Serum levels of prolactin, growth hormone, and cortisol in burn patients: correlations with severity of burn, serum cytokine levels, and fatality.

In this study, we measured serum prolactin (PRL), cortisol, growth hormone, interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha in patients admitted with small-to-moderate burn injuries. Serum samples were obtained at the time of admission from 49 adult male burn patients with ages ranging from 18 to 91 years and TBSA ranging from 0.001 to 60%. The levels of serum PRL, IL-8, IL-6, and IL-1beta correlated positively with the TBSA, whereas only serum IL-8 levels correlated positively with fatality. Each of these factors were increased at least 2-fold at the higher burn severity. Not surprisingly, there was a large degree of variability in the hormone and cytokine levels in this patient population, which presumably reflects individual levels of stress, as well as other physiological variables. We also studied relationships between serum hormone levels and serum cytokine levels in this context. Linear regression analysis revealed a significant positive correlation between the serum PRL level and the levels of IL-10, IL-6, and IL-8. These results indicate that PRL responds to burn injury at early time points and that a subset of cytokines are involved in the early response to burn injury.

Stress hormone secretion and gut signal transducer (STAT) proteins after burn injury in rats.

A burn injury triggers traumatic reactions characteristics of a stress. Here we investigated the early responses of prolactin (PRL), corticosterone (CS), and signal transducer and activator of transcription 5 (STAT5) in male Sprague-Dawley rats after burn injury. PRL and CS levels were determined in blood serum. STAT5 and phospho-STAT5 levels were determined in jejunum total protein extracts. The results confirmed an expected increase of CS between 4 and 6 h after the burn injury. Unexpectedly, PRL secretion was suppressed during the same time frame. These hormone levels returned to normal 6 to 8 h after burn injury. STAT5 was increased in the jejunum after burn injury, and its phosphorylation was increased between 8 and 11 h after burn injury. These changes in STAT5 were not temporally correlated with either the hormone changes that we observed or with previously documented changes of the gut function after burns.

Oral arginine improves blood pressure in renal transplant and hemodialysis patients.

BACKGROUND: Hypertension in kidney transplant (KT) patients may result from attenuated whole-body nitric oxide (NO) content and abnormal NO-mediated vasodilation. Increasing NO bioavailability with L-arginine (ARG) could theoretically restore the NO-mediated vasodilatory response and lower blood pressure. METHODS: In a prospective pilot study, 6 normotensive volunteers and 10 KT patients received oral supplements of ARG (9.0 g/d) for 9 days, then 18.0 g/d for 9 more days. Six hemodialysis (HD) and 4 peritoneal dialysis patients received the same dose for 14 days. Five KT patients received 30 mL/d of canola oil (CanO) in addition to ARG. Systolic (SBP) and diastolic (DBP) blood pressure, creatinine clearance (CCr), and serum creatinine (Cr) were measured at baseline, day 9, and day 18. In a subsequent study, 20 hypertensive KT patients with stable but abnormal renal function were randomized in a crossover study to start ARG-only or ARG+CanO supplements for two 2-month periods with an intervening month of no supplementation. SBP, DBP, CCr, and Cr were measured monthly for 7 months. RESULTS: In the pilot study, ARG reduced the SBP in HD patients from 171.5 +/- 7.5 mmHg (baseline) to 142.8 +/- 8.3 mmHg (p = .028). In the crossover study, SBP was reduced from baseline (155.9 +/- 5.0 mmHg), after the first 2 months (143.2 +/- 3.2 mmHg; p = .03) and subsequent 2 months (143.3 +/- 2.5 mmHg; p = .014) of supplementation. DBP was also reduced after supplementation in both studies. CanO had no effect on blood pressure. Renal function did not change. CONCLUSIONS: Oral preparations of ARG (+/-CanO) were well tolerated for up to 60 consecutive days and had favorable effects on SBP and DBP in hypertensive KT and HD patients.

Thermal injury increases the number of eosinophil progenitors in rat spleen and bone marrow.

We have investigated the effects of thermal injury upon myelopoiesis. IL-3, GM-CSF, and IL-5 were used to stimulate myeloid colony formation. IL-3 induces early myeloid progenitors and a more developed myeloid progenitor, the granulocyte-macrophage colony-forming unit (GM-CFU), to multiply and develop into mature myeloid cells. GM-CSF induces GM-CFU to become mature myeloid cells, while IL-5 induces eosinophil progenitors to become mature eosinophils. Stem Cell Factor (SCF) + IL-6 and FLT3 ligand, which have no effect on colony formation by themselves, were used to enhance the effects of IL-3 and GM-CSF, respectively. We found that thermal injury increased the number of early myeloid progenitors and GM-CFU in the spleen with either IL-3 or GM-CSF as a stimulant. Thermal injury increased the number of early myeloid progenitors in the bone marrow when GM-CSF, but not IL-3, was used to stimulate colony growth. Also, thermal injury increased the numbers of eosinophil progenitors in rat spleen and bone marrow and increased splenic levels of IL-5 mRNA.

The effect of burn injury on suppressors of cytokine signalling.

The newly identified suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway, and they act as a negative feedback loop by subsequently inhibiting the Jak/STAT pathway either by direct interaction with activated Jaks or with the receptors. In this study we investigated the expression and translation of SOCS proteins after burn injury. Thermal injury increased the expression of SOCS3 compared with sham at 4 h, 24 h, and 10 days after thermal injury in the liver. SOCS3 protein was increased at 4 and 24 h after thermal injury in the liver. Expression of SOCS1 mRNA was not detected in sham or burn liver. SOCS2 mRNA and cytokine-inducible SH2-containing protein (CIS) mRNA were detected at the same levels for both sham and burn at all time points in the liver. In the spleen there was a trend towards an increase in SOCS1 mRNA at all time points; thermal injury significantly decreased SOCS2 mRNA compared with sham at 4 h, SOCS3 mRNA was significantly increased at 24 h compared with 10 days, and CIS mRNA was detected at the same levels for both sham and burn at all time points. In conclusion, thermal injury causes elevations in SOCS3 within 4 h after a burn, reaching a maximum at 24 h post injury. Levels continue to be elevated for up to 10 days post injury. SOCS3 may be very important in regulating the balance between immunosuppression and inflammation after thermal injury.

Interleukin-6 signal transduction in human intestinal epithelial cells.

The gut is an important source of inflammatory cytokines, but there is scant information on the mechanisms of cytokine action in gut epithelium. We hypothesized that in human Caco-2 cells, IL-6 acts directly through stimulation of Stat phosphorylation and that bacterial lipopolysaccharide (LPS) causes Stat activation indirectly because of its ability to cause the autocrine secretion and action of interleukin (IL)-6. Stat1, Stat5a, and Stat5b, but not Stat3, were detected in Caco-2 cells. DNA-binding activity corresponding to activated Stat5 was stimulated in a biphasic manner by IL-6, with a transient early phase, followed by sustained activation between 8 and 48 h. LPS also stimulated Stat5-like binding, but there was no early phase of activation. Functional tests of Stat5 activation showed that IL-6 stimulated Stat5-dependent reporter gene transcription but had no effect on Stat1-dependent transcription. LPS did not stimulate Stat-dependent transcription, nor did it alter the transcriptional response to IL-6. Tyrosine phosphorylation of both Stat5a and Stat5b was induced by IL-6. We infer from these data that IL-6 acts on intestinal epithelia through a Stat5-mediated transcriptional mechanism, whereas LPS does not induce gene expression through autocrine activation of enterocyte Stat signaling. These data provide a basis for testing the in vivo regulation of gut signaling and the interaction of gut reticuloendothelial cells with epithelial signal transduction.

Nutritional immunomodulation leads to enhanced allograft survival in combination with cyclosporine A and rapamycin, but not FK506.

BACKGROUND: Recently, specific immunonutrients were found to increase experimental allograft survival when combined with cyclosporine A (CsA). This study compared the effect on rat cardiac allograft survival when nutritional immunomodulation was used with CsA, rapamycin (Rapa), or tacrolimus (FK506). METHODS: Intra-abdominal ACI to Lewis cardiac allografts were performed and assessed daily by palpation. Study groups included untreated controls and those receiving CsA, Rapa, or FK506. Rats were fed ad libitum with Impact diet (fortified with fish oil, arginine, and RNA) or standard rat food. Further study groups were transplanted that received a donor-specific transfusion in addition to immunosuppression and diet. RESULTS: Allograft survival was extended by combining Impact with CsA (45.3+/-19 days) and Rapa (165.3+/-52 days), but not FK506 (12.4+/-3.2 days). Mean graft survival in the Rapa/Impact group met criteria for functional tolerance. The addition of a donor-specific transfusion did not lead to graft survival advantages over similar groups not receiving a donor-specific transfusion. CONCLUSIONS: The use of immunonutrients improves transplant outcome in animals treated with short courses of CsA and Rapa, but not FK506. These findings highlight the potential differences in the effects of nutritional immunomodulation with different immunosuppressive drugs in the treatment of transplant patients.

The effect of nutritional immunomodulation on cardiac allograft survival in rats receiving mycophenolate mofetil, cyclosporine A, and donor-specific transfusion.

BACKGROUND: Immunosuppressive drugs continue to pose significant risks such as infection, toxicity, or neoplasia when used in long-term therapy. The investigation of newer and safer combined treatment strategies that decrease the need for these drugs is becoming increasingly important. Immunonutrients are known to have significant modulating effects on the immune system. Feeding with Impact, a commercially available diet enriched with arginine, omega-3 fatty acids, and RNA, recently has been shown to extend rat cardiac allograft survival when combined with a donor-specific transfusion (DST) and cyclosporine A (CsA). Because mycophenolate mofetil (MMF) is now commonly used in the clinical setting, the current study was designed to examine the effect on rat cardiac allograft survival when MMF was added to this immunosuppressive regimen. METHODS: Intra-abdominal ACI to Lewis heterotopic cardiac allografts were performed. Study groups included untreated controls and recipients receiving varying combinations of a DST (1 mL) on the day prior to engraftment, MMF 45 mg/kg/day from the day of transplant through postoperative day six, and CsA 10 mg/kg on the day prior to operation and 2.5 mg/kg from the day of transplant through postoperative day 6. Animals were fed ad libitum with Impact diet or standard lab chow. Graft survival was determined by cessation of a palpable heartbeat. RESULTS: Treatment with MMF led to a prolonged allograft survival over historical untreated controls. The combination of MMF with a donor-specific transfusion, Impact, or CsA was associated with an increase in graft survival over MMF alone. The addition of Impact to the combination of MMF and CsA resulted in further improvement. The most pronounced graft survival advantage was seen when Impact was combined with a DST and both of the immunosuppressive agents. One quarter of the animals in this group had a palpable donor heart beat at greater than 150 days, indicating functional tolerance in those animals. CONCLUSIONS: The administration of Impact diet to treatment groups in this study was associated with graft survival advantages when compared to most of the other study groups receiving a similar drug regimen and standard chow. These findings support the importance of nutritional influences on allograft survival, and highlight the potential of diet therapy when used with short courses of clinically relevant immunosuppressive drugs.

Nitric oxide promotes the internalization and passage of viable bacteria through cultured Caco-2 intestinal epithelial cells.

Nitric oxide (NO) may play an important role in the pathophysiology of intestinal barrier disruption. Our purpose was to investigate the effects of NO donors on the internalization and passage of bacteria through cultured intestinal epithelial cells. Human intestinal epithelial cell line Caco-2 cells were grown on microtiter plastic plates. The cells were incubated with Escherichia coli and sodium nitroprusside (SNP) or S-nitroso-N-acetyl-penicillamine (SNAP), as NO donors, at several concentrations. The numbers of viable bacteria internalized into the epithelial cells were measured. Caco-2 cells were also grown to confluency on membranes of bicameral systems. The cells were incubated with E. coli and SNP. The numbers of viable bacteria passed through the epithelial layer were determined. Viability of the bacteria and the intestinal epithelial cells after culture with SNP or SNAP were also determined. Both SNP and SNAP at .1 or 1 mmol/L increased the number of viable bacteria internalized into the enterocytes. Both 1 or 10 mmol/L SNP promoted bacterial passage through the intestinal epithelial layer. However, 10 mmol/L SNP decreased the number of viable Caco-2 cells and failed to increase the bacterial internalization into Caco-2 cells. Incubation of E. coli with SNAP at 10 mmol/L slightly decreased the number of viable bacteria and failed to increase the bacterial internalization into Caco-2 cells. We conclude that NO donors promote both the viable bacterial uptake and passage through the intestinal epithelial layer.

Effect of pentoxifylline on survival and intestinal cytokine messenger RNA transcription in a rat model of ongoing peritoneal sepsis.

OBJECTIVE: Septic animals receiving high-protein liquid diets have increased mortality and increased production of cytokines by the gut compared with animals receiving low-protein diets. The purpose of this study was to evaluate the ability of pentoxifylline to alter gut cytokine production in a rat model of prolonged acute peritonitis, to determine its effect on survival in such animals, and to determine whether alteration of gut cytokine production was associated with survival. DESIGN: Prospective, randomized animal study. SETTING: Research laboratory. SUBJECTS: Male Lewis rats weighing between 250 and 300 g. INTERVENTIONS: Anesthetized rats had placement of a gastrostomy, followed 1 wk later by implantation of a bacteria-filled osmotic minipump into the peritoneal cavity. Rats were fed a high-protein (20% total energy) enteral diet. Saline or pentoxifylline (5 or 20 mg/kg im) was administered daily beginning at the time of pump implantation. MEASUREMENTS AND MAIN RESULTS: Septic rats fed the high-protein liquid diet and given pentoxifylline in a dose of 5 mg/kg/day demonstrated improved survival compared with saline-treated animals or animals given the high dose (20 mg/kg/day) of pentoxifylline (p< .05). Administration of pentoxifylline at 5 mg/kg/day also down regulated the production of IL-6 messenger RNA (mRNA) in liver and lipopolysaccharide binding protein mRNA in the liver and intestine of septic animals given the high-protein liquid diet. CONCLUSION: Low-dose (but not high-dose) pentoxifylline administration reduced production of some, but not all, cytokines studied in the gut and liver in a rat model of acute peritonitis and this reduced production was associated with an improved survival in such animals.

Dietary amino acids as new and novel agents to enhance allograft survival.

Dietary supplementation with arginine was previously found to enhance cardiac allograft survival in rats when given with a donor-specific transfusion and a short low-dose course of cyclosporine. This study was performed to determine further the role of amino acid supplementation in prolonging allograft survival. Standard isocaloric, isonitrogenous diets were modified to contain 2 or 4% of energy from arginine, 2 or 4% from glutamine, 4% from glycine or the following combinations: 2% arginine with 2% glutamine, 2% arginine with 4% glutamine, or 1% arginine with 2% glutamine. These diets were started along with a donor-specific transfusion and a 7-d course of cyclosporine the day before cardiac transplantation from an ACI to Lewis strain rat. Median survival times in days for the groups were as follows: control without amino acids, 19.0; 2% arginine, 68.0; 4% arginine, 35.5; 2% glutamine, 28.5; 4% glutamine, 53.5; 4% glycine, 31.5; 2% arginine with 2% glutamine, 39.5; 2% arginine with 4% glutamine, 42.5 and 1% arginine with 2% glutamine, 35.5. Each experimental diet except 2% glutamine and 4% glycine significantly enhanced allograft survival (P < 0.05) with the 2% arginine diet being the best (91.6 +/- 32.3 d [mean +/- SEM] versus 20.1 +/- 3.2 d for control). It is concluded that both arginine and glutamine enhance the immunosuppressive effects of donor-specific transfusion and cyclosporine.

Dietary omega-3 and omega-9 fatty acids uniquely enhance allograft survival in cyclosporine-treated and donor-specific transfusion-treated rats.

BACKGROUND: Both laboratory and clinical studies have shown that dietary lipids may affect immunologic responses. This study was conducted to compare different classes of long-chain unsaturated fatty acids for their effect on allograft survival in animals receiving a donor-specific transfusion and a short course of low-dose cyclosporine (CsA). METHODS: Heterotopic ACI strain cardiac allografts were transplanted to Lewis strain rat recipients given diets with different lipid composition. In experiment 1, animals received CsA for 14 days and different diets were enriched with lipids with high concentrations of omega-3, omega-6, or omega-9 fatty acids. In experiment 2, animals received CsA for only 8 days and different diets were enriched with corn oil (omega-6), canola oil (omega-3 and omega-9), fish oil (omega-3) or a mixture of sunflower oil and fish oil (omega-3 and omega-9). RESULTS: In experiment 1, animals receiving the diet with 30% sunflower oil had the best allograft survival (200+/-42 days vs. 53+/-8 days for regular chow plus donor-specific transfusion and CsA, P<0.05). In experiment 2, diets containing canola oil (a mixture of omega-3 and omega-9 fatty acids) were associated with the best survival (P=0.0011 vs. regular chow). CONCLUSION: Dietary omega-3 and omega-9 fatty acids both enhanced cardiac allograft survival in a stringent rat strain combination. Canola oil is a convenient oil for administering both alpha-linoleic acid (omega-3) and oleic acid (omega-9) in a palatable form for human consumption. Further investigation of the potential usefulness of lipids in transplant therapy is warranted.

Arginine, fish oil, and donor-specific transfusions independently improve cardiac allograft survival in rats given subtherapeutic doses of cyclosporin.

BACKGROUND: Dietary supplementation with a fish oil and arginine-enriched immunoenhancing diet (Impact; Sandoz Nutrition, Minneapolis, MN) in a rat cardiac allograft model using donor-specific transfusion (DST) and cyclosporin (CsA) resulted in significant prolongation of cardiac allograft survival with many animals developing long-term tolerance. This study was done to determine whether arginine or fish oil was the active ingredient. METHODS: A standard AIN-76A diet was modified to include either 10% fish oil, 2% arginine, or 5% arginine with or without fish oil. Diets were fed to Lewis strain rats that received Ax C9935 Irish (ACI) heterotopic cardiac allografts beginning on day 1 and continuing indefinitely. A DST (1.0 mL ACI whole blood) was given with 10 mg/kg CsA on day 1 relative to transplant and 2.5 mg/kg/d on days 0 to 6. Groups of animals receiving AIN-76A diet fortified with 2% glycine and animals receiving a DST or DST/CsA and regular laboratory chow served as controls. RESULTS: Mean survival times +/- SEM in days were as follows: untreated, 7.1 +/- 0.4; CsA/2% glycine, 8.5 +/- 0.6; DST only, 9.6 +/- 1.1; DST/CsA, 26.6 +/- 6.4; CsA/2% arginine, 25.5 +/- 3.9; DST/CsA/2% arginine, 68.7 +/- 8.9; DST/CsA/5% arginine, 90.1 +/- 31.1; CsA/fish oil, 73.6 +/- 26.1; and DST/CsA/fish oil/5% arginine, 90.1 +/- 31.1. The effect of arginine was slightly dose dependent and was seen best in combination with DST, but the effect of fish oil was not enhanced by DST. CONCLUSIONS: Both fish oil and arginine dietary supplementation significantly improved allograft survival but through different mechanisms (DST vs non-DST dependent).

Minimal increase in gut-mucosal interleukin-6 during laparoscopy.

BACKGROUND: The gut clearly plays a significant role in postoperative recovery. Other investigators have shown an increase in gut-mucosal cytokines in septicemia and burn models. We tested the effects of laparotomy and laparoscopy on gut-mucosal IL-6 production. METHODS: A/J mice were randomized to three groups: control, laparotomy plus bowel manipulation (OBM), and laparoscopy plus bowel manipulation (LBM). Serum and gut-mucosal samples obtained at 4 and 8 h after surgery were analyzed for IL-6. RESULTS: We found that OBM is associated with increased serum and gut-mucosal IL-6 at both 4 and 8 h after surgery. In contrast, LBM showed a blunted response in serum IL-6 and no change in gut-mucosal IL-6 at both time intervals. CONCLUSIONS: We conclude that laparoscopy minimizes trauma to the peritoneal environment, thereby decreasing the gut's inflammatory response to operation. This differential response of the gut may partially explain the preservation of gut function following laparoscopy.

Diets and infection: composition and consequences.

This brief review focuses on the effects of nutrient composition of enteral diets on the outcome of surgical patients and experimental models of infection. Complete enteral diets containing combinations of immunonutrients (arginine, glutamine, RNA, omega-3 fatty acids), when given postoperatively or after trauma to surgical patients, can reduce hospital stay, overall costs, and the incidence of wound complications and acquired infections. Immunonutrient diets can also reduce the length of hospital stay when given to patients admitted to the surgical intensive care unit. A high protein diet is usually required for optimal benefit, although administration of high protein immunoenhancing diets may have adverse effects in animals with severe untreated peritonitis because of a sustained overproduction of cytokines.

Thermal injury functionally alters bone marrow-derived macrophages: a study of monocyte-hepatocyte interactions.

Thermal injury quantitatively and qualitatively alters hematopoiesis, including monocyte-macrophage lineage changes, resulting in altered mononuclear cell function. These bone marrow cells (BMCs) ultimately become fixed tissue macrophages (e.g., Kupffer cells). To study the effects of thermal injury on macrophage-hepatocyte interactions, rat BMCs were isolated 24 hours after burn injury, and myelopoiesis was induced by 7-day culture in granulocyte-macrophage colony-stimulating factor. Separate cultures included inflammatory mediators with growth factor function (IL-6 or PGE2). Cultured cells were incubated up to 96 hours with isolated normal hepatocytes (+/- lipopolysaccharide stimulation). The 96-hour exposure to postburn BMCs produced less of the acute phase proteins (APPs), C3 and transferrin, but more cytotoxicity as measured by 1-lactate dehydrogenase release. Sham BMCs cultured with added IL-6 caused higher APP release and minimal cytotoxicity, whereas burn BMCs stimulated lower APP release and retained cytotoxicity. In conclusion, myeloid cells regulate APP synthesis differently after thermal injury and may become more cytotoxic to hepatocytes.

Growth hormone attenuates the acute-phase response to thermal injury.

OBJECTIVE: To determine the effects of growth hormone (GH) on the hepatic acute-phase response (APR) in a burned rat model. SETTING: Laboratory. MATERIAL: Male Sprague-Dawley rats (weight, 300-350 g). INTERVENTIONS: Rats underwent a 40% total body surface area burn injury and received GH or saline solution daily by subcutaneous injection. Unburned rats served as controls. MAIN OUTCOME MEASURES: Hepatic messenger RNA (mRNA) expression and serum levels of alpha 1-acid glycoprotein and albumin were determined 2, 7, and 14 days after injury. RESULTS: The serum alpha 1-acid glycoprotein levels in GH-treated animals did not increase on days 2 and 7, whereas saline-treated animals showed a major increase. Hepatic mRNA expression increased dramatically on day 2 for burned groups; however, the mRNA pool levels of GH-treated animals showed a faster rate of decline to control levels on days 7 and 14. The albumin mRNA pool levels of GH-treated and control animals did not show significant differences, whereas the negative APR, indicated by loss of albumin mRNA, was more pronounced on day 7 in the saline-treated animals. By day 14, mRNA levels were comparable in all 3 groups. CONCLUSION: Growth hormone attenuated the positive APR, as indicated by a decrease in alpha 1-acid glycoprotein expression and production, and prevented the negative APR, as seen by an absence of a decline of albumin mRNA pool levels and serum concentration. We conclude that the beneficial effects of GH on thermal injury may be due in part to a modification of the APR.

Bone marrow and splenocyte coculture-generated cells enhance allograft survival.

BACKGROUND: Protocols that incorporate donor-specific cell infusions using bone marrow, spleen, or blood transfusion continue to enhance allograft survival and often lead to tolerance in experimental models. Clinical benefits from these modalities have not been as striking, leading to ongoing study in this field. We have explored culture techniques for the in vitro selection and development of cellular effectors capable of enhancing allograft survival. METHODS: Rat bone marrow or spleen cells cultured under a variety of conditions were screened for suppressor function. Bone marrow cells, nonadherent to plastic, cultured for 7 days with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and with or without splenocytes were found to contain predominantly myeloid lineage cells and had the ability to suppress phytohemagglutinin or mixed lymphocyte reaction-induced splenocyte proliferation. Standard donor-specific peripheral blood transfusion was compared with cultured donor-specific bone marrow cells, splenocytes, or marrow cells cultured with splenocytes (cocultured) administered intravenously at 1 x 10(7) cells/kg the day before an ACI to Lewis heterotopic heart transplant. Cyclosporine was administered at 10 mg/kg on day -1 and 2.5 mg/kg on days 0-6 relative to transplantation. RESULTS: Mean allograft survival in cyclosporine-treated animals was 8.5 days without and 16.6 days with a donor-specific blood transfusion. Cocultured cells extended allograft survival (39.5 days), whereas bone marrow or splenocytes cultured alone did not. With Percoll gradient separation, two predominant culture subfractions, one with potent suppressor function and another with stimulator function, were identified. Flow cytometric analysis showed mixed populations enriched for macrophages but also including dendritic cells in both subfractions. The suppressive fraction extended allograft survival to 20.8 days and the stimulatory fraction was less effective, yet remixing of both fractions regained the full allograft survival advantage. CONCLUSIONS: In this model, the coculture of bone marrow cells and splenocytes with granulocyte-macrophage colony-stimulating factor and lipopolysaccharide produced functionally divergent subpopulations that synergistically enhanced allograft survival. The development of cellular effectors with enhanced ability to prolong allograft survival using in vitro culture techniques is possible, and provides a new therapeutic option in the use of cell infusion-based therapies.

Causes of renal allograft loss in black vs. white transplant recipients in the cyclosporine era.

Black renal transplant recipients have a higher rate of allograft loss than white recipients. From 1 January 1984 to 1 January 1995, 463 transplants were performed at a single center and followed for a mean duration of 71 months. The causes of graft loss for white and black recipients, their age, gender, retransplantation rate, organ source, and HLA matching were compared. In the 150 black and 313 white recipients, graft loss rates in the first year were 20% in both groups, while after 1 yr there were 42 (28%) graft losses in blacks vs. 62 (20%) in whites (log-rank test p = 0.04). All diagnoses deemed causative of allograft loss were confirmed by biopsy. Chronic rejection resulting in graft loss occurred in 15% (n = 23) of black recipients compared to only 7% (n = 22) of white recipients (p = 0.002). There were no significant differences in the rate of death with a functioning kidney or other causes of graft loss between the two groups. A significant increase in HLA mismatches was noted in black recipients of cadaveric grafts compared to whites, but there was no difference between races in the rate of graft loss due to acute rejection. While the rate of graft survival remains lower in black recipients in the cyclosporine era, this is due entirely to late graft loss after 1-yr post-transplant due to chronic rejection.

Induction of endotoxin tolerance in rat bone marrow cells by in vivo infusion of tumor necrosis factor.

OBJECTIVE: To determine in a rat model whether a low-dose infusion of tumor necrosis factor (TNF) affects the production of the inflammatory cytokines TNF and interleukin (IL)-6, the immunosuppressive factor prostaglandin E2 (PGE2), and complement component C3 (C3) by isolated bone marrow-adherent and -nonadherent cells, cultured in the presence of lipopolysaccharide, a component of bacterial endotoxin. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of a university medical center. SUBJECTS: Sprague-Dawley rats (n = 18), 250 to 275 g. INTERVENTIONS: Animals received a continuous infusion of one of the following three treatments for 4 days: a) TNF in saline containing bovine serum albumin; b) saline containing bovine serum albumin; and c) saline alone. MEASUREMENTS AND MAIN RESULTS: After infusion, isolated bone marrow cells were cultured for 1 day and 3 days, with and without lipopolysaccharide (1 microgram/mL); culture supernatants were assayed for TNF, IL-6, PGE2, and C3. TNF infusion caused a decrease in the in vitro production of TNF, IL-6, and PGE2 by the lipopolysaccharide-stimulated adherent and nonadherent bone marrow cells. This tolerance to lipopolysaccharide stimulation was present after both 1 day and 3 days of culture. TNF infusion caused an increase in C3 production by the nonadherent cells. The production of TNF by adherent cells from saline-infused or bovine serum albumin-infused animals (controls) was greater in 3-day cultures compared with 1-day cultures, whereas the production of IL-6 and PGE2 was less. CONCLUSIONS: These results indicate that TNF infusion caused cells in the bone marrow to be tolerant to lipopolysaccharide stimulation or that TNF infusion programmed the cells to become tolerant to lipopolysaccharide stimulation on differentiation and/or maturation. The results also indicate that bone marrow cells may be regulated by TNF (probably indirectly) at different phases of maturation and/or differentiation with respect to the production of different mediators. Although TNF is considered to be an inflammatory cytokine, at low concentrations it may be an important down-regulator of the inflammatory response.