Effect of epidermal growth factor on sodium-dependent L-alanine transport in LLC-PK1 cells

We evaluated the role of epidermal growth factor (EGF) in the regulation of L-alanine transport in LLC-PK1 renal epithelia. After 2 h of incubation, EGF had no significant effect on L-alanine uptake by LLC-PK1 cells. However, prolonged (16 h) incubation with 2 and 20 ng/ml of EGF resulted in significant increases in sodium-dependent L-alanine uptake as compared with controls. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 20 ng/ ml) caused a marked increase in sodium-dependent L-alanine uptake after both 2 and 16 h of incubation, and the treatment with TPA (20 ng/ml) EGF (20 ng/ml) for 16 h resulted in significant acceleration of the TPA-stimulated increase in L-alanine uptake by LLC-PK1 cells. Coincubation with H-7 (20 microM) inhibited both EGF- and TPA-stimulated increases in L-alanine uptake, and genistein (20 microg/ml) blocked the stimulatory effect of EGF in L-alanine transport to the control level. Furthermore, coincubation with cycloheximide (20 microg/ml) for 16 h inhibited both EGF- and TPA-stimulated increases in L-alanine transport to a great extent. The sodium- independent L-alanine uptake was not affected by treatment with either EGF or TPA. These results suggest that the activation of protein kinase C through tyrosine kinase activation plays a role in the EGF effect of stimulating L-alanine transport in LLC-PK1 cells and that the effect is mainly due to increased protein de novo synthesis which occurs after protein kinase C activation.     

AFP-producing urothelial carcinoma of the bladder: a case report

We report a case of urothelial carcinoma of the urinary bladder with concurrent alpha-fetoprotein (AFP) elevation. A 60-year-old male was admitted for gross hematuria. Subsequent analyses revealed elevated serum AFP levels (970.20 ng/ml). He had no history of hepatitis, and hepatobiliary disease was not detected on computed tomography or ultrasound. Carcino-embryonic antigen was normal. The patient underwent radical cystectomy and was found to have a high-grade urothelial carcinoma of bladder on pathology. In addition, immunohistochemical staining of the tumor cells showed strong AFP positivity. Postoperatively, serum AFP levels decreased gradually to normal. In summary, urothelial carcinoma of the urinary bladder with AFP elevation is rare, and the mechanism and prognosis require further exploration.     

Pharmacokinetics of clonidine after epidural administration in surgical patients. Lack of correlation between plasma concentration and analgesia and blood pressure changes

The pharmacokinetics of epidural clonidine 150 micrograms was studied in 13 patients who had undergone abdominal hysterectomy. Plasma clonidine concentrations were measured up to 19 h in eight patients. In another five patients frequent blood sampling was performed only during the first 20 min to define early vascular uptake better. Peak plasma clonidine concentrations of 1.08 +/- 0.35 ng ml-1 (mean +/- s.d.) were reached between 5 and 10 min after injection. Plasma elimination half-life was 829 +/- 157 min and plasma clearance was 177 +/- 28 ml min-1. There was a significant decrease in arterial blood pressure within 10 min of the injection of clonidine. The maximum decrease in systolic blood pressure, from a pre-injection value of 135 +/- 24.7 to 99 +/- 14.4 mmHg (18.0 +/- 3.3 to 13.2 +/- 1.9 kPa), occurred at 60 min. Blood pressure remained significantly lower than the pre-injection value for 4 h. There was no change in heart rate. Verbal analogue pain scores, on a scale 0-10, decreased from a median of 7.6 before clonidine to 5.0 after 30 min (P less than 0.05). The median score at 60 min was 4.3. Thereafter, pain scores were not significantly different from the control score. We conclude that epidural clonidine 150 micrograms produces only moderate and short-lived postoperative analgesia. Absorption of clonidine from the epidural space into the blood is very rapid and may contribute to the hypotension that occurs.     

JAK2／STAT3信号通路在IL-6介导肾小管上皮细胞转分化中的作用

目的：研究Janus激酶（JAK2）和信号转导因子和转录活化因子（STAT3）途径的激活在IL-6介导人近端肾小管上皮细胞转分化中的作用。方法：（1）细胞培养及分组：待生长状况良好的HK-2细胞株60%-80%细胞贴壁后,无血清培养基同步24 h,然后根据分组给予相应的刺激。（2）指标检测：采用荧光定量PCR技术检测0 h、24 h、48 h7、2 h不同时间点α-SMA mRNA、E-cadherin mRNA的表达。采用Western blot方法检测25 ng/ml浓度的IL-6刺激24 h、48 h、72 h后α-SMA、E-cadherin蛋白的表达;检测0、5、10、25、50 ng/ml浓度的IL-6刺激30 min,以及IL-6（25 ng/ml）刺激0、15 min、30 min、60 min、120 min后P-STAT3蛋白的表达水平;检测AG490预处理细胞4 h,IL-6（25 ng/ml）分别刺激30 min后及48 h后P-STAT3、P-JAK2和α-SMA、E-cadherin蛋白的表达水平。结果：与刺激前比较,IL-6以时间依赖方式上调HK-2细胞α-SMA mRNA、蛋白的表达,下调E-cadherin mRNA、蛋白的表达;IL-6以时间和剂量依赖方式上调HK-2细胞P-STAT3的表达,高峰发生在30 min;AG490部分抑制STAT3、JAK2的磷酸化,部分抑制IL-6诱导的α-SMA蛋白表达的上调、部分抑制IL-6诱导的E-cadherin蛋白表达的下调。结论：HK-2细胞中,JAK2/STAT3信号通路的激活可能参与了IL-6介导的肾小管上皮细胞转分化的发生。     

Discriminatory immunohistochemical staining of urothelial carcinoma in situ and non-neoplastic urothelium: an analysis of cytokeratin 20, p53, and CD44 antigens

Distinction of urothelial carcinoma in situ (CIS) from reactive atypia on the basis of morphology alone may be difficult in some cases. Because this distinction is therapeutically and prognostically critical, we attempted to determine if an immunohistochemical panel would help in this differential diagnosis. The immunoprofile of 21 cases of CIS and 25 non-neoplastic urothelia (15 urothelial biopsies with reactive atypia from patients without a history of bladder cancer and 10 normal ureter sections from nephrectomies performed for renal cell carcinoma) was determined using antibodies against cytokeratin 20 (CK20), p53, and CD44 (standard isoform). In the normal urothelium CK20 showed patchy cytoplasmic immunoreactivity in only the superficial umbrella cell layer and CD44 stained only the basal cells. Nuclear immunoreactivity to p53 varied from negative to weak and patchy. Reactive urothelium also showed CK20 immunoreactivity in only the umbrella cell layer in all 15 cases, and p53 nuclear staining was predominantly negative with occasional weak positivity in the basal and parabasal intermediate cells. CD44 was overexpressed in the entire reactive urothelium in 9 cases (60%) or focally positive in intermediate cells in 6 cases (40%). In contrast, CIS showed intense CK20 and p53 positivity (81% and 57%, respectively) in the majority (>50%) of malignant cells. CD44 staining revealed residual basal cells with membranous reactivity in 44% of the cases of CIS; however, the neoplastic cells were immunonegative in all cases. At least one positive immunomarker (CK20 or p53) was abnormally expressed in all cases of CIS. Abnormal expression of CK20 (increased), p53 (increased), and CD44 (decreased) in urothelial CIS, and increased expression of CD44 in reactive atypia allows more confident distinction of urothelial CIS from non-neoplastic urothelial atypias. From a differential diagnosis perspective, use of a panel of all three antibodies with morphologic correlation would be essential.     

Behavior of the histamine level in induction of anesthesia with propofol and methohexital

In a study of the effect of intravenous anesthetics on plasma histamine levels, propofol and methohexital were administered to patients. Histamine determination was performed using an improved fluometric method specific for imidazole derivatives. As a primary step, the plasma histamine concentration was determined in 60 healthy, fasting probands and used as a comparative value. The mean value obtained from 60 examinations was 0.38 +/- 0.12 ng/ml, the median value was 0.37 ng/ml (Table 1). The next step consisted in determination of plasma histamine values in 20 patients 1 h following premedication with fentanyl. In this group, the mean value was 0.33 +/- 0.11 ng/ml, the median value 0.316 ng/ml (Table 2). In another 20 patients the plasma histamine concentration was determined 1 h following intramuscular injection of 1.4 microgram fentanyl +0.07 mg/kg droperidol (Thalamonal). In this group, the mean value was 0.373 +/- 0.11 ng/ml and the median value was 0.736 ng/ml. Subsequently, the effect of 2.5 mg/kg propofol (Disoprivan) or 1 mg/kg methohexital (Brevimytal) on plasma histamine levels was examined in a randomized, prospective study in 22 patients of ASA class I and II (Table 3, Fig. 2). Two minutes prior to injection of the test substances and 2, 4, 8, and 13 min following injection, plasma histamine levels, blood pressure, and heart rate were examined. In both groups, no changes in plasma histamine levels were observed during the period of examination. Comparison of the individual time columns within a group as well as intergroup comparisons revealed no statistically significant differences in either the t test or the Wilcoxon-Mann-Whitney U test.(ABSTRACT TRUNCATED AT 250 WORDS)     

The clinical usefulness of urinary basic fetoprotein (BFP) in patients with urological malignancies]

Basic fetoprotein (BFP) was measured by enzyme immunoassay in the urine of healthy adults and patients with benign or malignant urological diseases. Urinary BFP level in 34 healthy donors was very low (2.75 +/- 2.27 ng/ml). Since the BFP-positive rate is under 5% in healthy donors and, considering diagnostic efficiency for urological malignancies, a urinary BFP-concentration of 15 ng/ml was used as the cut off value. Urinary BFP was positive in 17.0% of 106 patients with benign urological diseases, in 51.9% of 52 patients with bladder cancer, in 75.0% of 8 patients with renal pelvic or ureteral cancer, in 25.0% of 20 patients with prostate cancer, and in 19.0% of 21 patients with renal cell carcinoma. In 60 patients with urothelial carcinomas, urinary BFP was higher in invasive diseases (greater than pT1) than in superficial diseases (p less than 0.05). The BFP level in urine also increased with a higher histological grade. The positive rate of urinary BFP was 78.9% in patients with invasive diseases and 66.7% in patients with grade 3 diseases. In 44 patients with urothelial carcinomas who underwent urinary cytological examination the positive rate was improved from 38.6% (17/44) to 81.4% (37/44) when measurement of urinary BFP was added. BFP in urine was found to be useful as a tumor marker for urothelial carcinomas. In addition, it was demonstrated that combined examination of urinary cytology with urinary BFP was more efficient for diagnosis of urothelial carcinomas.     

Release of immunoreactive serotonin following acid perfusion of the duodenum.

Cannulas were placed in the portal vein, hepatic vein, and infrarenal vena cava in eight anesthetized dogs. The duodenum of each dog was irrigated with saline (control) and 0.1 N HCI (50 ml in 10 min). Heparinized blood samples were taken from each cannula 5 min before, and 1, 5, 10, 30 and 60 min after each irrigation for measurement of immunoreactive serotonin concentrations. Serotonin concentrations did not change during saline irrigation of the duodenum. In contrast, serotonin release was consistently observed after duodenal acidification (pH 1.5-2.0). Portal venous serotonin concentrations were increased at 1 min (356 +/- 147 ng/ml) and following a biphasic pattern remained elevated at 30 and 60 min (499 +/- 131 and 489 +/- 187 ng/ml, respectively). Concentrations in the hepatic vein rose more slowly and to a lower peak (357 +/- 123 ng/ml at 10 min). Caval serotonin concentrations were increased at 10 min (342 +/- 121 ng/ml) but promptly returned to baseline levels. In the canine gastrointestinal tract, immunoreactive serotonin concentrations were highest in the duodenal bulb (15.4 mug/gm). This study demonstrated serotonin release from the duodenum following acid perfusion and documented that some of the released serotonin escaped hepatic inactivation. These findings support the possibility that serotonin may be a gastrointestinal hormone involved in the feedback inhibition of gastric acid secretion.     

Prolactin suppression of leukocyte chemotaxis in vitro.

Leukocyte chemotaxis in vitro was studied for cells from patients with pituitary adenomas. Leukocytes obtained preoperatively from two of three patients with elevated serum prolactin levels demonstrated chemotaxic alterations described in other malignant disease. Statistically significant suppression of chemotaxis occurred in the leukocytes of four of 12 specimens from normal donors at concentrations of 1000 ng/ml, and in four of eight specimens at 2000 ng/ml of prolactin in preincubation media. Thus prolactin concentration may influence the motility of leukocytes. The variable neoplastic behavior of morphologically similar pituitary adenomas may, in part, reflect a neurohormonally altered host response to the presence of these lesions.     

Research on the effect of atenolol on lidocaine pharmacokinetics in rats

The aim was to determine the influence of atenolol on lidocaine pharmacokinetics in rats for one hour interval of time (average of a dental intervention). The study was carried out on 2 groups of Wistar rats treated with saline solution (0.5 ml/kg), respectively with atenolol (1.5 mg/kg), administered orally 24 hours and 3 hours before intraperitoneal administration of lidocaine (1.5 mg/kg). Blood samples were collected before and 5, 10, 20, 30, 60 minutes after lidocaine administration. Lidocaine plasma concentrations were determined by HPLC. Some pharmacokinetic parameters of lidocaine were statistically significant higher (p < 0.05, ANOVA) for the rats treated with atenolol compared with control group: Cmax (196.97 +/- 2.15 ng/ml vs. 125.29 +/- 2.90 ng/ml), AUD (7734.07 +/- 129.06 ng/ ml x min vs. 4478.57 +/- 296.61 ng/ml x min), AUC1 after 5 minutes (314.23 +/- 6.59 ng/ml x min vs. 190.71 +/- 19.75 ng/ml x min). Tmax was 20 minutes, similar for both groups. CONCLUSION: local anesthesia with lidocaine might be enhanced in the presence of atenolol compared to controls.     

Comparison of metabolic responses to deep hypothermic total circulatory arrest and retrograde cerebral perfusion in an experimental model

An experimental study was designed to search the effectiveness of retrograde cerebral perfusion which is presently used as cerebral protection method for the surgery of arcus aorta. Twelve dogs were subjected to the study. Six of them were remained in total circulatory arrest at 20 degrees C for 60 min. Retrograde cerebral perfusion was done again at 20 degrees C for 1 h for the other six dogs.Tumor necrosis factor (TNF), P-selectin, Intracellular Adhesion Molecule (ICAM), Creatine Phosphokinase (CPK-BB) and tissue Adenosine triphosphate (ATP) levels were measured, before the cardiopulmonary bypass at 37 degrees C and during perfusion period at 5, 60 min and 4 h.Tissue ATP level for retrograde cerebral perfusion group was 3.99+/-0.7 mcmol/g tissue and 2.86+/-0.1 mcmol/g tissue for total circulatory arrest group at fourth hour (p<0.05). TNF level was significantly higher in total circulatory arrest group than retrograde cerebral perfusion group (p<0.05). The samples taken at fourth hour of reperfusion showed the TNF level was, 162.55+/-13.1 pcg/ml for total circulatory arrest group and this value was 12.5+/-3.4 pcg/ml for retrograde cerebral perfusion group.ICAM (Intracellular Adhesion Molecule) level was higher in total circulatory arrest group (18.75+/-3.6 ng/ml) when compared to retrograde cerebral perfusion group (8.75+/-1.8 ng/ml) (p<0.05).All parameters showed that retrograde cerebral perfusion preserved the brain functions better comparing with total circulatory arrest. The time necessary for aortic surgery may be provided by the retrograde cerebral perfusion technique.     

Liposomal inhibition of acrolein-induced injury in rat cultured urothelial cells

Purpose

To study the protection offered by empty liposomes (LPs) alone against acrolein-induced changes in urothelial cell viability and explored uptake of LPs by primary (rat) urothelial cells.

Methods

Acrolein was used as a means to induce cellular damage and reduce urothelial cellular viability. The effect of acrolein or liposomal treatment on cellular proliferation was studied using 5-bromo-2′-deoxy-uridine assay. Cytokine release was measured after urothelial cells were exposed to acrolein. Temperature-dependent uptake study was carried out for fluorescent-labeled LPs using confocal microscopy.

Results

Liposome pretreatment protected against acrolein-induced decrease in urothelial cell proliferation. LPs also significantly affected the acrolein-induced cytokine (interferon-gamma) release offering protection to the urothelial cells against acrolein damage. We also observed a temperature-dependent urothelial uptake of fluorescent-labeled LPs occurred at 37 °C (but not at 4 °C).

Conclusions

Empty LPs alone provide a therapeutic efficacy against acrolein-induced changes in urothelial cell viability and may be a promising local therapy for bladder diseases. Hence, our preliminary evidence provides support for liposome-therapy for urothelial protection and possible repair.     

In vitro cyclosporine toxicity. The effect of verapamil

The epithelial cell line LLC-PK1, which expresses many proximal tubular characteristics, was used to investigate the relationship between calcium, the calcium channel blocker verapamil, and cyclosporine toxicity. The LLC-PK1 cells took up cyclosporine when this was added in a concentration of 2 micrograms/ml, and this uptake was maximal at 30 min (112 +/- 3 ng cyclosporine/mg cell protein). At 12 micrograms/ml it inhibited the sodium glucose cotransporter, as assessed by phlorizin-inhibitable 14C-alpha-methyl glucopyranoside (alpha-MG) uptake (control 37.2 +/- 6.3, 12 micrograms/ml 21.2 +/- 1.1 mumol/hr/mg protein). Cyclosporine at 2 micrograms/ml did not affect cell growth after 5 days (control 945 +/- 60 micrograms cell protein per 25 cm2 flask, 2 micrograms/ml cyclosporine/ml 1046 +/- 32 micrograms protein/flask), even in the presence of 7.6 mM ionized calcium (862 +/- 37 micrograms protein/flask). Cyclosporine at 12 micrograms/ml inhibited cell growth (286 +/- 27 micrograms protein/flask), and raising the ambient ionized calcium concentration to 7.6 mM reduced cell growth further (91 +/- 6 micrograms protein/flask). Cyclosporine at concentrations of 2 and 12 micrograms/ml produced increasing cell vacuolation, as seen in vivo. Short-term uptake of 2 micrograms/ml cyclosporine could be inhibited by 1.0 mM and 0.5 mM verapamil (49 +/- 9.5 and 71 +/- 6.4 ng cyclosporine/mg cell protein, respectively, at 30 min). However, in the presence of 2 micrograms/ml cyclosporine 0.1 mM verapamil was toxic to the cells grown over five days (44 +/- 5 micrograms protein/flask). At 0.01 mM verapamil was not toxic to cell growth (921 +/- 29 micrograms protein/flask), but raising the medium calcium to 7.6 mM reduced cell growth (652 +/- 96 micrograms/ml). Inhibition of cyclosporine uptake did not occur with 0.01 mm verapamil (control 145.6 +/- 12.3 vs. 0.01 mM verapamil 150.4 +/- 3.8 ng cyclosporine/mg cell protein). The LLC-PK1 cell line represents a good in vitro model for cyclosporine renal tubular toxicity, as the in vivo observation of glycosuria and proximal tubular cell vacuolation in cyclosporine nephrotoxicity can be reproduced. In vitro this is shown to be associated with inhibition of sodium-dependent glucose cotransport. Verapamil inhibited cyclosporine uptake, but only at concentrations that were toxic to the cells. Verapamil potentiated rather than reduced the increased cyclosporine toxicity produced by increasing the medium calcium concentration. The suggested protective effect of verapamil against cyclosporine nephrotoxicity is therefore unlikely to be due to inhibition of cyclosporine uptake or of calcium entry into proximal tubular cells.     

Impact of different methods of estimating renal function on determining eligibility for cisplatin (CDDP)-based chemotherapy in patients with invasive urothelial carcinoma

Assessment of renal function is important to determine the appropriate dose for cisplatin (CDDP)-based chemotherapy. Many previous CDDP-based chemotherapy trials for bladder cancer have required a creatinine-clearance (Ccr) ≧60 ml/min for entry. However, there is little evidence on renal function assessed by estimated glomerular filtration rate (eGFR) using the 4-variable Modification of Diet in Renal Disease Study Equation (MDRD), which has recently been introduced, to determine the eligibility for CDDP-based chemotherapy. To evaluate the proportion of patients with invasive urothelial carcinoma(UC) who would be ineligible ("unfit") to receive CDDP-based chemotherapy based on eGFR criteria (eGFR ＜60 ml/min/1.73 m2), and to determine the side effects of chemotherapy in these "unfit" patients, we conducted a retrospective clinical study. Our study population consisted of 61 consecutive patients who underwent 100% dose CDDP-based chemotherapy for invasive UC with 24 h-Ccr≧50 ml/min between June 2001 and July 2009. We assessed renal function using 3 equations (eGFR, Ccr according to Cockcroft-Gault formula (C-G Ccr), and Ccr examined by 24-hour urine collection (24 h-Ccr)) as well. Mean values of eGFR, C-G Ccr, and 24 h-Ccr were 58.6, 68.9, and 82.8 ml/min, respectively (P＜ 0.001). In total, 29/61(48%) patients were ineligible ("unfit") to receive chemotherapy based on eGFR criteria. However, there was no difference in the frequency of side effects between eGFR ≧60 ml/min/ 1.73 m2 and eGFR ＜60 ml/min/1.73 m2 groups. Our observations suggest that 24 h-Ccr≧50 ml/min would be a reasonable cutoff for CDDP-based chemotherapy even when eGFR ＜60 ml/min/1.73 m2.     

Hormonal Milieu of the Seminiferous Tubules in the Normal and Cryptorchid Rat

The intratesticular concentration of androgens seems to be of more importance for normal spermatogenesis than the concentration of androgens in the peripheral blood.
Therefore, the levels of testosterone, 5α - dihydrotestosterone (DHT), androstenedione and FSH in the interstitial fluid of the rat testis were determined by radioimmunoassay. The following concentrations were obtained from the interstitial fluid of normal rats and from rats cryptorchid between 60 and 90 days of age: Testosterone 137 ± 25 (mean ± SEM) and 271 ± 34 ng/ml, DHT 11 ± 2 and 22 ± 5 ng/ml, androstenedione 8 ± 2 and 20 ± 4 ng/ml, respectively. The sum of testosterone and DHT in serum did not differ in the two groups. Serum FSH concentrations were significantly increased in the cryptorchid rats (942 ± 73 ng/ml) as compared to the control rats (600 ± 38 ng/ml). These elevated levels were also found in the testicular interstitial fluid (425 ± 33 ng/ml in controls, 641 ± 43 ng/ml in cryptorchid rats). It is thus evident that in the cryptorchid rat, both FSH and androgens are available to the seminiferouse tubules in significantly elevated amounts. Since these are the only hormones which are known to be of importance in spermatogenesis, the impaired Sertoli cell function and depletion of germinal cells in cryptorchidism is not due to an absolute shortage of stimulatory hormones.     

The inhibitory effects of colchicine on cell proliferation and mineralisation in culture

Colchicine is often used in the treatment of diseases such as familial Mediterranean fever (FMF) and gout. We have previously reported that patients with FMF who had colchicine on a daily basis and who had a total hip arthroplasty showed no heterotopic ossification after surgery. The mechanism by which colchicine causes this clinical phenomenon has never been elucidated. We therefore evaluated the effect of various concentrations of colchicine on cell proliferation and mineralisation in tissue culture, using rat and human cells with and without osteogenic potential. Cell proliferation was assessed by direct cell counts and uptake of (3H)thymidine, and mineralisation by measuring the amount of staining by Alizarin Red. Our findings indicate that concentrations of colchicine of up to 3 ng/ml did not affect cell proliferation but inhibition was observed at 10 to 30 ng/ml. Mineralisation decreased to almost 50%, which was the maximum inhibition observed, at concentrations of colchicine of 2.5 ng/ml. These results indicate that colchicine at low concentrations, of up to 3 ng/ml, has the capacity to inhibit selectively bone-like cell mineralisation in culture, without affecting cell proliferation. Further clinical and laboratory studies are necessary to evaluate the effects of colchicine on biological processes involving the proliferation of osteoblasts and tissue mineralisation in vivo, such as the healing of fractures, the formation of heterotopic bone and neoplastic bone growth.     

血小板源性生长因子BB对体外培养肌腱细胞增殖的影响

Objective To study the effect of platelet-derived growth factor-BB (PDGF-BB) in dif-ferent concentrations on proliferation of tendon cells cultured in vitro. Methods Rat tendon cells were cultured and identified in vitro. The rat tendon cells were cultured in PDGF-BB nutrient solution in different concentrations. They were then divided into 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB groups (cultured with 0.1 mL 0.5% PBS with addition of 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB respectively). Tendon cells in control group were cultured with 0.1 mL 0.5% FBS. Proliferation of tendon cells was detected by MTT test. The absorbance values of tendon cells in control group and 20 ng/mL PDGF-BB group before culture and after cultured for 12, 24, 36, 48, 60, 72 hs were determined. Results The isolated cells were identified to be rat tendon cells as they were Type Ⅰ collagen staining posi-tive and Type Ⅲ collagen staining negative. Compared with that of control group, the absorbance values of other groups were all increased, except for that of 250 ng/mL PDGF-BB group (P<0.05 or P<0.01). Besides, the absorbance value rose gradually with the increase of the concentration of PDGF-BB on, and then diminished gradually with the increase of the concentration of PDGF-BB from 20 ng/mL on. Tendon cells in 20 ng/ml PDGF-BB group began to increase in number when cultured for 12 hs, and it reached the highest level (0.53±0.04) at 48 h, which were obviously higher than those of control group at 24-72 h (P<0.01). The absorbance value of tendon cells in 20 ng/mL PDGF-BB group was significantly higher than that of control group at 24, 36, 48, 60, 72 h after culture (P<0.01 ). Conclusions PDGF-BB can promote the proliferation of tendon cells in a definite range of concentration and time.     

Influence of impaired renal function and Helicobacter pylori infection on serum pepsinogen concentrations]

It has been known that patients with chronic renal failure have elevated concentrations of serum pepsinogens, which are raised by Helicobacter pylori (H. pylori) infection of the stomach. This study was conducted to examine how renal dysfunction and H. pylori infection affect serum pepsinogen (PG) concentrations. The subjects consisted of 93 patients with renal disease (60 males and 33 females with a mean age of 55 +/- 1.3 years). Dialysis patients were not included. Twenty-four-hour urinary collection was performed, and creatinine clearance (Ccr) was calculated for all the subjects. Status of H. pylori infection was assessed by serum IgG antibody against H. pylori. Fasting serum PG I and PG II were measured by RIA. The subjects were divided into 4 groups based on Ccr: group A: Ccr > or = 71 ml/min; group B: 30-70 ml/min; group C: 11-30 ml/min; group D: Ccr < or = 10 ml/min. Regardless of the H. pylori status, serum PG I concentrations were elevated as the renal function declined; serum PG I levels of groups C and D (177.5 +/- 15.2 ng/ml and 234.0 +/- 32.2 ng/ml, respectively) were significantly higher than those of group A (66.1 +/- 9.6 ng/ml, p < 0.01); Group D also had a significantly higher concentration of PG I than group B (106.0 +/- 17.2 ng/ml, p < 0.01). The same tendency was found in serum PG II concentrations. However, the differences among the 4 groups were not statistically significant (16.2 +/- 2.3, 24.2 +/- 4.0, 28.3 +/- 3.5, 34.3 +/- 5.6 ng/ml for group A, B, C, and D, respectively). There was a negative correlation between Ccr and serum PG I concentrations (r = -0.45, p < 0.01). In comparison between H. pylori-infected patients and uninfected patients, serum levels of PG II were higher in the former patients than in the latter. This difference was significant in groups A and C. Serum PG I concentrations were the same between H. pylori-infected patients and their uninfected counterparts for the 4 groups. The present study has shown that serum PG I concentrations are raised by a loss of renal function, while PG II levels are elevated mainly by H. pylori infection of the stomach. It is concluded that renal function and H. pylori infection should be taken into account when serum PG concentrations are evaluated in patients with renal diseases.     

Right hepatectomy for combined primary neuroendocrine and hepatocellular carcinoma. A case report

INTRODUCTION

Cases of primary neuroendocrine tumors in the liver combined with hepatocellular carcinoma are scarce. Such cases could present either as combined-type tumor or collision type.

PRESENTATION OF CASE

A 51-year-old man presented with a mass in the right hemiliver. Serum level of alpha-fetoprotein was slightly elevated (2.3 ng/ml), with normal CA19-9 and CA125. The patient underwent right hepatectomy. The resected specimen showed a well-defined and heterogeneous gray-white to brown friable tumor, 20 cm in diameter. Microscopically, the tumor consisted predominantly of monotonous small- to medium-sized neoplastic cells arranged in trabeculea separated by sinusoidal spaces. Immunohistochemically, the tumor cells were strongly positive for synaptophysin and focally positive for chromogranin-A. Interestingly, the tumor cells showed patchy positive coarse granular staining of HerPar-1 involving about 1% of the tumor cells. Glypican-3 staining was negative. These immunohistochemical findings supported the diagnosis of combined high grade neuroendocrine carcinoma and hepatocellular carcinoma.

DISCUSSION

Cases of primary neuroendocrine tumors in the liver combined 82 with hepatocellular carcinoma are scarce. The uniqueness of this case lies in the fact that the neuroendocrine carcinoma component comprised more than 99% of the tumor area, and the minor hepatocellular carcinoma component was detected only by the immunohistochemical staining for HepPar-1.

CONCLUSION

To the best of our knowledge, this is the first case of combined neuroendocrine carcinoma and hepatocellular carcinoma in Egypt.     

Elevated hydrostatic pressure stimulates ATP release which mediates activation of the NLRP3 inflammasome via P2X<Subscript>4</Subscript> in rat urothelial cells

Partial bladder outlet obstruction (pBOO) is a prevalent urological condition commonly accompanied by increased intravesical pressure, inflammation, and fibrosis. Studies have demonstrated that pBOO results in increased NLRP3 inflammasome and caspase-1 activation and that ATP is released from urothelial cells in response to elevated pressure. In the present study, we investigated the role of elevated pressure in triggering caspase-1 activation via purinergic receptors activation in urothelial cells. Rat urothelial cell line, MYP3 cells, was subjected to hydrostatic pressures of 15 cmH₂O for 60 min, or 40 cmH₂O for 1 min to simulate elevated storage and voiding pressure conditions, respectively. ATP concentration in the supernatant media and intracellular caspase-1 activity in cell lysates were measured. Pressure experiments were repeated in the presence of antagonists for purinergic receptors to determine the mechanism for pressure-induced caspase-1 activation. Exposure of MYP3 cells to both pressure conditions resulted in an increase in extracellular ATP levels and intracellular caspase-1 activity. Treatment with P2X₇ antagonist led to a decrease in pressure-induced ATP release by MYP3 cells, while P2X₄ antagonist had no effect but both antagonists inhibited pressure-induced caspase-1 activation. Moreover, when MYP3 cells were treated with extracellular ATP (500 µM), P2X₄ antagonist inhibited ATP-induced caspase-1 activation, but not P2X₇ antagonist. We concluded that pressure-induced extracellular ATP in urothelial cells is amplified by P2X₇ receptor activation and ATP-induced-ATP release. The amplified ATP signal then activates P2X₄ receptors, which mediate activation of the caspase-1 inflammatory response.