Providing an obstetric fistula service.
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Ambulatory Care↗
Biomedical subjects
R C Kennedy
Publications and source records attributed to R C Kennedy.
HIV-1 gp120 proteins and gp160 peptides are toxic to brain endothelial cells and neurons: possible pathway for HIV entry into the brain and HIV-associated dementia.
Breakdown of the blood-brain barrier is commonly seen in patients with human immunodeficiency virus (HIV)-associated dementia, despite the lack of productive HIV-infection of the brain endothelium. Through this damaged blood-brain barrier, HIV and HIV-infected monocytes/macrophages infiltrate the brain and further infect microglia and brain macrophages. Neuronal cell death and dysfunction are the underlying cause of HIV-associated dementia, but no productive HIV-infection of neurons has been documented. It is likely that secreted viral products play a major role in blood-brain barrier damage and neuronal cell death. The aim of the present study was to examine the effect of HIV-1 gp160 peptides and gp120 proteins on brain microvascular endothelial cells and neurons from both human and rats. Four of the 7 gp160 peptides tested evoked significant neurotoxicity. Two different full-length recombinant HIV gp120 proteins (HIV-1CM235 gp120 and HIV-1MN gp120) also induced neuronal and brain endothelial cell death, and concentrations as little as 1 ng/ml evoked pronounced morphological changes in these cells and marked cytotoxicity. This study suggests that HIV proteins and peptides that are shed in vivo may be directly involved in blood-brain barrier damage and neuronal cell death in HIV-associated dementia.
AIDS Dementia Complex↗
Analysis of human lung endothelial cells for susceptibility to HIV type 1 infection, coreceptor expression, and cytotoxicity of gp120 protein.
The lung represents a potential target during HIV infection, and the onset of AIDS is associated with severe pulmonary complications in many patients. T-lymphocytes and alveolar macrophages form the majority of HIV-infected cells in the lung. However, other cell types in the lung could participate in HIV-mediated lung pathology and their role has not been investigated. The aims of this study were to determine if human lung microvascular endothelial cells (HLMEC) express HIV receptor and coreceptors, and if HIV can directly infect HLMEC. Specifically, we wished to determine if these cells constitute a viral reservoir in the lung, and if HIV-1 envelope proteins induce cytotoxic effects on HLMEC. Our results showed that by flow cytometry, HLMEC failed to express any CXCR4 or CCR5 on their surface. In contrast, RT-PCR revealed the presence of CXCR4 and CCR5 mRNA, but not CD4 in HLMEC. Two dual-tropic HIV-1 isolates failed to infect HLMEC in vitro, as determined by (1) p24 antigen capture ELISA, (2) reverse transcriptase assay, RT-PCR, and (3) DNA PCR. However, a recombinant HIV-1 gp120 preparation induced apoptotic cell death of HLMEC. These data support the hypothesis that no productive HIV-1 infection of HLMEC occurs in vitro. This suggests that in vivo, HLMEC may not be a major reservoir of HIV in the lung and the primary route for HIV invasion of the lung. Thus, while other mechanisms must play a role in HIV invasion and subsequent dissemination in the lung, lung endothelial cells do represent potential targets for the lethal effects of HIV viral proteins.
Apoptosis↗
Regulation of capsule gene expression by group A Streptococcus during pharyngeal colonization and invasive infection.
Capsular polysaccharide production by group A Streptococcus (GAS) is controlled by transcription of the has operon that encodes the enzymes uniquely required for synthesis of the hyaluronic acid polysaccharide. To investigate the regulation of capsule gene expression during infection, we developed a reporter strain of GAS in which the has operon promoter directed transcription of green fluorescent protein (GFP). Gfp expression was triggered within minutes after introduction of the reporter strain into the peritoneal cavity of mice, as evidenced by the recovery of highly fluorescent GAS from the peritoneum 1 h after challenge. Capsule gene expression was also stimulated in the bloodstream of infected mice, as intensely fluorescent bacteria were observed in blood samples collected after either intraperitoneal or intravenous challenge. Using a similar approach, we also observed rapid induction of capsule gene expression in bacteria inoculated into the pharynx of baboons. Compared to the inoculum, increased green fluorescence was recorded in bacteria recovered from throat swabs collected 1 h after inoculation in all five animals studied. We conclude that introduction of GAS into the pharynx or into deep tissues results in rapid induction of has operon expression, a critical adaptive response that enhances GAS survival in the infected host.
Animals↗
Epidemiology of herpesvirus papio infection in a large captive baboon colony: similarities to Epstein-Barr virus infection in humans.
The epidemiology of herpesvirus papio, a lymphocryptovirus similar to Epstein-Barr virus (EBV), was studied in a captive colony of >1900 baboons. Herpesvirus papio IgG antibody titers were measured by IFA. In total, 438 specimens from 296 baboons were assessed, including 116 serial specimens from 52 juveniles and 6 infants studied monthly for 1 year following birth and at age 18 months. Maternally derived antibody reached a nadir at 4 months of age. About 75% of animals at 12 months of age and >95% of animals after age 24 months demonstrated serologic evidence of herpesvirus papio infection. After age 3 years, the geometric mean titer was 1:60-75. The epidemiology of herpesvirus papio infection in baboons closely parallels that of EBV infection in humans. An animal model of lymphocryptovirus infection will facilitate investigations of human lymphocryptovirus biology.
Animals↗
Idiotype cascades associated with the CD4-HIV glycoprotein 120 interaction: immunization with anti-idiotypic antibodies induces anti-anti-idiotypic responses with anti-CD4 specificity and in vitro neutralizing activity.
Anti-idiotypic antibodies (Ab-2), which were generated in baboons against a mouse monoclonal antibody specific for the CD4 molecule expressed on human T cells, were used to produce anti-anti-idiotypic antibodies (Ab-3) in mice. This response induced by Ab-2 immunization of BALB/c mice was classified as anti-anti-idiotype (Ab-3) on the basis of the ability of the mouse Ab-3 to (1) specifically bind the baboon Ab-2 preparation, but not irrelevant baboon IgG preparations, (2) inhibit the binding of the anti-CD4 Ab-1 preparation to the baboon Ab-2, and (3) recognize a second baboon Ab-2, along with a rabbit Ab-2 specific for the monoclonal anti-CD4 Ab-1 preparation. The murine Ab-3 response also recognizes the CD4 molecule expressed on a human CD4+ T cell line, as determined by flow cytometry; recognizes the same epitopes on the CD4 molecule as the Ab-1; inhibits HIV-1 syncytium formation; and neutralizes HIV-1 primary isolates in vitro. These studies suggest that Ab-3 responses can be induced by anti-Id immunization, which serologically mimicks the antigen and Id specificities of the monoclonal anti-CD4 preparation used to generate the anti-Id. Thus, the Ab-3 response exhibits the characteristics of a population that represents the internal image of the Ab-1.
Animals↗
Bacterial determinants of persistent throat colonization and the associated immune response in a primate model of human group A streptococcal pharyngeal infection.
Group A streptococcal (GAS) pharyngitis and the subsequent bacterial colonization of the human throat elicit an immune response that may precipitate acute rheumatic fever in a susceptible host. To study the bacterial determinants that influence throat colonization and induction of humoral immunity, we characterized the behavior of GAS strains in a baboon model. An M-type 3 clinical isolate of GAS typical of strains that cause pharyngitis and invasive infection was recovered from the pharynx of six out of six baboons for at least 6 weeks after oral inoculation. By contrast, an isogenic mutant deficient in M protein failed to colonize most animals or was rapidly cleared. An isogenic mutant deficient in hyaluronic acid capsule colonized five out of six animals, but only persisted in the pharynx for 14-21 days. Colonized animals developed serum antistreptolysin O (SLO) and anti-M protein immunoglobulin (Ig)G. The kinetics of the antibody responses were similar to those seen after human infection. Peak titres increased with the duration of throat carriage. Colonization with GAS prevented recurrent colonization after challenge with the homologous wild-type strain, but not after challenge with a strain of different M protein type. Early clearance of the M protein-deficient strain was associated with increased susceptibility of this strain to phagocytic killing in non-immune serum, whereas clearance of the acapsular strain was associated with increased susceptibility to phagocytic killing in the presence of specific antibody. These studies support critical and distinct effects of the GAS M protein and capsule on throat colonization and induction of humoral immunity in a model that reproduces important features of pharyngeal colonization and immune response following human infection.
Animals↗
Maternal antibody transfer in baboons and mice vaccinated with a group B streptococcal polysaccharide conjugate.
Two animal models were used to study maternal transfer of antibody to a group B Streptococcus (GBS) type III polysaccharide-tetanus toxoid (III-TT) conjugate. The III-TT vaccine protected all 27 mouse pups born to vaccinated dams against a GBS challenge. In a separate study of vaccinated mouse dams and pups, maternal sera contained all 4 subclasses of polysaccharide-specific IgG, with IgG1 accounting for 83% of total IgG. Specific IgG subclass distribution (IgG1>>IgG2a=IgG2b=IgG3) in newborn pups closely resembled that in their mothers. Seven of 9 female baboons given the III-TT vaccine had 5- to 36-fold increases in specific antibody from baseline levels; they transferred 26%-185% of specific antibody to their offspring. Matched maternal and neonatal sera obtained at delivery were functionally equivalent in an in vitro opsonophagocytosis assay. These preclinical studies provide further evidence for effective immunogenicity of GBS conjugate vaccine and efficient transport of functionally active maternal antibody.
Animals↗
Analysis of human endothelial cells and cortical neurons for susceptibility to HIV-1 infection and co-receptor expression.
Neuronal cell death is believed to be the underlying cause of neurological diseases and AIDS dementia often seen in human immunodeficiency virus (HIV) infected patients. The means by which HIV invades the brain is still unknown and the mechanism of neuronal cell death remains to be elucidated. The aim of this study was to determine if direct infection of human brain endothelial cells and neurons play a role in viral invasion of the brain and neuronal cell death, respectively. To this effect, we evaluated human brain microvascular endothelial cells (HBMEC) and human cortical neurons (HCN) for the expression of HIV co-receptors and their susceptibility to HIV-1 infection. While both HBMEC and HCN failed to express any CXCR4 and CCR5 on their cell surface, as assessed by flow cytometry, RT - PCR revealed the presence of CXCR4 and CCR5 mRNA in HBMEC but not in HCN. Two dual tropic HIV-1 primary isolates failed to infect both cell types as determined by p24 antigen capture ELISA, RT - PCR and DNA PCR. These data support the hypothesis that no productive infection of HBMEC and HCN occurs in vitro and suggest that other cell types are the primary focus of HIV-1 infection in the brain.
Brain↗
Adenovirus keratitis: a role for interleukin-8.
PURPOSE: Adenovirus type 19 (Ad19) infection of the human cornea results in a chronic, multifocal, subepithelial keratitis. Existing evidence suggests that early subepithelial corneal infiltrates are composed of polymorphonuclear neutrophils. In this study, the capacity of Ad19-infected human corneal stromal fibroblasts (HCFs) to produce neutrophil chemotactants (chemokines) was tested. METHODS: HCFs grown from human donor corneas and passaged thrice were infected with a corneal isolate of Ad19 or mock-infected with virus-free media. Bioactivity of the cell supernatants was tested by a neutrophil chemotaxis assay. Supernatants were assayed by enzyme-linked immunosorbent assay for the neutrophil chemotactants interleukin-8 (IL-8) and GRO-alpha. Corneal facsimiles were generated with HCFs and collagen type I, infected with Ad19, and assayed by immunohistochemistry. RESULTS: Ad19 infection of HCFs increased neutrophil chemotaxis from a baseline of 0.4+/-0.7 cells/high-powered field (hpf; mock-infected) to 21.8+/-2.3 cells/hpf (Ad19-infected). Chemotaxis was reduced by the addition of neutralizing antibodies against IL-8 and GRO-alpha. Infection of HCFs induced quantities of IL-8 protein 300- and 1000-fold over mock-infected controls at 4 and 24 hours, respectively (33 versus 11,813 pg/mL at 4 hours, and 57 versus 76,376 pg/mL at 24 hours, P< or = 0.001 for both). In contrast, GRO-alpha protein levels were only sevenfold higher at 24 hours postinfection (118 pg/mL in mock-infected controls versus 880 pg/mL in Ad19-infected cell supernatants). Neither chemokine was induced by infection of an immortalized human corneal epithelial cell line. Immunohistochemistry of infected corneal facsimiles demonstrated IL-8 in the extracellular matrix within 3 days after infection. CONCLUSIONS: Production of chemokines in infected tissues facilitates an early innate immune response to infection, and in the infected corneal stroma represents an elementary defense mechanism. Interleukin-8 may play a role in the development of subepithelial infiltrates in adenovirus keratitis.
Adenoviridae↗
DNA cancer vaccination strategies target SV40 large tumour antigen in a murine experimental metastasis model.
Two plasmids encoding SV40 Tag under the control of different promoters have been examined for their ability to induce complete protection against murine experimental metastasis induced with an SV40-transformed tumour cell line. BALB/c mice immunized with a plasmid encoding SV40 Tag under the control of the SV40 promoter (pSV3neo) exhibited no detectable levels of anti-SV40 Tag antibody and were only partially protected from tumour foci development in the lungs after Intravenous tumour challenge. In contrast, mice receiving a plasmid encoding SV40 Tag under the control of the CMV promoter (pCMV-Tag) demonstrated high levels of anti-SV40 Tag antibody. These mice were completely protected from lung tumour foci development after challenge. Since antibody responses were induced only by the immunization which provided complete protection from metastatic tumour challenge, these data support the notion that antibody may play an important role in protection against experimental pulmonary metastasis within this model. Our results demonstrate that DNA immunization may serve as a possible immunotherapeutic strategy against cancers expressing tumour-specific or tumour-associated antigens.
Animals↗
Comparison of simian virus 40 large T antigen recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis.
In this report we examine the ability of a recombinant tumor antigen preparation to prevent the establishment of experimental pulmonary metastasis. Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was injected into BALB/c mice followed by challenge with an intravenous injection of syngeneic SV40-transformed tumorigenic cells. The experimental murine pulmonary metastasis model allows for the accurate measurement of metastatic lessions in the lungs at various times after the challenge, using computer-assisted video image analysis. Following challenge, lung metastasis and survival data for the groups of mice were obtained. Animals immunized with recombinant SV40 T-Ag showed no detectable sign of lung metastasis and survived for more than 120 days after challenge. Antibodies specific for SV40 T-Ag were detected in the serum of immunized mice by enzyme-linked immunosorbent assay. Splenocytes obtained from mice immunized with recombinant SV40 T-Ag did not lyse syngeneic tumor cells, indicating that no cytotoxic T lymphocyte response was induced. Control mice developed extensive lung metastasis and succumbed to lethal tumor within 4 weeks after challenge. These data indicate that immunization with the recombinant SV40 T-Ag induces protective, T-Ag-specific immunity in an experimental pulmonary tumor metastasis model.
Animals↗
DNA vaccination strategies against infectious diseases.
DNA immunisation represents a novel approach to vaccine and immunotherapeutic development. Injection of plasmid DNA encoding a foreign gene of interest can result in the subsequent expression of the foreign gene products and the induction of an immune response within a host. This is relevant to prophylactic and therapeutic vaccination strategies when the foreign gene represents a protective epitope from a pathogen. The recent demonstration by a number of laboratories that these immune responses evoke protective immunity against some infectious diseases and cancers provides support for the use of this approach. In this article, we attempt to present an informative and unbiased representation of the field of DNA immunisation. The focus is on studies that impart information on the development of vaccination strategies against a number of human and animal pathogens. Investigations that describe the mechanism(s) of protective immunity induced by DNA immunisation highlight the advantages and disadvantages of this approach to developing vaccines within a given system. A variety of systems in which DNA vaccination has resulted in the induction of protective immunity, as well as the correlates associated with these protective immune responses, will be described. Particular attention will focus on systems involving parasitic diseases. Finally, the potential of DNA immunisation is discussed as it relates to veterinary medicine and its role as a possible vaccine strategy against animal coccidioses.
Animals↗
Immunoglobulin variable regions as idiotype vaccines.
This article focuses on the use of immunoglobulin variable regions, including Id, anti-Id, anticlonotypes, and Id engineering as putative vaccines and vaccine strategies for infectious diseases; and specific discussion of Id systems involving antigenic determinants associated with potentially pathogenic organisms.
Animals↗
Fetal immunization of baboons induces a fetal-specific antibody response.
Neonates face a high risk of infection because of the immaturity of their immune systems. Although the transplacental transfer of maternal antibodies to the fetus may convey improved postnatal immunity, this transfer occurs late in gestation and may fail to prevent in utero infection. Both fetal immunization and in utero exposure to antigen can result in a state of immunologic tolerance in the neonate. Tolerance induction of fetal and premature infant lymphocytes has become a paradigm for neonatal responsiveness. However, fetal IgM responses have been demonstrated to maternal immunization with tetanus toxoid and to congenital infections such as rubella, toxoplasma, cytomegalovirus and human immunodeficiency virus. Moreover, 1-week-old infants can respond to standard pediatric vaccination, and neonates immunized with polysaccharide antigens do not develop immunologic tolerance. Here, direct immunization of the baboon fetus with recombinant hepatitis B surface antigen produced a specific fetal IgG antibody response. No specific maternal antibody response was detected, eliminating the possibility of vertical antibody transmission to the fetus. Some infants also responded to later vaccinations with hepatitis B surface antigen, indicating that no immunological tolerance was induced by prior fetal immunization. These results characterize the ability of the fetal immune system to respond to in utero vaccination. We demonstrate that active fetal immunization can serve as a safe and efficient vaccination strategy for the fetus and neonate.
Animals↗
Genetic vaccines.
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Acquired Immunodeficiency Syndrome↗
Comparison and characterization of immunoglobulin G subclasses among primate species.
Little information is available on the immunoglobulin G (IgG) subclasses expressed in the sera of nonhuman primate species. To address this issue, we compared the IgG subclasses found in humans (IgG1, IgG2, IgG3, and IgG4) to those of nonhuman primates, such as baboons and macaques. Cross-reactive antihuman IgG subtype-specific reagents were identified and used to analyze purified IgG from sera by solid-phase enzyme-linked immunosorbent assay. Protein A-purified human IgG obtained from sera was composed of IgG1, IgG2, IgG3, and IgG4, whereas baboon and macaque IgG was composed of IgG1, IgG2, and IgG4. Protein G-purified human IgG was composed of IgG1, IgG2, IgG3, and IgG4, whereas baboon and macaque IgG was composed of IgG1, IgG2, and IgG4. To test the possibility that baboon and macaque IgG3 is actually present, but is outcompeted for binding to proteins A and G by the other more abundant IgG subclasses, we repurified the IgG from sera that did not bind either protein A or protein G. We found a baboon IgG3 population in the sera that did not bind protein A, but bound protein G. No IgG3 subtype was detectable in macaque sera. These data suggest that baboon sera, like human sera, contain four IgG subtypes, whereas macaque sera exhibit only three of the human subclass analogs. In addition, the IgG subtype-specific reagents were shown to be useful in determining the IgG subclass distribution following vaccination of baboons with hepatitis B surface antigen.
Animals↗
Quantitation of tumor foci in an experimental murine tumor model using computer-assisted video imaging.
A method is described in which both number and diameter of lung tumor foci obtained from an experimental murine tumor model of pulmonary metastasis are quantified using a digital imaging device. Lungs obtained from mice previously inoculated with tumorigenic cells are stained with India ink and Fekete's solution. Using appropriate exposure settings and top-lighting, a charged-coupled device digital camera is used to obtain the image of the organ. The image is then analyzed by the associated video imaging analysis software. Diameter of tumor foci on the image is expressed in image pixels and foci are quantitated. Density and size threshold parameters are set, thereby eliminating the variability resulting from the subjective nature of manual enumeration of tumor foci. The method is objective and reproducible and should be useful for studies where quantifying tumor foci diameter and number directly from the organ is required.
Animals↗