Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment.

Induction of meiotic and post-meiotic alterations of male germ cells in vitro has been the target of several research efforts since 1960. However, to date, the establishment of an ideal culture system in which spermatogonial stem cells can be maintained and directed to proliferate and undergo meiosis and complete spermiogenesis does not exist. This is attributed to the difficulties concerning the isolation and purification of defined subpopulations of germ cells and the establishment of male germ cell lines. In addition, there is no adequate knowledge regarding the optimal biochemical conditions that promote the survival and differentiation of germ cells in long-term cultures. This review focuses on the methodologies that have been proved sufficient to achieve differentiation of cultured male germ cells. Furthermore, the factors regulating spermatogenesis and the technical prerequisites to achieve differentiation of cultured male germ cells are described. Finally, the role of in vitro cultures of immature diploid germ cells in the therapeutic management of men negative for haploid cells in their testes and the subsequent potential genetic and epigenetic risks are discussed.

Clinical evaluation of the efficacy of an antithrombin agent "Argatroban" combined with exercise therapy on increase of the skeletal muscle blood flow.

Argatroban has selective antithrombin activity and widely used for treatment of ASO. In this study we investigated vasodilating activity of Argatroban besides antithrombin activity in ASO patients. Three patients who have undergone F-P bypasses previously which were all occluded received 10 mg or 20 mg of Argatroban per day intravenously for 4 weeks. Skin temperature were measured before and after administration of Argatroban at the point of 1, 2, 4 weeks which increased 2.3-6.0 degrees C after administration of Argatroban. Subjective symptoms were also improved and these patients became to be able to walk 1.5-3.3 km. These patients were also given PGE, intravenously, however, temperature increase was less than 1.1 degrees C. These results showed that Argatroban has not only antithrombin activity but also significant vasodilating activity resulting in increase of skeletal muscle blood flow.

Density of phenotypic markers on BAL T-lymphocytes in hypersensitivity pneumonitis, pulmonary sarcoidosis and bronchiolitis obliterans with organizing pneumonia.

In vitro studies have shown that the density of surface antigens reflects the degree of activation of T-cells. We therefore studied the density of surface antigens on T-cells from bronchoalveolar lavage fluid (BALF) and blood in patients with sarcoidosis, hypersensitivity pneumonitis (HP) and bronchiolitis obliterans with organizing pneumonia (BOOP). BALF cells were stained with anti-CD3, anti-CD4, anti-CD8 and anti-human leucocyte antigen-DR(HLA-DR) monoclonal antibodies, and were analysed by cytoflowmetry. The density was evaluated by measuring the Mean Channel fluorescence intensity of the stained cells. The results demonstrated a significant increase in the CD3 density in patients with hypersensitivity pneumonitis (108.2 +/- 20.2 MC), compared with those with pulmonary sarcoidosis (51.2 +/- 12.6), BOOP (74.5 +/- 29.3), and healthy controls (57.1 +/- 11.5). Similar results were obtained for the CD4 and CD8 density in patients with HP. Although the number of HLA-DR positive cells was increased, the density was lower in patients with sarcoidosis (57.4 +/- 11.6) and hypersensitivity pneumonitis (57.4 +/- 14.8), than in healthy controls (72.2 +/- 15.1). Comparable changes were not observed in the peripheral blood. These results suggest that T-cell activation in hypersensitivity pneumonitis may be associated with an increase in the CD3, CD4, and CD8 density on BALF T-cells.

Proliferative characteristics of fibroblast lines derived from open lung biopsy specimens of patients with IPF (UIP).

We compared the doubling time of fibroblasts derived from idiopathic pulmonary fibrosis (usual interstitial pneumonia) (IPF [UIP]) lung tissues and control fibroblasts, cultured in usual growth medium, and examined the response of these fibroblasts to platelet-derived growth factor (PDGF) and prostaglandin E2 (PGE2). Ten fibroblast lines from open lung biopsy specimens of patients with IPF (UIP) and ten control fibroblast lines from surgically resected lung tissue of patients with limited lung diseases were established. The average doubling time of fibroblast lines was 32.0 +/- 6.0 h (mean +/- SD) in UIP and 33.2 +/- 10.4 h in controls, showing no difference between the two groups. To examine the responses of fibroblasts to PDGF and PGE2 and the differences between fibroblasts derived from fibrotic tissues with different intensity of fibrosis, lung specimens from five patients with IPF were subdivided into two groups, higher-intensity fibrotic lesions (H) and lower-intensity fibrotic lesions (L). The fibroblast lines were established separately. 3H-thymidine uptake with or without PDGF or PGE2 was examined. Results were expressed as the index of thymidine incorporation into the fibroblasts. There were no differences in the doubling times and the responses to PDGF and PGE2 between H and L. There were no differences between control and H regarding their response to PDGF. In response to PGE2, the growth inhibition for H was significantly decreased compared with the control (p less than 0.05). There was no difference in growth inhibition between H and L. The finding that PGE2 inhibits fibroblast proliferation less in UIP lung tissue suggests that fibroblasts from UIP were functionally altered cells or, to some extent, out of normal regulation. These results suggest an abnormal proliferation of fibroblasts observed in IPF (UIP).

[Increased oxidant production by peripheral blood granulocytes in patients with idiopathic pulmonary fibrosis].

Oxidant production by peripheral granulocytes stimulated with phorbol myristate acetate (PMA) was investigated in 11 patients with idiopathic pulmonary fibrosis (IPF), 6 with interstitial pneumonia associated with collagen vascular disease (IP-CVD), 8 with sarcoidosis and 12 healthy subjects. Oxidant production was examined by flow cytometry using dichlorofluorescein diacetate. The reactivity of granulocytes to PMA was assessed according to the ratio between mean fluorescent intensity of granulocytes stimulated maximally with PMA and that without PMA, (stimulation index: S.I.). The concentration of PMA that induced half maximal fluorescent intensity of granulocytes (PC 1/2 max) was used as the index of sensitivity. The S.I. was 7.2 +/- 0.45 in IPF, 6.3 +/- 0.6 in IP-CVD, 6.0 +/- 0.71 in sarcoidosis, and 5.8 +/- 0.2 in healthy subjects. However differences between groups were not significant. PC 1/2 max was 7.3 +/- 2.1 ng/ml in IPF, 9.1 +/- 3.0 ng/ml in IP-CVD, 12 +/- 6.9 ng/ml in sarcoidosis and 16.1 +/- 5.8 ng/ml in healthy subjects. There was significant difference between IPF and healthy subjects (p less than 0.05) indicating that peripheral granulocytes in patients with IPF are more highly sensitive to PMA than healthy subjects.

[Multidisciplinary treatment of head and neck cancer].

UNLABELLED: This presentation deals with multidisciplinary treatment of head and neck cancer, especially focusing on maxillary sinus carcinoma (MSC) and nasopharyngeal carcinoma (NPC). Since 1982, a new multidisciplinary treatment incorporating neo-adjuvant chemotherapy has been introduced in the treatment of MSC. The Neo-adjuvant chemotherapy includes cisplatin (CDDP) + peplomycin (PEP), adriamycin (ADR) analogs + CDDP + PEP, and CDDP +5-FU. Two courses of chemotherapy were given intraarterially with the interval of 2 weeks. Routinely, radiotherapy of 40 Gy by Linac was given to the primary tumor site, concomitantly combined with 5-FU intraarterial injections only during the first 10 days, 2 weeks after the end of chemotherapy. Additional treatment was performed according to the extent of the residual tumors. The 5-year survival rate for the 28 patients treated with this therapy was 55%. The 5-year survival rate by T classification was 100% for T2, 76% for T3 and 0% for T4 cases. The preservation rate of maxillo-facial structures and functions was 82%. Concerning NPC, neo-adjuvant chemotherapy included CDDP + PEP, ADR + CDDP + PEP and ADR + cyclophosphamide + PEP. Two courses of chemotherapy were performed, followed by radiotherapy of 60 Gy by Linac. Then intracavitary 60Co therapy was performed, followed by adjuvant chemoimmunotherapy. The 5-year survival rate for 21 patients treated with this therapy was 44%. CONCLUSIONS: (1) The 5-year survival rate was better for patients with MSC who were treated with multidisciplinary treatment incorporating intraarterial neo-adjuvant chemotherapy than that for patients who received other treatment so far. Furthermore, the highest preservation rate of maxillo-facial structures and functions was achieved in the neo-adjuvant chemotherapy group. However, survival rates for T4 cases were very poor, so another approach should be taken. (2) The 5-year survival rate was also better for patients with NPC who were given multidisciplinary treatment than for patients who received other treatment to date. However, there was no decrease of distant metastases, which we aimed initially, despite the introduction of neo-adjuvant chemotherapy.