures received 2 artificial tooth materials (polymethyl methacrylate (PMMA)/double-cross linked PMMA fillers; 35%/59% (SR Antaris DCL, SR Postaris DCL); experimental 48%/46%). At baseline and after 12 months, impressions of the dentures were poured with improved stone. After laser scanning, the casts were superimposed and matched. Maximal vertical loss (mm) and volumetric loss (mm(3)) were calculated for each tooth and log-transformed to reduce variability. Volumetric loss was related to the occlusally active surface area. Linear mixed models were used to study the influence of the factors jaw, tooth, and material on adjusted (residual) wear values (alpha=.05). RESULTS: Due to drop outs (n=5) and unmatchable casts (n=3), 69% of all teeth were analyzed. Volumetric loss had a strong linear relationship to surface area (P<.001); this was less pronounced for vertical loss (P=.004). The factor showing the highest influence was the subject. Wear was tooth dependent (increasing from incisors to molars). However, these differences diminished once the wear rates were adjusted for occlusal area, and only a few remained significant (anterior versus posterior maxillary teeth). Another influencing factor was the age of the subject. CONCLUSIONS: Clinical wear of artificial teeth is higher than previously measured or expected. The presented method of analyzing wear of artificial teeth using a laser-scanning device seemed suitable.

Skin problems associated with pegylated liposomal doxorubicin-more than palmoplantar erythrodysesthesia syndrome.

Liposomal pegylated doxorubicin is an encapsulation form of doxorubicin, with an improved pharmacokinetic profile and the ability to selectively accumulate into tumor tissue. As a result, the tolerated dose of the drug can be increased, followed by a reduced incidence of neutropenia and cardiotoxicity in comparison to doxorubucin treatment. However, a common adverse dose-schedule limiting effect of the treatment is palmoplantar erythrodysesthesia syndrome. In this retrospective study we included six patients hospitalised in the University Hospital of Zurich during the last 2 years, in connection with side effects caused by pegylated liposomal doxorubicin. These patients received this chemotherapeutic agent for treatment of various malignancies such as breast cancer, ovarian cancer, mycosis fungoides and cutaneous B-cell lymphoma. Three of six patients in this study developed classical palmoplantar erythrodysesthesia, one developed palmoplantar erythrodysesthesia associated with extensive bullous disease, one developed eruption of lymphocyte recovery syndrome and one developed intertrigo like dermatitis with stomatitis. Pegylated liposomal doxorubicin induces various skin reactions including palmoplantar erythrodysesthesia syndrome. However, the exact clinical presentation might depend on pre-existing skin diseases.

Cholesterol and Fatty Acids Regulate Dynamic Caveolin Trafficking through the Golgi Complex and between the Cell Surface and Lipid Bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection.

Isolated primary human cells from different donors vary in their permissiveness-the ability of cells to be infected and sustain the replication of human immunodeficiency virus type 1 (HIV-1). We used replicating HIV-1 and single-cycle lentivirus vectors in a population approach to identify polymorphic steps during viral replication. We found that phytohemagglutinin-stimulated CD4(+) CD45RO(+) CD57(-) T cells from healthy blood donors (n = 128) exhibited a 5.2-log-unit range in virus production. For 20 selected donors representing the spectrum of CD4 T-cell permissiveness, we could attribute up to 42% of the total variance in virus production to entry factors and 48% to postentry steps. Efficacy at key intracellular steps of the replicative cycle (reverse transcription, integration, transcription and splicing, translation, and budding and release) varied from 0.71 to 1.45 log units among donors. However, interindividual differences in transcription efficiency alone accounted for 64 to 83% of the total variance in virus production that was attributable to postentry factors. While vesicular stomatitis virus G protein-mediated fusion was more efficacious than CCR5/CD4 entry, the latter resulted in greater transcriptional activity per proviral copy. The phenotype of provirus transcription was stable over time, indicating that it represents a genetic trait.

Oral tongue squamous cell carcinoma (OTSCC): alcohol and tobacco consumption versus non-consumption. A study in a Portuguese population

There has been an increase in the incidence of carcinoma of the tongue, particularly among alcohol and tobacco non-users. However, the number of studies that would allow a better understanding of etiological factors and clinical features, particularly in the Portuguese population, is very limited. This study was based on patients with squamous cell carcinoma of the anterior two thirds of the tongue that were treated at the Department of Head and Neck Surgery of the ¿Instituto Portugues de Oncologia de Lisboa - Francisco Gentil" IPOLFG) in Lisbon, Portugal, between January 1, 2001 and December 31, 2009. The patients were divided in alcohol and tobacco users and non-users in order to evaluate the differences between these 2 groups based on gender, age, tumor location, denture use, and tumor size, metastasis and stage. Of the 354 cases, 208 were users and 146 were non-users. The main location in both groups was the lateral border of the tongue. Denture use showed no significant effect in both study groups. It was possible to conclude that patients who did not drink or smoke were older and presented with smaller tumor size, lower incidence of ganglion metastasis and lower tumor stage compared with alcohol and tobacco users.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Extraintestinal manifestations in inflammatory bowel disease: frequency and associated risk factors in the nationwide Swiss IBD cohort study (SIBDCS)

Background: Data on the frequency of extraintestinal manifestations (EIM) in Crohnʼs disease (CD) and ulcerative colitis (UC) are scarce. Goal: to evaluate prevalences, forms of EIM and risk factors in a large nationwide IBD cohort. Methods: Data from validated physician enrolment questionnaires of the adult Swiss IBD cohort were analyzed. Logistic regression models were used to identify EIM risk factors. Results: 950 patients were included, 580 (61%) with CD (mean age 43yrs) and 370 (39%) with UC (mean age 49yrs), of these, 249 (43%) of CD and 113 (31%) of UC patients had one to 5 EIM. The following EIM were found: arthritis (CD 33%, UC 21%), aphthous stomatitis (CD 10%, UC 4%), uveitis (CD 6%, UC 4%), erythema nodosum (CD 6%, UC 3%), ankylosing spondylitis (CD 6%, UC 2%), psoriasis (CD 2%, UC 1%), pyoderma gangrenosum (CD and UC each 2%), primary sclerosing cholangitis (CD 1%, UC 4%). Logistic regression in CD identified the following items as risk factors for ongoing EIM: active disease (OR 1.95, 95% CI 1.17-3.23, P=0.01), positive IBD family history (OR 1.77, 95% CI 1.07-2.92, P=0.025). No risk factors were identified in UC patients. Conclusions: EIM are a frequent problem in CD and UC patients. Active disease and positive IBD family history are associated with ongoing EIM in CD patients. Identification of EIM prevalence and associated risk factors may result in increased awareness for this problem and thereby facilitate their diagnosis and management.

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).