Successful Introduction of Human Renovascular Units into the Mammalian Kidney.

Background: Cell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in formation of vascular networks.

Evaluating the synergistic neutralizing effect of anti-botulinum oligoclonal antibody preparations.

Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease.

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge.

Chimeric vaccine composed of viral peptide and mammalian heat-shock protein 60 peptide protects against West Nile virus challenge.

The protective efficacy and immunogenicity of a chimeric peptide against West Nile virus (WNV) was evaluated. This virus is the aetiological agent of West Nile fever, which has recently emerged in the western hemisphere. The rapid spread of WNV throughout North America, as well as the constantly changing epidemiology and transmission of the virus by blood transfusion and transplantation, have raised major public-health concerns.

Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA.

Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein.

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations.

A live attenuated West Nile virus strain as a potential veterinary vaccine.

This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody.

Development of RT-PCR for turkey meningoencephalitis virus and partial sequence analysis of the NS5 gene.

Turkey meningoencephalitis virus (TMEV) causes paralysis and mortality in turkeys. Because the classical diagnostic methods are complicated, we developed the RT-PCR as a new molecular diagnostic method. Since the nucleic acid sequence of TMEV is unknown, the first step in developing the RT-PCR relied on conserved sequences of viruses belonging to the Flaviviridae family, in which TMEV has been classified serologically.

PCR Identification and Differentiation of Baboon Cytomegalovirus From Other Human and Nonhuman Primate Cytomegalovirus.

Background: Differential identification of baboon alpha, beta, and gamma herpesviruses is an essential technology in order to monitor xenozoonotic transmission of baboon viruses to foreign species organ and/or cell recipients. We present polymerase chain reaction techniques that will differentiate known baboon cytomegaloviruses (CMV) from their closely related counterparts found in humans. Methods and Results: Polymerase chain reaction techniques for identification of the known beta herpesviruses commonly present in the baboon are reported.

Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice.

The neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan.

Divergent envelope E2 alphavirus sequences spanning amino acids 297 to 352 induce in mice virus-specific protective immunity and antibodies with complement-mediated cytolytic activity.

We have proposed a general algorithm for identification of potential immunoprotective domains (cassettes) on the envelope E2 polypeptide of alphaviruses (H. Grosfeld, B. Velan, M.

Adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant and aluminium hydroxide on neutralizing antibody, antibody-isotype and delayed-type hypersensitivity responses to Semliki Forest virus in mice.

Outbred mice were inoculated subcutaneously with inactivated Semliki Forest virus (SFV) in saline and combinations of the virus with complete Freund's adjuvant (CFA) aluminium hydroxide (Al) and dimethyl dioctadecyl ammonium bromide (DDA). The immune response was evaluated for delayed-type hypersensitivity, for total ELISA antibodies and antibody-isotypes and for neutralizing antibodies. DDA was the most efficient adjuvant in inducing DTH, CFA the second and Al induced a DTH response that was only slightly higher (statistically not significant) than that induced by the inactivated virus without adjuvants.

Protection by dehydroepiandrosterone in mice infected with viral encephalitis.

Dehydroepiandrosterone (DHEA) has a significant protective effect in mice infected with West Nile virus (WNV), Sindbis virus neurovirulent (SVNI) and Semliki Forest virus (SFV). Mice injected subcutaneously (SC) with a single injection of DHEA (1 g/kg) on the same day or one day pre or post infection with WNV resulted in 40-50% mortality as compared to 100% in control injected mice (p less than 0.05).

Serologically defined linear epitopes in the E2 envelope glycoprotein of Semliki Forest virus.

A set of 41 overlapping peptides, representing the complete sequence of SFV-E2 protein were synthesized and analyzed in the ELISA test against murine anti-SFV sera. No single peptide was recognized by all antisera. Eight peptides were found to be highly reactive with hyperimmune anti-SFV sera.

Semliki Forest virus E2 envelope epitopes induce a nonneutralizing humoral response which protects mice against lethal challenge.

Along the 422 amino acids of the Semliki Forest virus (SFV) E2 envelope glycoprotein, we identified 13 peptide cassettes (ranging in size from 15 to 25 amino acids and designated A through N) that contain hydrophilic sequences flanked by amino acid sequences conserved in the E2 envelopes of the alphavirus family. Six peptide blocks containing either a single cassette or two to three contiguous cassettes (A, BC, DE, FG, HIK, and LMN) were produced in Escherichia coli as recombinant proteins fused to the N terminus of beta-galactosidase. All of the SFV E2 recombinant polypeptides except A-beta-galactosidase were recognized on Western blots (immunoblots) by anti-SFV polyclonal antisera.

Determination of human IgG and IgM class antibodies to West Nile virus by enzyme linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) was developed and used for the detection of IgG and IgM antibodies to West Nile virus in human sera. Thirteen paired sera of clinical cases and 24 control sera taken randomly from a blood bank repository were tested. The sera were reacted in microtiter plates coated with PEG-treated WNV antigen.

Control of protein synthesis in Semliki forest virus-infected cells.

Protein synthesis in Semliki forest virus-infected chicken embryo cells was studied by labeling them with [35S]methionine for short periods at different times after infection, with or without synchronization of protein synthesis by the hypertonic block technique. The rate of host-cell protein synthesis declined almost linearly in inverse correlation to the increase in the amount of virus specific RNA. At 5.

Sequential translation of nonstructural proteins in cells infected with a Semliki Forest virus mutant.

Four nonstructural proteins with apparent molecular weights of 70,000 (ns-70), 86,000 (ns-86), 78,000 (ns-78), and 60,000 (ns-60) were translated in cells infected with Semliki Forest virus ts-1 mutant and maintained at the restrictive temperature. After synchronization of the initiation of protein synthesis these proteins were synthesized in the above order, suggesting that they are translated as a polyprotein starting from one initiation site. Two short-lived intermediates with apparent molecular weights of 155,000 and 135,000 were regularly detected.

Transitional control in Semliki forest virus infected cells.

The cistron of the 42 S RNA, which codes for other than structural proteins, is poorly translated in the middle of the growth cycle. The translational control operating in the infected cells is not expressed in cell-free protein synthesising systems programmed with the 42 S RNA, since mostly non-structural proteins are made in vitro. One of our temperature-sensitive mutants of SFV, ts-1, directs the synthesis of two non-structural proteins with molecular weights of 78,000 (ns-78) and 86,000 (ns-86) but only at the restrictive temperature.

Tryptic peptide analysis on nonstructural and structural precursor proteins from Semliki Forest virus mutant-infected cells.

Analysis of [35S]methionine-labeled tryptic peptides of the large proteins induced by temperature-sensitive mutants of Semliki Forest virus was carried out. The 130,000-molecular-weight protein induced by ts-2 and ts-3 mutants contained the peptides of capsid protein and of both major envelope proteins E1 and E2. The ts-3-induced protein with molecular weight of 97,000 contained peptides of the capsid and envelope protein E2 but not those of E1.

Replication of Semliki Forest virus.

Replication of Semliki Forest virus, a typical alphavirus, takes place in the cytoplasm of many eukaryotic cells. The virus genome, the 42 S RNA, directs the synthesis of at least two RNA-dependent RNA polymerases. By the aid of these enzymes complementary 45 S RNA is synthesized; it serves as a template for the synthesis of positive RNA strands with sedimentation values of 45 S and 26 S.