Cell Interactions in Three Dimensional Tissue Culture

三维组织培养中的细胞相互作用

• 批准号: 7734733
• 负责人: Leonid B. Margolis
• 金额: $ 151.07万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734733
• 关键词: Activated Lymphocyte; Acyclovir; Address; Affect; Amyloid Fibrils; Binding; CCR5 gene; CD3 Antigens; CD8B1 gene; CXCR4 gene; Cell Communication; Cell Count; Cell-Free System; Cells; Chronic; Clinical; Clinical Trials; Colorectal; Complex; Cultured Cells; Cyclophilins; Cytomegalovirus; DNA; DNA-Directed DNA Polymerase; Data; Dendritic Cells; Development; Down-Regulation; Endogenous Factors; Enzymes; Event; Evolution; Flow Cytometry; Goals; Green Fluorescent Proteins; HIV; HIV-1; HLA-DR Antigens; Herpesviridae; Human; Human Herpesvirus 6; IL2RA gene; IL3RA gene; ITGAX gene; Immune; Immunologic Deficiency Syndromes; Immunophenotyping; Infection; Integration Host Factors; Interferon Gamma/Interleukin-2; Interleukin-1 beta; Interleukin-15; Lead; Life; Lymphoid Tissue; Mediating; Memory; Microbe; Modeling; Myelogenous; Outcome; Parasites; Pathogenesis; Patients; Peptidylprolyl Isomerase; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Prevention; Production; Prostatic; RNA-Directed DNA Polymerase; Recombinants; Reverse Transcription; Role; Seminal fluid; Sexual Transmission; Signal Transduction; Simplexvirus; Site; Small Inducible Cytokine A3; System; T-Cell Depletion; T-Lymphocyte; Testing; Therapeutic; Tissues; Tonsil; Tonsillar Tissue; Toxoplasma gondii; Up-Regulation; Vagina; Variant; Viral; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; base; cellular targeting; concept; cytokine; deoxyguanosine triphosphate; design; human tissue; in vivo; inhibitor/antagonist; lymph nodes; macrophage; pathogen; prevent; response; simulation; stromal cell-derived factor-1beta; tissue culture; transmission process; virus pathogenesis

Non-HIV microbes affect HIV pathogenesis by mechanisms that have yet to be elucidated. In general, host factors, in particular imunoactivation together with viral evolution are factors that largely determine the course of HIV infection and non-HIV microbes may affect these phenomena. We addressed these problems by studying pathogenesis of HIV and its interaction with other viruses in various human tissues infected ex vivo. 1. Critical role of tissue activation in HIV infection First, we focused on the role of immunoactivation in HIV-1 pathogenesis. Paradoxically, HIV infection that leads to immunodeficiency depends on tissue immunoactivation. We propose that immune activation in secondary lymphoid tissues is central to the pathogenesis of immune deficiency in chronic HIV-1 infection. In vivo it may predict the pace of CD4 depletion and the onset of clinical immune deficiency. Earlier we found that in HIV-infected lymph nodes there is a significant perturbation of cytokine secretion in particular of interleukin-1beta, interleukin-2, interferon-gamma and interleukin-15 as well as MIP-1alpha and SDF-1beta. Also, there was a profound upregulation of the activation marker, CD38, in naive, central memory, and effector CD4+ and CD8+ T cells. To study this phenomenon we used a system of blocks of human lymphoid tissue and of cervico-vaginal tissue cultured ex vivo. HIV-1 infection was associated with the activation of both HIV-1-infected and uninfected (bystanders) T cells in infected tissues creating a vicious cycle that facilitates HIV own production by creating new cellular targets for viral infection and therefore contributing to the T cell depletion. 2. Effect of non-HIV pathogens on HIV infection in human lymphoid tissue Co-pathogens may contribute to immunoactivation, thus affecting HIV disease. We investigated HIV response to coinfection with some of HIV-1 copathogens, human cytomegalovirus (HCMV or HHV-5) and the cellular parasite Toxoplasma gondii. This required optimization of the human tonsillar tissue to support hCMV replication. hCMV DNA, flow cytometry of viral-positive cells and expression of the green fluorescent protein CMVPT30-gfp in a recombinant strain of hCMV demonstrated that hCMV readily replicated in tissue blocks. Immunophenotyping of HCMV-infected cells showed the preferential infection of activated lymphocytes (CD3+ HLA-DR+ and CD3+ CD25+) and also of plasmocytoid and myeloid dendritic cells (CD3- CD123+ CD11c+). The number of these cells significantly increased in HIV-1-coinfected tissues. Accordingly, HCMV replication was enhanced 2- to-3 fold. This upregulation occurred in tissues infected with either CXCR4- or CCR5-utilizing HIV-1. Thus, HIV-1 creates new targets for hCMV, which may explain the strong association of hCMV with HIV-1 infection in vivo. Human lymphoid tissue ex vivo also provides a model to study HIV interaction with non-viral HIV pathogens in particular of a parasite Toxoplasma gondii. We found that live T. gondii preferentially inhibits CCR5-utilizing HIV-1 replication in coinfected tissues. This effect was reproduced by treatment of the tissue blocks with recombinant C-18, a T. gondii-encoded cyclophilin that binds to CCR5. Thus, T.gondii may influence the outcome of viral infection by preferentially suppressing CCR5-utilizing HIV-1 variants. These studies revealed that HIV- copathogens may differentially generate positive and negative signals for HIV replication, thus affecting selection of particular HIV variants eventually changing the course of HIV disease. 3. Up- and downregulation of HIV-1 replication in cervico-vaginal tissue Endogenous factors also generate such signals. We participated in the identification of a semen factor that facilitates HIV- replication, and we showed that it upregulates HIV replication in infected T cells, macrophages, ex vivo human tonsillar tissues, and cervico-vaginal tissues. This factor consists of naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase, which form amyloid fibrils. Thus, these fibrils may play an important role in sexual transmission of HIV and could represent new targets for its prevention. Whether non-HIV microbes affect formation of such fibrils now has to be investigated. Natural infection with non-HIV-1 microbes can be used for development of new anti-HIV strategies. Here, we exploit the unique ability of herpesvirus (HHV) kinases to phosphorylate acyclovir (ACV), the first step in transforming this otherwise inert compound into an inhibitor of HHV DNA polymerases. Surprisingly, we found that in a cell-free system, phosphorylated ACV directly suppresses HIV reverse transcriptase. We showed that HIV-1 RT incorporates ACV-TP into the nascent HIV-1 DNA chain with a level of efficiency similar to that of its natural equivalent dGTP. Incorporation of ACV-TP results in the formation of a dead-end complex and traps RT at the site of termination leading to the complete termination of reverse transcription. Accordingly, we found that in tissues coinfected with HHVs that are capable of phosphorylating ACV, HIV-1 was inhibited. That was demonstrated in cervico-vaginal tissue as well as in other tissues, where the critical events of HIV-1 pathogenesis and transmission occurs in vivo, tonsils, lymph nodes, and colorectal tissue. Various HHVs, including the ubiquitous HHV-6 detected in all but one tissue ex vivo, may mediate HIV-1 suppression by ACV. No anti-HIV ACV activity was detected in either the HHV-free tissue or in HHV-free cell cultures. However, when HHV-6-infected cells were added to the HIV-1-infected (HHV-free) cell cultures, ACV became a potent suppressor of HIV-1 replication. In summary, the following mechanism seems to be responsible for ACV suppression of HIV-1 in human tissues ex vivo, the majority of which carry one or several HHVs, including HHV-6: ACV is monophosphorylated by herpesviral enzymes in HHV-infected cells and is then cellular kinases convert it into ACV-TP, which suppresses HIV in coinfected tissues by inhibiting HIV-1 RT. Our results suggest that ACV may be therapeutically beneficial for various HIV-1-infected patients, since the majority of humans are already infected with various HHVs that transform ACV at least during reactivation. However, clinical trials are needed to test this concept in vivo. In general, the combination of ACV with an endogenous HHV infection to suppress HIV may constitute a new principle of anti-HIV therapy; a binary weapon in which one inert component is converted by another, endogenous component, into an active therapeutic compound.

非HIV微生物影响HIV发病机制的机制尚未阐明。一般来说，宿主因素，特别是免疫激活和病毒进化在很大程度上决定了HIV感染的过程，而非HIV微生物可能影响这些现象。我们通过研究艾滋病毒的发病机制及其在体外感染的各种人体组织中与其他病毒的相互作用来解决这些问题。

项目成果

Real-time PCR assay of individual human immunodeficiency virus type 1 variants in coinfected human lymphoid tissues.

对共感染的人淋巴组织中的个体人类免疫缺陷病毒 1 型变种进行实时 PCR 测定。

• DOI: 10.1128/jcm.41.5.2126-2131.2003
• 发表时间: 2003
• 期刊: Journal of clinical microbiology
• 影响因子: 9.4
• 作者: Ito,Yoshinori;Grivel,Jean-Charles;Margolis,Leonid
• 通讯作者: Margolis,Leonid

A naturally occurring variation in the proline-rich region does not attenuate human immunodeficiency virus type 1 nef function.

富含脯氨酸区域中自然发生的变异不会减弱人类免疫缺陷病毒 1 型 nef 功能。

• DOI: 10.1128/jvi.78.18.10197-10201.2004
• 发表时间: 2004
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Rucker,Elke;Munch,Jan;Wildum,Steffen;Brenner,Matthias;Eisemann,Jutta;Margolis,Leonid;Kirchhoff,Frank
• 通讯作者: Kirchhoff,Frank

Interactions between human immunodeficiency virus type 1 and vaccinia virus in human lymphoid tissue ex vivo.

离体人体淋巴组织中 1 型人类免疫缺陷病毒与牛痘病毒之间的相互作用。

• DOI: 10.1128/jvi.00326-07
• 发表时间: 2007
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Vanpouille,Christophe;Biancotto,Angelique;Lisco,Andrea;Brichacek,Beda
• 通讯作者: Brichacek,Beda

Candidate microbicides block HIV-1 infection of human immature Langerhans cells within epithelial tissue explants.

• DOI: 10.1084/jem.192.10.1491
• 发表时间: 2000-11-20
• 期刊: The Journal of experimental medicine
• 作者: Kawamura T;Cohen SS;Borris DL;Aquilino EA;Glushakova S;Margolis LB;Orenstein JM;Offord RE;Neurath AR;Blauvelt A
• 通讯作者: Blauvelt A

Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo.

非感染性 X4（而非 R5）人类免疫缺陷病毒 1 型病毒颗粒会抑制离体人体淋巴组织中的体液免疫反应。

• DOI: 10.1128/jvi.78.13.7061-7068.2004
• 发表时间: 2004
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Fitzgerald,Wendy;Sylwester,AndrewW;Grivel,Jean-Charles;Lifson,JeffreyD;Margolis,LeonidB
• 通讯作者: Margolis,LeonidB