Biomedical subjects

R B Markham

Publications and source records attributed to R B Markham.

At least 19 recordsLinked to original sources

PubMed1996

Conditional reduction of human immunodeficiency virus type 1 replication by a gain-of-herpes simplex virus 1 thymidine kinase function.

The development of an effective vaccine for human immunodeficiency virus type 1 (HIV-1) would be a major advance toward controlling the AIDS pandemic. Several disparate strategies for a safe and effective HIV vaccine have been proposed. Recent data suggest that loss-of-function live-attenuated virus could be a safe lentivirus vaccine. Here, we propose a gain-of-function approach that can complement loss-of-function in enhancing the safety profile of a live-attenuated virus. We describe an example in which ganciclovir (GCV) was used to treat effectively nef(-)HIV-1 engineered to express herpes simplex virus (HSV-1) thymidine kinase (TK). This treatment was found to be highly efficient in controlling HIV-1 spread in tissue culture and in a small animal (hu-PBL-SCID) model. We demonstrate that one distinct advantage of GCV-HSV-TK treatment is the elimination of integrated proviruses, a goal not easily achieved with other antiretrovirals.

AIDS Vaccines↗

PubMed1996

Selective transmission of human immunodeficiency virus type 1 variants to SCID mice reconstituted with human peripheral blood monoclonal cells.

The relative infectiousness of laboratory and primary human immunodeficiency virus type 1 (HIV-1) variants was evaluated in in vitro cell cultures of peripheral blood mononuclear cells or MT-2 cells and in Hu-PBL-SCID mice. HIV(MN) and syncytium-inducing primary isolates were preferentially transmitted to cells in tissue culture. HIV(Ba-L) and non-syncytium-inducing (NSI) primary isolates were more infectious in Hu-PBL-SCID mice. Phylogenetic analysis of env sequences derived from the primary isolates, from the cell cultures, and from five Hu-PBL-SCID mice was performed by using methods designed for resolving differences among closely related sequence pairs. This analysis demonstrated preferential transmission of an evolutionarily related subset of NSI variants to Hu-PBL-SCID mice. The pattern of selective transmission of a restricted range of NSI variants that is observed in the clinical setting is maintained in Hu-PBL-SCID mice and not in tissue culture systems. The Hu-PBL-SCID mouse model system, when used with appropriate phylogenetic analysis methodologies, will be useful for identifying and characterizing the more infectious HIV-1 variants that should be targeted for vaccine development.

Amino Acid Sequence↗

PubMed1995

Human immunodeficiency virus type 1 env and p17gag sequence variation in polymerase chain reaction-positive, seronegative injection drug users.

Variation in gene sequences from human immunodeficiency virus type 1 (HIV-1) p17gag and the third hypervariable region (V3 loop) of env was examined in a cross-sectional study of single specimens from 4 HIV-1 polymerase chain reaction-positive, seronegative injection drug users. As observed in sexually transmitted disease, clones of HIV-1 genes isolated from an individual were remarkably homogeneous, with amino acid variation in the envelope region of 0.3%-0.9% and in p17gag of 0.0%-0.7%. Intersubject variation was much greater (at the amino acid level, 15% for the envelope and 4.3% for p17gag), with conservation in the envelope only of regions at the base of the V3 loop. Since at least some subjects likely acquired infection intravenously, the ability of virus to survive in seminal fluid or on mucosal surfaces cannot explain the observed homogeneity, which may depend on direct interactions between the virus and the infected cell.

Amino Acid Sequence↗

PubMed1995

Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization.

Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin-labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic.

Bacteremia↗

PubMed1994

Maternal IgG1 and IgA antibody to V3 loop consensus sequence and maternal-infant HIV-1 transmission.

Maternal-infant transmission of HIV-1 occurs in 13-40% of pregnancies. Studies on transmission of maternal immunity to HIV antigens have used antigens from viruses not representative of clinical isolates and have been conflicting. Using a consensus peptide sequence based on HIV isolates found in Haiti, we found that Haitian mothers who transmitted infection to their offspring had significantly higher mean concentrations of IgG1 antibodies to the V3 loop of the primary neutralising domain of the viral envelope (gp 160) than non-transmitters (p = 0.02). Concentrations of IgA antibody to this domain were similar in transmitters and non-transmitters.

Amino Acid Sequence↗

PubMed1993

A model of human immunodeficiency virus encephalitis in scid mice.

Human immunodeficiency virus (HIV)-associated dementia complex is a common and devastating manifestation of the late phases of HIV infection. The pathogenesis of dementia complex is poorly understood and effective treatments have not been developed, in part because of the lack of an appropriate animal model. Mice with severe combined immunodeficiency (scid mice), which accept xenografts without rejection, were intracerebrally inoculated with human peripheral blood mononuclear cells and HIV. One to 4 weeks after inoculation, the brains of these mice contained human macrophages (some of which were HIV p24 antigen positive), occasional multinucleated cells, and striking gliosis by immunocytochemical staining. Human macrophages also were frequently positive for tumor necrosis factor type alpha and occasionally for interleukin 1 and VLA-4. Cultures of these brains for HIV were positive. Generally, human macrophages were not present in the brains of control mice, nor was significant gliosis, and HIV was not recovered from mice that received HIV only intracerebrally. Pathologically, this model of HIV encephalitis in scid mice resembles HIV encephalitis in humans and the data suggest that the activation of macrophages by infection with HIV results in their accumulation and persistence in brain and in the development of gliosis. This model of HIV encephalitis should provide insights into the pathogenesis and treatment of this disorder.

AIDS Dementia Complex↗

PubMed1993

Biologic effects after a single dose of poly(I):poly(C12U) in healthy volunteers.

Poly(I):poly(C12U) (mismatched double-stranded RNA; atvogen), an interferon inducer, is active against human immunodeficiency virus in vitro. To determine the extent and duration of the biologic effects of poly(I):poly(C12U), we administered a single dose of the drug to healthy volunteers in a randomized, double-blind, placebo-controlled 2-week crossover study. We analyzed blood for alpha and gamma interferons, neopterin, 2',5'-oligoadenylate synthetase, lymphocyte surface markers, lymphocyte proliferation after exposure to soluble antigens and mitogens, and natural killer cell activity. Minimal biologic effects were observed after administration of a single 200-mg dose to four volunteers; therefore, the dose was increased to 600 mg in 10 subjects. Only neopterin levels and symptoms were greater after administration of 600 mg of poly(I):poly(C12U) than after administration of placebo (Wilcoxon signed rank sum test, P = 0.06). A definite response in 2',5'-oligoadenylate synthetase activity, however, was seen in a few subjects. Neither alpha nor gamma interferon was detectable in serum after poly(I):poly(C12U) dosing. The neopterin changes after administration of poly(I):poly(C12U) were similar at both poly(I):poly(C12U) dose levels, with an early decrease at 6 h, a peak at 1 day, and a gradual decrease toward the baseline over the following 3 days. A mild flu-like syndrome occurred in one-half of the subjects following administration of poly(I):poly(C12U) and in only one subject following administration of placebo. This syndrome resolved within 16 h after poly(I):poly(C12U) dosing. We conclude that poly(I):poly(C12U) does not induce measurable levels of interferon and causes only minimal biologic or toxic effects among those parameters measured after administration of a single dose in the 200- to 600-mg dose range in health volunteers.

2',5'-Oligoadenylate Synthetase↗

PubMed1993

Immune complexes from immunized mice and infected cystic fibrosis patients mediate murine and human T cell killing of hybridomas producing protective, opsonic antibody to Pseudomonas aeruginosa.

We examined the basis for the absence in cystic fibrosis (CF) patients of opsonic antibodies to the mucoid exopolysaccharide (MEP) antigen surrounding Pseudomonas aeruginosa that infect these patients. Opsonic antibodies to MEP are found in sera of the minority of CF patients that remain noncolonized into the second to fourth decades of life and protect rodents from chronic P. aeruginosa endobronchial infections. High titers of nonopsonic antibodies to MEP are found in P. aeruginosa-infected CF patients. Immunization of mice with doses of MEP that provoke only nonopsonic antibodies elicited CD3+, CD8+, T cell receptor alpha beta receptor+, major histocompatibility complex-unrestricted cytotoxic lymphocytes specific for hybridoma cells producing opsonic but not nonopsonic antibodies. Cytotoxicity was dependent on immune complexes on the surface of the T cells. Normal murine T cells could be activated by concanavalin A and sensitized with immune complexes for cytotoxic killing of hybridoma targets. CF patients infected with P. aeruginosa had serum immune complexes that sensitized concanavalin A-activated human T cells to kill murine hybridoma cells producing opsonic but not nonopsonic antibody. These results could explain the absence in infected CF patients of MEP-specific opsonins, an occurrence that accompanies the persistence of this infectious state.

Animals↗

PubMed1992

Mouse model for exoerythrocytic stages of Plasmodium falciparum malaria parasite.

Research on the exoerythrocytic (EE) stages of human malaria parasites has been hindered because of the lack of an easily available suitable animal model. We report here an approach to produce mature EE-stage Plasmodium falciparum parasites by using severe combined immunodeficient (scid) mice with transplanted human hepatocytes. Transplantation of human hepatocytes into scid mice (scid hu-hep), their subsequent intravenous infection with P. falciparum sporozoites, and the development of mature liver-stage merozoites was achieved. Immunofluorescent staining of scid hu-hep kidney tissue sections demonstrated the presence of circumsporozoite protein (early during infection), merozoite surface antigen 1, and liver schizont antigen 1. The scid hu-hep model can serve as a source of human malaria liver-stage parasites, decreasing the need for nonhuman primates. Use of this model will facilitate characterization of EE-stage antigens and the assessment of stage-specific chemotherapeutic agents and candidate vaccines.

Animals↗

PubMed1992

Effect of donor and recipient immunization protocols on primary and secondary human antibody responses in SCID mice reconstituted with human peripheral blood mononuclear cells.

We have examined the ability of mice with severe combined immunodeficiency (SCID mice) reconstituted with human peripheral blood mononuclear cells (PBMC) to generate human antibody responses after specific immunization. SCID mice reconstituted with cells from a keyhole limpet hemocyanin (KLH)-naive donor are unable to generate specific human antibody responses after immunization with that antigen. After KLH immunization, SCID mouse recipients of human PBMC from a KLH-immune subject develop specific human antibody levels exceeding those of the donor. Human antitetanus antibody titers in reconstituted, immunized mice are also equivalent to those of the donor, provided that the mice are immunized within days of human cell transplantation. The ability of reconstituted mice to generate high titers of specific human antibody is lost within 35 days of human cell reconstitution, even though titers of total human immunoglobulin (Ig) are preserved. SCID mice reconstituted with tetanus-immune donor cells fail to generate IgA responses after booster immunization, and IgM responses are low or nonexistent. These data indicate that early exposure of the adoptive recipients of human cells to antigen is required to transfer specific human humoral responses. These findings are also consistent with a requirement for persistence of antigen for the maintenance of B-cell memory. The ability to achieve specific human antibody levels equivalent to those obtained with humans indicates that reconstituted mice may be useful for the evaluation of human antibody-mediated mechanisms of resistance to infection. The data indicate, however, that cells from immunized donors will have to be used for such studies.

Animals↗

PubMed1992

Comparison of polymerase chain reaction and culture for detection of Borrelia burgdorferi in naturally infected Peromyscus leucopus and experimentally infected C.B-17 scid/scid mice.

Culture and the polymerase chain reaction (PCR) were compared for detection of Borrelia burgdorferi infection in wild-caught Peromyscus leucopus and experimentally inoculated C.B-17 scid/scid (severe combined immunodeficient) mice. PCR targeted highly conserved regions of the ospA gene and could detect one to five cultured organisms and 10 to 50 copies of molecularly cloned ospA DNA. Organs (kidney, spleen, and urinary bladder) and/or ear biopsy samples were obtained from 108 captured P. leucopus mice, and tissues were obtained from 7 experimentally inoculated mice. A simple sample-processing procedure with proteinase K and detergent treatment was used in the PCR analysis. Overall, B. burgdorferi was detected in 29 of 108 (27%) P. leucopus mice by culture and in 31 of 108 (29%) mice by PCR. As assessed by the kappa statistic, agreement between PCR and culture was high for ear and bladder (kappa = 0.80 and 0.65, respectively) and low for kidney and spleen (kappa = 0.37 and 0.03, respectively). While concordant results were obtained from 98 animals, PCR detected B. burgdorferi from 6 additional mice for which cultures were negative and culture detected B. burgdorferi from 4 animals which were PCR negative. Further phenol-chloroform extraction of DNA in a limited number of samples improved the sensitivity of PCR compared with that of culture. These results indicate that PCR may be as sensitive as culture for detecting B. burgdorferi in ear samples and that PCR analysis is suitable for establishing the infection status of animals in mark-release-recapture studies.

Animals↗

PubMed1991

The role of cytophilic IgG3 antibody in T cell-mediated resistance to infection with the extracellular bacterium, Pseudomonas aeruginosa.

Previous studies have demonstrated that T lymphocytes from mice immunized with a high m.w. polysaccharide Ag from Fisher-Devlin immunotype I Pseudomonas aeruginosa can adoptively transfer protection against challenge with the homologous bacterial strain to susceptible mice. This T cell-mediated resistance has been found to be B cell dependent, although serum from immunized mice is incapable of passively transferring protection to nonimmune mice. The current studies demonstrate that T cells from immunized mice possess receptors that permit them to be adsorbed to IgG3-secreting hybridomas, but not to IgM-secreting hybridomas. Cross-linking of antibody on the surface of immune T cells results in release of a soluble factor that inhibits bacterial growth. Treatment of T cells to remove cytophilic antibody eliminates their ability to adoptively transfer protection to nonimmune mice, and the protective ability can be restored by co-incubating the T cells with monoclonal P. aeruginosa-specific IgG3 antibody before adoptive transfer to nonimmune mice. These observations are consistent with a model in which T lymphocytes from immunized mice are activated by cross-linking of FcR for IgG3 to secrete an antibacterial lymphokine. The ability of IgG3 at low antibody concentrations to act synergistically with T lymphocytes to inhibit bacterial growth could explain the evolutionary selection of this antibody isotype as the predominant subclass of IgG secreted in response to bacterial capsular polysaccharide Ag.

Animals↗

PubMed1988

Suppressor T cells regulating the cell-mediated immune response to Pseudomonas aeruginosa can be generated by immunization with anti-bacterial T cells.

We previously demonstrated that immunization with low (10 micrograms) doses of high m.w. polysaccharide from the gram-negative bacterium Pseudomonas aeruginosa generates T cells that suppress the ability of antibacterial T cells (Tab) to protect against bacterial infection. The current studies indicate that Ts cells with properties identical to those elicited by low dose polysaccharide immunization can be generated by immunization with Tab. Tab-elicited Ts cells can abrogate in vivo induction and in vitro and in vivo expression of antibacterial T cell activity. Tab-elicited Ts are Ag-specific and H-2 restricted in their suppressor activity. Non-immune T cells fail to elicit suppressor activity. These studies provide additional evidence that the protective T cell response to P. aeruginosa is controlled by a network of T cells that are probably recognizing idiotypic determinants on P. aeruginosa-immune T and B cells.

Animals↗

PubMed1988

T cells recognizing polysaccharide-specific B cells function as contrasuppressor cells in the generation of T cell immunity to Pseudomonas aeruginosa.

The cellular interactions involved in the development of T cell-mediated immunity to Pseudomonas aeruginosa have been examined. T cell immunity can be generated by immunizing mice with 10 micrograms of P. aeruginosa polysaccharide (PS) plus the antimitotic agent vinblastine sulfate. Vinblastine is required to inactivate a population of Ts cells generated by immunization with 10 micrograms of PS alone. Immunization with either live bacteria or with higher dose (50 micrograms) of PS without vinblastine also generates T cell immunity; these protocols activate a population of Lyt-1+, 2-, I-J+ T cells which, like vinblastine, counteract the effect of Ts cells. Immunization with 10 micrograms PS alone fails to activate this T cell subpopulation. When administered at the time of immunization, this subpopulation can render the tolerogenic 10-micrograms immunization protocols immunogenic. Like previously described contrasuppressor T cells, this T cell subpopulation exhibits an affinity for the lectin Vicia villosa. We have determined, however, that the T cells that act as contrasuppressor cells in this system are directly activated by PS-immune B cells and not by PS Ag. Furthermore, their activity can be removed by adsorption to PS-specific B cell hybridomas. Our studies indicate an important role for B cells in the development of T cell immunity to P. aeruginosa and suggest that a complex idiotype network controls the development of this response.

Agranulocytosis↗

PubMed1988

Exposure of mice to live Pseudomonas aeruginosa generates protective cell-mediated immunity in the absence of an antibody response.

In previous studies we have elicited T cell-mediated protective immunity to the extra-cellular Gram-negative bacterium Pseudomonas aeruginosa by administering P. aeruginosa polysaccharide Ag and the anti-mitotic agent vinblastine sulfate to BALB/c mice. The current studies indicate that T cells which inhibit the growth of P. aeruginosa in vitro and protect granulocytopenic mice from P. aeruginosa infection can be generated by exposure of BALB/c mice to as few as 10(2) live bacteria without simultaneous administration of vinblastine. The in vitro inhibition of bacterial growth and mouse protection are P. aeruginosa immunotype specific. Exposure to 10(6) live bacteria is required to elicit a detectable antibody response. These findings indicate a potential role for T cells in resistance to P. aeruginosa infection in the large majority of individuals who lack anti-P. aeruginosa antibody.

Animals↗

PubMed1987

In vitro T cell-mediated killing of Pseudomonas aeruginosa. V. Generation of bactericidal T cells in nonresponder mice.

We have previously reported that BALB/c mice immunized with 10 micrograms of a Pseudomonas aeruginosa polysaccharide antigen (PS) and 100 micrograms vinblastine sulfate develop T cell-mediated protective immunity, but fail to generate an antibody response. Vinblastine functions in this system to remove a suppressor cell that normally inhibits expression of this form of immunity after PS immunization. T cells from CB.20 mice immunized with the 10 micrograms of PS and 100 micrograms vinblastine fail to kill P. aeruginosa in vitro. These mice are allotype congenic with BALB/c mice, differing at loci closely linked to the IgH-1 locus. Immunization of CB.20 mice with 10 micrograms PS and 100 micrograms vinblastine results in the appearance of T cells which suppress in vitro bactericidal activity of BALB/c T cells. In the current study we found that T cell-mediated bactericidal activity can be generated in CB.20 mice by increasing the dose of vinblastine given at the time of PS immunization. The phenotype of the CB.20 bactericidal T cell generated by high dose vinblastine is identical to that of the BALB/c bactericidal T cell, and the CB.20 bactericidal T cell can adoptively transfer protective immunity to granulocytopenic mice. After immunization of BALB/c and CB.20 mice with PS alone, approximately one log fewer CB.20 T cells than BALB/c T cells are required to suppress bacterial killing in vitro. Furthermore, the number of CB.20 T cells required to suppress in vitro bacterial killing is directly correlated with the dose of vinblastine administered at the time of immunization. Increasing the immunizing dose of PS overcomes suppressor activity and allows the generation of bactericidal T cells in BALB/c mice without a requirement for vinblastine. CB.20 mice fail to generate bactericidal T cells after immunization with high doses of PS. These results indicate that CB.20 and BALB/c mice both possess the full repertoire of T cells required to express bactericidal T cell activity and that the differences in their responses reflect only quantitative differences in suppressor cell activity.

Animals↗

PubMed1986

In vitro T cell-mediated killing of Pseudomonas aeruginosa. IV. Nonresponsiveness in polysaccharide-immunized BALB/c mice is attributable to vinblastine-sensitive suppressor T cells.

We reported previously that BALB/c mice immunized with a polysaccharide (PS) antigen isolated from immunotype 1 Pseudomonas aeruginosa and vinblastine sulfate develop T cell-mediated protective immunity, despite their failure to produce specific antibody. In vitro, Lyt-1-,2+, I-J+ T cells from vinblastine- and PS-immunized mice kill P. aeruginosa by secretion of a bactericidal lymphokine. BALB/c mice immunized with PS alone generate neither protective antibodies nor a protective T cell response. The current studies indicate that T cells from mice immunized with PS alone significantly suppress the bactericidal activity of T cells from mice immunized with vinblastine and PS. The suppressor T cells are of the same Lyt-1-,2+, I-J+ phenotype as the bactericidal T cells. Suppression is mediated by a soluble product of these suppressor T cells which both inhibits T cell proliferation and interferes with the production or release of the bactericidal lymphokine. Cyclophosphamide, used in other systems to remove suppressor T cells, fails to enhance bacterial killing and does not inhibit suppressor cell activity. These studies indicate that immunization with PS elicits responses in two functionally distinct subgroups of Lyt-1-,2+, I-J+ T cells, and that these cells are distinguishable by their sensitivity to vinblastine sulfate.

Animals↗

PubMed1986

T lymphocyte-mediated protection against Pseudomonas aeruginosa infection in granulocytopenic mice.

BALB/c mice immunized with Pseudomonas aeruginosa immunotype 1 polysaccharide develop protective T cell immunity to bacterial challenge. In vitro, T cells from immunized mice kill P. aeruginosa by production of a bactericidal lymphokine. The present study demonstrates that adoptive transfer of T cells from immunized BALB/c mice to granulocytopenic mice resulted in 97% survival on challenge with P. aeruginosa, compared with 17% survival with adoptive transfer of T cells from nonimmune BALB/c mice. This protection is specifically elicited by reexposure to the original immunizing antigen; adoptive recipients cannot withstand challenge with immunotype 3 P. aeruginosa. However, the adoptive recipients do survive simultaneous infection with both P. aeruginosa immunotypes 1 and 3. Adoptive transfer of T cells from the congenic CB.20 mice, which are unable to kill P. aeruginosa in vitro, provides only 20% protection to granulocytopenic mice. These studies indicate that transfer of specific immune T lymphocytes can significantly enhance the resistance to P. aeruginosa infection in granulocytopenic mice.

Agranulocytosis↗