Sirolimus-associated infertility: case report and literature review of possible mechanisms.

The mammalian-target-of-rapamycin/mTOR-inhibitor sirolimus as a component of the immunosuppressive strategy after solid organ transplantation is effective at preventing allograft rejection. However, recent reports indicate that sirolimus is associated with altered sex hormone levels and impaired sperm quality parameters. Herein, we report on a case of sirolimus-associated infertility in a young male heart-lung transplant recipient and provide a detailed synopsis of potential mechanisms by which sirolimus may negatively influence spermatogenesis. Testicular immunohistochemistry, the course of sex hormone and sperm quality parameters of our patient support the hypothesis that mTOR might act as an important key regulator in the reproductive system. Fortunately, due to withdrawal of sirolimus as part of the maintenance, immunosuppression improved sperm quality and sex hormone parameters could be observed. Recently, these improvements even resulted in a spontaneous pregnancy of the patient's wife more than 1 year after the drug was withdrawn. In our view, oligospermia as a possible and at least partly reversible side-effect of mTOR inhibitors has to be taken into consideration, particularly, when administrated to young male patients.

Pharmacokinetics and selectivity of aminolevulinic acid-induced porphyrin synthesis in patients with cervical intra-epithelial neoplasia.

Photodynamic therapy (PDT), due to its tumor selectivity, represents an alternative approach to diagnose and treat cervical intra-epithelial neoplasia (CIN) without altering normal surrounding tissue. Our aim was to investigate the pharmacokinetics and the selectivity of 5-aminolevulinic acid (5-ALA)-induced porphyrin fluorescence after topical administration, to obtain basic clinical data for future diagnostic fluorescence imaging and PDT protocols for CIN. Twenty-eight non-pregnant women with a cytological diagnosis of low-grade or high-grade squamous intra-epithelial lesions were included. An aqueous solution containing 3% 5-ALA was topically applied 1 to 6 hrs prior to conization using a cervical cap. After excision, porphyrin-induced fluorescence was quantified in dysplastic (n = 14) and normal epithelium (n = 28) by means of quantitative fluorescence microscopy. High values of porphyrin fluorescence were found in squamous epithelium between 150 and 450 min, with a maximum at 300 min following administration of 5-ALA. Ratios of porphyrin fluorescence of dysplastic vs. surrounding normal epithelium were 1.3 and 1.21 for CIN 1 (n = 3) and CIN 2 (n = 3), respectively. In CIN 3 patients (n = 8), this ratio was 2.35; the best selectivity of 5-ALA-induced porphyrin fluorescence in CIN 3 lesions (ratio 3) was observed with a topical administration time of between 150 and 250 min. Our results demonstrate that patients with CIN 3 show higher 5-ALA-induced fluorescence compared with normal epithelium. The optimal administration time of topically applied 5-ALA was between 3 and 4 hr. Our data suggest that topical ALA-PDT and photodynamic diagnosis might be suitable for detecting CIN.

Immunopathological observations after xenogeneic liver perfusions using donor pigs transgenic for human decay-accelerating factor.

BACKGROUND: Donor pigs transgenic for human decay-accelerating factor (hDAF) were used in a xenogeneic ex vivo liver perfusion model to study the effect of this modification on the development of hyperacute rejection. METHODS: Three transgenic pigs were hepatectomized after hypothermic portal and transaortal gravity perfusion. Livers from six nontransgenic pigs served as controls. All livers were perfused for 3 hr with human blood from two donors diluted to a hematocrit of 30%. Particular importance was placed on the use of an optimal perfusion technique incorporating the floating suspension of the organs in a waterbath and intermittent external pressurization. Biochemical, physiological, and immunological parameters were assessed. Tissue specimens taken before and after perfusion were analyzed using routine histology, electron microscopy, and immunohistology. RESULTS: Complement activation was more pronounced in the control group. AP50 and CH50 values fell to about 60% of the initial levels in control experiments, whereas they remained at 80% of the initial levels during perfusion of hDAF livers. After 180 min, pig tumor necrosis factor alpha levels were 7862+/-1645 pg/ml for unmodified livers and 2830+/-734 pg/ml in the hDAF group. Human tumor necrosis factor alpha levels were similar in both groups. Control livers showed marked morphological alterations and distinct deposition of complement factors, whereas livers expressing hDAF showed no signs of hepatocellular necrosis and almost no complement deposition beyond C3 activation. CONCLUSIONS: These results confirm that the transgenic expression of the human complement regulatory protein hDAF reduces complement activation and prevents hyperacute rejection in a xenogeneic liver perfusion model over the 3-hr evaluation period used in this study.

Hypothernic storage of pig hepatocytes: influence of different storage solutions and cell density.

For clinical use of bioartifical liver devices a constant supply of primary liver cells has to be provided. Hypothermic storage of isolated pig hepatocytes could support large-scale stocking of cells. Freshly isolated pig hepatocytes from slaughterhouse livers were stored at 4 degrees C for 24, 48, and 72 h three different solutions: Leibovitz L-15 + 5% polyethylene glycol (PEG), University of Wisconsin (UW) solution, and a simplified UW solution. After storage, cells were cultured for 2 weeks in the collagen sandwich configuration. Viability of hepatocytes was 65, 85, and 83% after 24 h storage, 21, 74, and 70% after 48 h, and 5, 65, and 59% after 72 h in Leibovitz L-15 medium, UW, and the simplified UW, respectively. After storage in L-15 medium, cells attached poorly to collagen matrices and exhibited ultrastructural lesions. Functional performance in this group, as judged by albumin secretion and cytochrome P450-dependent activity in subsequent culture, decreased rapidly as a function of storage time, with zero values after 48 h storage. In contrast, hypothermia of hepatocytes in both UW solutions resulted in well-preserved cells with respect to ultrastructural appearance, attachment rates, and functional performance during culture. No significant differences were observed between the original and the simplified UW solution. Higher cell concentrations up to 5 x 10(7) cells/ml improved viability of hepatocytes on warmup. In terms of cell supply for hybrid artificial liver support, hypothermic storage of hepatocytes at 4 degrees C could mean an alternative to the cryopreservation of cells, which usually results in a substantial loss of cells and vital function of cells. Thus, pig hepatocytes could be stored at 4 degrees C for several days and meet the logistical need of bioartificial liver devices while avoiding the hazards of cell freezing.

Expression of human decay accelerating factor (hDAF) in transgenic pigs regulates complement activation during ex vivo liver perfusion--immunopathological findings.

Ex vivo perfusions of human decay accelerating factor-expressing transgenic (n = 3), and nontransgenic (n = 6) porcine livers with human blood revealed a higher degree of organ damage in non-transgenic pig livers. Transgenic livers were protected from immunohistologically detectable complement deposition, despite corresponding IgM and IgG deposits in both groups. Complement activation and consumption of C3 and C4 turned out to be lower in transgenic pig livers. In contrast to livers of normal landrace pigs, livers from genetically manipulated pigs showed no morphological alterations after perfusion.

Viable porcine hepatocytes from slaughterhouse organs.

A modified enzymatic isolation technique for the successful harvesting of porcine liver cells from slaughterhouse organs is introduced. Digestion of the left medial liver lobe (n = 74) resulted in 1.0 +/- 0.3 x 10E7 viable hepatocytes per gram tissue and an overall yield of 1.92 +/- 0.5 x 10E9 cells per isolation (viability: 93 +/- 2%).