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The refinement of gamete micromanipulation techniques has made conception possible for couples with male factor infertility who otherwise would remain childless. Moreover, intracytoplasmic sperm injection (ICSI) has ensured that such refractory cases can now generate offspring as successfully as in couples that merely require in vitro insemination. However, despite the now sterling record of ICSI it does not assure a successful outcome for every patient. This can be due, for instance, to the inability of the spermatozoon to activate the oocyte, and applies obviously in cases where spermatozoa are absent from the ejaculate or testicular biopsy. In the present paper we describe in detail the reasons for such failure and review the options that may help overcome it. In particular, we outline the treatment protocol for the situation in which spermatozoa are unable to induce oocyte activation. Further, we report on the clinical outcome achieved with spermatozoa retrieved from the testis, and in cases of extreme oligozoospermia we also explore the option of replicating a single spermatozoon while gaining information on its genomic content. For the most extreme situation in which men have no identifiable germ cells, we will discuss the current status of efforts to accomplish neo-gametogenesis through embryonic stem cell differentiation.

The antibody HMB45 is used to diagnose lymphangioleiomyomatosis, a hyperproliferative disorder of lung smooth muscle cells with mutations in both alleles of either TSC1 or TSC2. A subset of these tumor cells expresses the melanoma-associated antigens gp100 and melanoma antigen recognized by T cells (MART-1). To explore the feasibility of targeting tumors in lymphangioleiomyomatosis by melanoma immunotherapy, we therefore assessed melanoma target antigen expression and existing immune infiltration of affected tissue compared with normal lung and melanoma as well as the susceptibility of cultured lymphangioleiomyomatosis cells to melanoma reactive cytotoxic T lymphocytes in vitro. Tumors expressed tyrosinase-related proteins 1 and 2 but not tyrosinase, in addition to gp100 and MART-1, and were densely infiltrated by macrophages, but not dendritic cells or T cell subsets. Although CD8(+) lymphocytes were sparse compared with melanoma, cells cultured from lymphangioleiomyomatosis tissue were susceptible to cytotoxic, gp100 reactive, and major histocompatibility complex class I restricted CD8(+) T cells in functional assays. Responder T cells selectively clustered and secreted interferon-gamma in response to HLA-matched melanocytes and cultured lymphangioleiomyomatosis cells. This reactivity exceeded that based on detectable gp100 expression; thus, tumor cells in lymphangioleiomyomatosis may process melanosomal antigens different from melanocytic cells. Therefore, boosting immune responses to gp100 in lymphangioleiomyomatosis may offer a highly desirable treatment option for this condition.

Tocopherols are lipophilic antioxidants found in vegetable oils. Here, we examined the growth inhibitory effect of a gamma-tocopherol-enriched tocopherol mixture (gammaTmT) against CL13 murine lung cancer cells grown in culture and as subcutaneous tumors in A/J mice. We found gammaTmT had no effect after 2 days and weakly inhibited the growth of CL13 in culture after 5 days (28% growth inhibition at 80 microM). Dietary treatment with 0.1 and 0.3% gammaTmT for 50 days inhibited the growth of CL13 tumors in A/J mice by 53.9 and 80.5%, respectively. Histopathological analysis revealed an increase in tumor necrosis compared to control tumors (80 and 240% increase by 0.1 and 0.3% gammaTmT, respectively). Dietary treatment with gammaTmT dose-dependently increased gamma- (10.0-37.6-fold) and delta-tocopherol (8.9-26.7-fold) in the tumors of treated mice compared to controls. Dietary treatment with gammaTmT also increased plasma gamma- (5.4-6.7-fold) and delta-tocopherol (5.5-7-fold). Whereas others have demonstrated the cancer preventive activity of gammaTmT against mammary and colon cancer, this is the first report of growth inhibitory activity against lung cancer. Further studies are needed to determine the underlying mechanisms for this anticancer activity, and to determine if such activity occurs in other models of cancer.

Cigarette smoke is a risk factor for colon cancer, but the importance of dose and interaction with genetic susceptibility remain poorly understood. We used data from a population-based case control study, to examine the association between cigarette smoking and colon cancer in African Americans and whites, and colon cancer and polymorphisms in GSTM1 and GSTT1. A total of 554 cases of primary colon cancer and 874 controls were included in this analysis. We found no association between cigarette smoking (ever versus never) and colon cancer in African Americans (odds ratio (OR)=0.93, 95% confidence interval (CI)=0.65-1.33). In contrast, there was an increased risk of cigarette smoking in whites (OR=1.43, CI=1.05-1.94). There was a small increased risk of colon cancer for individuals with GSTM1 null (African Americans, OR=1.43, CI, 0.98-2.09; whites, OR=1.19, CI, 0.90-1.58) and a decreased risk of colon cancer for individuals with GSTT1 null (African Americans, OR=0.59, CI: 0.40-0.86; whites, OR=0.72, CI: 0.53-1.00). There were weak interactions between GSTT1 null and cigarette smoking in whites, and GSTM1 null genotype and cigarette smoking in African Americans. GSTT1 and GSTM1 polymorphisms may be weakly related to colon cancer risk and there may be racial differences in gene-smoking interactions.

Despite repeated high-risk exposure to infectious HIV-1, some individuals remain HIV-1 seronegative and apparently uninfected. The use of nonhuman primate model systems to study SIVmac transmission may help to elucidate the factors responsible for protection in exposed, seronegative (ESN) humans. In an earlier vaccination study, three control rhesus macaques that were exposed to three sequential intravaginal challenges with pathogenic SIVmac251 failed to show evidence of infection after 5 years of observation. 51Cr release assay results suggested that these animals had low-level cytotoxic T lymphocyte responses to SIVmac proteins. We hypothesized that these responses might be an important component of protection from mucosal challenge. We performed an additional intravaginal challenge of all three macaques and monitored SIV-specific T cell responses in peripheral blood, using the sensitive enzyme-linked immunospot (ELISpot) assay. After the fourth challenge, one animal became infected; this animal did not mount a strong SIV-specific T cell response. Two other macaques remained uninfected as determined by peripheral blood mononuclear cell (PBMC) coculture, polymerase chain reaction (PCR), and branched DNA (bDNA) analysis of peripheral blood and lymphoid tissues, but demonstrated boosting of SIV-specific T cell responses after challenge. These results support a protective role for SIVmac-specific T cells in repeatedly exposed, persistently seronegative rhesus macaques.

Human immunodeficiency virus type 1 (HIV-1)-infected subjects treated early after infection have preserved HIV-1-specific CD4+ T-cell function. We studied the effect of highly active antiretroviral therapy (HAART) on the frequency of HIV-1-specific CD8+ T cells in patients treated during early (n = 31) or chronic (n = 23) infection. The degree of viral suppression and time of initiation of treatment influenced the magnitude of the CD8+ T-cell response. HIV-1-specific CD8+ T cells can increase in number after HAART in subjects treated early after infection who have episodes of transient viremia.