Moses S Kapembwa

Oral contraceptive use induces upregulation of the CCR5 chemokine receptor on CD4+ T cells in the cervical epithelium of healthy women.

Heterosexual transmission of human immunodeficiency virus type I (HIV-1) is the predominant mode of infection worldwide. Increased risk of HIV-1 transmission has been reported with oral contraceptive use. To elucidate the underlying mechanism of this observation, intraepithelial endocervical T lymphocytes from women using oral contraceptives were analysed for expression of activation and chemokine receptors. T lymphocytes from the cervical epithelium and peripheral blood of women using combined oral contraceptives (COC) and those using no contraceptive method (NONE) were compared. Cervical T lymphocytes were obtained with a cytobrush and in parallel, mononuclear leukocytes were separated from blood by centrifugation over a ficoll-hypaque gradient. Cellular activation markers and HIV-1 chemokine co-receptors, CXCR4 and CCR5, were analysed by flow cytometry. Activation markers (CD69, CD25 and HLA-DR) on T cells were expressed at higher levels in the cervical epithelium than peripheral blood T cells but were no different in those women using COC. CXCR4 was widely expressed on cervical and on blood T cells, but was not influenced by COC use. By contrast, the number of T cells expressing CCR5 increased in women using COC (P<0.05). The level of cervical CCR5 expression per cell was shown to increase on both activated CD4(+) (CD69(+), P<0.05; HLA-DR(+), P<0.01) and CD8(+) (CD69(+), P<0.05; HLA-DR(+), P<0.05) T lymphocytes compared with COC use. These data show that with COC use, the expression of CCR5 on CD4(+) T lymphocytes is increased. Furthermore, the cell surface density of CCR5 is increased on both CD4(+) and CD8(+) T lymphocyte subsets. These findings suggest a mechanism by which oral contraceptive use can increase the risk of HIV-1 transmission.

Chemokine Receptor Expression on Mucosal Dendritic Cells from the Endocervix of Healthy Women

Dendritic cells (DCs) may be an initial target of human immunodeficiency virus (HIV) during heterosexual transmission. An analysis of DCs in the intraepithelial layer of the endocervix of the female genital tract from healthy women showed that ~20% expressed CD1a, and 30% expressed cutaneous leukocyte antigen (CLA). Langerin, a molecule associated with Langerhans DCs, was on CD1a-positive and -negative DCs and on CLA-positive cells. CCR5 and CXCR4 were detected on CD1a-positive and -negative cervical DCs. These findings suggest that DCs in the genital tract are potential targets for macrophage-tropic and lymphotropic strains of HIV.

Higher levels of activation markers and chemokine receptors on T lymphocytes in the cervix than peripheral blood of normal healthy women

Heterosexual transmission of human immunodeficiency virus (HIV-1) is the predominant mode of infection world-wide. To better understand sexual transmission of HIV-1 in women we have analysed virus co-receptor and cellular activation marker expression on T lymphocyte subsets from the cervical epithelium and have made comparisons with peripheral blood T cells. Intraepithelial cervical T lymphocytes were obtained with a cytobrush, immunolabelled and analysed by flow cytometry. Activation markers (CD69, CD25 and HLA-DR) were found to be more highly expressed on cervical than on blood T lymphocytes. These higher levels of activation on cervical T lymphocyte subsets could facilitate HIV-1 infection. CXCR4 was expressed at marginally higher levels than CCR5 on T cells from the cervical epithelium and peripheral blood. Thus, the preferential transmission of macrophage tropic strains of HIV-1 following sexual contact cannot be explained solely on the expression of chemokine co-receptors by T lymphocyte subsets.

Evaluation of the cervical cytobrush sampling technique for the preparation of CD45+ mononuclear cells from the human cervix.

A cytobrush technique developed to prepare mononuclear cells from the intraepithelial layer of the endocervix has been evaluated. Specimens yielded approximately 4-6x10(6) cells, of which 10-15% were CD45+. Between 10% and 15% of these CD45+ cells were mononuclear leukocytes. The non-leukocyte cell fraction exhibited high levels of autofluorescence and for flow cytometry analysis, it was necessary to exclude these cells by gating. Macrophages constituted approximately 60% and T lymphocytes, 40% of the mononuclear cells in cytobrush samples. The CD4/CD8 T-cell ratio was similar to that observed in blood. In 9 of 13 specimens, B lymphocytes constituted less than 1% of the mononuclear cell fraction suggesting that the mononuclear cells were derived from the intraepithelial compartment rather then the deeper lamina propria. Lack of B lymphocytes also indicates minimal blood contamination in these samples, a conclusion supported by labelling for the red blood cell (RBC) glycoprotein glycophorin A. However, the need to monitor all samples for possible blood contamination was indicated by 4 of 13 samples in which B lymphocytes accounted for 2-8% of the mononuclear cells.

Ex vivo analysis of HIV-1 co-receptors at the endocervical mucosa of women using oral contraceptives

Combined oral contraceptives may alter the microenvironment of the female genital tract and, thus, influence susceptibility of endocervical cells to HIV‐1 transmission. The mechanism for this effect is unknown but might involve combined oral contraceptive up‐regulation of chemokine receptors on CD4+ endocervical cells. We measured chemokine co‐receptor (CCR5 and CXCR4) expression on cervical intraepithelial CD4+ T lymphocytes, macrophages and dendritic cells using flow cytometry in 32 healthy women, 16 of whom were combined oral contraceptive users and 16 non‐users. All women tested negative for sexually transmitted infections. Combined oral contraceptive users showed a higher proportion of CCR5+ CD4+ T lymphocytes compared with combined oral contraceptive non‐users (P < 0.05). However, expression of both co‐receptors on cervical intraepithelial macrophages and dendritic cells was no different between the two groups. Up‐regulation of CCR5 on cervical intraepithelial CD4+ T lymphocytes offers a potential explanation by which women receiving combined oral contraceptives may be at increased risk of HIV transmission.

Macrophages are increased in cervical epithelium of women with cervicitis

Background: Sexually transmitted diseases (STIs) are major causes of morbidity in women. The mechanisms involved in establishment of genital mucosal infection are poorly defined. Objective: To investigate changes in cervical epithelial (CE) CD45+ cell subpopulations in women with microscopic evidence of cervicitis (n=9) and those without (n=12). Methods: CE samples were obtained using cytobrush including matched venous blood. CE and peripheral blood (PB) mononuclear cells were analysed by flow cytometry for CD3+, CD4+, CD8+, CD14+,CD19+, and HLA-DR+ expression. Results: Women with cervicitis had increased CE macrophages compared with those without (p<0.05). MHC class II+ cells were predominant in all cervical samples. Considerably fewer B lymphocytes were found in cervical samples in both groups of women. No changes were observed in cervical T lymphocyte subsets. However, a relative CD8+ lymphocytosis in PB was noted in women with cervicitis. Conclusion: The increased numbers of CE macrophages in women with cervicitis may have important implications for pathogenesis of STIs including human immunodeficiency virus infection.

Advance refusals: does the law help?

Advance refusals of life-sustaining treatment involve three potentially conflicting interests: those of the patient; those of the doctor; and those of the law. The states interest in protecting life can clash with the patients right to self determination which, in turn, can conflict with the doctors desire to act in the patients best interests. Against this background, we present the case of a patient who was treated (arguably) contrary to his advance refusal but in accordance with English law.