Ovariectomy does not induce osteopenia through interleukin-6 in rhesus monkeys (Macaca mulatta)

To characterize the role of interleukin-6 (IL-6) in estrogen (E2)-depletion bone loss, we utilized a nonhuman primate model of human skeletal physiology. Adult female rhesus monkeys were sham-operated (S; n = 5), ovariectomized (ovx; n = 10), or ovx followed by E2 replacement (ovx + E2; n = 10) and evaluated for the indicated parameters at 0, 3, 6, and 9 months post-ovx. Lumbar spine bone mineral density (BMD) decreased by 3 months and continued to decline through 9 months in the ovx, but not in the ovx + E2 or S groups. Middle and distal radius BMD was decreased at 9 months in the ovx, but not in the ovx + E2 or S groups. The S group had marked fluctuations in bone remodeling parameters, and cytokine levels in S animals were consistent with menstrual cycling, and therefore only those values in the ovx and ovx + E2 groups are reported. Serum osteocalcin and skeletal-specific alkaline phosphatase were elevated in the ovx group compared with the ovx + E2 group. There was no difference in serum or bone marrow plasma IL-6 levels between the ovx and ovx + E2 groups. Similarly, there was no difference in basal or phorbol ester-stimulated IL-6 levels of peripheral blood mononuclear cell or bone marrow cell culture supernatants between groups. There was no difference in serum or bone marrow soluble IL-6 receptor between groups. However, the bone marrow plasma soluble IL-6 receptor levels were transiently increased from baseline at 3 months in the ovx but not in the ovx + E2 group. In summary, there was no bone loss in the ovx + E2 group, although the serum and bone marrow IL-6 levels were similar to those of the ovx group. These data suggest that modulation of IL-6 is not the key mechanism through which estrogen deprivation mediates bone loss in rhesus monkeys.     

Intracavernosal injection of vasoactive intestinal polypeptide (VIP) does not induce erection in man per se

Summary Vasoactive intestinal polypeptide (VIP) was injected intracavernously in five normal subjects. Injections of 1, 5, 10 or 20 g were given with a tourniquet at the base of the penis. In no instance was full tumescence or erection achieved. Visual sexual stimulation and vibration provoked full erection after all doses. Detumescence occurred in four after stimulation, but one man dad a 2-h-long erection after 20 g of VIP. VIP is most probably the transmitter involved in relaxation of the trabecular smooth muscle. As it is involved to a lesser degree in dilatation of the penile artery, some degree of nervous stimulation is then required to obtain erection.     

Neurotoxicity after spinal anaesthesia induced by serial intrathecal injections of magnesium sulphate An experimental study in a rat model

We have previously demonstrated in a rat model that the lumbar intrathecal injection of 0.02 ml 6.3% magnesium sulphate, a concentration iso-osmolar with rat plasma, produces a state of spinal anaesthesia and general sedation which reversed completely after 6 h, without evidence of neurotoxicity, immediately or during the week thereafter. Using the same model and five groups of six animals in each, we administered the same volume and concentration of magnesium sulphate and compared its clinical effects with those of 0.02 ml 12.6% magnesium sulphate, 0.02 ml 2% lignocaine and 0.02 ml 0.9% sodium chloride solution, given as a series of 15 injections on alternate days for a period of 1 month. The animals were then killed and their spinal cords and meninges examined histologically. No significant differences were noted in the times of onset, durations of sensory and motor blockade and the times to full recovery throughout the entire period of 1 month's observation in the animals receiving intrathecal 6.3% magnesium sulphate. In the group receiving 12.6% magnesium sulphate, the time of onset of sensory and motor blockade was shorter and the duration of both parameters was significantly longer than in the former group. Full clinical recovery and resumption of normal eating and drinking took place in both groups throughout the entire series of 15 successive intrathecal injections. Identical, mild, uniform histopathological changes in the spinal cord were seen in all the five groups, including the group in which only the intrathecal catheter was implanted. The complete recovery and benign consequences of repeated intrathecal injections of iso-osmolar magnesium sulphate in a rat model indicate a lack of neurotoxicity and provide an impetus for further trials in larger animal species, before initial clinical trials of this substance, given intrathecally, in humans.     

Proliferating cell nuclear antigen (PCNA) immunostaining as an alternative to bromodeoxyuridine (BrdU) immunostaining for brain tumours in paraffin embedded sections

Summary Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and BrdU using anti-PCNA and anti-BrdU monoclonal antibodies, respectively, was performed in 16 human brain tumours, including 3 glioblastomas multiforme, 2 anaplastic astrocytomas, 1 cerebellar astrocytoma, 2 recurrent meningiomas, 4 non-recurrent meningiomas, 3 neurinomas and 1 medulloblastoma. Patients with brain tumours received an injection of bromodeoxyuridine (BrdU) intravenously during surgery, and tumour specimens were fixed in 70% ethanol and embedded in paraffin. The percentage of positive cells for PCNA was compared with a BrdU labelling index using adjacent paraffin-embedded sections. The percentage of PCNA-positive cells was correlated with the BrdU labelling index and the histological malignancy of the brain tumours. The correlation coefficient was 0.84. This suggests that the immunohistochemical staining for PCNA in paraffin sections is a good alternative to the BrdU labelling index.     

p38丝裂原活化蛋白激酶在全脑缺血再灌注损伤大鼠海马神经元DNA修复中的作用

目的 探讨p38丝裂原活化蛋白激酶(p38 MAPK)在全脑缺血再灌注损伤大鼠海马神经元DNA修复中的作用.方法 清洁级雄性SD大鼠108只,采用四血管阻断法建立大鼠全脑缺血再灌注模型,随机分为3组(n=36):假手术组(S组)仅暴露双侧颈总动脉和椎动脉;缺血再灌注组(IR组)侧脑室注射1%DMSO溶液5μl,30 min后行全脑缺血再灌注;p38 MAPK抑制剂SB203580干预组(SB组)侧脑室注射SB203580溶液5 μl(溶于1%DMSO溶液),30 min后行全脑缺血再灌注.分别于再灌注2、6、12、24、48和72 h时各组处死6只大鼠,提取海马组织观察神经元病理学结果,计算神经元凋亡指数(AI),测定磷酸化的p38 MAPK蛋白及Ku70蛋白表达水平.结果 与S组比较,IR组和SB组各时点AI升高,p-p38 MAPK蛋白表达上调,p-Ku70蛋白表达下调(P(0.05或0.01),病理损伤明显;与IR组比较,SB组各时点AI降低,p-p38 MAPK蛋白表达下调,p-Ku70蛋白表达上调(P<0.01),病理损伤程度减轻.结论 p38 MAPK可能通过下调DNA修复酶Ku70蛋白的表达,使海马神经元DNA修复功能受损,导致神经元凋亡,参与全脑缺血再灌注损伤.     

The non-steroidal anti-inflammatory agent, indoprofen, does not protect against hydrochloric acid induced lung injury in the rat

The effects of pulmonary instillation of hydrochloric acid on solute flux from the lung (measured as the clearance of 99mTcDTPA) together with an index of oedema formation (the ratio of lung wet weight to lung dry weight) was measured in rats. There was a significant increase in 99mTcDTPA clearance (P less than 0.001) and also in lung wet:dry weight ratio (P less than 0.01) 4-5 h following acid challenge. There was no difference in the effects of challenge with acid of pH1 and pH2. In addition, intravenous injection of indoprofen (20mg/kg) a non-steroidal anti-inflammatory agent failed to produce any beneficial effect on the variables under study when given after acid (pH2) challenge. There was also histological evidence of an influx and sequestration of granulocytes into the lung. Despite this, the plasma concentration of thiobarbituric acid reactive material (used as an index of cell-derived, oxidant-free radical production) was significantly reduced in all acid-treated groups. These data show that hydrochloric acid will initiate a severe inflammatory response in the lung of the rat and that the non-steroidal anti-inflammatory agent indoprofen when given after the injury produced no evidence of a beneficial effect on this inflammatory response.     

Up-regulation of organic anion transporter 1 protein is induced by chronic furosemide or hydrochlorothiazide infusion in rat kidney.

BACKGROUND: Thiazide and loop diuretics are secreted from the proximal tubule via the organic anion transport system to reach their principal sites of action. Recently, a multispecific organic anion transporter 1 (OAT1) was identified in rat kidney and was localized to the basolateral membrane of the S2 segment in the proximal tubule. We postulated that interactions between thiazide or loop diuretics and OAT1 may play a role in the adaptation to long-term diuretic use, and investigated whether OAT1 is regulated in vivo by chronic administration of diuretics at the protein level. METHODS: Semi-quantitative immunoblotting and immunohistochemistry were carried out in kidneys from male Sprague-Dawley rats using a polyclonal peptide-derived antibody to OAT1. Furosemide (12 mg/day/rat, n = 6), hydrochlorothiazide (3.75 mg/day/rat, n = 6) or vehicle (1.7% ethanolamine, n = 6) were infused subcutaneously for 7 days using osmotic minipumps. Experimental and vehicle-control rats were pair-fed, and two bottles of drinking water were provided, one containing tap water and the other containing a solution of 0.8% NaCl with 0.1% KCl. RESULTS: Overt diuretic responses were observed to both furosemide and hydrochlorothiazide infusions. There were no differences in body weight or creatinine clearance between the experimental and control rats. Although OAT1 protein abundance in cortical homogenates was increased by furosemide infusion (271 +/- 35 vs 100 +/- 15%, P < 0.05), Na-K-ATPase alpha1 subunit protein abundance was not affected (113 +/- 14 vs 100 +/- 8%, P = 0.42). Immunohistochemical localization in tissue sections confirmed a strong increase in OAT1 expression in the basolateral membrane of the S2 segment of proximal tubule. OAT1 protein abundance in cortical homogenates was also increased by hydrochlorothiazide infusion (181 +/- 25 vs 100 +/- 7%, P < 0.01), whereas Na-K-ATPase alpha1 subunit protein abundance was not affected (105 +/- 4 vs 100 +/- 4%, P = 0.34). CONCLUSION: Chronic furosemide or hydrochlorothiazide infusion caused increases in OAT1 protein abundance in rat kidney. These results suggest that OAT1 may be up-regulated in vivo by substrate stimulation at the protein level.     

Prostate-specific antigen (PSA) protein does not affect growth of prostate cancer cells in vitro or prostate cancer xenografts in vivo

BACKGROUND: Prostate-specific antigen (PSA) is produced in high amounts by normal and malignant prostate cancer cells. PSA is a serine protease with substrates that include semenogelin I and II, insulin-like growth factor binding protein 3, fibronectin, and laminin. PSA, via its enzymatic activity, may play a role in growth, invasion, and metastasis of prostate cancer cells. Recent data also suggest that the PSA protein itself, independent of enzymatic activity, may also function as an endothelial cell-specific inhibitor of angiogenesis. METHODS: Human (PC3, DU145) and rat (AT2, AT6) prostate cancer cell lines were transfected with the full PSA gene encoding preproPSA protein. PSA-producing clones of each cell line were selected and the amount of enzymatically active PSA produced by each cell line determined using a PSA-specific fluorescent peptide substrate. In vitro and in vivo growth characteristics of PSA-producing transfectants were compared to neomycin controls and wild type cells. RESULTS: All selected clones produced and secreted PSA (5-120 ng/ml/10(5) cells). None of the PSA-transfected cell lines produced detectable amounts of enzymatically active PSA. Production of enzymatically inactive PSA by prostate cancer cell lines did not alter growth kinetics in vitro. PSA-producing xenograft doubling times in vivo were similar to neomycin controls and wild type. CONCLUSION: Although recent reports suggest the PSA protein itself may be antiangiogenic, our results demonstrate that production of PSA protein by prostate cancer cells does not significantly alter growth in vitro or in vivo.     

Alfuzosin (10 mg) does not affect blood pressure in young healthy men

OBJECTIVES: Alfuzosin 10mg is a uroselective alpha(1)-adrenoceptor antagonist used to treat lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Recent studies have suggested the potential efficacy of alfuzosin in the treatment of distal ureteral stones and prostatitis syndrome, two conditions frequently encountered in young patients. The objective of this study was to evaluate the effect of 10mg alfuzosin on blood pressure (BP) and heart rate (HR) in young healthy volunteers. MATERIALS AND METHODS: In a randomized, double-blind, placebo-controlled, crossover study, the effect of alfuzosin 10mg on BP and HR was evaluated in 14 male volunteers (mean age: 28 yr; range: 24-30). BP<135/85 obtained in two separated measurements was a main inclusion criterion. Patients were then randomized to alfuzosin (10mg once a day) or placebo for 1 wk, followed by a washout week, and then crossed over to the other treatment. Patients were instructed to self-measure systolic (SBP) and diastolic (DBP) blood pressure and HR every hour between 8am and 8pm during the first and the last day of each cycle treatment. RESULTS: All 14 enrolled volunteers completed the study. No significant difference in either SBP, DBP, or HR was observed between the placebo and alfuzosin groups at baseline. Alfuzosin did not affect SBP, DBP, or HR. No hypotensive episode (SBP reduction >10%) was recorded during each treatment. CONCLUSIONS: This study shows that alfuzosin 10mg is well tolerated by young healthy subjects and may therefore be safely administered to young normotensive patients affected by distal ureteral stones and prostatitis.     

Utilization of elderly kidney donors (>70 years) does not affect graft survival in the medium term

BackgroundThe need for organs for renal transplantation has encouraged the use of grafts from increasingly older donors. Earlier studies performed in Spain have shown the suitability of donors aged 60–65 years. In this single-center study, we evaluated our results using donors >70 years old.MethodsWe evaluated 401 primary transplantations performed from January 2000 to December 2009. Their initial immunosuppression was a tacrolimus-based (n = 324), cyclosporine-based (n = 70) or calcineurin inhibitor–free (n = 7) regimen patients. Recipients were classified according to the donors age: <50 (42.6%); 50–70 (39.7%) and >70 (17.5%) years.ResultsThere were no differences in recipient or donor gender, time on dialysis, cold ischemia, delayed graft function, or acute rejection episodes. However, the mean age was higher among patients who received grafts from donors >70 years old; 42.5 ± 12.4 years for <50, 58.1 ± 8.2 years for 50–70, and 65.7 ± 7.2 years for >70; (P = .000). The serum creatinine at 12 months was increased according to the age of the donor; 1.4 ± 0.6, 1.8 ± 0.6, 70 and 1.7 ± 0.5 mg/dL, respectively (P = .001). The graft survival rates at 5 years were 81%, 74%, and 70%, respectively (P = .519). Upon multivariate analysis only HLA-DR mismatches, delayed graft function, and acute rejection episodes were associated with graft loss. Patient survival rates (86%) at 5 years were similar among recipients from donors aged 50–70 and >70 years, but higher (96%) for those who received a graft from a donor <50 years (P = .003).ConclusionsNearly 20% of donors were >70 years old in our study. Their kidneys displayed excellent short-term outcomes.     

Expression of dentin matrix protein 1 (DMP1) in nonmineralized tissues

Dentin matrix protein 1 (DMP1) is an Arg-Gly-Asp-containing acidic phosphoprotein that was originally identified from a rat incisor cDNA library and was thought to be a dentin-specific protein. DMP1 was later shown to express in a number of hard tissue-forming cells, including osteoblasts, osteocytes, ameloblasts, and cementoblasts, and was considered to play important roles in mineralization. Further, DMP1 gene expression was also detected in fetal bovine brain and in newborn mouse brain. These findings indicate the possibility of DMP1 expression in other soft tissues. In the present study, to clarify the significance of DMP1 expression in nonmineralized tissues, we made a specific antibody to mouse DMP1 peptides and demonstrated that DMP1 protein was localized in mouse brain, pancreas, and kidney by immunohistochemistry. Further DMP1 mRNA was detected in nonmineralized mouse tissues including liver, muscle, brain, pancreas, and kidney by RT-PCR. Based on the evidence that the localization and the expression of DMP1 are not restricted to mineralized tissues, we assume that DMP1 may have functions other than the regulation of mineralization.     

Effect of photodynamic therapy (PDT) on the expression of pro-apoptotic protein Bak in nasopharyngeal carcinoma (NPC)

BACKGROUND AND OBJECTIVE: The stimulatory effects of low intensity laser therapy (LILT) have been widely published for the treatment of chronic ulceration. In contrast to this previous work, the current study investigated its potential efficacy (by using a dosage of 9 J/cm2) in the management of acute wounds. For this purpose, uncomplicated postoperative wounds after minor podiatric surgery were examined. STUDY DESIGN/MATERIALS AND METHODS: The study was designed as a controlled group study. Ethical approval was granted by the University of Ulster's Research Ethics Committee. Patients (n = 9) presenting with a total of 12 wounds after minor surgical procedures (partial/total nail avulsions/electrosurgery) were recruited from the Podiatry Teaching Clinic, Northern Ireland. Patients attended the clinic once per week for assessment and treatment. Weekly irradiation was performed by using a CBM Master 3 (CB Medico, Copenhagen, Denmark) diode laser (GaAlAs). The physical parameters of the output of this unit were as follows: wavelength, 830 nm; average power output, 30 mW; spot size, 0.1 cm2; irradiance, 300 mW/cm2; continuous wave output. Wound assessment and recording of pain levels were conducted weekly. Wound measurement was completed by using planimetry and digitising methods. RESULTS: Current findings indicated no statistically significant differences between Laser and Control groups for wound closure (P = 0.28 digitising; P = 0.49 planimetry) nor for pain levels reported (P = 0.88). CONCLUSION: It would seem that LILT provides no advantages in the management of minor postoperative wounds over current practice. Despite no apparent benefit of infrared laser at this dosage in the management of acute stage wounds, further research is required to determine its potential efficacy in the management of other wound types.     

Hypochlorhydria‐induced calcium malabsorption does not affect fracture healing but increases post‐traumatic bone loss in the intact skeleton

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post‐traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr?/? mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr?/? and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium‐enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro‐computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme‐linked immunosorbent assay. Fracture healing was unaffected in Cckbr?/? mice. However, Cckbr?/? mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr?/? mice. Therefore, under conditions of hypochlorhydria‐induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914–1921, 2016.

     

Glycochenodeoxycholate (GCDC) inhibits cytokine induced iNOS expression in rat hepatocytes

BACKGROUND: Although the accumulation of hydrophobic bile acid (e.g., glycine conjugated chenodeoxycholic acid, GCDC) is considered to be an important factor contributing to cholestatic liver dysfunction, its pathogenesis is poorly understood. The purpose of this study was to examine the effect of the bile salt GCDC on the regulation of iNOS expression, a key immune modulator during liver inflammation. MATERIALS AND METHODS: GCDC significantly decreased cytokine-stimulated iNOS promoter activity, and both iNOS mRNA and protein expression. GCDC decreased iNOS promoter activity by preventing IkappaB degradation and inhibiting NF-kappaB DNA-binding activity. To explore the role of iNOS in bile salt induced apoptosis, we also examined the effect of NO on caspase-3 activity. RESULTS: GCDC strongly induced caspase-3 activity, and this increase was abrogated by both exogenous NO exposure and endogenous NO synthesis. Furthermore, adenoviral iNOS (AdiNOS) pre-treatment decreased acute cholestatic-induced liver injury in a rat bile duct ligation model. CONCLUSIONS: These findings indicate a novel signaling pathway where potentially toxic bile salts down-regulate hepatic iNOS expression. This blockade of the iNOS mediated antiapoptotic phenotype may have important implications in certain liver disorders.     

Photodynamic therapy with photofrin in combination with Buthionine Sulfoximine (BSO) of human glioma in the nude rat

High concentrations of cellular glutathione (GSH) within tumour cells may reduce the ability of photodynamic therapy (PDT) to selectively destroy tumour, consequently, a means of improving the therapeutic ratio of PDT in brain tumour is necessary. Therefore, we hypothesize that PDT in combination with Buthionine Sulfoximine (BSO), an agent which lowers cellular glutathione, can significantly enhance destruction of U87 and U251n tumour cells. PDT was performed using Photofrin as a photosensitiser in combination with BSO administration on male Fisher rats with intracerebral U87 and on non-tumour rats (administered at different optical doses in combination with Photofrin). In vitro experimentation utilising colony forming, cell cytotoxicity, and matrigel artificial basement membrane invasion assays showed significant enhancement of tumour kill and significant reduction of migration in tumour cells treated with BSO in combination with Photofrin PDT in comparison with individual therapies for both U87 and U251n cell lines. In vivo combination PDT-BSO treatment of U87 tumour rats exhibited significantly more tumour necrosis than individual treatments. In conclusion, our data suggests BSO enhances Photofrin PDT treatment of human glioma.     

Hyperstosis induced by the bisphosphanate (2-PEBP) in the oophoretomized rat

Summary To prevent the high turnover bone remodeling associated with acute estrogen deficiency, the bisphosphonate [2-(2-pyridinyl) ethylidene-BP] (2-PEBP) was administered to oophorectomized (OX) rats. Three groups of 15 rats each (250 g) were studied. Group (Gp) A was sham operated, Gp B was OX, and Gp C received 2-PEBP (1.72 mg/kg/day) intraperitoneally for 3 days commencing 4 days postoophorectomy. Oophorectomy was confirmed with serum estradiol measurements. Blood samples were collected on days −7, 0, 7, 14, 21, and 28 for ionized calcium (Ca²⁺), PTH, and serum bone gla protein (BGP). Rats received tetracycline for bone histomorphometric labeling. All results were compared to Gp A. Body weight increased significantly in Gps B and C (P<0.005 by day 28). There was no significant difference in Ca²⁺, and PTH levels in Gps B and C were similar to Gp A. BGP levels were significantly higher on day 28 in Gp B (P<0.05). In Gp C, BGP levels were significantly decreased on days 7,21, and 28 (P<0.03). Gp B revealed increased bone turnover without loss of bone volume (BV/TV). BV/TV was significantly increased in Gp C despite a decrease in parameters of bone formation and normal osteoclast number. In conclusion, 2-PEBP in the OX rat inhibited bone resorption more than formation with resultant hyperostosis. Serum BGP appeared to be a good marker of the changes observed on bone histomorphometry.