Hillary D. White

Flow Cytometric Analysis of Leukocytes in the Human Female Reproductive Tract: Comparison of Fallopian Tube, Uterus, Cervix, and Vagina

PROBLEM: The tissues of the human female reproductive tract (Fallopian tube, uterus, cervix, and vagina) may play different roles in the provision of mucosal immunity. The purpose of this study was to develop a uniform method suitable for quantitative comparison of the leukocytes from all these tissues.

Unique CD8+ T cell-rich lymphoid aggregates in human uterine endometrium.

Using confocal scanning laser microscopy of viable tissue sections, we have demonstrated organized lymphoid aggregates (LA), that have a unique structure, in the stratum basalis of uterine endometrium. These LA consist of a core of B cells surrounded by more numerous T cells and an outer halo of monocytes/macrophages. The T cells in the LA were almost exclusively CD8+CD4‐. These CD8+ LA, in terms of both their T cell and B cell components, were either small or absent during the early proliferative stage of the menstrual cycle, significantly larger in size at mid‐cycle and during the secretory phase, and absent in post‐menopausal women, suggesting that their development is hormonally influenced. This new finding of a menstrual cycle‐dependent, phenotypically unique, organized immune cell structure may lead to new insights into the mechanisms by which the endometrium accepts a semiallogeneic graft while providing resistance to infectious organisms. J. Leukoc. Biol. 61: 427–435; 1997.

Mucosal immunity in the human female reproductive tract : Cytotoxic T lymphocyte function in the cervix and vagina of premenopausal and postmenopausal women

PROBLEM: To investigate the mucosal immune system in the cervix and vagina of premenopausal women in terms of immune cells present and cytolytic capacity of mucosal CD3+ T cells in the lower reproductive tract.

A method for the dispersal and characterization of leukocytes from the human female reproductive tract.

PROBLEM: The isolation of human female reproductive tract (RT) cells that maintain viability and are representative of the entire population is essential for a thorough evaluation of mucosal immunity in the reproductive tract mucosa. Here, we describe the isolation of RT cells in high yields and with high viability from the Fallopian tube, uterine endometrium, endocervix, ectocervix and vagina.
 METHOD OF STUDY: This cell dispersion method uses an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase (PHC), and employs a 250‐μm mesh screen to facilitate cell dispersion.
 RESULTS: The yields of cells isolated per gram of tissue in the presence of this PHC cocktail were compared and found to be strikingly higher relative to the yields obtained with other enzyme cocktails or in the absence of enzymes. Flow cytometry was used to characterize leukocyte subsets isolated from uterine endometrium in the presence of the various enzyme cocktails. The common leukocyte antigen marker CD45, pan T‐cell marker CD3, monocyte/macrophage marker CD14 and B‐cell marker CD19 were retained after exposure to the PHC cocktail of enzymes. The expression of CD8 and CD4 was lost after exposure to added enzymes but regained after culture overnight.
 CONCLUSION: These studies demonstrate the feasibility of using enzymatic digestion for the isolation of whole populations of Fallopian tube, endometrial, cervical and vaginal cells, including leukocyte subsets in high yields, and provide a foundation for investigating mucosal immune cell function in the human female RT.

Human Immunodeficiency Virus–Specific and CD3-Redirected Cytotoxic T Lymphocyte Activity in the Human Female Reproductive Tract: Lack of Correlation between Mucosa and Peripheral Blood

CD8(+) T cell phenotype and function were assessed in the female reproductive tracts (FRTs) of 3 human immunodeficiency virus (HIV)-positive patients who had undergone hysterectomy. FRT cytotoxic T lymphocyte (CTL) lytic activity from 1 patient (patient 872) was detected by using CD3-dependent redirected-lysis assay and HIV-specific assay, concomitant with the presence of CD8(+) cells. In contrast, samples from the 2 other HIV-positive patients (patients 1356 and 1364), who also were asymptomatic for HIV-associated illnesses, demonstrated no CTL activity in any solid tissue tested by either assay, despite activity by autologous peripheral blood mononuclear cells (PBMC). This absence of CTL activity was correlated with a relative absence of CD8(+) cells in the FRT, whereas CD8(+) cells were present in PBMC. Thus, CTL activity in PBMC may fail to correlate with mucosal activity. The finding of CTL activity in the FRT of patient 872 represents the first description of CTL in upper and lower FRT tissues of an HIV-positive woman.

Treatment of pain in fibromyalgia patients with testosterone gel: Pharmacokinetics and clinical response.

To test our hypothesis that testosterone deficiency plays an important role in chronic pain, a Phase I/II pilot study was initiated with 12 fibromyalgia patients to verify that a daily dose for 28days with transdermal testosterone gel would 1) significantly and safely increase mean serum testosterone concentrations from low baseline levels to mid/high-normal levels, and 2) effectively treat the pain and fatigue symptoms of fibromyalgia. Pharmacokinetic data confirmed that serum free testosterone concentrations were raised significantly above baseline levels, by assessment of maximum hormone concentration (Cmax) and area under the curve (AUC) parameters: free testosterone Cmax was significantly raised from a mean of 2.64pg/mL to 3.91pg/mL (p<0.05), and 24hour free testosterone AUC was significantly raised from a mean of 35.0pg-hr/mL to 53.89pg-hr/mL. Assessment of the typical symptoms of fibromyalgia by patient questionnaire and tender point exam demonstrated significant change in: decreased muscle pain, stiffness, and fatigue, and increased libido during study treatment. These results are consistent with the hypothesized ability of testosterone to relieve the symptoms of fibromyalgia. Symptoms not tightly related to fibromyalgia were not improved.