Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus

Summary.  We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats. Received January 14, 2000 Accepted August 4, 2000     

Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts

BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.     

An abnormal signal transduction pathway in CD4-CD8- double-negative lymph node cells of MRL lpr/lpr mice.

We describe here a novel 26-kDa posphoprotein (p26), associated to the T cell receptor of CD4-CD8- lymph node cells of lpr/lpr mice, but not present in significant amounts in control mouse cells including polyclonally activated T cells. lpr p26 is constitutively phosphorylated on a tyrosyl residue. It is most likely a member of the G protein family and displays high GTP-binding and GTPase activities both unsensitive to interleukin 2. Bordetella pertussis toxin has no effect on the spontaneously enhanced GTP hydrolysis. The traits associated to p26 could contribute to the distinctive features of lpr/lpr double-negative cells.     

Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance

Therapeutic application of broadly reactive anti-T cell antibodies can lead not only to potent immunosuppression but also to profound and long-lived T cell depletion. We reasoned that a strategy that almost exclusively targets activated cytopathic donor reactive T cells and spares immunoregulatory networks might prove to be an exceptionally potent and highly selective means of producing long-term engraftment and tolerance. Herein we show that the combined administration of rapamycin and agonist IL-2- and antagonist IL-15-related cytolytic fusion proteins provides for long-term engraftment/tolerance in exceptionally stringent allotransplant models by (1) limiting the early expansion of activated T cells, (2) preserving and even exaggerating their subsequent apoptotic clearance, and (3) further amplifying the depletion of these activated T cells by antibody-dependent mechanisms, while (4) preserving CD4+CD25+ T cell-dependent immunoregulatory networks.     

Autologous primary muscle-derived cells transfer into the lower urinary tract

The goal of these experiments was to establish the basic methodology for future clinical applications of muscle-derived cells (MDC) tissue engineering and gene transfer for the treatment of urological dysfunction. Primary MDC isolated via preplating techniques from adult female SD rats were transduced with retrovirus encoding the expression of beta-galactosidase reporter gene. The MDC were injected into the right and left lateral walls of the bladder and proximal urethra of the autologous animals (n = 6) with a 10 microl Hamilton micro syringe. The amount of injected MDC ranged from 1 to 2 x 10(6) cells. The injected tissue was harvested after 7, 14, and 28 days, sectioned and examined histologically for beta-galactosidase and immunohistochemically for fast myosin heavy chain specific to skeletal muscle. The tissues were also stained for anti-CD4 and anti-CD8 antibodies to assess for cellular immune reaction. We have detected a large number of autologous MDC expressing beta-galactosidase and positively stained for fast myosin heavy chain in the bladder and urethral wall. Many injected myoblasts and myotubes were also seen in the bladder and urethral wall at each time point. Staining of lymphocytes with anti-CD4 and anti-CD8 antibodies was negative after MDC injection at each time point. We have demonstrated the long-term survival of autologous MDC and MDC mediated gene transfer into the bladder and urethral wall. Autologous MDC and MDC mediated gene transfer may be a promising treatment to augment bladder and urethral sphincter function.     

IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF THYROGLOBULIN IN HUMAN FOLLICULAR ADENOMA

An electron microscopic immunohistochemical localization of thyroglobulin (TG) using PAP methods has been made in 15 cases of cold follicular adenoma. All cases of follicular adenoma showed organ specific functions such as synthesis, storage, reabsorption, and hydrolysis of thyroglobulin except for an area composed of follicular cells with trabecular arrangement. Immuno-reaction product for TG was precisely demonstrated in follicular lumina, subapical vesicles and reabsorbed colloid droplets. The reaction product observed in the follicular lumen was clearly demarcated from the cytoplasm of the follicular cells by the apical plasma membrane. The subapical vesicles ranging approximately from 50 mμ to 300 mμ in diameter were rarely observed in follicular adenoma and some of them fused with the reabsorbed colloid droplets. The reabsorbed colloid droplets usually had the intense reaction product and hydrolyzed colloid droplets had a vacuole containing floccular low electron dense materials. There is no reaction product in rough endoplasmic reticulum and Golgi complexes.