Uptake dynamics of scrapie agent in the intestinal villous epithelium of suckling and weanling Syrian hamsters

In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrP(Sc) ) decreases significantly after weaning. In this study, the dynamics of PrP(Sc) uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrP(Sc) -positive villous epithelium cells immunohistochemically. The number of PrP(Sc) -positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrP(Sc) -positive cells with increasing age might be a common phenomenon among the superfamily Muridae.     

Transplantation of adipose stromal cells, but not mature adipocytes, augments ischemia-induced angiogenesis

Therapeutic angiogenesis has emerged as a promising therapy to treat patients with ischemic diseases. Transplantation of bone marrow cells (BMCs) is reported to augment collateral development in ischemic organs either by differentiating into vascular cells or by secreting angiogenic cytokines. Recent evidence suggests that adipose tissues secrete a number of humoral factors and contain pluripotent stem cells. Here, we evaluated the therapeutic potential of adipose tissue-derived cells to promote angiogenesis in a mouse model of hind limb ischemia. Stromal vascular fraction cells (SVFs) were isolated from inguinal adipose tissue. Endothelial-like cells or smooth muscle-like cells could be obtained from the culture of SVFs in the presence of growth factors. Freshly isolated BMCs, SVFs, or mature adipocytes were transplanted into the ischemic hind limb of mice. SVFs significantly augmented collateral development as determined by the restoration of blood perfusion and capillary density of the ischemic muscle. Angiogenic effects of SVFs were as potent as those of BMCs. Mature adipocytes showed no proangiogenic effects. The ischemic muscle contained endothelial cells or smooth muscle cells that derived from the transplanted SVFs and BMCs. These results suggest that SVFs might be used to promote angiogenesis in ischemic tissues.     

Prevalence and genetic properties of Salmonella enterica serovar typhimurium definitive phage type 104 isolated from Rattus norvegicus and Rattus rattus house rats in Yokohama City, Japan

Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.     

Construction of recombinant herpes simplex virus type I expressing green fluorescent protein without loss of any viral genes

For use in various applications in research on herpes simplex virus type 1, we attempted to generate recombinant HSV-1 expressing green fluorescent protein (GFP) without any loss of viral genes. Our results were as follows. (i) A recombinant HSV-1 (YK333), in which a GFP expression cassette driven by the Egr-1 promoter was inserted into the intergenic region between UL3 and UL4, was constructed. (ii) YK333 replicated as well as wild-type HSV-1 F strain in Vero cells. (iii) As one application of the recombinant YK333 for research on HSV-1, we developed a system to detect anti-herpetic activity, termed a fluorescence-based anti-viral assay. The 50% inhibitory concentration of ganciclovir for YK333 determined using our newly developed assay was comparable to that determined using a plaque reduction assay. YK333 will be a convenient tool for herpes simplex virus research, including such applications as monitoring of viral replication in vitro and in vivo, and rapid screening of potential anti-herpetic agents.     

Analysis of the factors motivating HCV-infected patients to accept interferon therapy

ABSTRACT: BACKGROUND: The aims of this study were to analyze factors motivating the acceptance of interferon (IFN) therapy and to clarify the prevalence of oral mucosal diseases in hepatitis C virus (HCV)-infected Japanese patients treated with IFN. FINDINGS: A total of 94 HCV-infected patients who were admitted to our hospital for IFN therapy were asked questions regarding their motivation to accept IFN therapy and were investigated for the presence of oral lichen planus (OLP) before and during IFN treatment. Recommendation and encouragement from other people were the most common factors motivating the acceptance of IFN therapy (49/94, 52.13%). The other motivators were independent decision (30.85%), economic reasons (5.32%), and others. According to multivariate analysis, three factors - sex (male), retreatment after previous IFN therapy, and independent decision to accept IFN therapy - were associated with patients after curative treatment of hepatocellular carcinoma (HCC). The adjusted odds ratios for these three factors were 26.06, 14.17, and 8.72, respectively. The most common oral mucosal lesions included OLP in 11 cases (11.70%). One patient with OLP had postoperative squamous cell carcinoma of the tongue. The rate of sustained virological response (SVR) was 45.45% in cases with OLP and 54.55% in cases without OLP. There were no patients who discontinued IFN therapy because of side effects such as oral mucosal diseases. CONCLUSIONS: We should give full explanation and recommend a course of treatment for a patient to accept IFN therapy. The system to support liver disease as well as oral diseases is also necessary for patient treated for IFN therapy.     

Transmission of simian immunodeficiency virus carrying multiple cytotoxic T-lymphocyte escape mutations with diminished replicative ability can result in AIDS progression in rhesus macaques

Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations that result in escape from CTL recognition with viral fitness costs. The replication in vivo of such viruses carrying not single but multiple escape mutations in the absence of the CTL pressure has remained undetermined. Here, we have examined the replication of simian immunodeficiency virus (SIV) with five gag mutations selected in a macaque possessing the major histocompatibility complex haplotype 90-120-Ia after its transmission into 90-120-Ia-negative macaques. Our results showed that even such a "crippled" SIV infection can result in persistent viral replication, multiple reversions, and AIDS progression.     

Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells

Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.     

Increased expression of highly sulfated keratan sulfate synthesized in malignant astrocytic tumors

Keratan sulfate (KS) proteoglycans are expressed on a subpopulation of microglia in normal adult brain. We previously showed the up-regulated expression of KS in one of glioblastoma cell lines using anti-KS antibody (5D4). However, it has not been clarified whether KS is expressed in brain tumors and is involved in their malignancy. In this study, 54 astrocytic tumors were investigated about KS-expression using Western-blot with 5D4. In six of 14 anaplastic astrocytomas (43%) and 23 of 34 glioblastomas (68%), KS was detected by 5D4. KS was hardly detected by 5D4 in diffuse astrocytoma, suggesting that KS-expression is significantly expressed in malignant astrocytic tumors. In immunohistochemistry, KS is highly expressed in cell surface of malignant astrocytic tumors. Taken together, KS might be associated with the malignancy of astrocytic tumors, and be useful for a prognostic factor of astrocytic tumors.     

Pigment Epithelium-derived Factor (PEDF) Ameliorates Advanced Glycation End Product (AGE)-induced Hepatic Insulin Resistance In Vitro by Suppressing Rac-1 Activation

Advanced glycation end products (AGEs) could be implicated in insulin resistance. However, the molecular mechanisms underlying this are not fully understood. Since pigment epithelium-derived factor (PEDF) blocks the AGE-signaling pathways, we examined here whether and how PEDF improves insulin resistance in AGE-exposed hepatoma cells, Hep3B cells. Proteins were extracted from Hep3B cells, immunoprecipitated with or without insulin receptor substrate-1 (IRS-1) antibodies, and subjected to Western blot analysis. Glycogen synthesis was measured using [ (14)C]-d-glucose. AGE induced Rac-1 activation and increased phosphorylation of IRS-1 at serine-307 residues, JNK, c-JUN, and IkappaB kinase in association with decreased IkappaB levels in Hep3B cells. PEDF or overexpression of dominant negative Rac-1 blocked these effects of AGE on Hep3B cells. Further, AGEs decreased tyrosine phosphorylation of IRS-1, and subsequently reduced the association of p85 subunit of phosphatidylinositol 3-kinase with IRS-1 and glycogen synthesis in insulin-exposed Hep3B cells, all of which were inhibited by PEDF. Our present study suggests that PEDF could improve the AGE-elicited insulin resistance in Hep3B cells by inhibiting JNK- and IkappaB kinase-dependent serine phosphorylation of IRS-1 via suppression of Rac-1 activation. PEDF may play a protective role against hepatic insulin resistance in diabetes.     

Accumulation of MAC387+ macrophages in paracortical areas of lymph nodes in rhesus monkeys acutely infected with simian immunodeficiency virus

We investigated the histological features of lymph nodes, focusing on monocytes/macrophages, in rhesus monkeys (Macaca mulatta) acutely infected with simian immunodeficiency virus (SIV). In monkeys infected with a pathogenic SIV, SIVmac239, MAC387(+) newly blood-derived macrophages markedly increased in number at paracortical areas at 11 to 14 days postinoculation, concomitant with the peak of the primary SIV antigenemia. The MAC387(+) macrophages densely gathered around high endothelial venules and formed cell clusters with CD3(+) T lymphocytes, tingible body macrophages, and plasmacytoid monocytes. In the cell clusters, CD3(+) T lymphocytes which closely adhered to the MAC387(+) macrophages enlarged in size, suggesting a histological manifestation of T-lymphocyte activation by macrophages. By 54 days postinoculation, when SIV antigenemia became undetectable, the MAC387(+) macrophages decreased in number and the cell cluster disappeared from paracortical areas. In contrast, the monkeys infected with a nef-deleted mutant of SIVmac239 showed lower levels of SIV antigenemia and lower numbers of MAC387(+) macrophages in paracortical areas than those infected with SIVmac239. These results indicate that MAC387(+) macrophages accumulate in paracortical areas for the period of the intense primary SIV antigenemia and may play an important role in activating naive T lymphocytes.