Urothelial dysplasia and inflammation induced by Schistosoma haematobium total antigen instillation in mice normal urothelium

Objectives

Squamous cell carcinoma of the urinary bladder has been associated with Schistosoma haematobium infection in many parts of Africa. The epidemiologic association is based on case control studies and on the close correlation of urinary bladder cancer incidence with prevalence of S. haematobium infection within different geographic areas. A parasite-tumor linkage is further suggested by the predominance of squamous cell (as opposed to transitional cell) morphology of bladder carcinomas seen in S. haematobium-endemic areas. The cellular mechanisms linking S. haematobium infection with cancer formation are not yet defined. In the present study, we hypothesized that the parasite antigens might induce alterations in urothelium.

Materials and methods

We investigated the effects of S. haematobium total antigen in CD-1 mice normal bladders after intravesical administration of the parasite antigens. The bladders were analyzed histopathologically 20 and 40 weeks after treatment.

Results

Intravesical instillation of S. haematobium total antigens induces the development of urothelial dysplasia and inflammation.

Conclusions

In our work, we demonstrate for the first time that S. haematobium antigens are the direct cause of alterations in urothelium.     

Transient receptor potential vanilloid type 2 (TRPV2) expression in normal urothelium and in urothelial carcinoma of human bladder: correlation with the pathologic stage

OBJECTIVE: To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. METHODS: Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. RESULTS: Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. CONCLUSIONS: Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.     

Immunohistochemical demonstration of blood group antigens in neoplastic and normal human urothelium: a comparison with standard red cell adherence

The specific red cell adherence test as a method to detect blood group antigen deletion in urothelial malignancy has been reported to yield approximately 40 per cent false negative results in 0 blood group patients. Our study of multiple sections of 8 normal ureters from blood group 0 patients and more than 220 specimens of transitional cell cancer taken from 48 patients reveals that the immunoperoxidase technique is more specific than the specific red cell adherence method in predicting subsequent invasion in blood group O(H) patients presenting with superficial transitional cell carcinomas (71 compared to 29 per cent) but is no more specific for tumors containing A or B antigens. However, immunoperoxidase staining does improve discernment of underlying histologic detail and, thereby, facilitates recognition of false positive antigen testing associated with squamous and adenomatous metaplasia. Areas of squamous and adenomatous metaplasia in specimens we tested were frequently antigen positive in invasive tumors. Therefore, we believe that these areas must be disregarded in determining antigen deletion in transitional cell carcinomas.     

5'-FITC标记的PCNA反义寡核苷酸在人膀胱癌细胞BIU-87中的分布及稳定性分析

目的观察硫代修饰型及天然型PCNA反义寡核苷酸在脂质体介导下或直接转染人膀胱癌细胞BIU-87后在细胞内的分布及其稳定性。方法将异疏氰酸荧光素（5＇－FITC）标记的18mer硫代磷酸化修饰型及未修饰型PCNA反义寡核苷酸在脂质体介导下或直接转染人膀胱癌细胞BIU－87，应用荧光显微镜观察转染细胞从细胞内的时相分布。结果修饰型反义核酸直接转染细胞后30分钟，少数细胞胞浆荧光呈离散型、点状分布，4小时后有少数细胞胞核有强荧光聚积。在脂质体介导下，荧光细胞数目明显增加，4小时后绝大多数细胞迅速积聚于细胞核，4～8小时后，细胞核内荧光强度进一步增强，12小时后细胞荧光减弱甚至消失。而非修饰型反义核酸在直接转染或脂质体介导下均发现荧光在3小时后消失。结论脂质体增加PCNA修饰型反义寡核苷酸与膀胱癌细胞BIU－87结合的数量，同时使其在细胞核内分布聚积明显；PCNA反义寡核苷酸硫代修饰具有更好的稳定性。     

An immunoperoxidase study of S-100 protein distribution in normal and neoplastic tissues

The presence of S-100 protein was immunohistochemically studied in many types of formalin-fixed and paraffin-embedded tumors (260 cases). Peripheral nerve tumors, i.e., schwannomas, neurofibromas, granular cell tumors, and neurogenic sarcomas were demonstrated to contain variable amounts of S-100 protein in the tumor cell cytoplasm and nuclei. In ganglioneuromas and ganglioneuroblastomas, neoplastic Schwann cells or satellite cells were positive for S-100 protein. About one-half of the cases of carcinoid tumors stained weakly for S-100 protein. In addition to these nervous tissue and carcinoid tumors, chondrosarcoma, chordomas, pleomorphic adenomas of the salivary gland, and Langerhans cell granulomatosis were also shown to produce S-100 protein. In many types of breast tumors and other lesions, S-100 protein positive cells were likely to correspond to the distribution of myoepithelial cells. These results indicate that S-100 protein is not strictly specific to nervous tissue and its tumors; however, the immunohistochemical demonstration of S-100 protein can be a useful diagnostic tool in tumor diagnosis.     

Effects of ulinastatin (urinary trypsin inhibitor) on ATP, intracellular pH, and intracellular sodium transients during ischemia and reperfusion in the rat kidney in vivo

Purpose. To investigate the effects of ulinastatin on renal ischemia–reperfusion injury, we monitored the dynamic changes in ATP, intracellular pH (pHi), and intracellular sodium (Nai) in rats in vivo. Methods. Renal ischemia was induced by clamping the abdominal aorta for 30 min followed by reperfusion for 60 min. Ulinastatin, 50 000 U·kg⁻¹ (UTI group), or normal saline (NS group) was infused for 30 min before ischemia. ³¹P- and double quantum ²³Na-NMR were used to monitor ATP, pHi, and Nai. Results. During ischemia, ATP was rapidly depleted and Nai increased to the same extent in both groups. After 60 min reperfusion, Nai in the NS group was almost restored to the preischemic baseline level (117.2 ± 7.4% of the baseline value), but the recovery of ATP was incomplete (60.9 ± 7.7%). The recovery of Nai in the UTI group began earlier than in the NS group with better recovery of ATP. The pHi values showed severe acidosis in the NS group compared with the UTI group during ischemia and reperfusion. As for ultrastructural findings, after 60 min reperfusion, the mitochondria were less swollen and less disorganized with respect to the membrane and the cristae in the UTI group. Conclusion. The transcellular sodium gradient is restored before the ATP level is normalized during postischemic reperfusion. Ulinastatin might protect mitochondrial conformation during ischemia, and facilitate functional recovery of the ionic pump after reperfusion. Received: April 7, 2000 / Accepted: September 13, 2000     

Control of intracellular pH in rat calvarial osteoblasts: Coexistence of both chloride-bicarbonate and sodium-hydrogen exchange

Summary Intracellular pH was monitored continuously in cultured rat calvarial osteoblasts using the pH-sensitive fluorescent dye bis carboxyethyl carboxyfluorescein (BCECF), loaded into the cells as its membrane permeant ester. Recovery from an intracellular acid load generated by exposure to NH₄Cl was unaffected by the anion exchange inhibitors 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disuphonic acid (SITS) and 4,4′-diisothiocyanatostilbene 2,2′-disuphonic acid (DIDS) (100 μM), but blocked by the sodium-hydrogen exchange inhibitor amiloride (1 mM) and dependent on external sodium, suggesting that recovery is brought about by a sodium-hydrogen exchanger in the plasma membrane. The cells do, however, possess a SITS-sensitive chloride-bicarbonate exchanger, because iso-osmotic replacement of chloride by gluconate leads to intracellular alkalinization, that is inhibited by SITS, but independent of external sodium. Parathyroid hormone brings about an intracellular acidification, which may be due to an inhibition of sodium-hydrogen exchange.     

Expression of the epidermal growth factor receptor family in normal and malignant urothelium

OBJECTIVE: To compare the immunohistochemically assessed expression of the epidermal growth factor receptor (EGFR) family in normal and malignant bladder urothelium, and suggest new hypotheses about their function in the development and progression of transitional cell carcinoma (TCC). PATIENTS AND METHODS: EGFR, ERBB2, ERBB3 and ERBB4 were evaluated immunohistochemically in normal urothelium (NU, 15), primary non-metastasized invasive TCC (NMC, 19) and in primary invasive TCCs with corresponding metastases (MC, 51, both specimens). RESULTS: All NU samples expressed ERBB4, none expressed ERBB2 and two expressed EGFR; all staining was uniform throughout all cell layers. ERBB2 expression increased and ERBB4 decreased from normal samples to carcinomas. There was no difference between NMCs and MCs in ERBB2, ERBB3 and ERBB4, but the NMCs expressed more EGFR than both NU and MC samples. There were no associations with T category, grade or survival. All combinations of expression levels for the four receptors were detected, with no dominant profile. CONCLUSION: We hypothesise that: (i) ERBB4 is important for differentiation in NU; (ii) ERBB2 is up-regulated with carcinogenesis in the urinary bladder but does not discriminate between bladder cancer with or without metastases; (iii) EGFR may be a marker of indolent disease. A current hypothesis, that superficial layers of NU do not express EGFR and thus protect the basal cells from the mitogenic effect of urinary EGF, is challenged.     

Growth, differentiation and senescence of normal human urothelium in an organ-like culture

OBJECTIVE: To examine the kinetics of growth, differentiation and senescence of normal human urothelium in an organoid-like culture model. MATERIALS AND METHODS: Micro-dissected normal human urothelium explants were grown on porous membranes pretreated with various matrix components. Between 5 and 30 days of culture, cell proliferation was assessed by BrdU incorporation. Differentiation was evaluated on the basis of cytokeratin (Ck) and uroplakin (UP) expression. Epidermal growth factor family mRNA expression was monitored during explant outgrowth. Senescence was assessed by measuring endogenous beta-galactosidase activity and p16(INK4a) mRNA expression. RESULTS: Collagen IV was the most efficient matrix component for urothelial cell expansion. BrdU incorporation by urothelial cells was 5% between 15 and 30 days, corresponding to steady-state urothelium in vivo. Heparin-binding EGF (HB-EGF), Amphiregulin (AR) and Transforming Growth Factor alpha (TGF alpha) expression correlated with increased cell proliferation. UPII expression was stable throughout culture. P16(INK4a) mRNA expression and beta-galactosidase activity increased on day 25, giving signs of senescence. CONCLUSIONS: This model retains many characteristics of the urothelium in vivo. It can be used for pharmacological studies between 15 to 25 days and to study mechanisms such as wound healing, proliferation and senescence.     

对不同月龄雄性大鼠成骨细胞内钙和钙通道电流的实验研究

目的研究不同月龄的雄性大鼠成骨细胞内钙浓度及钙通道电流是否存在差异.方法采用二次酶消化法分离不同月龄(分别为1、2、3、5、7、9、11、13、15)雄性大鼠的原代成骨细胞,通过激光扫描共聚焦技术测定细胞内游离钙浓度([Ca2+]i)(以平均荧光强度表示),同时应用全细胞膜片钳技术记录成骨细胞膜钙电流(ICa)的变化.结果共聚焦结果显示随月龄增加,成骨细胞内[Ca2+]i逐渐降低,但相邻2月龄组之间细胞内[Ca2+]i无显著性差异(P＞0.05);1、2、3月龄组与11、13、15月龄组成骨细胞内[Ca2+]i有显著性差异(P＜0.05),5、7、9月龄组与各组相比均无差异(P＞0.05).应用全细胞膜片钳技术发现刺激电压为+10 mV时,2、7、13月龄组鼠ICa分别为(-392.77±97.07)pA、(-330.33±33.86)pA和(-287.68±71.01)pA,13月龄组鼠与2月龄组鼠相比,ICa明显降低(P＜0.05),而7月龄组鼠与2月龄组鼠和13月龄组鼠相比,ICa均没有明显差异(P＞0.05).结论不同月龄大鼠成骨细胞内[Ca2+]i存在差异,其机制可能与细胞膜上钙通道活性改变相关.     

Expression of PH-20 in normal and neoplastic breast tissue

BACKGROUND: Tumor metastasis involves a sequence of interrelated steps, of which penetration beyond the basement membrane is an essential component. Hyaluronic acid (HA) is a major structural component of the complex proteoglycans found in extracellular matrices and basement membranes. Hyaluronidase (PH-20) degrades HA, resulting in the disruption of basement membrane integrity and possible tumor dissemination. MATERIALS AND METHODS: Total RNA was extracted from samples (n = 51) of normal breast tissue (n = 12), ductal carcinoma in situ (DCIS) (n = 12), infiltrating ductal breast adenocarcinoma (n = 13), and metastatic breast cancer to lymph nodes (n = 14). RT-PCR was used to determine the relative level of PH-20 in each specimen. RESULTS: PH-20 was detected in 41/51 (80.4%) of the specimens evaluated. PH-20 was present in 12/12 (100%) normal breast tissues; 8/12 (66.7%) DCIS; 13/13 (100%) invasive breast cancers; and 8/14 (57.1%) metastases. Of those specimens in which PH-20 was detected, there were increased levels of PH-20 in metastatic breast cancer to lymph nodes compared to DCIS and invasive breast cancer. Stratification of specimen by race revealed that African American women had higher levels of PH-20 with invasive and metastatic beast cancer. CONCLUSIONS: Increased levels of PH-20 are noted in invasive and metastatic breast cancer compared to DCIS. Tumors from African American women with invasive and metastatic breast cancer demonstrated higher levels of PH-20 than Caucasians. Varying levels of PH-20 in mammary tissue may contribute to early invasion and metastasis of breast cancer.     

The content and distribution of glycosaminoglycans in the ejaculates of normal and vasectomized men

Summary. The glycosaminoglycans (GAGs) of sperm and seminal plasma of normal men and seminal plasma of vasectomized individuals have been identified and quantified by two dimensional (2D) electrophoresis. The sperm contains predominantly CSC and HS as well as significant amounts of DS which achieves a high level in the sperm of the youngest man, while HA and LSC are either undetectable or present in small quantities. In normal seminal plasma, characteristically, DS is essentially lacking whereas CSC is the major GAG and HA and LSC account for relatively high percentages. Interestingly, in the ejaculates of vasectomized men the DS content is relatively prominent and the HA concentration varies widely. The oversulfated chondroitin sulfates CSD/CSE were detected in 7 of the 37 specimens. Their presence in a normal human body fluid is reported for the first time and the previous observation of the youthful DS/CSC switch is expanded to this study.     

腰椎间盘内弹性蛋白分布的观察与定量分析

目的 :研究弹性蛋白在正常与突出腰椎间盘内的分布变化和形态学改变 ,探讨椎间盘不同部位弹性蛋白分布与椎间盘功能的关系以及椎间盘退变突出的原因。方法 :扫描电镜观察成人正常与突出腰椎间盘内不同部位弹性蛋白的分布。应用高效液相色谱测量椎间盘内相应部位弹性蛋白含量。结果 :弹性蛋白仅占正常椎间盘干重的1 92 7%± 0 2 47% ,存在于椎间盘各部位 ,从椎间盘浅部到深部、从纤维环周缘到髓核内部 ,弹性蛋白纤维密度逐渐降低 ,含量逐渐减少。突出椎间盘弹性蛋白纤维密度降低 ,含量明显减少 ,仅占椎间盘干重的 0 945 %±0 492 % ,尤以纤维环深部降低明显 ,弹性蛋白纤维上出现磨损、裂隙、不规则空洞等。结论 :弹性蛋白纤维参与了腰椎间盘框架的构成 ,在腰椎间盘内含量少 ,分布不均匀 ,与椎间盘的功能密切相关。突出椎间盘弹性蛋白的含量减少 ,其分布改变及病理形态学改变 ,可能是椎间盘退变突出的重要物质基础。     

Intracellular distribution of calcium and zinc in normal, hyperplastic, and neoplastic human prostate: X-ray microanalysis of freeze-dried cryosections

X-ray microanalysis has been performed on freeze-dried cryosections of normal, hyperplastic, and neoplastic human prostates. Needle biopsies from prostates suspected to be malignant were collected from ten patients. Seven of these patients had fully developed nodular hyperplasia, whereas the remaining three specimens were histologically classified as moderately differentiated adenocarcinomas. Prostates with normal light microscopic appearance were obtained from two brain-dead kidney donors. The concentration of calcium in secretory vesicles was found to be several orders of magnitude higher than the concentration of magnesium and zinc in all of the glands studied, which is consistent with calcium being the major cation secreted by the prostatic acinar cells. Some elderly prostates and neoplastic prostates revealed even lower mean zinc:calcium ratios in secretory vesicles. Even though both elements varied considerably, the wide variation of the zinc:calcium ratios was due mainly to variations in the concentration of zinc. Large, electron-dense bodies occasionally were found in the cytoplasm of prostatic acinar cells. These bodies contained high concentrations of sulphur which by far was the dominating element.     

持续性非卧床腹膜透析患者继发性甲状旁腺功能亢进相关因素分析

目的 探讨持续性非卧床腹膜透析(CAPD)患者继发性甲状旁腺功能亢进(SHPT)的患病率及相关因素.方法 分析2011年7月至2012年1月在本院腹膜透析中心规律随访的CAPD患者数据库资料.所有患者根据全段甲状旁腺激素(iPTH)分为甲状旁腺功能亢进组及甲状旁腺功能非亢进组.通过Logistic回归分析SHPT的相关影响因素.结果 共纳入639例患者,SHPT的患病率为46.95％.Logistic多因素回归分析结果显示,低血红蛋白、低血钙、高血磷、高碱性磷酸酶、高血肌酐、高标准化蛋白分解代谢率(nPCR)及低钙透析液是SHPT的独立影响因素.结论 CAPD患者中SHPT患病率较高,钙磷代谢异常、高蛋白饮食、肾性贫血及低钙透析液是影响SHPT发生的独立因素.     

正常与畸形愈合跟骨应力分布的比较研究

 目的 探讨在静力载荷作用下,正常与畸形愈合跟骨的应力分布差异。方法 将正常跟骨有限元模型的后关节面由内向外、由前向后楔形切除,模拟骨折后距下关节面塌陷的畸形愈合模型。切除前B?hler角为35°,切除后为0°。自足跟及跟腱附着点处分别垂直向上对模型施加320 N和160 N的载荷,观察畸形愈合跟骨的Vonmises应力分布,并与正常模型进行比较。结果 畸形愈合跟骨的距下关节面应力降低,应力分布特点为从正常的跟骨前中关节面为主要应力区转为跟骨外侧紧靠跟骰关节处,外侧主要应力区要高于内侧,是支撑载荷的主要部位。从跟骨结构的细化比较来看,首先,距下关节面顶部的塌陷造成跟骨主要应力区部分后移,转至跟腱附着点处由正常的(1.51±0.22) MPa增至(3.11±0.24) MPa,而距下关节面顶部的应力由正常的(6.71±0.37) MPa减至(2.83±0.49) MPa。其次,跟骨前侧近跟骰关节处应力明显加大,由正常的(0.46±0.15) MPa增至(2.13±0.15) MPa,载距突应力由5.18 MPa减少至1.41 MPa。结论 跟骨骨折距下关节面塌陷是多数病理变化的主因,在临床治疗中应先解决距下关节面的塌陷问题,恢复跟骨正常高度,跟骨内部应力的分布对解决跟骨骨折后期出现的相关部位疼痛至关重要。     

Pharmacodynamic and pharmacokinetic studies and prostatic tissue distribution of fosfomycin tromethamine in bacterial prostatitis or normal rats

In this study, we assessed the therapeutic effects of fosfomycin tromethamine (FT) in a bacterial prostatitis (BP) rat model. The BP model was induced by Escherichia coli and was demonstrated after 7 days microbiologically and histologically. Then, 25 BP rats selected were randomly divided into five treatment groups: model group, positive group, FT‐3 day group, FT‐7 day group and FT‐14 day group. Ventral lobes of prostate from all animals were removed, and the serum samples were collected at the end of the experiments. Microbiological cultures and histological findings of the prostate samples demonstrated reduced bacterial growth and improved inflammatory responses in FT‐treatment groups compared with the model group, indicating that FT against prostatic infection induced by E. coli showed good antibacterial effects. Moreover, plasma pharmacokinetics and prostatic distribution of fosfomycin were studied and compared in BP and normal rats. The concentrations of fosfomycin in samples were analysed by liquid chromatography–tandem mass spectrometry. There were no differences in plasma pharmacokinetic parameters between two groups. But significantly higher penetration of fosfomycin into prostatic tissues was found in BP rats. We therefore suggested that FT had a good therapeutic effect on BP and it might be used in curing masculine reproductive system diseases.     

Short-term primary cultures in studies of post-natal maturation of the rat proximal tubule - proton and bicarbonate transport

This is a review of recent work based on an in vitro model which has allowed us to investigate the postnatal maturation of renal epithelial cells. Renal proximal tubule cells from 8- to 40-day-old Sprague-Dawley rats were studied after 48 h of primary culture. The regulation of intracellular pH (pHi) was measured by quantitative fluorescence microscopy using 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis or acidosis using 15 mM ammonium salt. The ability of the cells to recover from both acidosis and alkalosis improved during post-natal maturation. The improvement in recovery from intracellular acidosis could be entirely accounted for by an increase in the rate of Na⁺/H⁺ exchange. The capacity for Na⁺/H⁺ exchange was independent of the cellular growth rate, but depended on cellular differentiation. A developmental increase in the activity of Cl^–/HCO₃-exchange between 12 and 14 days of age was also demonstrated. No developmental change was seen in either steady-state pHi (7.27–7.35) or in cytoplasmic buffer capacity (37.6–44.4 mM/pHi). The characteristics of transporter maturation revealed by these experiments are very similar to those observed in isolated perfused proximal tubules of developing rabbits.