Recently, tetherin has been identified as an effective cellular factor that prevents the release of human immunodeficiency virus type 1. Here, we show that the production of virus-like particles induced by viral matrix proteins of Lassa virus or Marburg virus was markedly inhibited by tetherin and that N-linked glycosylation of tetherin was dispensable for this antiviral activity. Our data also suggest that viral matrix proteins or one or more components that originate from host cells are targets of tetherin but that viral surface glycoproteins are not. These results suggest that tetherin inhibits the release of a wide variety of enveloped viruses from host cells by a common mechanism.

More than two decades have passed since genetically modified HIV was used for gene delivery. Through continuous improvements these early marker gene-carrying HIVs have evolved into safer and more effective lentiviral vectors. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production. Accordingly, lentivector technologies now have widespread use in basic biology and translational studies for stable transgene overexpression, persistent gene silencing, immunization, in vivo imaging, generating transgenic animals, induction of pluripotent cells, stem cell modification and lineage tracking, or site-directed gene editing. Moreover, in the present high-throughput ‘-omics’ era, the commercial availability of premade lentiviral vectors, which are engineered to express or silence genome-wide genes, accelerates the rapid expansion of this vector technology. In the present review, we assess the advances in lentiviral vector technology, including basic lentivirology, vector designs for improved efficiency and biosafety, protocols for vector production and infection, targeted gene delivery, advanced lentiviral applications and issues associated with the vector system.

The main objective of this review was to assess the role of dairy cattle and their products in human infections with Shiga toxin-producing Escherichia coli (STEC). A large number of STEC strains (e.g., members of the serogroups O26, O91, O103, O111, O118, O145, and O166) have caused major outbreaks and sporadic cases of human illnesses that have ranged from mild diarrhea to the life-threatening hemolytic uremic syndrome. These illnesses were traced to O157 and non-O157 STEC. In most cases, STEC infection was attributed to consumption of ground beef or dairy products that were contaminated with cattle feces. Thus, dairy cattle are considered reservoirs of STEC and can impose a significant health risk to humans. The global nature of food supply suggests that safety concerns with beef and dairy foods will continue and the challenges facing the dairy industry will increase at the production and processing levels. In this review, published reports on STEC in dairy cattle and their products were evaluated to achieve the following specific objectives: 1) to assemble a database on human infections with STEC from dairy cattle, 2) to assess prevalence of STEC in dairy cattle, and 3) to determine the health risks associated with STEC strains from dairy cattle. The latter objective is critically important, as many dairy STEC isolates are known to be of high virulence. Fecal testing of dairy cattle worldwide showed wide ranges of prevalence rates for O157 (0.2 to 48.8%) and non-O157 STEC (0.4 to 74.0%). Of the 193 STEC serotypes of dairy cattle origin, 24 have been isolated from patients with hemolytic uremic syndrome. Such risks emphasize the importance and the need to develop long-term strategies to assure safety of foods from dairy cattle.

Nuclear reprogramming of somatic tissue enables derivation of induced pluripotent stem (iPS) cells from an autologous, non-embryonic origin. The purpose of the current study was to establish efficient protocols for lineage-specification of human iPS cells into functional glucose-responsive, insulin-producing progeny. We generated human iPS cells, which were then guided with recombinant growth factors that mimic the essential signaling for pancreatic development. Reprogrammed with four stemness factors, human fibroblasts were here converted into authentic iPS cells. Under feeder-free conditions, fate-specification was initiated with activin A and Wnt3a that triggered engagement into definitive endoderm, followed by priming with FGF10 and KAAD-cyclopamine. Addition of retinoic acid, boosted by the pancreatic endoderm inducer indolactam V (ILV), yielded pancreatic progenitors expressing PDX1, NGN3 and NEUROD1 markers. Further guidance, under IGF-1, HGF and DAPT, was enhanced by glucagon like peptide-1 (GLP-1) to generate islet-like cells that expressed pancreas-specific markers including insulin and glucagon. Derived progeny demonstrated sustained expression of PDX1, and functional responsiveness to glucose challenge secreting up to 230 pM of C-peptide. A pancreatogenic cocktail enriched with ILV/GLP-1 offers a proficient means to specify human iPS cells into glucose-responsive hormone-producing progeny, refining the development of a personalized platform for islet-like cell generation.

Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet, iPS generation from patients with type 2 diabetes (T2D) has not been demonstrated. Here, we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4, SOX2, KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions, reprogrammed cells underwent dedifferentiation with mitochondrial restructuring, induction of endogenous pluripotency genes - including NANOG, LIN28, and TERT, and down-regulation of cytoskeletal, MHC class I- and apoptosis-related genes. Notably, derived iPS clones acquired a rejuvenated state, characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors, including Indolactam V and GLP-1, redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus, in elderly T2D patients, reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications.

Nuclear reprogramming of adult somatic tissue enables embryo-independent generation of autologous, patient-specific induced pluripotent stem (iPS) cells. Exploiting this emergent regenerative platform for individualized medicine applications requires the establishment of bioequivalence criteria across derived pluripotent lines and lineage-specified derivatives. Here, from individual patients with type 1 diabetes (T1D) multiple human iPS clones were produced and prospectively screened using a battery of developmental markers to assess respective differentiation propensity and proficiency in yielding functional insulin (INS)-producing progeny. Global gene expression profiles, pluripotency expression patterns, and the capacity to differentiate into SOX17- and FOXA2-positive definitive endoderm (DE)-like cells were comparable among individual iPS clones. However, notable intrapatient variation was evident upon further guided differentiation into HNF4α- and HNF1β-expressing primitive gut tube, and INS- and glucagon (GCG)-expressing islet-like cells. Differential dynamics of pluripotency-associated genes and pancreatic lineage-specifying genes underlined clonal variance. Successful generation of glucose-responsive INS-producing cells required silencing of stemness programs as well as the induction of stage-specific pancreatic transcription factors. Thus, comprehensive fingerprinting of individual clones is mandatory to secure homogenous pools amenable for diagnostic and therapeutic applications of iPS cells from patients with T1D.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus originally identified in human prostate cancers (33). Small numbers of XMRV-infected cells have been observed in prostatic stromal cells but not in prostate carcinoma (33). Another study identified XMRV proviral DNA in 6 and 23% of prostate tumors when analyzed by real-time PCR and immunostaining, respectively (27). While initial studies associated XMRV almost exclusively in men who were homozygous for a variant of RNase L (R462Q), which is known to have reduced antiviral activity (33), more recent work failed to link XMRV infection and RNase L mutation (4). XMRV has also been reported in patients with chronic fatigue syndrome (CFS) (17). A total of 67% of CFS patients were positive for XMRV proviral DNA, whereas only 3.7% of healthy subjects were positive for XMRV. Subsequent testing by several other groups found no evidence of infection with XMRV in CFS patients or in healthy controls (30). In Europe, no XMRV was detected in 139 prostate cancer patients in an Irish cohort (4), while no or very few XMRV-specific DNA, RNA, or antibodies were detected in Germany or the United Kingdom cohort of CFS (7, 10, 34).These conflicting data make it unclear to what degree XMRV infects humans and whether it plays a role in human diseases. If an etiological link is confirmed, detection and prevention of XMRV would provide novel intervention strategies for early diagnosis and treatment of both diseases. Moreover, since XMRV or XMRV-specific antibodies were detected in apparently healthy subjects, it would be critical to monitor XMRV contamination in clinical products for transfusion and transplantation.For a better understanding of XMRV transmission, tissue tropism, and pathogenicity, studies of XMRV infection in animal models are crucial. Laboratory mice have provided important small animal model systems for many human diseases, due to their availability, size, low cost, ease of handling, and fast reproduction rate, and extensive studies have been carried out in mice to study the pathogenesis of closely related murine leukemia viruses (MLVs) (5,11,20,23,32). However, studies of XMRV pathogenesis in a mouse model have been hampered by the lack of functional receptor for XMRV in standard laboratory mice derived from Mus musculus species.XMRV is closely related to xenotropic MLVs (X-MLVs) (33). The X-MLVs and polytropic MLVs (P-MLV) use Xpr1 as a receptor for cell entry (1, 31, 37), and so does XMRV (6, 13, 36). Xpr1 has four known variant receptor alleles in mice, Xpr1 n , Xpr1 sxv , Xpr1 c , and Xpr1 p , and each has a different susceptibility for P-MLV and/or X-MLV (35). P-MLV uses Xpr1 n as receptor and most cells from Mus musculus laboratory mice express this receptor (35). Wild mice of the Eurasian genus Mus, such as Mus dunni, express the Xpr1 sxv allele and are susceptible to both P-MLV and X-MLV, whereas the Asian mouse species Mus castaneus expresses Xpr1 c and is susceptible only to X-MLV (19). Mus pahari is another Asian wild mouse species. T...

SUMMARYStreptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1) has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of β-cells in damaged islets.