Search PubMed⌕ Search

Biomedical subjects

R C Kennedy

Publications and source records attributed to R C Kennedy.

At least 37 records · Page 2Linked to original sources
PubMed1998

Anatomy of mammalian conjunctival lymphoepithelium.

Ocular surface immune mechanisms are subservient to the fine function of the eye. A clear cornea with a smooth, well-lubricated facade is prerequisite to lucid vision. Hence, corneal inflammation and post-inflammatory scarring are intolerable, and the cornea contains a minimum of lymphoid elements. Although conjunctival dysfunction and consequent tear film deficiency can malign the corneal surface, conjunctival inflammation is tolerated to a considerable degree. In contrast to the human cornea, human conjunctiva contains an abundance of lymphoid tissue. Certain aspects of human conjunctival immunology elicit little debate. Langerhans cells are abundant in conjunctival epithelium. Isolated CD8+ suppressor/cytotoxic T cells predominate in conjunctival epithelium, while T cells in the substantia propria distribute equally between CD4+ T helper cells and CD8+ cells. Yet the presence of plasma cells in human conjunctiva, the expression of secretory component by human conjunctival epithelium, and the function of human conjunctival lymphoid follicles are in dispute. Confusion may derive in part from the use of inappropriate animal models; rodent conjunctiva does not appear to be a worthy facsimile for human conjunctiva. Discrepancies between different human studies likely result from variance in subject age, biopsy site and extent, histologic or histochemical technique, and perhaps the degree of inflammation present at the time of biopsy. Careful immunohistochemical and in situ molecular assays on well-defined loci within the conjunctiva of comparable human subjects may resolve such questions in the future. Organized mucosa-associated lymphoid tissue is rigorously defined as mucosal lymphoid follicles with an ultrastructurally distinct overlying lymphoepithelium. Based on available evidence, the epithelium overlying mammalian conjunctival lymphoid follicles does not contain distinct M cells. Whether zonal differences in morphology reflect real differences in the capacity to sample tear film antigens for presentation to the mucosal immune system remains to be established.

Animals↗
PubMed1998

Sequence comparisons of non-human primate HIV-1 coreceptor homologues.

Infection of non-human primate peripheral blood mononuclear cells (PBMCs) in vitro with primary human immunodeficiency virus type 1 (HIV-1) isolates is extremely inefficient and often unattainable. The mechanism of resistance to infection by primary HIV-1 isolates in chimpanzee and baboon PBMCs is unknown. In this study, two HIV-1 coreceptors, CCR5 and CXCR4, were sequenced from chimpanzee and baboon PBMCs to determine if any sequence variations or mutations in these genes could be responsible for resistance to HIV infection. Primers were designed from the human coreceptor sequences and were able to amplify the CCR5 and CXCR4 genes from these non-human primate cells. No 32 base pair deletion (delta32) mutations were found in any of the non-human primate samples tested. CXCR4 sequence analysis showed chimpanzee and baboon share 99.7 and 98% nucleotide sequence homology and 100 and 98.9% amino acid sequence homology, respectively, compared to the human sequence. CCR5 sequence analysis demonstrated that chimpanzee and baboon share 99.6 and 98% nucleotide homology and 100 and 98% amino acid homology, respectively, with the human sequence. These data indicate that no variations in these coreceptor gene sequences exist that can explain the lack of susceptibility to infection with primary HIV-1 isolates in non-human primate PBMCs.

Amino Acid Sequence↗
PubMed1998

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen.

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.

Animals↗
PubMed1998

Cross-clade inhibition of human immunodeficiency virus type 1 primary isolates by monoclonal anti-CD4.

A murine monoclonal antibody (MAb) with human CD4 specificity was tested for the ability to inhibit primary human immunodeficiency virus type 1 (HIV-1) isolates clades A through E. Human peripheral blood mononuclear cells (PBMC) were used as target cells for infectivity. The HIV-1 primary isolates were examined for the capacity to infect PBMC targets in the presence or absence of the anti-CD4 MAb, designated P1. P1 broadly inhibited clade A, C, D, and E isolates, based on a reduction of HIV-1 p24 antigen concentrations compared with untreated controls. Little to no virus-inhibiting activity was observed with a primary HIV-1 clade B isolate, designated BZ167. Additionally, a second primary clade B isolate was efficiently inhibited from infecting PBMC targets by P1. The data indicate that P1 exhibits group-specific inhibiting activity against non-clade B primary HIV-1 isolates in vitro.

Animals↗
PubMed1998

T cell syncytia induced by HIV release. T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber.

A chemotaxis chamber has been developed to analyze both the velocity and the directionality of individual T cells in gradients of high molecular mass molecules over long periods of time. Employing this chamber, it is demonstrated that syncytia induced by HIV in SUP-T1 cell cultures release two T cell chemoattractants with approximate molecular masses of 30 and 120 kDa. Neither uninfected single cells nor polyethylene glycol-induced syncytia release detectable chemoattractant, suggesting that these chemoattractants are linked to HIV infection. Soluble gp120 functions as a T cell chemoattractant and the addition of anti-gp120 antibody to syncytium-conditioned medium blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. The addition of anti-CD4 antibody to syncytium-conditioned medium also blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. These results demonstrate that HIV-induced T cell syncytia release a low and a high molecular mass T cell chemoattractant, and suggest that the high molecular mass factor is gp120 and that it functions through the CD4 receptor.

CD4 Antigens↗
PubMed1998

Immunotherapy of SV40 induced tumours in mice: a model for vaccine development.

Various vaccination strategies were compared for their ability to elicit antigen-specific tumour immunity, using the SV40-BALB/c murine tumour system. Specifically, mice were injected with baculovirus-derived recombinant SV40 Tag (rTag), synthetic peptides corresponding to B cell epitopes on SV40 Tag or a plasmid DNA construct encoding the gene for SV40 Tag. In vivo tumour immunity was determined by a lethal tumour challenge with syngeneic SV40-transformed tumour cells. SV40 Tag-specific antibody titres were induced in mice immunized with rTag or Tag synthetic peptides. Partial tumour protection was observed in mice that were immunized with SV40 Tag peptides, where as complete tumour immunity was observed in mice immunized with rTag. Although protective tumour immunity was also observed in mice immunized with DNA, negligible levels of antibodies to SV40 Tag were detected. Examination of the cytotoxic T lymphocyte (CTL) activity in mice injected with the SV40 Tag-DNA construct revealed Tag-specific lysis of syngeneic SV40-transformed tumour cells. Conversely, little to no CTL activity was detected in mice immunized with rTag. However, antigen-specific antibodies from rTag immunized mice were capable of mediating antibody-dependent cell-mediated cytotoxicity against SV40-transformed cells. These data indicate that the immune mechanisms elicited for protection against SV40 induced tumours in mice appeared to be dependent on the vaccination strategy employed and included both humoral and cell-mediated immune responses.

Animals↗
PubMed1997

Purification and characterization of secretory IgA from baboon colostrum.

In this report, we describe a method for purifying secretory immunoglobulin A (sIgA) from baboon (Papio anubis) colostrum. The colostrum was first clarified by centrifugation and then analyzed with various anti-human Ig-specific immunologic reagents. Cross-reactive IgA in the baboon colostrum was identified by ELISA. Western blot analysis also demonstrated cross-reactive epitopes associated with human IgA1, IgA2, secretory component (SC), and joining (J) chain. To purify the sIgA, colostrum was separated into 4 distinct fractions by gel filtration chromatography. Analysis of the individual fractions by ELISA indicated that the IgA elutes over one peak. The IgA fraction was compared with purified human sIgA on SDS-PAGE, and exhibited heavy (H) chains, light (L) chains, SC, and J chain. The baboon colostrum was also analyzed by ELISA for specific IgG H and L chain epitopes utilizing monoclonal antibodies (MAbs). No significant quantity of IgG was detected in the baboon colostrum or in the individual 4 fractions, while L chain reactivity was observed in the sIgA fraction. The sIgA fraction was pooled, concentrated, and was found to contain approximately 7 mg/ml sIgA. To determine if the baboon sIgA was dimeric like human sIgA, the purified sIgA was sized by molecular sieve chromatography. The molecular size of the sIgA preparation (350 kDa) was determined empirically by comparison to known molecular species used to calibrate the column. In addition, native SDS-PAGE indicated that baboon sIgA, like human sIgA, migrates between IgG and IgM, suggesting it has a dimeric form. The purified baboon sIgA preparation should prove useful in the future study of mucosal immune responses induced in non-human primate species and for the generation of sIgA-specific immunological reagents.

Animals↗
PubMed1997

Development of an experimental murine pulmonary metastasis model incorporating a viral encoded tumor specific antigen.

A SV40 murine tumor model was developed and characterized involving intravenous inoculation of BALB/c mice with syngeneic SV40-transformed kidney fibroblasts (mKSA cells). Following intravenous inoculation with mKSA cells, viable tumor cells were recovered from primary organ cell culture of the brain, spleen, lungs, and kidneys of tumor bearing mice. The presence of mKSA tumor cells in these tissues was confirmed by morphological identification and by immunofluorescence directed to SV40 large tumor antigen (T-ag). Additionally, a computer assisted method was used to enumerate and quantitate the size of tumor foci. Tumor foci were observed in the lungs and were quantifiable based on both size and number. The number and size of foci observed in the lungs of tumor bearing mice was dependent on the dose of mKSA cells and time post-inoculation. Ultimately, the tumor burden in inoculated mice was found to be lethal. Quantification of tumor foci in the lung, survival data, and detection of metastasis to organs at sites distal to tumor cell inoculation, provides specific reference points for use in examining the mechanism(s) of the immune response to tumors expressing viral antigen and in evaluating immunologic based therapies within this new SV40 murine tumor model. The methods described herein can be applied for the development of new animal models of metastasis that express viral encoded tumor-specific antigens.

Animals↗
PubMed1997

Nonhuman primate models to evaluate vaccine safety and immunogenicity.

When considering preclinical studies to evaluate the safety and immunogenicity of putative vaccine candidates, such as nucleic acid vaccines, species most closely related to humans should be considered. Phylogenetically, the great apes (chimpanzees, orangutans, gorillas, and gibbons) are most closely related to humans. However, the great apes, which diverged from humans over 5 million years ago, represent endangered or threatened species that limits their utility in preclinical studies. In addition, cost considerations for using great apes in biomedical studies represents another serious limitation. The Old World monkeys, (macaques, baboons, mandrills, and mangabeys), diverged from humans over 15 million years ago. A number of the Old World monkey species including rhesus, cynomolgus, and African green monkeys, have also been employed in biomedical research to evaluate vaccine safety and immunogenicity. New World monkeys (aotus, owl, cebus monkeys, and marmosets) are the most phylogenetically divergent from humans, yet they have also been utilized to develop nonhuman primate models for a number of human infectious diseases and tumors. The advantages and disadvantages in selecting a particular nonhuman primate species for studies to evaluate DNA vaccine safety and immunogenicity are briefly discussed. Comparative immunology, reproductive physiology, endogenous infectious agents, and cost considerations are briefly described.

Animals↗
PubMed1997

Differential display to identify and isolate novel genes expressed during spermatogenesis.

Spermatogenesis is a complex differentiation process in which diverse stage-specific proteins are co-ordinately expressed. Previously, subtractive hybridization and differential hybridization have been used in the identification of differentially expressed mRNAs. Although these techniques have been successfully used they require large amounts of RNA and are time consuming. To overcome these problems we have made use of the recently described mRNA differential display technique. The technique is an effective method which can identify and separate cDNAs that are differentially expressed between various cell-types. By comparing RNA from testes of mature (> 60 days old) and prepubertal (15-16 days old) mice we have identified nine differential cDNA bands expressed in mature testes. The differential display cDNA band DDC8 was used screen a testis cDNA library and the full length cDNA was isolated and sequenced. DDC8 cDNA is 1965 bp with an open reading frame of 533 amino acids which codes for a predicated hydrophilic protein with a calculated molecular weight of 62.04 kDa. RNase protection assays indicate DDC8 to be expressed during the postmeiotic stages of spermatogenesis and database searches using both nucleotide and amino acid sequences show DDC8 to have similarities to structural, cytoskeletal and associated proteins.

Amino Acid Sequence↗
PubMed1997

The baboon as a nonhuman primate model for assessing the effects of maternal immunization with Haemophilus influenzae type b polysaccharide vaccines.

These studies were performed to assess the utility of the baboon as a nonhuman primate model to evaluate vaccines for use in humans. Specifically, we examined the antibody response of baboons immunized during the third trimester of pregnancy with Haemophilus influenzae type b (Hib) polyribosylribitol phosphate (PRP) conjugate and unconjugated polysaccharide vaccines. Some of the vaccinated mothers failed to respond to a single immunization with unconjugated Hib PRP. Specific Hib PRP immunoglobulin G (IgG) but not IgM antibodies cross the baboon placenta and are detected in the offspring. Higher-titer baboon anti-Hib PRP did not express two previously defined cross-reactive human anti-Hib PRP idiotypes and was biased towards lambda light-chain expression. Spectrotype analysis indicated that baboon anti-Hib PRP was restricted in heterogeneity and oligoclonal.

Animals↗
PubMed1996

Titration of a vaccine stock preparation of human immunodeficiency virus type 1SF2 in cultured lymphocytes and in chimpanzees.

A large stock preparation of the HIV-1SF2 isolate has been derived after serial passage in human peripheral blood mononuclear cells (PBMCs). This viral stock has a titer of 10(4.9) TCID50 in human PBMCs and 10(4.2) TCID50 in chimpanzee PBMCs. By inoculation into animals the 50% chimpanzee infectious dose titer was found to be about 10(2.3). Virus isolation from animals was achieved on most occasions within 1-4 weeks after inoculation and then became transient. Viral RNA and DNA PCR analyses confirmed the virus infection of the chimpanzees. Anti-HIV antibody levels in the inoculated animals ranged from 1:400 to 1:6400 as measured by ELISA. About 680 vials of this stock preparation, frozen at -190 degrees C, are available for future studies of vaccines and antiviral therapies.

AIDS Vaccines↗
PubMed1996

Characterization of baboon class I major histocompatibility molecules. Implications for baboon-to-human xenotransplantation.

Increasingly strong medical and political pressures are stimulating consideration of the transplantation of baboon organs and cells into humans. Critical to the success of these xenotransplants is management of the immune system such that graft rejection and, in the case of bone marrow transplantation, graft-versus-host disease do not result in transplant failure. The polymorphic products of the major histocompatibility complex (MHC) are the primary barrier to successful allotransplantation, and here we describe class I MHC molecules from baboon (Papio anubis) to gain an understanding of how similarities and differences between baboon and human MHC molecules might affect xenograft survival and function. Comparative analyses of our five novel baboon class I molecules with defined HLA class I molecules demonstrate that the baboon class I molecule are up to 90% identical. Disparity between baboon class I proteins and their human homologues lies predominately at positions in the antigen-binding groove, while C-terminal portions of the class I heavy chain are more conserved between the two species. Such concentration of cross-species differences within the alpha1 and alpha2 domains involves a majority of substitutions at positions demonstrating polymorphism in human alleles; the location of substitutions distinguishing baboon and human molecules thus resembles the positioning of human class I allopolymorphisms. Because this preliminary characterization indicates that both baboon and human T cells with be restricted by xenogeneic class I molecules, immune responses triggered during baboon-to-human transplantation should mimic those arising during MHC mismatched human allotransplantation.

Amino Acid Sequence↗
PubMed1996

Protection against a lethal challenge with SV40-transformed cells by the direct injection of DNA-encoding SV40 large tumor antigen.

Plasmid DNA encoding the large tumor antigen (T- ag) of SV40 was used to actively immunize mice to assess the induction of SV40 T-ag-specific immunity. Mice were injected with the naked DNA i.m., and immune responses were compared to those elicited in mice immunized with the recombinant SV40 T-ag protein. Compared to immunization with the recombinant protein, naked DNA induced weak antibody responses to SV40 T-ag. No increase in natural killer cell activity was observed following either recombinant protein or nucleic acid vaccination. However, the recombinant SV40 T-ag failed to induce SV40 T-ag-specific CTL responses, whereas the plasmid DNA encoding SV40 T-ag elicited CTL activity specific for SV40 T-ag. The SV40 T-ag-specific CTL lysed in vitro only syngeneic target cells (H-2(d)) expressing SV40 T-ag, indicating that the CTL are MHC restricted. Both the recombinant protein and naked DNA preparations induced immune responses that were protective against a lethal challenge with syngeneic SV40-transformed cells. A comparison of recombinant protein versus nucleic acid immunization indicates that both humoral and cell-mediated immune responses may play a role in SV40 T-ag immunity. These data indicate that active immunization with genes encoding tumor-specific antigens may be an efficacious strategy for the induction of tumor immunity.

Animals↗
PubMed1996

Nuclei number in human embryos co-cultured with human ampullary cells.

A study was undertaken to evaluate the effect on nuclei number in human embryos cultured in vitro with primary cell lines of human Fallopian tube epithelium. The development of 203 surplus human embryos, cultured in standard culture medium (Earle's balanced salt solution + 15% A5) with or without ampullary cells, was observed from day 2 to day 5.5 post-insemination. The expanded blastocysts in both culture conditions were analysed for nuclei numbers per blastocyst. Embryos transferred to co-culture at the 2-cell stage had an average of 120.7 nuclei per blastocyst, which was significantly higher than the average of 62.9 nuclei per blastocyst (P = 0.023) for the embryos transferred to co-culture at the 4-cell stage. The embryos cultured in the control medium had an average of 42.1 nuclei per blastocyst, which was significantly less than co-cultured embryos (P = 0.04). Severely fragmented embryos (grades 3 and 4) did not show recovery in co-culture. Our results show that when human embryos are transferred to co-cultures before the 4-cell stage, the blastulation rate and the cell number per embryo increase significantly compared to the embryos cultured in standard culture medium. The possible effect of co-culture on embryonic gene expression is discussed.

Adult↗
PubMed1996

Mode of action of a lysostaphin-like bacteriolytic agent produced by Streptococcus zooepidemicus 4881.

Electron microscopy of zoocin A-treated sensitive streptococcus cells revealed cytoplasmic disruption and ultimately complete rupture of the cell wall. Culture viability and optical density were shown to decrease rapidly and simultaneously in Streptococcus pyogenes FF22 but less quickly in the relatively more resistant Streptococcus mutans 10449. Zoocin A was shown to cleave hexaglycine in a colorimetric cell-free microtiter assay system, and it is concluded that the killing action of zoocin A, like that of lysostaphin, is most probably the result of direct cleavage of the peptidoglycan cross-links in the cell wall. The relationship between sensitivity to zoocin A and the peptidoglycan cross-linkage structure of Streptococcus zooepidemicus, Lactococcus spp., S. pyogenes, Streptococcus gordonii, Streptococcus oralis, S. mutans, and Streptococcus rattus has been evaluated.

Anti-Bacterial Agents↗