Improved recovery of cryopreserved canine islets by use of beraprost sodium.

Cryopreservation of pancreatic islets provides many advantages for clinical transplantation. Unfortunately, the freezing and thawing processes lead to a significant loss of islets. In this study, an attempt was made to increase the yield and viability of islets after cryopreservation and thawing. By using canine islets, we evaluated whether beraprost sodium (BPS), a stable prostacyclin analog, protects islets during the freeze-thaw processes and improves the recovery of frozen-thawed islets. Canine islets were frozen and thawed by the procedures used routinely for storage of human islets. In this study, we deliberately used islets of lower purity (60+/-3.6%), which is undesirable for cryopreservation. The recovery of viable islets after thawing is poorer with islets of lower purity than with highly purified islets. BPS was added to both the cryopreservation solutions containing dimethyl sulfoxide (DMSO) and the thawing solution containing sucrose. After thawing, the islet recovery (islet number after thawing divided by islet number before freezing) was 71.1+/-12.7% with 1 nM BPS, 77.8+/-5.6% with 10 nM BPS, 79.3+/-6.7% with 100 nM BPS, and 69.2+/-7.2% in control preparations without BPS. Islet viability assessed by supravital staining was 57.5+/-5.6%, 64.7+/-7.0%, 67.5+/-6.5%, and 57.7+/-4.9% with 1 nM, 10 nM, and 100 nM BPS and controls, respectively. Both islet recovery and viability were significantly better with 10 nM and 100 nM BPS than with the controls (p<0.03). After 3 days in culture, islet numbers in the 10 nM and 100 nM BPS groups were significantly higher and showed better insulin-release responses than those from the 1 nM BPS and control groups. Histologically, islet structure was well preserved in the 10 nM and 100 nM BPS groups, whereas many islets of the control group were smaller and fragmented. Electron microscopic examination revealed that 10 nM and 100 nM BPS preserved the microstructure of islet cells, and signs of apoptosis or necrosis were rare. It was concluded that BPS improved the recovery and viability of canine islets after cryopreservation and thawing. BPS would be a useful agent for improving the recovery of cryopreserved human islets for clinical transplantation.     

贝前列腺素钠对糖尿病大鼠肾脏保护作用研究

目的 观察贝前列腺素钠（Beraprost Sodium,BPS）对糖尿病肾病大鼠肾脏功能及形态学方面的影响.方法 将30只SD大鼠随机分为正常对照组（NC）,糖尿病组（DM）,BPS治疗组（BPS）.STZ腹腔注射建模成功后,BPS组给予贝前列腺素钠0.6mg·kg-1·d-1灌胃,其余饲养条件三组皆同.灌胃12w结束时检测大鼠血糖、24h尿量、肾重体重比（KW/BW）、24 h尿白蛋白（UA1b/2A h）和肌酐清除率（Ccr）,并观察3组大鼠肾脏光镜及电镜下病理表现.结果 DM组血糖、尿量、KW/BW、UA1b/24 h和Ccr水平均较NC组升高（均P〈0.01）,BPS组除血糖外各项指标较DM组降低（均P〈0.01）;在光镜及电镜下观察到BPS组病理变化均较DM组有明显改善.结论 贝前列腺素钠对糖尿病大鼠肾脏具有保护作用.     

贝前列素钠对糖尿病大鼠相关炎症因子的影响

目的探讨贝前列素钠对糖尿病大鼠炎症因子的影响及其保护作用。方法30只SD大鼠随机分为正常对照组、糖尿病组,贝前列素钠组。正常对照组喂以普通饲料,其他各组大鼠均喂以高脂饲料,贝前列素钠组给予0．6m。g／（kg·d）,每日2次。观察8周,最终纳入实验各组6只,分别比较3组大鼠治疗前后白介素6（IL-6）、肿瘤坏死因子-a【（TNF-a）、髓过氧化物酶（MPO）、超敏C反应蛋白（hs-CRP）水平的影响。结果给药8周后,糖尿病组IL．6、TNF-a、MPO、hs．CRP水平均较对照组显著升高（P〈0．01）。贝前列素钠组较糖尿病组IL-6、TNF-a、MPO、hs-CRP水平显著降低（P〈0．05,P〈0．01）。结论贝前列素钠的干预可以降低糖尿病大鼠的炎症因子水平,减轻炎症损伤。     

Alterations in macrophage collagenase secretion induced by gold sodium thiomalate

The effects of gold sodium thiomalate (GST) on the production of specific collagenase by thioglycolate-elicited macrophages was investigated. Our studies demonstrated that GST administration can significantly decrease collagenase production in a dose-dependent manner. These effects were observed with levels of GST attainable in serum or synovial tissue during routine chrysotherapy. In addition, GST altered lysozyme secretion by activated macrophages in a pattern distinct from that of collagenase alteration. These effects of enzyme secretion were not secondary effects of GST on viability, general protein secretion, or the specific assay procedures utilized, and were not attributable to the thiomalate moiety. Thus, GST may exert its therapeutic effect in rheumatoid arthritis through interference with the production of degradative proteolytic enzymes, which are important effector molecules mediating tissue destruction.     

Assessment of islet cell viability using fluorescent dyes

Summary A rapid fluorometric method has been developed to evaluate the viability of isolated islet cells. The assay differentiates between viable and nonviable cells by the simultaneous use of the inclusion and exclusion dyes acridine orange and propidium iodide. When viewed by fluorescent microscopy, viable cells fluoresce green, while nonviable cells fluoresce bright red. Although the acridine orange and propidium iodide assay measures membrane integrity, the results of this assay correlate with other measures of cell viability. Compared to trypan blue exclusion, this assay is easier to read, more stable, and has fewer staining artifacts. The assay enables the rapid estimation of the viability of a population of islet cells prior to time-consuming experiments rather than retrospectively. This assay can also be used with intact islets. Stained islets can be divided into three distinct groups: green fluorescing islets contain insulin, red fluorescing islets contain little or no insulin and a third class of islets containing some non-viable cells fluoresce red, green, and yellow. The yellow colour is due to the superimposition of red and green fluorescing cells.     

Increased detection of interleukin-5 in sputum by addition of protease inhibitors.

The measurement of interleukin (IL)-5 in sputum is problematic, with interfering factors affecting immunoassay. The authors investigated whether sputum proteases could be acting as interfering factors by studying the effect of protease inhibitors (PI) on sputum IL-5 measurement. Induced sputa from 20 subjects with asthma were divided into aliquots, processed with and without protease inhibitors (in low and high concentrations) and the levels of IL-5 (spiked and endogenous) measured by enzyme immunoassay were compared. The concentration of sputum IL-5 was significantly increased by PI, with median (interquartile range) levels processed with no, low and high PI concentrations being 0 (0), 41.8 (75.6) and 66.1 (124.4) pg x mL(-1), respectively. There was also a significant increase in percentage recovery of spiked IL-5. Although high concentrations of PI reduced cell viability, there was no effect on total or differential cell counts and low concentrations of PI had no effect on cell counts or viability. Levels of endogenous interleukin-5 in sputum of asthmatic subjects can be significantly increased by the addition of protease inhibitors, and samples which would be regarded as negative for interleukin-5 without protease inhibitors may instead have considerable amounts of interleukin-5 detected.     

Enhanced neutrophil superoxide anion production and its modification by beraprost sodium in spontaneously hypertensive rats

To clarify the function of polymorphonuclear leukocytes (PMN) in spontaneously hypertensive rats (SHR) and the effect of beraprost sodium (BS) on these functions, we examined superoxide anion (O₂⁻) production and adherent activity by PMN, as well as modification of these functions by BS ex vivo and in vitro. In study 1, we measured PMN functions in 4-week-old SHR and Wistar-Kyoto (WKY) rats. In study 2 (ex vivo), 14-week-old SHR received vehicle (n = 6) and BS (30 μg/kg/day [n = 6] and 100 μg/kg/day [n = 7]) once daily for 4 weeks. In study 3 (in vitro), PMN from 18-week-old SHR were incubated with BS (0.1 and 1 μmol/L) and theophylline (200 μmol/L), which is reported to inhibit the PMN O₂⁻ production. Systolic blood pressure, platelet counts, and PMN O₂⁻ production stimulated by phorbol ester myristate acetate were significantly elevated in 4-week-old SHR compared with WKY (P < .05). Beraprost sodium decreased the ex vivo PMN O₂⁻ production, serum superoxide dismutase activity, and platelet counts (P < .05); however, BS did not reduce the in vitro PMN O₂⁻ production. These data support our hypothesis that the enhanced PMN function contributes to the cardiovascular damages during the early phase of SHR, and that BS has merit for preventing the O₂⁻ related organ damages in this model.     

贝前列素钠治疗2型糖尿病下肢动脉病变的随机对照研究

目的 比较贝前列素钠和阿司匹林治疗糖尿病合并下肢动脉病变的疗效及安全性.方法 采用随机、对照研究方法将2009年11月至2010年8月30例糖尿病合并下肢动脉病变的患者按照2：1随机分为贝前列素钠组（A组,40μg每日3次,n=20）及阿司匹林组（B组,100 mg每日1次,n=10）,服药12周.随机分组后分别在0、12周及24周进行无痛行走距离、最大行走距离的测定;0、12周测量踝肱指数（ABI）及血常规、肝肾功能、血脂及心电图.整个研究过程中对不良反应进行了评价.组间比较采用t检验、X2检验或秩和检验.结果 2组在年龄、性别、糖尿病慢性并发症无明显差异.A组疼痛、麻木及冷感症状的改善显著优于B组（X2=5.96,P＜0.05）;与基线（0周）时（276、1000 m）比较,A组在12周及24周无痛行走距离中位数分别为350 m（Z=-2.94,P＜0.05）和315 m（Z=-2.88,P＜0.05）,最大行走距离分别为1713 m（Z=-3.73,P＜0.05）和1600 m（Z=-3.58,P＜0.05）,均显著增加;B组在12周时无痛及最大行走距离分别为400 m（Z=-2.13,P＜0.05）及1075 m（Z=-2.54,P＜0.05）,较0周时显著增加,但24周时无痛及最大行走距离分别为390 m（Z=-0.81,P＞0.05）和1025 m（Z=-0.21,P＞0.05）,较0周无显著变化.两组间行走距离比较在0、12、24周均无显著差异,但12周时A组和B组无痛行走距离增加值的中位数分别为125、30 m（Z=-2.87,P＜0.05）,24周时分别为82.5、0 m（Z=-3.31,P＜0.05）,差异均有统计学意义;12周时A组和B组最大行走距离增加值的中位数分别为500、50 m（Z=-3.20,P＜0.05）,24周时分别为300、10 m（Z=-3.02,P＜0.05）,差异均有统计学意义.两组肝功能、肾功能、血常规、ABI在治疗前后无明显差异.结论口服前列腺素钠可安全有效地用于糖尿病合并下肢动脉病变的治疗.     

Effects of beraprost sodium (Dorner) in patients with diabetes mellitus complicated by chronic arterial obstruction

The effects of the prostaglandin I2 derivative beraprost sodium (Dorner) on ankle pressure index (AP; ankle joint-to-upper extremity systolic pressure ratio), subjective symptoms, and intermittent claudication were investigated in diabetic patients with arteriosclerosis obliterans (ASO). Forty patients (25 men and 15 women), mean age 63.9 years, were enrolled in this study. ASO was grade I in 30 patients, grade II in seven, grade III in one, and grade IV in two according to the Fontaine classification. They were administered six tablets (20 microg/tablet) of beraprost sodium daily for 6 months. At 3 and 6 months, API had significantly increased and symptoms such as coldness, numbness, and lack of feeling in the lower extremities were significantly improved. Ten evaluable patients increased ambulatory distance by approximately threefold, suggesting an improvement in intermittent claudication. Adverse reactions were experienced by five (12.5%) of the 40 patients (one case each of headache, dull headache, pain in the posterior region of the neck, heartburn, stomach discomfort, and anemia), but all were mild and resolved without treatment. Beraprost sodium was shown to improve API and symptoms in the lower extremities in diabetic patients with ASO, suggesting that it is useful in treating peripheral circulatory disorders in such patients.     

Increased immunostaining of collagenase and TIMP in eruptive xanthoma

Remodeling of the extracellular matrix is an important function of interstitial collagenase. The activity of this enzyme forms the initial and rate limiting step in collagen degradation; moreover, this enzyme appears representative of a family of connective tissue metalloproteinases. Conversely, a widely distributed glycoprotein, tissue inhibitor of metalloproteinases (TIMP), may be an important regulator of matrix degradation. To study the roles of collagenase and TIMP in pathologically altered dermal connective tissue, immunohistochemistry was used to localize collagenase and TIMP in an eruptive xanthoma, a chronic tuberous xanthoma, and normal skin. Normal skin and the chronic tuberous xanthoma showed mild diffuse staining of both proteins throughout the dermis. In contrast, intense dermal staining of both collagenase and TIMP was present in the eruptive xanthoma. Thus, the marked accumulation of lipid in dermal macrophages was associated with a significant increase in matrix collagenase and TIMP. This increase may reflect direct production of these two proteins by macrophages. Alternatively, it may be due to increased production by fibroblasts stimulated by macrophage-derived cytokines. The balance of degradative and inhibitory activities in the extracellular matrix may regulate the extent and nature of dermal remodeling.     

Perfusion lung scan as a prognostic indicator of response to beraprost sodium in idiopathic pulmonary arterial hypertension

STUDY OBJECTIVE: To study whether there is any difference in the clinical characteristics between the two patterns of perfusion lung scan of idiopathic pulmonary arterial hypertension (normal vs. diffuse, multiple ill-defined defects) and whether the perfusion lung scan patterns of these patients would predict the effect of long-term use of beraprost sodium. METHODS: We evaluated 27 patients who used beraprost sodium for over 3 months, and noted a diffuse patchy pattern in 13 cases and a normal pattern in the remaining 14 cases. We judged that beraprost sodium was effective when at least two of the following conditions were met: improvement in symptom of dyspnea, more than 10% decrease in peak velocity of tricuspid valve regurgitation by echocardiography (Vmax), or more than 10% increase in 6-min walking distance. RESULTS: At baseline there was no difference between the two groups in dyspnea, hemodynamic parameters, and 6-min walking distance. After the use of beraprost sodium, the normal group showed improvement in dyspnea, 6-min walking distance, and Vmax. But the diffuse patchy group showed no improvement. The use of beraprost sodium in the normal group was effective in 10 out of 14 cases, but was effective in only two out of 13 cases in the diffuse patchy group. CONCLUSION: Perfusion lung scan pattern in patients with idiopathic pulmonary arterial hypertension is a useful prognostic indicator of the response to beraprost sodium.     

Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.     

Comparison of the acute pulmonary vasodilating effect of beraprost sodium and nitric oxide in congenital heart disease.

BACKGROUND: Congenital heart disease patients who have pulmonary hypertension (PH) require an evaluation for pulmonary vascular reactivity before surgical repair. In the present study the acute pulmonary vasodilating effects of 100% oxygen (O2), beraprost sodium (BPS) and 40 ppm inhaled nitric oxide (iNO) during cardiac catheterization were compared. METHODS AND RESULTS: There were 90 patients who underwent cardiac catheterization for evaluation of PH (mean age, 16.5+/-16 years). The baseline mean pulmonary artery (mPA) pressure was 69.6+/-14.8 mmHg and the pulmonary arteriolar resistance (Rpa) was 13.8+/-8.3 Wood unit m2. Change in pulmonary vascular reactivity was defined as a decrease in mPA or Rpa>20% from baseline. The response to 100%O2, iNO and BPS during cardiac catheterization was 84%, 72.7% and 64%, respectively. Pair comparisons among each hemodynamic parameter showed no difference between the acute vasodilating effect of BPS and iNO. In some patients BPS showed a stronger effect than iNO in lowering Rpa. CONCLUSIONS: BPS has a similar pulmonary vasodilating effect to iNO and can be used as an acute pulmonary vasodilating agent during cardiac catheterization with potential benefits over iNO.     

Increased islet beta cell replication adjacent to intrapancreatic gastrinomas in humans

Aims/hypothesis

Type 1 and type 2 diabetes are characterised by a beta cell deficit. Islet hyperplasia has been described in patients with Zollinger–Ellison syndrome secondary to gastrin-producing tumours (gastrinomas), and gastrin therapy has increased beta cell mass in rodents and human islets in vitro. In the present studies we addressed the following questions: (1) In pancreas specimens from gastrinoma cases, is the fractional beta cell area increased? (2) If so, is this restricted to tumour-adjacent islets or also present in tumour-distant islets? (3) Is new beta cell formation (beta cell replication and islet neogenesis) increased and beta cell apoptosis decreased in pancreas specimens from gastrinoma cases?

Methods

Pancreas was obtained at surgery from four patients with Zollinger–Ellison syndrome caused by pancreatic gastrinomas and 15 control subjects at autopsy.

Results

Islet fractional beta cell area (p<0.001), islet size (p<0.001) and beta cell replication (Ki67 staining) (p<0.05) were increased in islets adjacent to the tumours, but not in tumour-distant pancreas, compared with control subjects. We did not observe any differences in beta cell apoptosis or in the number of insulin-positive cells in ducts either adjacent to or distant from the tumour.

Conclusions/interpretation

One or more factors released by human gastrinomas increase beta cell replication in islets immediately adjacent to the tumour, but not in tumour-distant islets. While these findings demonstrate that adult human beta cells can be driven into the cell cycle, they caution against the therapeutic usefulness of gastrin, since islets located >1 cm away from the gastrinomas did not exhibit changes in beta cell turnover, despite markedly elevated systemic gastrin levels sufficient to cause severe gastrointestinal symptoms.     

Improvement of collagenase distribution with the ductal preservation for human islet isolation

A delivery of collagenase at the islet-exocrine interface is crucial for successful human islet isolation. In this study, we investigated how the ductal preservation method at the procurement site affected collagenase distribution. At first, we analyzed human islet isolation data among groups using Serva collagenase with or without ductal injection (DI) or using new Liberase MTF with DI. Then, to assess the distribution of collagenase, human pancreata were classified into two groups: without DI (no DI, n = 5) and with DI at the procurement site (DI, n = 5). Collagenase with 1% marking dye was perfused in the same manner as in our clinical isolation. The distension of the pancreas and the microscopic distribution of the dyed collagenase in pancreas sections were examined. For microscopic analysis, islets were counted and classified into three criteria: unreached, dye didn’t reach the islet surface; surface, dye resided on the surface of the islet but not inside; and inside, dye was found inside the islet. As a result, DI groups substantially improved islet yields. In addition, Liberase MTF with DI significantly improved efficacy of pancreas digestion. All pancreata were well distended macroscopically. However, microscopically, the majority of islets in the no DI group were untouched by the dyed collagenase. Ductal preservation substantially improved dyed collagenase delivery on the surface of islets. In conclusion, delivery of collagenase on the surface of islets was unexpectedly insufficient without DI, which was substantially improved by DI. Thus, ductal preservation is a potent method to improve collagenase delivery and islet yields.     

Improvement of collagenase distribution with the ductal preservation for human islet isolation

A delivery of collagenase at the islet-exocrine interface is crucial for successful human islet isolation. In this study, we investigated how the ductal preservation method at the procurement site affected collagenase distribution. At first, we analyzed human islet isolation data among groups using Serva collagenase with or without ductal injection (DI) or using new Liberase MTF with DI. Then, to assess the distribution of collagenase, human pancreata were classified into two groups: without DI (no DI, n = 5) and with DI at the procurement site (DI, n = 5). Collagenase with 1% marking dye was perfused in the same manner as in our clinical isolation. The distension of the pancreas and the microscopic distribution of the dyed collagenase in pancreas sections were examined. For microscopic analysis, islets were counted and classified into three criteria: unreached, dye didn't reach the islet surface; surface, dye resided on the surface of the islet but not inside; and inside, dye was found inside the islet. As a result, DI groups substantially improved islet yields. In addition, Liberase MTF with DI significantly improved efficacy of pancreas digestion. All pancreata were well distended macroscopically. However, microscopically, the majority of islets in the no DI group were untouched by the dyed collagenase. Ductal preservation substantially improved dyed collagenase delivery on the surface of islets. In conclusion, delivery of collagenase on the surface of islets was unexpectedly insufficient without DI, which was substantially improved by DI. Thus, ductal preservation is a potent method to improve collagenase delivery and islet yields.     

贝前列素钠预处理对兔心肌缺血/再灌注损伤的早期拮抗作用

目的:探讨贝前列素钠（beraprost sodium,BPS）预处理对兔心肌缺血/再灌注(I/R)损伤的早期拮抗作用。方法: 将36只日本大耳白兔随机分为3组,即A组（假手术组）、B组（I/R组:缺血40 min后,再灌注90 min）和C组（BPS预处理组:以15 μg/kg,2次/d,［1］连续喂养4周,最后1次喂养后3 h内进行I/R）。以上3组均开胸暴露心脏,A组在左冠状动脉左室支处穿线不结扎,观察130 min结束实验;B组及C组开胸后结扎左冠状动脉左室支40 min,心电图若出现Ⅱ导联ST段升高及结扎周围心肌的颜色变紫或变深显示已形成心肌梗死(MI)模型。然后进行以下检测:①再灌注90 min后,随机选取6只日本大耳白兔,立即取结扎点下损伤的心肌组织,用100 g/L福尔马林固定后,部分进行HE染色在光镜下观察心肌形态学改变;部分用免疫组化染色法检测心肌细胞中Bax、Bcl-2的表达。②再灌注4 h后,取右心房（或股静脉）血应用电化学发光免疫法检测血浆中心肌中肌酸激酶(CK-MB)的变化。结果: ①HE染色显示,A组的心肌结构正常,B组的心肌损伤较严重,C组的心肌损伤较轻。②免疫组化染色法检测显示,A组Bcl-2的表达呈阳性,Bax的表达呈阴性;B组Bax的表达呈强阳性,Bcl-2的表达呈弱阳性;C组Bax的表达呈弱阳性,Bcl-2的表达介于A组与B组之间。③用电化学发光免疫法检测心肌酶学结果,CK-MB升高者3个组依次为B组>C组>A组,B组CK-MB的水平均明显高于C组与A组（P<0.01）。结论: BPS预处理对兔心肌I/R损伤早期具有拮抗作用。     

Osteoporotic bone microstructure by collagenase etching.

Collagenase etching has been used to show the microstructure of bone from patients suffering from primary osteoporosis. Both polished and unpolished surfaces of trabecular bone from femoral heads were treated with collagenase solution before study in the scanning electron microscope. The polished surfaces show the mineral component of this bone as small rounded units approximately 10-20 nm across, which aggregate to form a continuous phase of contiguous spheroidal particles approximately 100 nm across. Lamellations are clearly seen to be due to the removal of collagen fibres up to approximately 200 nm across, fibres in adjacent lamellae being arranged approximately perpendicular to each other. The unpolished surfaces also show small rounded units, which aggregate into rods of mineral approximately 100 nm across. Although these rods form a connected system, they are loosely packed, compatible with their being interspersed with the collagen fibres in vivo. This model for the detailed microstructure of bone is consistent with specimens from a number of other sources and shows no features unique to osteoporosis.