Hepatocyte polyunsaturated fatty acid enrichment increases acute phase protein synthesis.

BACKGROUND: The production of acute phase proteins by the liver is critical for homeostasis and recovery after injury. Polyunsaturated fatty acids, specifically of the n-3 family, have demonstrated anti-inflammatory properties that promote recovery; however, the effects of these fatty acids on acute phase protein synthesis have not been fully evaluated. METHODS: The synthesis of the acute phase proteins alpha-2 macroglobulin and lipopolysaccharide binding protein was studied in hepatocyte-Kupffer's cell cocultures from Wistar rats. Cultures were endotoxin stimulated after enrichment with albumin complexed arachidonic acid (20:4n-6) or docosahexaenoic acid (22:6n-3). Protein synthesis was analyzed by [35S]-methionine labeling or Western blotting. Culture interleukin-6 levels were determined. RESULTS: Both polyunsaturated fatty acids increase hepatocyte synthesis of acute phase proteins alpha 2-macroglobulin and lipopolysaccharide binding protein compared with controls; however, the response in the docosahexaenoic acid (22:6n-3) treated cultures was significant (P < .05 vs control). Interleukin-6 was also increased in the polyunsaturated fatty acid cultures compared with controls (P < .05) vs control). Cellular phospholipids were significantly enriched with the individual supplemented fatty acids (P < .05 vs bovine serum albumin). CONCLUSIONS: Polyunsaturated fatty acids have the capability to increase in vitro acute phase protein synthesis. This may contribute to the observed anti-inflammatory effect of n-3 polyunsaturated fatty acid enrichment.

The effect of preexisting long-term diabetes on the outcome after islet transplantation in rats.

Studies of islet transplantation conducted immediately following diabetes induction may not accurately reflect the clinical situation. Long term preexisting diabetes with generalized microvasculature complication might adversely affect the outcome after islet transplantation. The present study testing this hypothesis by evaluating the effect of long-term preexisting diabetes on glucose-induced insulin secretion up to 6 months after transplantation of two different quantities of islets. One thousand two hundred or 2,400 islets were isotransplanted into the left renal subcapsular space at 10 days (acute diabetes), 3 months (chronic diabetes), or 6 months (long-term diabetes) after diabetes induction by streptozotocin in the rat. In addition, one group of diabetic rats in which normoglycemia was maintained with exogenous insulin treatment for 6 months was then transplanted with 1,200 islets. Intravenous glucose tolerance tests were performed at 10, 90, and 180 days after islet transplantation. Islet transplantation normalized the basal blood glucose levels within 24-48 h in all transplanted groups that remained normal for the entire study period of 6 months, with no differences among acute, chronic, or long-term diabetes. Basal plasma insulin levels were also normalized in all transplanted groups. Diabetic (acute, chronic, or long-term) rats transplanted with 2,400 islets achieved normal glucose-induced insulin secretion at 10 and 90 days after transplantation. In contrast, glucose-induced insulin secretion was impaired in rats transplanted with only 1,200 islets, with no differences among acute, chronic, and long term diabetes. However, at 180 days after transplantation, long term diabetic rats transplanted with 2,400 islets had impaired insulin secretion compared to normal controls. Insulin-treated long-term diabetic rats transplanted with 1,200 islets had normal glucose-induced insulin secretion at 10 days after transplantation. However, at 90 and 180 days after transplantation, insulin-treated long-term diabetic rats had impaired glucose-induced insulin secretion which was not different from nontreated transplanted long-term diabetic rats. It is concluded that long-term preexisting diabetes has no impact on the early outcome after islet transplantation. However, it may adversely affect the long-term function of the transplanted islets. Also, transplantation of a sufficient islet mass is the critical factor in achieving complete glucose homeostasis.

The role of neutrophils in peritoneal adhesion formation.

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.

Immunosuppressive effect of 2'-deoxycoformycin (Pentostatin) for rat islet allotransplantation.

Adenosine deaminase (ADA) is an important enzyme for proper function of lymphocytes and congenital absence of ADA results in a form of severe combined immunodeficiency syndrome. 2'-Deoxycoformycin (Pentostatin, DCF) irreversibly inhibits ADA and therefore has been suggested as an immunosuppressive drug. The present study evaluated the immunosuppressive effect of DCF for islet allotransplantation in rats. Isolated islets (1,500 islets) from Lewis rats were transplanted into the kidney subcapsular space of streptozotocin-induced diabetic Wistar-Furth rats. DCF was administered IP either as a single injection at 1 mg/kg/wk, 1 mg/kg twice weekly, 5 mg/kg/twice weekly or 1 mg/kg/day, or as a continuous infusion at 0.8 or 1 mg/kg/day. Daily administration of DCF at 0.8 mg/kg in both methods, single daily injection or continuous infusion, resulted in a lymphopenia and a decrease in concanavalin A stimulation of splenic lymphocytes. However, DCF (in all doses) was not effective in preventing islet allograft rejection as evaluated by measuring the duration of normoglycemia following islet transplantation and by microscopic examination of the islet grafts. In fact, the duration of normoglycemia following islet transplantation was 7.5 +/- 0.9 and 9.0 +/- 1.0 days in rats receiving DCF in single daily injection or continuous infusion, respectively. This was not significantly different from control nontreated transplanted rats (8.5 +/- 0.7 days). Increasing the dose of DCF to 1 mg/kg, administered by continuous infusion, resulted in 100% mortality. For comparison, cyclosporine A (20 mg/kg, IP daily injection for 14 days) prolonged islet allograft survival to 27.3 +/- 1.5 days (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of peritoneal macrophages reduces postoperative peritoneal adhesion formation.

Postoperative adhesion formation results from a fibroproliferative inflammatory reaction. Macrophages are critical in the final resolution of the inflammatory process and tissue repair, including modulation of proliferation and differentiation of fibroblasts and secretion of neutral proteases like plasminogen activator. We, therefore, studied the influence of peritoneal macrophage enhancement on postoperative adhesion formation in five groups of rabbits. Group 1 was a control with normal peritoneum. Animals in group 2 had increased macrophage population in their peritoneum by intraperitoneal injection of protease peptone 3 days before adhesion induction. In group 3, animals were treated by protease peptone as in group 2 and then depleted of the increased macrophage population by peritoneal lavage before adhesion induction. In group 4 macrophages were transplanted from animals enriched as in group 2 into a nonenriched peritoneum at the time of adhesion induction. Group 5 had a normal peritoneum with peritoneal lavage before adhesion induction. Peritoneal adhesions were induced at laparotomy by repairing a peritoneal defect in two different models. It was found that enhancement of peritoneal macrophages by protease peptone reduced markedly the degree of postoperative adhesion formation. After depletion of the enhanced peritoneal macrophages by peritoneal lavage the degree of adhesion formation was equivalent to that of controls. Finally, macrophage transplantation into a nonenhanced macrophage peritoneum also reduced the degree of postoperative adhesion formation. It is concluded that enhancement of peritoneal macrophages reduces postoperative peritoneal adhesion formation.

Immune privilege of the testis for islet xenotransplantation (rat to mouse).

The testis has been suggested as an immune privileged site for islet transplantation. The present study evaluated this hypothesis by transplanting islets from Wistar Furth rats into (a) the testes; (b) the subcapsular space of the kidneys; or (c) the cryptorchid abdominal testes of streptozotocin-induced diabetic Swiss ND4 mice. Transplantation of 800 rat islets into the cryptorchid testes normalized blood glucose for 9.3 +/- 1.4 (Mean +/- SD) days, not significantly different from that of the scrotal testis site (12.4 +/- 1.3), or when the subcapsular space of the kidneys was used (11.5 +/- 1.2). When mouse islets were isotransplanted into the cryptorchid testes of diabetic mice, normoglycemia was maintained for the entire 3 month study period. Histologic examination of the islet xenograft-bearing cryptorchid testes at day 7 post transplantation and 2 days after returning to hyperglycemia revealed lymphocyte infiltration surrounding and inside the graft. No lymphocyte infiltration was seen in the isograft bearing-testes at 3 mo after transplantation. Cyclosporine A (CsA, 15 mg/kg/day) administration to the islet xenograft recipient slightly prolonged the normoglycemic period to 13.7 +/- 1.8 days (p < 0.01). Increasing CsA dose to 25 mg/kg induced a 66% (4/6) mortality, and did not further prolong the normoglycemic period. Using a lower number of rat islets (200 or 400 islets), prolonged graft survival was achieved in some (4 out of 20) animals when the cryptorchid testis was used. In contrast, transplantation of 400 rat islets into the subcapsular space of the kidneys was not associated with prolonged graft survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the number of islets on the outcome after transplantation in streptozotocin-induced diabetic rats.

This study tested the hypothesis that normalization of glucose homeostasis after islet transplantation is correlated to the number of islets, and by increasing this number a complete normalization of glucose homeostasis could be achieved, 1,200 or 2,400 islets were transplanted into the left kidney subcapsular space in streptozotocin-induced diabetic rats. Intravenous glucose tolerance tests were performed at 10 days, 3 and 6 months after transplantation. Transplantation of both 1,200 and 2,400 islets normalized the basal blood glucose levels within 24-48 hours, which remained normal for the entire study period of 6 months. Basal plasma insulin levels and body weight were also normalized in both transplanted groups. Transplantation of 2,400 islets achieved normal glucose-induced insulin secretion at 10 days after transplantation and for the following 6 months. In contrast, glucose intolerance was present in rats transplanted with only 1,200 islets. It is concluded that complete glucose homeostasis after islet transplantation is dependent on the number of transplanted islets and can be achieved by increasing this number.

Deleterious effect of cyclosporins on the ischemic kidney in the rat and the protection by the calcium antagonist verapamil.

Cyclosporine A (CsA) nephrotoxicity has been suggested to be aggravated in the presence of ischemia, as occurs after renal transplantation. Cyclosporine G (CsG) may be less nephrotoxic than CsA. This study evaluated in the rat (1) the effect of CsA and CsG on blood flow and the function of the kidney subjected to 60 min of warm ischemia and (2) the protective effect of the calcium antagonist verapamil (VP). After left nephrectomy, ischemia was induced in the right kidney by the clamping of the kidney pedicle for 60 min, which resulted in a significant increase in serum creatinine (SCr) to 2.30 +/- 0.25 mg/dL by Day 1 with 25% mortality by Day 7. The administration of CsA or CsG (20 mg/kg i.v. daily for 7 days) after 60 min of renal ischemia significantly increased SCr and mortality compared with ischemia alone. In another set of experiments, 60 min of warm ischemia was applied to the right kidney and RBF was measured in both kidneys with a laser Doppler flowmeter. Blood flow in the ischemic kidney returned to the preischemic level by 15 min after the removal of the vascular clamp in the control animals. In contrast, in animals treated with CsA, a significant decrease in RBF was seen in both kidneys; however, blood flow in the ischemic kidney was significantly lower than that in the nonischemic kidney. CsG also decreased RBF in both kidneys, although in the left (nonischemic) kidney, RBF remained significantly higher with CsG than with CsA.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentafraction-Du Pont versus albumin for resuscitation of a lethal intestinal ischemic shock in rats.

This study compares the effect of pentafraction-Du Pont 6% (PF), albumin 5% (ALB), and Ringer's lactate (RL) on plasma volume (PV) expansion and survival in a lethal intestinal ischemic shock model. Shock was induced by exteriorizing the small intestines and occluding the mesenteric vessels for 75 min. Changes in PV were estimated using hematocrit (Hct). The solutions were administered continuously for 6 hr in volumes to maintain a stable Hct, or 15 ml/100 g body weight (bwt) for PF and ALB and 44 ml/100 g bwt for RL. Hct and bwt were measured hourly during the infusion and at 24 h. Untreated animals in shock developed hemoconcentration (Hct 62%) corresponding to a PV of 41% of baseline preshock level, with 9% (3/35) of the animals surviving 72 hr. RL expanded PV to 87% of preshock level, with a 57% (20/35) 72 hr survival rate and 46% (16/35) surviving greater than 7 days. Only 34% as much ALB was needed to induce a greater PV expansion of 101% with a 72 hr survival rate of 51% (18/35) and 46% (16/35) surviving greater than 7 days. When PF was used, PV expanded to 109% of preshock level with survival rates improving to 80% (28/35) at 72 hr and 71% (25/35) greater than 7 days compared to RL and ALB (P less than 0.05). There were no significant differences in survival rates between RL and ALB treated animals. We conclude that PF improves survival following intestinal ischemic shock compared to ALB and RL. PF is a safe and effective alternative to albumin for resuscitation in this shock model.