IMMUNOTOXICITY OF ZIDOVUDINE PLUE SULFAMETHOXAZOLE-TRIMETHOPRIM EXPOSURE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 艾滋病毒感染者患有免疫缺陷综合征，这已经得到了很好的描述。 然而，B淋巴细胞在这一人群中的命运仍然被低估。 尽管IgG滴度升高，但HIV感染者产生特异性抗体以响应新抗原的能力受损。 先前在小鼠中的工作表明，同时暴露于齐多夫定（ZDV）和磺胺甲恶唑-甲氧苄啶（SMX-TMP）会导致协同免疫毒性，特别是产生抗原特异性抗体应答的能力。 我们小组的初步临床数据显示，ZDV + SMX-TMP暴露影响了接种后HIV感染受试者的流感特异性血清IgG滴度，并消除了IgG滴度和CD 4+计数之间通常观察到的正相关性。 在感染HIV的个体中，应答T细胞非依赖性B细胞有丝分裂原的B细胞的增殖和抗体产生减少。 我们的假设是，暴露于ZDV加SMX-TMP有助于在HIV感染个体中观察到的B细胞缺陷。 我们的目的是1）评价从用ZDV加SMX-TMP治疗的受试者中分离的B和T细胞的离体增殖、抗体产生和细胞因子分泌，和2）区分这种免疫毒性是否影响先天性B细胞功能、T细胞辅助或两者。 受试者将从肯塔基州大学蓝草护理诊所招募，受试者为HIV感染者，接受包括ZDV在内的联合抗逆转录病毒治疗，CD 4+计数大于350个细胞/L，病毒载量不可检测。 最终分析将纳入15例提供知情同意书的患者。 每例受试者将接受为期30天的SMX-TMP实验疗程（每日1片双倍规格片剂）。 在治疗开始和结束时，将通过静脉穿刺采集每例受试者的血液。 将通过Ficoll梯度纯化分离外周血单核细胞用于分析。 将比较治疗前后的数据，每例受试者作为其自身对照。 细胞将与不同类型的有丝分裂原一起培养，以刺激增殖和抗体应答，如下所示：美洲商陆抗原（T细胞依赖性B细胞有丝分裂原）、金黄色葡萄球菌科万（T细胞非依赖性B细胞有丝分裂原）和植物血凝素A（T细胞有丝分裂原）。 将通过血细胞计数法测定增殖。 将通过采集的培养物上清液的ELISA测定来测量抗原特异性抗体应答，定量IgG和IgM的滴度。 将通过ELISPOT技术测定抗原特异性抗体分泌细胞的数量。 此外，将通过利用细胞计数珠阵列技术和流式细胞术测定T细胞分泌细胞因子的能力，包括肿瘤坏死因子-α、干扰素-g、白细胞介素（IL）-2和IL-4。 这些数据将通过在体外环境中检查B和T细胞的功能性，为该药物在人体中的毒性机制提供证据。 参与该人群患者治疗的临床医生将受益于该药物组合可能引起的额外免疫抑制的知识。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. HIV-infected individuals endure an immunodeficiency syndrome that has been well-described. However, the fate of B lymphocytes in this population remains underappreciated. Despite having elevated IgG titers, HIV-infected individuals have an impaired ability to produce specific antibodies in response to neo-antigens. Previous work in mice suggests that simultaneous exposure to zidovudine (ZDV) and sulfamethoxazole-trimethoprim (SMX-TMP) causes a synergistic immunotoxicity, specifically to the ability to produce an antigen-specific antibody response. Pilot clinical data from our group shows that ZDV plus SMX-TMP exposure impacts influenza-specific serum IgG titers in HIV-infected subjects post-vaccination, and eliminates the positive correlation typically observed between IgG titers and CD4+ counts. Decreased proliferation and antibody production have been demonstrated in B cells responding to T cell-independent B cell mitogens in individuals infected with HIV. Our hypothesis is that exposure to ZDV plus SMX-TMP contributes to the B cell defects observed among HIV-infected individuals. We aim to 1) evaluate ex vivo proliferation, antibody production, and cytokine secretion of B and T cells isolated from subjects treated with ZDV plus SMX-TMP and 2) differentiate whether this immunotoxicity affects innate B cell function, T cell help, or both. Subjects will be recruited from the University of Kentucky Bluegrass Care Clinic who are HIV infected, receiving combination antiretroviral therapy that includes ZDV, have CD4+ counts greater than 350 cells/¿l, and undetectable viral loads. Fifteen patients who grant informed consent will be included in the final analysis. Each subject will be given a 30-day experimental course of SMX-TMP (1 double-strength tablet daily). Blood will be obtained from each subject by venipuncture at the beginning and at the end of treatment. Peripheral blood mononuclear cells will be isolated by Ficoll gradient purification for analysis. Data from before and after treatment will be compared, with each subject serving as his or her own control. Cells will be cultured with mitogens of different types to stimulate proliferative and antibody responses as follows: pokeweed antigen (T cell-dependent B cell mitogen), Staphylococcus aureus Cowan (T cell-independent B cell mitogen), and phytohemagglutinin A (T cell mitogen). Proliferation will be assayed by hemocytometric enumeration. Antigen-specific antibody responses will be measured by an ELISA assay of the collected culture supernatants, quantifying titers of IgG and IgM. The number of antigen-specific antibody secreting cells will be determined by an ELISPOT technique. Further, T cells will be assayed for their ability to secrete cytokines, including tumor necrosis factor-a, interferon-g, interleukin (IL)-2, and IL-4 by utilizing cytometric bead array technology and flow cytometry. These data will provide evidence as to the mechanism of this drug toxicity in humans by examining the functionality of B and T cells in this in vitro context. Clinicians involved in the treatment of patients in this population will benefit from the knowledge of additional immunosuppression this drug combination may cause.