A novel intranuclear RNA vector system for long-term stem cell modification.

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells, while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction.

The effect of complement regulatory protein expression on pig endothelial cells to porcine endogenous retrovirus lyses by human sera.

INTRODUCTION: Expression of human complement regulatory proteins (CRP) on pig endothelial cells (PEC) has been useful to avoid hyperacute rejection by human sera. On the other hand, porcine endogenous retrovirus (PERV) from PEC transfectants with CRP may acquire resistance to human sera. In this study, we investigated the effects of the transfected CRP on PERV neutralization and/or lysis by human sera. METHODS: cDNA of membrane cofactor protein (MCP: CD46), decay accelerating factor (DAF: CD55), and CD59 were transfected to PEC lines by lipofection. The expressions of these CRPs were verified by FACS analysis. The PEC lines with human CRPs were then transfected with the LacZ gene and PERV subtype B (PERV-B) to investigate PERV infectivity by LacZ pseudotype assay. Culture supernates of PEC were inoculated to HEK293 cells with or without 10% human sera. The inoculated 293 cells were then histochemically stained to count the LacZ-positive blue foci and calculated the rate of reduction of LacZ-positive cells by serum. RESULTS: PERV from the PEC with DAF or CD59 showed a resistance to human sera compared with those of control PEC (DAF: 59.6% +/- 5.3%, CD59: 61.1% +/- 3.9% vs control: 31.3% +/- 3.6%; P < .01). However, PEC with MCP did not cause such an effect (28.8% +/- 2.5%). CONCLUSIONS: While expression of DAF and CD59 on PEC changed its PERV responsiveness to human sera, MCP did not improve it.

Regulatory properties of the integrated long terminal repeat of the feline immunodeficiency virus.

To examine the regulatory properties of feline immunodeficiency virus (FIV) long terminal repeat (LTR) integrated into host chromatin, Crandell feline kidney cells were stably transfected with the FIV LTR that directs the bacterial chloramphenicol acetyltransferase (CAT) gene. Using these cells, we examined the effects of treatment with several chemical agents, infection with feline viruses, or transfection with effector plasmids expressing FIV gene products on FIV LTR-directed gene expression. Among them, treatment with the phorbol ester (a strong activator of protein kinase C), forskolin (an inducer of cyclic-AMP), 5-azacytidine (a DNA methylation antagonist), or infection with feline herpesvirus type 1 (FHV-1), resulted in induction of CAT activity in the cells. These results suggest that the integrated FIV LTR is stimulated by cellular transcriptional factors induced by phorbol ester, forskolin and FHV-1, and is also inactivated by DNA methylation. Furthermore, this permanent cell line can be used as a screening system of activators of the FIV LTR.

Antigenic analysis of feline calicivirus capsid precursor protein and its deleted polypeptides produced in a mammalian cDNA expression system.

An entire open reading frame in a cDNA encoding the capsid protein gene of feline calicivirus (FCV) was subcloned into a mammalian expression vector. After transfection of the constructed plasmid (pMCV-II) into COS-7 cells, the expressed protein was detected by indirect immunofluorescence assay (IFA) using a panel of monoclonal antibodies (MAbs) to the capsid protein of FCV. All of the MAbs reacted with the transfected COS-7 cells in IFA. The 76 kDa capsid precursor protein was demonstrated in an immunoblot analysis, indicating that the translated precursor protein was not processed into the matured capsid protein in this expression system. Two in-frame deleted and a frameshift mutated cDNAs were generated by using restriction sites within the capsid protein coding sequence in pMCV-II to analyze the antigenic sites of the protein. The results of IFA using the MAbs and COS-7 cells transfected with the deleted or mutated cDNAs suggested that three neutralizing epitopes had a conformational nature and that the other four linear epitopes were related to 74 amino acid residues between positions 381 and 454 in the protein, in which high variation was known to be present among three strains of FCV.

Detection of feline immunodeficiency virus transcripts by quantitative reverse transcription-polymerase chain reaction.

We developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure to estimate replication status of feline immunodeficiency virus (FIV) in peripheral blood mononuclear cells (PBMCs) of cats. Primers used for the RT-PCR were designed to detect only multiple spliced transcripts of FIV and allowed us to detect the specific transcripts with high specificity. By using limiting-cell-dilution RT-PCR, we demonstrated that the specific transcripts were quantitatively detected in a single infected cell in a background of 1 x 10(6) uninfected cells without Southern blot hybridization. Furthermore, the transcripts were observed efficiently in all PBMCs of the chronically FIV-infected cats when examined by this RT-PCR technique. There results demonstrated that this RT-PCR method is applicable for specific detection of the FIV-specific transcripts in the PBMCs and for estimation of the viral replication status in vivo.

Epitopes and nuclear localization analyses of canine distemper virus nucleocapsid protein by expression of its deletion mutants.

A series of nucleocapsid protein (NP)-deleted genes of the Onderstepoort strain was constructed in order to locate antigenic regions of the NP of canine distemper virus. The expression of proteins from 5'-deleted NP genes was examined in COS-7 cells by indirect immunofluorescence assay using three monoclonal antibodies (MAbs), c-5, f-5 and h-6, and a rabbit serum against NP. These MAbs reacted with two regions of NP. Amino acid residues from 1 to 80, and 337-358, were necessary and sufficient for formation of the epitopes identified by MAbs f-5 and h-6, and c-5, respectively. The proteins translated from intact or 3'-deleted genes were found to be localized in the nuclei of COS-7 cells, whereas the proteins from the 5'-deleted genes were mainly detected in the cytoplasm. These results suggested that 80 amino acid residues at the N-terminus are required for transportation of NP into the nucleus.

Influence of nasal allergic reactions on the clearance of middle ear effusion.

In order to investigate the influence of nasal allergic reactions on the clearance of middle ear effusion, an animal model of nasal allergy and otitis media with effusion was produced in the same guinea pigs simultaneously by passive sensitization with serum of homologous animals containing IgE antibodies (for nasal allergy) and by inoculation of immunocomplex into the tympanic cavity (for otitis media with effusion). Usually, middle ear effusion appeared within 2 to 3 days and disappeared within 7 to 9 days after the inoculation of immunocomplex. Three days after the inoculation of immunocomplex, intranasal antigen challenge was performed three times daily and continued until the animals were killed. Disappearance of middle ear effusion appeared to be delayed in animals in which nasal allergic reactions were induced. Middle ear effusion was not found in those ears that were not inoculated with immunocomplex. Findings of the present study indicate that IgE-mediated allergic reactions of the mucous membrane lining the nose, nasopharynx, and eustachian tube constitute a factor indicative of a chronic state of disease, rather than a cause of otitis media with effusion.

Staphylococcus epidermidis and Staphylococcus aureus in otitis media with effusion.

Bacteriologic investigation of middle ear effusion (MEE), external ear canal, and the nasopharynx was carried out on 458 patients with otitis media with effusion. Staphylococcus epidermidis was the most common bacteria in MEE, even after excluding the contaminants from the external ear canal, which had the same value of minimal inhibitory concentration as the paired MEE. The bacterial agreement of S epidermidis between MEE and the nasopharynx was extremely rare in contrast with Haemophilus influenzae, Streptococcus pneumoniae, and Branhamella catarrhalis, although the organism was also frequently isolated from the nasopharynx. Staphylococcus aureus, having the same minimal inhibitory concentration as that in the nasopharynx, was more frequently found in MEE than S epidermidis. The results suggest that S epidermidis found in MEE is not a pathogen, but rather a contaminant in many instances. Staphylococcus aureus seems to be a causative agent in otitis media with effusion.

Antibodies to Borna disease virus in infected adult rats: an early appearance of anti-p10 antibody and recognition of novel virus-specific proteins in infected animal brain cells.

The time course for appearance of antibodies to Borna disease virus (BDV) major antigens, p40, p24, p18 and p10 were investigated in BDV-inoculated adult rats by Western blotting. Anti-p10 antibodies were detected in sera as early as anti-p40 and -p24 antibodies at four or five weeks after inoculation. Furthermore, in addition to these major antigens of BDV, the rat serum could detect additional 80-, 58-, 43-, 20-, and 16-kDa proteins in BDV-infected cultured cells and/or animal brain cells by Western blot analysis. Of these proteins, the 20- and 16-kDa proteins were shown to be related to p24 protein by their reactivity with anti-p24 monoclonal antibody. Interestingly, the 58- and 24-kDa were found only in BDV-infected animal brain cells but not in cultured cells. The results in this study could provide a useful information on the mechanism for the viral replication and pathogenesis.

Comparative functional analysis of the various lentivirus long terminal repeats in human colon carcinoma cell line (SW480 cells) and feline renal cell line (CRFK cells).

Basal promoter activities of various lentiviral long terminal repeats (LTRs) in a human colon carcinoma cell line (SW480 cells) and a feline renal cell line (CRFK cells) were examined by the chloramphenicol acetyltransferase (CAT) assay using the LTR-CAT reporter plasmids. In SW480 cells, the basal promoter activities induced by LTRs of visna virus, caprine arthritis-encephalitis virus (CAEV), and simian immunodeficiency virus (SIVAGM) were moderate, and those induced by LTRs of human immunodeficiency virus (HIV) type 1 (HIV-1) and HIV type 2 (HIV-2) were low. However, the activity induced by the LTR of feline immunodeficiency virus (FIV) was extremely low. In CRFK cells, the basal promoter activities induced by LTRs of visna virus, CAEV and SIVAGM were relatively high, and those induced by LTRs of HIV-1, HIV-2 and FIV were moderate. From these data, although the structure of the LTR of FIV is reported to be similar to that of visna virus and CAEV, the function of the LTR of FIV is rather quite different from that of the LTR of these viruses.

Quantification of feline immunodeficiency virus (FIV) proviral DNA in peripheral blood mononuclear cells of cats infected with Japanese strains of FIV.

The polymerase chain reaction (PCR) method was applied for measurement of the proviral DNA copy number of feline immunodeficiency virus (FIV) in peripheral blood mononuclear cells (PBMC) of cats experimentally and naturally infected with FIV. In experimentally infected cats except one cat infected with the Petaluma strain, FIV-specific DNAs were efficiently amplified with the PCR method under the conditions used in this study. In the naturally FIV-infected cats, the specific DNAs were also amplified. We established a quantitative method for measurement of proviral DNA copy number in PBMC from cats infected with TM2-type of FIV strains, and found that the number was variable among the six cats examined, ranging from 10(4.0) to 10(5.7) copies per 10(5) PBMCs. This method can be applicable to cats naturally infected with FIV of TM2-type. Proviral DNA quantitation developed here could be useful as an additional parameter to evaluate the relationships among the proviral load, immune response and development of the clinical symptoms, and to monitor efficacy of antiviral therapy in vivo.

Expression of the nucleocapsid protein gene of the canine distemper virus.

We constructed a cDNA clone of canine distemper virus (CDV) encoding an entire nucleoprotein (NP) gene, by means of the reverse transcription-polymerase chain reaction (RT-PCR). The cloned NP gene was inserted into the eucaryotic expression vector, pRVSV. After transfection of the plasmid into Vero cells, we examined the expression of CDV-specific NP antigen by means of indirect immunofluorescence assay (IFA) and Western blotting, using various antibodies against NP of CDV and an antiserum against NP of measles virus. The CDV-NP specific antigen was detected in the nuclei of the cells transfected with pRV-ON, by means of IFA with antibodies specific to the NP.

Comparison of the Rev transactivation of feline immunodeficiency virus in feline and non-feline cell lines.

The Rev protein of feline immunodeficiency virus (FIV) differentially transactivates the expression of viral structural proteins by allowing the accumulation of unspliced and singly spliced viral mRNA in cytoplasm via the Rev response element (RRE) at the end of env. To investigate the role of rev gene of FIV for the virus life cycle and cell tropism, we constructed the Rev expression plasmids, and functional activity of the Rev was assayed by using chloramphenicol acetyltransferase (CAT) assay system in feline and non-feline cell lines. Although the FIV Rev protein showed high transactivity to result in enhanced CAT production in a feline cell line, the productions of the CAT in non-feline cell lines were significantly lower than that in the feline cell line. These results indicate that specific cellular factor(s) present in feline cell line is required for the FIV Rev full-action and also suggest that the Rev action plays one of the important roles in determining the FIV cell tropism.

Persistence of high virus neutralizing antibody titers in cats experimentally infected with feline immunodeficiency virus.

The development of virus neutralizing (VN) antibody is one of the most effective host defense mechanisms against virus infection. In the present study, we developed a new VN assay against feline immunodeficiency virus (FIV) using a feline T-lymphoblastoid cell line, MYA-1 cells, based on inhibition of viral reverse transcriptase production. This assay is applicable to strains of FIV which can not infect CRFK cells. By using the assay, we examined long-term responses of VN antibody in cats experimentally infected with FIV. VN antibody titers increased progressively during first 30 weeks post inoculation and remained at high titers thereafter for 7 years of observation periods.

The molecular cloning and sequence of an open reading frame encoding for non-structural proteins of feline calicivirus F4 strain isolated in Japan.

The nucleotide sequence of the 5'-end of feline calicivirus (FCV) Japanese F4 strain genome was determined. This region had 5311 bases and contained a large open reading frame (ORF1) encoding the non-structural proteins. The nucleotide sequence of the ORF1 region was highly conserved as compared with that of FCV F9 strain. When the deduced amino acid sequence of the ORF1 was compared with those of FCV F9 and CFI strains, the sequence was also highly conserved (88.9% and 88.8%, respectively). Functional motifs of the non-structural proteins were common to these strains. There were 2C polypeptide-, 3C cysteine protease- and 3D RNA-dependent RNA polymerase-like regions. The N-terminal region of 2C-like region continued upstream from the region identified by Neill [Virus Res. 17: 145-160]. Furthermore, the presence of 2B-like region was suggested in the upper stream of the 2C-like region, although the function of the region is unknown. When Kyte and Dolittle hydrophobicity profiles of the predicted amino acid sequences of the ORF1s of FCV F4 and F9 were computed and compared, both the profiles had striking similarities. In the region between residues 950-1000, there was a high rate of basic amino acid residues, suggesting that the polypeptide in this region of FCV may have a nucleic acid-binding function.

Seroepidemiological survey of feline retrovirus infections in cats in Taiwan in 1993 and 1994.

In order to investigate the prevalence of infections with three feline retroviruses feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and feline syncytial virus (FSV) in Taiwan, we collected a total of 75 blood samples from cats from veterinary hospitals, a breeding cattery and a homeless shelter in 1993 and 1994. We examined the presences of anti-FIV and FSV antibodies and FeLV-p27 antigen in these samples by the indirect immunofluorescence and/or enzyme-linked immunosorbent assays. All of the serum samples positive for FIV were obtained from homeless cats and the overall FIV positive rate was 4%. The overall positive rates of FSV and FeLV were 28% and 1.3%, respectively. From these results, together with previous seroepidemiological surveys by others, it was revealed that the prevalence of FIV and FeLV infections appeared to be lower in Taiwan than in the United States or Japan. In contrast, the prevalence of FSV infection in Taiwan was as high as that in Japan.

Establishment of carrier-state infection of a feline renal cell line with feline syncytial virus.

Although feline syncytial virus (FSV) is normally highly cytopathogenic in Crandell feline kidney cells, a non-cytopathic persistent infection was established in the cells after cocultivation of the initially infected cells with uninfected cells 4 times. More than 90% of the persistently infected cells were positive for FSV antigen, and electron microscopy showed that the culture produced morphologically normal FSV. Virus from the carrier culture was infectious, however, the titer of the virus from the culture was lower than that from the cytocidally infected CRFK cells.

Type I allergic reactions of the middle ear and eustachian tube: an experimental study.

To clarify the role of type I allergic reactions in etiology and pathogenesis of otitis media with effusion and to determine whether or not the middle ear is an allergic "shock" organ, we made animal models of nasal allergy in guinea pigs by passive sensitization with serum of homologous animals containing specific IgE antibodies. We also examined the eustachian tube, tympanic cavity (histologically), and tubal function after the induction of type I allergic reactions of the nose. However, the involvement of histologic changes was limited only up to the area near the pharyngeal orifice. The tubal dysfunction evoked by nasal allergic reactions was transient, culminating in no middle ear effusion. Upon direct antigen-challenge into the tympanic cavity, allergic changes were observed in the mucosa lining the tympanic bulla, even though no microscopic effusion was present. Findings of the present study suggest that type I allergic reactions of the nose are not an etiologic factor for otitis media with effusion, although the middle ear is potentially an allergic shock organ.

[Membranous nephropathy in Japanese children].

Since 1973, 15 patients, consisting of 8 boys and 7 girls, were diagnosed as having membranous nephropathy (MN). The average age at detection was 8.2 years (2-14 years). The presenting symptom was edema in 1, pyrexia in 1 and upper respiratory infection in 1 case, in the all other cases, abnormal urinalysis was detected by the school or chance urinalysis. Surface antigen of hepatitis B virus (HBs) was positive in 6 patients and negative in 9. Anti-nuclear antibody (ANA) was positive in 3 and negative in 11. In one patient, ANA was not tested. One patient who was negative for ANA was diagnosed as having SLE 4 years later. At the last follow-up, 10 patients continued to have urinary abnormalities. Among these was one case positive for HBs antigen who went into end-stage renal failure. In the other 14 patients, the serum creatinine level was below 1.4 mg/dl. All patients showed a normal mesangium or mild mesangial proliferation. The patient diagnosed as having SLE. 4 years later showed mesangial deposits at the first renal biopsy. In our experience, most patients with MN were detected by the school or chance urinalysis and six of the these had positive HBs antigen. Lupus nephritis must be ruled out in making a diagnosis of idiopathic MN.

[Henoch-Schönlein purpura nephritis (HSPN) in children: comparison of the incidence and severity between two 12-year groups].

From 1971 to 1982, we treated 286 patients of HSP in Kitasato University Hospital. In these 286 patients, 137 developed purpura nephritis (47.9%). During the second 12-year period (from 1985 to 1996), we treated 34 HSPN patients among 189 HSP patients (18.0%). The clinico-pathological evaluations and comparisons in 30 cases from 1971 to 1982 and in 11 cases from 1985 to 1996 were performed, using the ISKDC grading. The numbers of patients of HSP and the incidence of HSPN both decreased in the more recent 12-year group. Furthermore, the severity of the renal histopathological findings decreased in the more recent group as well.