Leptin and leptin receptor in human seminal plasma and in human spermatozoa

Leptin, a 167 amino acid peptide, is known to influence the gonads via direct and indirect effects. Recent studies provide contradictory proposition about the peripheral impact of leptin in the male gonads. Thus, we examined leptin and its receptors in human seminal plasma and in human ejaculated spermatozoa by Western blot technique and fluorescence microscopy. In seminal plasma we found a free leptin band (16 kDa) by an anti-leptin polyclonal antibody. Incubation of seminal plasma with recombinant leptin caused a statistically significant increase in the amount of free leptin (p < 0.01) and supports this finding. Furthermore, a soluble leptin receptor (145 kDa) was found in human seminal plasma in the same specimen. We also detected a 145-kDa leptin receptor isoform in ejaculated spermatozoa as a possible target of leptin action in the male genital tract, which was localized at the tail of spermatozoa by immunofluorescence microscopy only. This receptor was significantly associated with the intactness of sperm plasma membranes. Spermatozoa with deteriorated membranes contained 49.2 +/- 6.9% leptin receptor signal intensity compared with spermatozoa having intact membranes (p < 0.01). This finding is difficult to interpret and may be caused by a leakage of OB-R due to loss of membrane integrity. In conclusion, these data provide further hints for a peripheral role of leptin in the male genital tract, possibly, by an interaction between leptin and spermatozoa via sperm leptin receptors.     

rhbFGF和rhEGF对成纤维细胞的促增殖作用

目的：掌握人皮肤成纤维细胞的培养技术，了解该细胞的生长特性；研究rhbFGF和rhEGF在不同浓度下对人皮肤成纤维细胞增殖的影响，为临床准确定量应用这两种因子修复创面、加速创面愈合提供理论依据。方法：体外培养人皮肤成纤维细胞，用不同浓度的rhbFGF和rhEGF干预细胞，四甲基偶氮唑盐比色法（MTT）检测细胞的活性，选择最适浓度生长因子干预的细胞，用流式细胞仪检测细胞的DNA倍体情况。结果：不同浓度的rhbFGF和rhEGF分别对成纤维细胞干预48h后，显示rhbFGF在50μg／L浓度时MTT值最大，rhEGF在80μg／L浓度时MTT值最大；并检测出在最适浓度下细胞倍体的含量。结论：应用最佳浓度的rhbFGF和rhEGF可以更快的促进创面愈合，减少瘢痕的形成。     

成人胰岛大容量分离技术的改进及胰岛功能检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能,为利用同种异体胰岛移植治疗1型和部分2型糖尿病提供理论依据和技术基础。方法 采用改良的自动分离技术连续分离28例人胰岛,然后用连续性密度样度离心法纯化胰岛。胰岛收获量以国际标准的胰岛当量(islet equivalent,IEQ)表示。胰岛功能的测定分别为体外测定胰岛的胰岛素／DNA比率;静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内胰岛功能鉴定并随后进行腹腔糖耐量试验,连续测定移植鼠血糖水平及其体内C肽浓度。结果 28例成人胰腺分离的胰岛收获量为5000～1030000IEQ／胰腺,平均为291635IEQ／胰腺,前13例平均每个胰腺收获49123IEQ,平均每克组织收获846IEQ。平均纯度为87％,随着技术的改进后15例的分离结果则分别为：平均每个胰腺501813IEQ,平均每克组织7003IEQ,平均纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常鼠。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的实验基础。     

Hypercalcemia and human nature

Patients on hemodialysis may develop severe and symptomatic hypercalcemia if skeletal buffering is ineffective. We report a case of persistent hypercalcemia with apparent extrarenal vitamin D synthesis. Associated aluminium intoxication was suggested on desferrioxamine challenge and adynamic uremic osteodystrophy confirmed on bone biopsy. Plasma calcitriol did not suppress with corticosteroids but did with ketoconazole. No other evidence for underlying granulomatous disease was found. We discuss our approach to less usual causes of hypercalcemia, and emphasise the pitfalls associated with factitious disorders.     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prionrelated material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.     

Inhibiting action human glioblastoma extract on haemolytic complement of human and xenogeneic sera

The action of some extracts of human glioblastoma of the haemolytic complement of human and xenogeneic sera has been examined. The study suggests that the antigenic products of some human glioblastomas may induce an inhibiting effect on the haemolytic complement of human, as well as xenogeneic (rat and rabbit), sera. The inhibiting action on the xenogeneic sera is indicative of the presence of species-specific antigens or tumor cells, comparable to that of normal tissues.     

雌二醇、三苯氧胺对乳腺癌和子宫内膜细胞增殖的影响

目的　通过雌二醇 (E2 )、三苯氧胺 (TAM )作用于乳癌及子宫内膜细胞 ,研究这两种因素对雌激素受体 (ER)阳性细胞的增殖作用 ,探讨乳癌TAM耐受的产生与细胞来源的关系。方法1× 1 0 - 6 mol/LTAM、1× 1 0 - 8mol/LE2 作用于乳癌及子宫内膜细胞 ,利用3H TdR示踪细胞增殖动力学。结果　TAM对子宫内膜细胞增殖 (6 .32 % )的抑制作用与对照组 (6 .40 % )比较差异无显著性 (P <0 .0 1 ) ,TAM对乳癌细胞增殖 (45 .84% )的抑制作用与对照组 (52 .72 % )比较差异有显著性 (P <0 .0 1 ) ,但其对TAM耐受的乳癌细胞增殖 (9.64 % )与对照组 (6 .32 % )比较有显著刺激作用 ;E2 对 3种细胞增殖的刺激作用与对照组比较差异都有显著性。E2 与TAM联合作用于子宫内膜及乳癌细胞与单独使用E2 时相比 ,表现为抑制细胞增殖的作用 ;但它们联合作用于TAM耐受乳癌细胞时表现出比E2 更大的刺激细胞增殖的作用。结论　E2 对 3种来源细胞都表现为刺激增殖的作用 ,但TAM抑制乳癌细胞增殖的作用则依赖E2 的浓度水平而不同     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prionrelated material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prion-related material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.     

Detection of human papillomavirus DNA and p53 gene mutations in human prostate cancer

The relationship between integration with human papillomavirus (HPV) and p53 gene mutations in tissues of prostate cancer was examined. Tissue samples analyzed were obtained by total prostatectomy (29 stage B cancer cases) and from autopsy (22 endocrine therapy-resistant metastatic disease cases). HPV DNA was detected in 8 of 51 (16%, 5 in stage B and 3 in autopsy cases) by polymerase chain reaction (PCR) using consensus primers on L1 region. Genotypes of HPV were entirely type 16. Structural abnormalities of p53 gene were detected in 7 of the 22 autopsy cases (32%) by PCR-single-strand conformation polymorphism analysis and direct sequencing. No p53 gene mutation was found in stage B cancer cases. Analysis of mutation spectra revealed clear differences between Japanese and Westerners. There was a significant difference in the mutation frequency between stage B and autopsy cases (P < 0.01, Fisher's exact test). One case showed both integration of HPV and p53 gene mutation in different cancer foci. However, the other cases revealed an inverse correlation between the presence of HPV DNA and p53 gene mutations. These data show that p53 genetic alteration is correlated with the progression of prostate cancer, in contrast to the integration of HPV that may occur in a relatively early stage. In conclusion, this study may indicate that either p53 gene mutation or the presence of HPV's oncogenic protein E6 is involved in the development of prostate cancer. © 1996 Wiley-Liss, Inc.     

Formation of amyloid in encapsulated human pancreatic and human stem cell-generated beta cell implants

Detection of amyloid in intraportal islet implants of type 1 diabetes patients has been proposed as cause in their functional decline. The present study uses cultured adult human islets devoid of amyloid to examine conditions of its formation. After intraportal injection in patients, amyloid deposits <15 µm diameter were identified in 5%–12% of beta cell containing aggregates, 3–76 months posttransplant. Such deposits also formed in glucose-controlling islet implants in the kidney of diabetic mice but not in failing implants. Alginate-encapsulated islets formed amyloid during culture when functional, and in all intraperitoneal implants that corrected diabetes in mice, exhibiting larger sizes than in functioning nonencapsulated implants. After intraperitoneal injection in a patient, retrieved single capsules presented amyloid near living beta cells, whereas no amyloid occurred in clustered capsules with dead cells. Amyloid was also demonstrated in functional human stem cell-generated beta cell implants in subcutaneous devices of mice. Deposits up to 35 µm diameter were localized in beta cell-enriched regions and related to an elevated IAPP over insulin ratio in the newly generated beta cells. Amyloid in device-encapsulated human stem cell-generated beta cell implants marks the formation of a functional beta cell mass but also an imbalance between its activated state and its microenvironment.     

Adenoviral-mediated overexpression of membrane-bound human FasL and human decoy Fas protect pig islets against human CD8+ CTL-mediated cytotoxicity

Pig islets are considered to be most suitable source of islets for xenotransplantation into patients with type 1 diabetes mellitus. However, cellular rejection, especially CD8+ CTL-mediated cytotoxicity, remains a formidable barrier preventing long-term xenograft survival. Our previous study demonstrated that human CD8+ CTLs were highly detrimental to xenograft cells and that this strong cytotoxicity of human CTLs was mediated mainly by the Fas/FasL apoptotic pathway. Furthermore, we exploited novel methods for inhibiting human CD8+ CTL-mediated xenocytotoxicity with overexpression of membrane-bound human FasL and human decoy Fas antigen in xenografted cells. In the present study, we assessed the cytoprotective effects of these novel inhibitory molecules overexpressed by an adenoviral-mediated system in pig islets. Isolated pig islets were transfected with adenovirus vector encoding either human decoy Fas or membrane-bound human FasL genes. Thirty percent to 60% of transfected pig islets expressed these molecules producing 60% to 88% suppression of CTL killing compared with parental pig islets. These data indicated that pig islet grafts isolated from transgenic pigs with either membrane-bound human FasL or human decoy Fas antigen genes may control the innate cellular response to xenografts, and creating a window of opportunity to facilitate xenograft survival.