Isolation, culture, and characterization of endocrine cells from 6-month-old porcine pancreas

Porcine endocrine cells were isolated from pancreas of 6-month-old pigs by two-step enzymatic digestion procedures. They were separated by the density gradient (isopycnic) centrifugation method using Histopaque-1077. Isolated cells were cultured and divided into two groups: suspension cells and adhesion cells. Suspension cells maintained their cell numbers on and after 7 days in culture. Approximately 1 x 10(7) cells were obtained from single pancreas of a 6-month-old pig. The cultured suspension cells took up dithizone (DTZ) staining 14 days after isolation in culture and indicated the presence of beta-cells. In in vitro study, the suspension cells were capable of secreting insulin into the culture medium. The suspension cells were tested for insulin and glucagon staining by Western blot analysis. These results indicated the maintenance of endocrine cell function after isolation. However, cultured adhesion cells failed to maintain their function during culture. In in vivo study, the suspension cells were transplanted into diabetes-induced nude mice. Reduction in blood glucose level was obtained after transplantation. Intraperitoneal glucose tolerance test (IPGTT) results showed a normal pattern of blood glucose clearance. After 1 week, the transplanted endocrine cells were detected with anti-insulin antibody by immunostaining and it showed the presence of viable beta-cells under the renal capsule of nude mice. Collectively, our results suggest that isolated and cultured suspension porcine endocrine cells maintained their endocrine function. These endocrine cells can be used as isolated islets for further study, including transplantation experiments.     

Metallic stents for malignant and benign ureteric obstruction

OBJECTIVE: To report our experience of using metallic stents to treat ureteric obstruction caused by malignant or benign disease. PATIENTS AND METHODS: Nine patients with obstruction in 11 ureters caused by malignant or benign disease (mean age 61 years, range 35-82, mean follow-up 7 months, range 3-11) were treated using metallic stents. A balloon-expandable metallic stent was used in one patient and self-expandable metallic stents in the remaining eight. All stents were inserted via a percutaneous antegrade approach. RESULTS: Of the 11 ureters, nine remained patent with no further manipulation during the follow-up of 3-11 months. An additional stent was placed in continuity with the first in two ureters of two patients at 4 and 5 weeks after the first procedure because of persistent obstruction. After the second intervention, their obstruction was improved. Transient vesico-ureteric reflux occurred in two of three stented distal ureters, but the reflux resolved spontaneously within 2 months after stent implantation. Ureteric patency was maintained in all patients and no major complications related to stenting occurred during the follow-up. Two patients died from cervical cancer at 3 and 5 months after stenting. CONCLUSION: In patients with difficult ureteric obstructions a metallic stent provides a safe and effective alternative to an indwelling double-pigtail catheter or percutaneous nephrostomy.     

Correlation between spinal cord blood flow and arterial diameter following acute spinal cord injury in rats

Summary Simultaneous measurements of spinal cord blood flow and arterial diameter at areas adjacent to a site of spinal cord injury were carried out to determine changes in CO₂ reactivity and autoregulation. The spinal cord injury was made at T10 level by the epidural clip compression method. A spinal window was drilled at an area either 7 mm caudal or 7 mm rostral to the injury site for the measurement of spinal cord blood flow and arterial diameter at the same time. Spinal cord blood flow was decreased at both spinal windows, especially at the rostral window. Arterial diameter was also decreased significantly at both sites. The ischaemic zone evaluated histologically tended to expand more diffusely in the rostral direction than in the caudal direction.In the pre-injury stage, both CO₂ reactivity and autoregulation were present in the spinal cord. Following the clip injury, CO₂ reactivity and autoregulation were both impaired in the areas 7 mm adjacent to the impact site. Correlation coefficients suggested that the rostral spinal cord tended to sustain more injury than the caudal spinal cord.The histologically proven spinal cord ischaemia following the injury may have resulted from the decreased arterial diameter and impaired CO₂ reactivity and dysautoregulation of the spinal cord.     

Effect of intravenous cyclophosphamide in systemic lupus erythematosus: relation to lymphocyte subsets and activation markers

Since the mechanism mediating the beneficial effect of intravenous cyclophosphamide (IVCY) in systemic lupus erythematosus (SLE) is unknown, we investigated lymphocyte subsets and markers of activated lymphocytes in patients received IVCY, and compared the results with the effect of steroid pulse. In 55 patients with SLE, 34 patients receiving IVCY [21 cases (61.8%) were responsive] and 25 patients received steroid pulse [21 cases (84.0%) were responsive] (four patients who were resistant to steroid pulse therapy were transferred to IVCY). When the lymphocyte subsets and markers of activated lymphocytes were compared in the responsive and unresponsive group of IVCY, soluble CD4 levels and the ratio of HLA-DP-positive T cells were significantly higher in the unresponsive group. Further, the changes of these markers and costimulatory molecules [LFA-1 (CD11a), ICAM-1 (CD54), CD40 and CD40-ligand (CD154)] were also examined in the responsive patients. The ratio of HLA-DP-positive T cells did not change in the IVCY-responsive group, while it decreased in the steroid pulse therapy-responsive group. The ratio of CD11a on T cells increased and CD54 on B cells decreased in the IVCY-responsive group. The ratio of CD154 on T cells increased in the steroid pulse-responsive group, while it decreased in the IVCY-responsive group. These results suggest that the effect of IVCY is different to that of steroid pulse therapy and mainly related to B cell activation, and that these markers may contribute to predict the responsiveness of IVCY.     

DNA-daunorubicin complexes specifically suppress in vitro spontaneous anti-DNA antibody production in lymphocytes of patients with systemic lupus erythematosus

Elevated production of anti-DNA antibody in patients with systemic lupus erythematosus (SLE) is a central problem in the pathogenesis of tissue injury. In the present study, we attempted to manipulate anti-DNA antibody production through the antigen-cytotoxic drug conjugates, DNA-daunorubicin complexes. The effect of DNA-daunorubicin complexes was determined by examining SLE lymphocytes for spontaneous in vitro production of anti-DNA antibody. These complexes, at 2 μg/ml, suppressed anti-DNA antibody production, but not total IgG production, which suggests that specific suppression of anti-DNA antibody production was achieved at this concentration. We believe that the DNA-daunorubicin complexes affected mainly B cells, since such suppression was obtained by treating B cells, as well as B plus T cells. Furthermore, the complexes had no effect on the proliferative responses of SLE T cells to DNA, phytohemagglutinin, or concanavalin A. These results indicate that DNA-daunorubicin complexes may have the potential for selectively suppressing anti-DNA antibody production in patients with SLE.     

Endogenous Androgens Diminish Food Intake and Activation of Orexin A Neurons in Response to Reduced Glucose Availability in Male Rats

Sex steroids modify feeding behavior and body weight regulation, and androgen reportedly augments food intake and body weight gain. To elucidate the role of endogenous androgens in the feeding regulation induced by reduced glucose availability, we examined the effect of gonadectomy (orchiectomy) on food intake and orexin A neuron’s activity in the lateral hypothalamic/perifornical area (LH/PFA) in response to reduced glucose availability (glucoprivation) induced by 2-deoxy-d-glucose (2DG) administration in male rats. Rats (7W) were bilaterally orchiectomized (ORX group) or sham operated (Sham group). Seventeen days after the surgery, food intake response to 2DG (400 mg/kg, i.v.) was measured for 4 h after the infusion. The same experiment was performed for the immunohistochemical examination of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the arcuate nucleus (Arc). Food intake induced by glucoprivation was greater in the ORX group than the Sham group, and the glucoprivation-induced food intake was inversely correlated with plasma testosterone concentration. Glucoprivation stimulated c-Fos expression of the orexin A neurons at the LH/PFA and c-Fos expression in the dorsomedial Arc. The number and percentage of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the dorsomedial Arc were significantly higher in the ORX group than the Sham group. This indicates that endogenous androgen, possibly testosterone, diminishes the food intake induced by reduced glucose availability, possibly via the attenuated activity of orexin A neuron in the LH/PFA and neurons in the dorsomedial Arc.