Deep venous thrombosis in surgical intensive care unit: prevalence and risk factors.

BACKGROUND: Critically ill patients are at high risk for developing venous thromboembolism. The objective of this study was to determine the prevalence of, and risk factors for, lower extremity deep vein thrombosis (DVT) among critically ill surgical patients in Thailand. MATERIALS AND METHODS: Patients older than 15 years who were admitted to a surgical intensive care unit (ICU) of a tertiary care hospital were enrolled. Bilateral lower extremity compression Doppler ultrasonographic examination was performed to detect DVT within 14 days of ICU admission. Demographic data, primary disease, operative intervention, co-morbidities, acute physiology and chronic health evaluation (APACHE) II score and the length of ICU stay were tested for association with the presence of DVT. RESULTS: Among the 190 first-time admitted ICU patients with a mean APACHE II score of 9.2 +/- 6.0 (range, 0-29), 20 patients had DVT (prevalence of 10.5%). Thromboprophylaxis was not given to any patient. The only independent and significant risk factor for DVT was a longer ICU stay. Age, sex, APACHE II score, presence of comorbidities and operative intervention were not associated with the presence of DVT. CONCLUSION: The prevalence of DVT in critically ill patients in a Thai surgical ICU was approximately 10.5%. Further research is needed to evaluate the risks and benefits of venous thromboprophylaxis in Thai patients.

Green tea extract prolongs allograft survival as an adjunctive therapy along with low dose cyclosporine A.

The current immunosuppressive drugs are successful in prolonging allograft survival but fail to achieve transplantation tolerance or prevent chronic rejection. Consequently, there is ongoing research to develop novel combinatorial therapies that are more efficacious in prolonging allograft survival as well as induce tolerance toward the transplanted organ. The present study aims to study the efficacy of green tea extract (GTE) in combination with low dose cyclosporine A (CyA) in prolonging allograft survival in mice. Numerous studies have reported the anti-inflammatory and immunomodulatory properties of GTE and its various catechin components. GTE is also known to attenuate CyA induced nephrotoxicity. Therefore, we hypothesized that GTE alone or in combination with CyA will prolong graft survival. Our study demonstrates that GTE in combination with low dose CyA significantly prolongs graft survival as well as increase the production of immunosuppressive cytokine, IL-10. GTE also decreases CyA induced high TGF-beta production, which is incriminated in CyA induced nephrotoxicity. We also observed that GTE inhibits both nonspecific and antigen-specific proliferation of T cells in vitro. These results indicate the potential of GTE as an adjunctive therapy in combination with CyA to prolong allograft survival and to reduce CyA induced nephrotoxicity.

Cyclosporine inhibition of angiogenesis involves the transcription factor HESR1.

PURPOSE: Angiogenesis is critical in normal development and in tumor growth. Experimentally, cyclosporine A (CyA) inhibits angiogenesis in an in vivo mouse model and an in vitro capillary tube model. The mechanisms behind its antiangiogenic effects are not well characterized. To determine which nuclear factor, if any, may be involved in the antiangiogenic effects of CyA, we performed a microarray analysis of human aortic endothelial cells (HAEC) subjected to CyA and another calcineurin inhibitor, FK 506. METHODS: HAEC were divided into four groups: (1) HAEC incubated with CyA 2 microg/mL; (2) HAEC incubated with CyA 10 microg/mL; (3) HAEC incubated with FK 506 1 microg/mLl for 24 h; and (4) HAEC as control. We used Affymetrix GeneChip U133-A for gene expression analysis and validated our results with quantitative reverse transcription-polymerase chain reaction. RESULTS: At a 2 microg/mL dose, CyA treated HAEC revealed a 44-fold increase in the expression of hairy enhancer of split-related protein 1 (HESR1) and 1.73-fold down-regulation of transcripts encoding for the vascular endothelial growth factor (VEGF) receptor (VEGFR2). At 10 microg/mL, the expression of the HESR1 transcript was 57-fold higher than control, and VEGFR2 exhibited a 1.93-fold down-regulation. Quantitative reverse transcription-polymerase chain reaction confirmed a significant (P < 0.0001) increase in expression of HESR1 in CyA treated cells. In contrast, the expression level of HESR1 was not affected by the FK 506 treatment. CONCLUSION: CyA demonstrate antiangiogenic activities linked to an overexpression of HESR1 transcription factor, and down-regulation of VEGFR2. Thus, use of high-dose CyA may provide a novel treatment in angiogenesis dependent disease.

An interleukin-6-neutralizing antibody prevents cyclosporine-induced nephrotoxicity in mice.

INTRODUCTION: Chronic use of cyclosporine A (CyA) induces nephrotoxicity primarily due to endothelial dysfunction. In our previous studies, potential mechanisms were identified in vitro and implicated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and interleukin-6 (IL-6) as key components in causing endothelial dysfunction. In this study, we tested the hypothesis that NADPH oxidase activity and IL-6 are key components in renal damage in an in vivo model. METHODS: Male mice C57B/6 mice from Jackson Laboratory (Bar Harbor, ME) at 6-8 wks were subjected to a low-salt diet throughout the trial. After 1 week on a low-salt diet, the mice were injected daily with treatments in 50 muL vehicle composed of 75% cremaphor (Sigma, St. Louis, MO) and ethanol for 5 wks. A vehicle-alone group was also set aside. Mice were weighed and 25 mg/kg/day cyclosporine (Novartis Pharma, St. Louis, MO) was injected daily. Apocynin (Calbiochem, Gibbstown, NJ) 20 mg/kg were injected either alone or concomitantly with CyA. Another group of mice were administered IL-6 antibody (Cat no. MAB406; R&D Systems, Minneapolis, MN) at 2 mug/day along with CyA. The kidneys were removed en bloc immediately and submitted in formalin for paraffin sections. Trichrome stains were performed. Slides were blinded and 10 photographs of cortical areas per treatment group were taken, which covered an estimate of 10% surface area in random fashion. Areas of renal damage, which were determined by tubular necrosis, were identified and quantified by amount of necrosis per photograph. Each photograph was divided into 10 blocks, and the number of blocks that contained necrotic tubules per photo was recorded. RESULTS: The two control mice (low salt only) had no damage. The four vehicle mice had trace amounts of tubular necrosis. CyA treatment group demonstrated the highest amount of damage (29/70; 41%). CyA with apocynin, a specific NADPH oxidase inhibitor, was found to have 36% (22/60) damage, whereas the CyA with IL-6 antibody only was observed to have 15% (6/40) damage. Comparing imaging analysis, there was no difference between mice treated with CyA alone and with CyA with apocynin. However, the amount of damage in mice treated with CyA and IL-6 antibody was found to be significantly lower than both CyA and CyA with apocynin. CONCLUSIONS: CyA action as a calcineurin inhibitor has allowed prolongation of kidney transplants, but its chronic use has led to devastating consequences such as allograft nephropathy. Previously, we have identified potential mechanisms of CyA-induced endothelial dysfunction in vitro. The current study identifies increased IL-6 expression as a mechanism by which CyA induces renal damage and that the use of an IL-6-neutralizing antibody may be useful in reducing CyA-induced renal damage.

Ginger extract inhibits LPS induced macrophage activation and function.

BACKGROUND: Macrophages play a dual role in host defence. They act as the first line of defence by mounting an inflammatory response to antigen exposure and also act as antigen presenting cells and initiate the adaptive immune response. They are also the primary infiltrating cells at the site of inflammation. Inhibition of macrophage activation is one of the possible approaches towards modulating inflammation. Both conventional and alternative approaches are being studied in this regard. Ginger, an herbal product with broad anti inflammatory actions, is used as an alternative medicine in a number of inflammatory conditions like rheumatic disorders. In the present study we examined the effect of ginger extract on macrophage activation in the presence of LPS stimulation. METHODS: Murine peritoneal macrophages were stimulated by LPS in presence or absence of ginger extract and production of proinflammatory cytokines and chemokines were observed. We also studied the effect of ginger extract on the LPS induced expression of MHC II, B7.1, B7.2 and CD40 molecules. We also studied the antigen presenting function of ginger extract treated macrophages by primary mixed lymphocyte reaction. RESULTS: We observed that ginger extract inhibited IL-12, TNF-alpha, IL-1beta (pro inflammatory cytokines) and RANTES, MCP-1 (pro inflammatory chemokines) production in LPS stimulated macrophages. Ginger extract also down regulated the expression of B7.1, B7.2 and MHC class II molecules. In addition ginger extract negatively affected the antigen presenting function of macrophages and we observed a significant reduction in T cell proliferation in response to allostimulation, when ginger extract treated macrophages were used as APCs. A significant decrease in IFN-gamma and IL-2 production by T cells in response to allostimulation was also observed. CONCLUSION: In conclusion ginger extract inhibits macrophage activation and APC function and indirectly inhibits T cell activation.

Three-dimensional aortic aneurysm model and endovascular repair: an educational tool for surgical trainees.

OBJECTIVES: Endovascular aortic aneurysm repair (EVAR) is a current valid treatment option for patients with abdominal aortic aneurysms (AAAs). The success of EVAR depends on the selection of appropriate patients, which requires detailed knowledge of the patient's vascular anatomy and preoperative planning. Three-dimensional (3D) models of AAA using a rapid prototyping technique were developed to help surgical trainees learn how to plan for EVAR more effectively. METHOD: Four cases of AAA were used as prototypes for the models. Nine questions associated with preoperative planning for EVAR were developed by a group of experts in the field of endovascular surgery. Forty-three postgraduate trainees in general surgery participated in the present study. The participants were randomly assigned into two groups. The 'intervention' group was provided with the rapid prototyping AAA models along with 3D computed tomography (CT) corresponding to the cases of the test, while the control group was provided with 3D CTs only. RESULTS: Differences in the scores between the groups were tested using the unpaired t test. The mean test scores were consistently and significantly higher in the 3D CT group with models compared with the 3D CT group without models for all four cases. Age, year of training, sex and previous EVAR experience had no effect on the scores. CONCLUSION: The 3D aortic aneurysm model constructed using the rapid prototype technique may significantly improve the ability of trainees to properly plan for EVAR.

Comparison of risk-scoring systems in predicting hospital mortality after abdominal aortic aneurysm repair.

OBJECTIVE: To compare the Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity, Portsmouth adjustment (P-POSSUM), the Hardman index and the Glasgow aneurysm score (GAS) in the prediction of hospital mortality after abdominal aortic aneurysm (AAA) repair. METHODS: Medical charts of 146 AAA patients treated between January 1996 and January 2007 were reviewed. The P-POSSUM, Hardman index and GAS were calculated for each patient. The scores were tested and compared for their discriminatory ability to predict hospital death. RESULTS: Of the 146 patients with ruptured and unruptured AAAs (133 underwent open repair, five underwent extra-anatomical bypass and eight underwent endovascular aneurysm repair), 18 died (12%) after AAA repair. The areas under the receiver operating characteristic curves for the GAS, Hardman index and P-POSSUM for predicting hospital mortality were 0.740, 0.730 and 0.886, respectively. The area under the receiver operating characteristic curve for the P-POSSUM was significantly higher than those of other scores. CONCLUSION: In the present study, the P-POSSUM was the best predictor of hospital mortality for patients undergoing AAA repair.

Effect of 6-gingerol on pro-inflammatory cytokine production and costimulatory molecule expression in murine peritoneal macrophages.

BACKGROUND: Pro-inflammatory cytokines produced primarily by macrophages are key elements in many surgical conditions including sepsis, ischemia-reperfusion injury, and transplant rejection. Herbal products are being used as alternative treatments in such inflammatory conditions. Ginger is known for its ethno-botanical applications as an anti-inflammatory agent. 6-gingerol is one of the active ingredients of ginger that imparts ginger with its anti-inflammatory properties. We hypothesized that the anti-inflammatory effect of 6-gingerol is because of inhibition of macrophage activation, more specifically by an inhibition of pro-inflammatory cytokines and antigen presentation by lipopolysaccharide (LPS) activated macrophages. METHODS: To study the effect of 6-gingerol on pro-inflammatory cytokines, we measured the liberation of TNF-alpha, IL-1beta, and IL-12 by murine peritoneal macrophages exposed to several doses of 6-gingerol in the presence of LPS stimulation. We also studied the effect of 6-gingerol on the cell surface expression of B7.1, B7.2, and MHC II. Finally, we examined the APC function of the 6-gingerol treated macrophages by a primary mixed lymphocyte reaction. RESULTS: 6-gingerol inhibited the production of pro-inflammatory cytokines from LPS stimulated macrophages but had no effect on the LPS-induced expression of B7.1, B7.2, and MHC II. The APC function of LPS stimulated macrophages was also unaffected by 6-gingerol treatment. CONCLUSION: Our data indicate that 6-gingerol selectively inhibits production of pro-inflammatory cytokines from macrophages but does not affect either the APC function or cell surface expression of MHC II and costimulatory molecules. We, thus, provide a mechanistic insight into the anti-inflammatory properties of 6-gingerol that may be useful to treat inflammation without interfering with the antigen presenting function of macrophages.

Ultrastructural changes in cirrhotic and noncirrhotic patients due to hepatectomy.

BACKGROUND/PURPOSE: Alterations at the ultrastructural level can be identified prior to histological change in the early phase of irreversible cell damage. The aim of this investigation was to compare the ultrastructural changes in cirrhotic and noncirrhotic liver in response to ischemic and reperfusion injury due to hepatectomy. METHODS: Hepatic resections using the same technique were performed in cirrhotic and noncirrhotic patients. Three biopsy specimens (Tru cut) from each patient, in the unresected part of the liver, were studied by transmission electron microscopy: immediately after laparotomy, before releasing of the porta hepatis clamp (ischemic phase), and 30-45 min after reperfusion. RESULTS: All patients did well after surgery, except for 1 cirrhotic patient who died of liver failure. There were no significant differences in operative time, blood loss, and inflow occlusion times in any of the 15 patients. We found that morphological changes were the same in the 10 non-cirrhotic and 4 cirrhotic patients. Changes during the ischemic phase included nuclear membrane deformity, focal chromatin condensation at the nuclear margin, and swelling of both mitochondria and endoplasmic reticulum. In the reperfusion phase, there were early irreversible changes in the nuclei of some hepatocytes and intramitochondrial particles and increased vacuolization in cytoplasm. Endothelial cells, Kupffer cells, bile canaliculi, and Ito cells were not affected in either the ischemic or the reperfusion phase. However, in the 1 cirrhotic patient who died of liver failure, there were marked swelling and dilated cristae in mitochondria during the ischemic phase and deformity of Ito cells during the reperfusion phase. CONCLUSIONS: In this, the first report of ultrastructural changes due to hepatectomy in cirrhotic patients, we found that the changes were the same as those in non-cirrhotic patients, except for the one cirrhotic patient who had postoperative liver failure.

The effect of allo-injury in an in vitro model of allograft microvasculature.

Endothelial cells are critical to the integrity of allograft vasculature and can be damaged by alloreactive T cells or soluble mediators of alloreactivity. The biochemical effects of T cell-mediated damage to the endothelial cells have been studied, but not the structural and morphological effects of allo-injury on endothelial cells in the allograft. We utilized an assay that reproduces microvasculature in vitro to study the effect of alloreactivity on endothelial cells. In this assay, endothelial cells are induced into capillary-like networks that simulate microvascular capillaries. We studied the effect of allogeneic T cells and of soluble mediators from both mixed lymphocyte cultures (MLCs) and rejected heart allograft tissue on the in vitro capillaries. We found that both allogeneic T cells and soluble mediators inhibit the formation of the in vitro endothelial capillaries, suggesting that they cause a mild-to-moderate dysfunction of the endothelial cells. The inhibitory effect of the soluble mediators seems to be mediated, at least partly, by IFN-gamma, since this effect was prevented by antibody to IFN-gamma. Furthermore, pre-incubation of the in vitro capillaries with IFN-gamma appeared to magnify the effect of allogeneic T cells, as shown by a complete breakdown of well-formed in vitro capillary networks. Our experiments suggest that the in vitro capillary-tube model reflects structural injury to allograft vasculature by alloreactive T cells and their soluble mediators.

Augmentation of ischemia/reperfusion injury to endothelial cells by cyclosporin A.

Ischemia/reperfusion (I/R) carries significant injury to endothelial cells in transplanted organs and is an important factor in chronic rejection. Immunosuppressive drugs, notably cyclosporin A (CyA) and FK506, can potentially augment this injury. Here, our goal was to determine the combined effects of I/R and CyA or FK506 on endothelial cells. Transformed mouse endothelial cells (SVEC 4-10) were subjected to ischemia or I/R for 2-24 hours by incubating cells in 100 per cent N2 (ischemia) followed by 5 per cent CO2 and 95 per cent O2 (reperfusion) for 24 hours. In separate experiments, CyA or FK506 was added to cells subjected to ischemia or I/R. Nonviable cells were determined by Trypan blue exclusion assay. All experiments (done in triplicate) were analyzed by Student's t test. Increasing ischemia times resulted in a greater number of nonviable cells (2% nonviable cells at 0 hours and 57% at 24 hours of I/R). Addition of CyA significantly increased the number of nonviable cells when compared with the control (I/R only) group (P = 0.014). Interestingly, FK506 did not increase the percentage of nonviable cells compared with the control group (P = 0.2). Unlike FK506, CyA augments I/R injury to endothelial cells in vitro. These findings could be relevant in chronic rejection and transplantation.

Initial angiogenic response in reduced renal mass after transplantation.

INTRODUCTION: Shortage of organs is a major problem in kidney transplantation and requires novel strategies to increase the number of kidney transplants. To reduce the shortage of kidneys, we have proposed transplantation of two halves of one kidney into two recipients (hemirenal transplantation, HRT) and have shown its feasibility in pig and human kidneys. However, reduced renal mass can lead to progressive renal failure in rodents and can reduce the longevity of kidney transplants in humans. Recent studies suggest that derangement of angiogenesis plays a role in the progressive renal failure after reduction in renal mass in rodents. However, since the renal physiology of rats is different from that of large animals, we studied angiogenesis in reduced renal mass transplants in pigs and determined if the reduction in renal mass has the same effect in large animals as that in rodents. MATERIALS AND METHODS: Kidney autotransplantation was performed in domestic outbred swine. Heminephrectomy of the autotransplanted kidney and nephrectomy of the contralateral kidney were performed 1 week after transplantation to reduce the renal mass. Four weeks after transplantation, the pigs were sacrificed and the hemirenal and control nephrectomy specimens were processed for morphometric analysis of glomerular capillary density and immunohistochemical analysis of VEGF expression. Soluble extracts from the kidneys were tested in an in vitro angiogenesis assay to determine their activity to influence angiogenesis. Statistical analysis with ANOVA was performed on the glomerular capillary density in kidney specimens. RESULTS: All these parameters of angiogenesis were increased in the reduced renal mass autotransplants as compared to normal kidneys or whole kidney autotransplants. Glomerular capillary density was increased significantly after reduction in renal mass. VEGF expression also was increased progressively by the third week after reduction in renal mass. Soluble extract from the reduced renal mass transplants significantly increased the in vitro angiogenesis. CONCLUSION: This is the first study to demonstrate that angiogenesis is increased in the initial stages of reduction in renal mass after transplantation in a large animal model. Increased angiogenesis was found in this model earlier than reported in small animal models (2 weeks in pigs versus 6 weeks in rats). Taken together with other studies, our data suggest that derangement in angiogenesis could play an important role in long-term graft function after hemirenal transplantation.

beta1-integrin-ligand disengagement induces in vitro capillary tube disruption mediated by p38 MAPK activity.

BACKGROUND: Disrupting cell-matrix interactions may lead to capillary injury as seen in sepsis and transplant rejection. Previously, we demonstrated capillary disruption mediated by beta1-integrin-ligand disengagement. We now determine whether p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways are involved in this capillary injury. METHODS: Endothelial capillaries on Matrigel were preincubated with a p38 MAPK inhibitor (SB203580), ERK pathway inhibitor (PD98059), or dimethyl sulfoxide. Subsequently, a beta1-integrin blocking (P5D2) or an irrelevant antibody was added. After 24 hours, capillary integrity was quantified as capillary intersections/well. Antibody-treated cell lysates then were immunoprecipitated with either a phospho-p38 MAPK or phospho-ERK1/2 antibody. Kinase activity was measured with ATF-2 and Elk-1 fusion proteins as substrates for p38 MAPK and ERK, respectively, followed by Western blotting. RESULTS: P5D2 disrupted capillary tubes. Increased p38 MAPK activity at 8 hours and ERK activity at 2 and 8 hours were seen in P5D2-treated lysates. Preincubation with SB203580, but not with PD98059 or DSMO, significantly reduced capillary tube disruption. CONCLUSIONS: The beta1-integrin-ligand disengagement resulted in capillary disruption and stimulated p38 MAPK and ERK activity. In spite of activation of both pathways, the p38 MAPK but not the ERK pathway inhibitor prevented beta1-integrin antibody effects. Inhibiting p38 MAPK may mitigate capillary injury associated with sepsis and transplant rejection.

Amelioration of cyclosporin A effect on microvasculature by endothelin inhibitor.

BACKGROUND: We have previously shown that endothelial injury by cyclosporin A (CyA) is associated with an increased endothelin-1 (ET-1) release. We now sought to determine, in an animal model of angiogenesis, if inhibiting the effect of ET-1 on endothelial cells (ECs) would reverse the CyA-mediated endothelial injury in an animal model of angiogenesis. METHODS: An angiogenic mixture of Matrigel (0.5 ml), fibroblast growth factor (1 ng/ml), vascular endothelial growth factor (100 ng/ml), and heparin (64 unit/ml) was injected as a subcutaneous plug in the flank of C3H mice (n = 5). In experimental groups CyA (20 mg/ml), CyA, and BQ 123 (ET-A receptor antagonist), CyA and PD 142893 (ET-A and ET-B receptor antagonist), or CyA and ET-1 antibody were added to the angiogenic mixture. Angiogenesis in the mixture was quantified by modified planimetric point counting method in skin/Matrigel cross-sections stained with factor VIII to highlight endothelial neocapillaries. Mean +/- SD of angiogenic area was analyzed with analysis of variance and Bonferroni test. The survival curves obtained by Kaplan-Meier analysis were compared between the groups, and the statistical significance of survival and mortality rates was computed by log rank's and Fisher's exact test, respectively. RESULTS: The mean +/- SD of angiogenic area in control animals (without CyA in the angiogenic mixture) was 56.76 +/- 4.2. CyA inhibited angiogenesis in the subcutaneous angiogenic plug. Adding CyA to the angiogenic mixture significantly reduced angiogenic area (5.33 +/- 1.4, P <.001) while vehicle for CyA had no such effect (56.33 +/- 3.8, P =.10). Polyclonal ET-1 antibody or PD 142893 ameliorated the effect of CyA, whereas BQ 123 did not. The mean angiogenic areas in animals with ET-1 antibody, PD 142893, or BQ 123 in the angiogenic mixture were 57.20 +/- 7.5 (P =.06), 46.00 +/- 11.5 (P = 1.0), 8.60 +/- 2.9 (P <.001), respectively. CONCLUSIONS: Our data show that blocking ET-B receptors specifically ameliorates the microvascular injury to the neocapillaries in angiogenesis caused by CyA. Antiendothelin-1 antibody and ETR antagonist (PD 142893) could, therefore, reduce the ill effects of CyA on microvascular endothelium.

Morphological and biochemical effects of immunosuppressive drugs in a capillary tube assay for endothelial dysfunction.

Immunosuppressive drugs common in clinical transplantation are known to have untoward effects on the vascular system. The effects of some drugs, notably cyclosporin A (CyA), have been studied on the vascular system, while those of others have not. In the vascular system, endothelial cells are the predominant cell type exposed to intravascular concentrations of immunosuppressive drugs. We therefore studied the effects of drugs common in clinical transplantation on endothelial cells in a capillary tube assay. The endothelial cells in the capillary tubes are morphologically more similar to those in the microvasculature than endothelial cells in monolayers. We studied the kinetics and extent of capillary tube formation and prostacyclin (PGI2) and endothelin-1 (ET-1) release from the in vitro capillaries to determine the morphological and biochemical effects of five immunosuppressive agents on endothelial function. We found a significant difference in the morphological and biochemical effects of the two common calcineurin inhibitors, CyA and tacrolimus (FK506) on capillary morphology in vitro. The former had a pronounced injurious effect on the morphology of the in vitro capillaries, while the latter did not. CyA also significantly increased ET-1 release by the capillaries, but FK506 did not. Mycophenolate mofetil (MMF) was the only other agent that had a moderately injurious effect on the morphology of the in vitro capillaries. Sirolimus (rapamycin) and dexamethasone, similar to FK506, had no effect on the capillary morphology. All these agents, except dexamethasone, increased PGI2 release. Our data suggest that CyA adversely affects the morphology of the microvasculature and that this is mediated, at least partly, by an increased ET-1 release by endothelial cells exposed to CyA. These findings describe a novel effect of CyA and MMF on endothelial cells that could be relevant to understanding the mechanisms of immunosuppressive drug-mediated endothelial injury in clinical transplantation.

Role of endothelin-1 in microvascular dysfunction caused by cyclosporin A.

BACKGROUND: Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is implicated in cyclosporin A (CyA) vasculopathy. Previously we have demonstrated, in an in vitro model of endothelial capillaries, that CyA inhibits the formation of the capillaries and, in high doses, disrupts the capillaries. This study addresses the role of ET-1 in CyA-induced endothelial dysfunction of the in vitro capillaries. STUDY DESIGN: Endothelial cells (ECs) were cultured on a laminin-rich matrix, Matrigel, to form capillary-like networks. The ECs were treated with CyA either before capillary tube formation or after capillary tubes had formed. ppET-1 gene expression was studied by reverse transcriptase polymerase chain reaction. To determine if ET-1 was involved in the CyA-mediated disruption of the in vitro capillaries, ET-1 binding to the endothelial cells was blocked by ET-1 antibody and ET receptor antagonists. The effects of exogenous ET-1 were also studied. The results were quantified by counting the number of capillary networks, and the statistical significance was determined with ANOVA. RESULTS: ppET-1 was expressed in ECs during capillary tube formation, but disappeared once capillary tubes had matured. The ppET-1 gene expression reappeared when the capillary tubes were exposed to CyA. Exogenous ET-1 partially reversed the inhibition of tube formation by cyclohexamide, allowing initiation of tube formation. CyA-mediated capillary dysfunction was completely prevented by an anti-ET-1 antibody and an ET-B receptor antagonist. CONCLUSIONS: Endothelin-1 plays a significant role in CyA-induced endothelial dysfunction and may play a role in allograft vasculopathy. Blocking of ET-1 is a strategy to prevent endothelial dysfunction caused by CyA.

Early hemodynamic changes after renal transplantation: determinants of low central venous pressure in the recipients and correlation with acute renal dysfunction.

BACKGROUND: Transplantation of organs leads to several physiologic changes in the recipients who, during their anephric state on chronic hemodialysis, have increased total body water and several electrolyte imbalances. Several abnormal parameters are observed in the physiology of the renal recipient. The central venous pressure (CVP) in the recipient invariably declines despite vigorous fluid resuscitation for reasons that are not clear at the present time. MATERIAL/METHODS: We studied 77 kidney transplants retrospectively, in which we observed a significant decline in central venous pressure (CVP) in the immediate posttransplant period. This phenomenon occurred despite aggressive fluid management and positive fluid balances averaging nearly four liters. Our analysis included the time course of the phenomenon itself as well as a detailed comparison of various parameters in the recipient and the renal graft for possible correlation with this consistent decline in CVP. RESULTS: Neither the absolute CVP nor the drop in CVP appeared to influence the rate of ATN. Interestingly, we found that the kinetics of the decline in CVP were remarkably similar in recipients of both cadaveric and living-related kidneys. CONCLUSIONS: This finding suggests that the reperfusion injury or a related effect may be responsible for the clinical phenomenon presented in this study.

Critical evaluation of radiolabeled lymphocytes to detect acute renal transplant rejection in a large animal model.

BACKGROUND: Renal transplant rejection cannot be diagnosed with certainty by non-invasive techniques. These techniques lack the specificity to differentiate rejection from other causes of renal dysfunction such as ATN and calcineuria toxicity. Since rejection involves lymphocytes, which the other courses of dysfunction do not, radiolabeling lymphocytes is an attractive technique to diagnose rejection non-invasively. MATERIAL/METHODS: We report our experience with this technique in a pig model of renal transplantation. We studied two groups of pigs, one with renal autografts and the other with allografts. We optimized radiolabeling of lymphocytes and also the technology for detection of these lymphocytes. The uptake of the radiolabeled lymphocyte was compared between the two groups by surface detection and, at the end of the experiment, with scintigraphy of renal tissues. RESULTS: Despite adequate labeling and viability of lymphocytes, only 1-2% of injected lymphocytes 'homed' to the renal grafts. Furthermore, although the detection technology was optimized, a poor signal to noise ratio interfered with the detection of the labeled lymphocytes. Due to these problems rejection could not be differentiated from ATN in this model. CONCLUSIONS: We conclude that increasing the specific activity of the lymphocytes and improving the signal to noise ratio will enhance the specificity and sensitivity of this technique and facilitate non-invasive diagnosis of rejection.

In vitro immunomodulatory effects of herbal products.

Immunosuppressive drugs have been developed from natural products such as soil and fungi, which are also the sources of some commonly used herbal products. However, the effect of herbal products on immune response has not been investigated. Because these products can affect the host immune system they can induce either rejection or tolerance after a transplant procedure. To investigate the effects of ten commonly used herbal products on transplant-related immune function we performed in vitro lymphocyte proliferation tests using phytohemagglutinin, mixed lymphocyte culture (MLC) assay, and interleukin (IL)-2 and IL-10 production from MLC. Dong quai, ginseng, and milk thistle had nonspecific immunostimulatory effects on lymphocyte proliferation, whereas ginger and green tea had immunosuppressive effects. Dong quai and milk thistle increased alloresponsiveness in MLC, whereas ginger and tea decreased these responses. The immunostimulatory effects of dong quai and milk thistle were consistently seen in both cell-mediated immune response and nonspecific lymphoproliferation, whereas that of ginseng was not. The immunosuppressive effect of green tea and ginger were mediated through a decrease in IL-2 production, but the immunostimulatory effects of dong quai and milk thistle were not. We conclude that green tea, dong quai, ginseng, milk thistle, and ginger have effects on in vitro immune assays that may be relevant in transplantation in humans.

Immunostimulatory effect of Silybum Marianum (milk thistle) extract.

BACKGROUND: Herbal products are increasingly used for their effects on the immune system. Milk Thistle, a commonly used herbal product is known to inhibit growth of certain tumors, although the mechanism of this effect remains unknown. Previously we have shown that Milk Thistle extracts stimulate neurons in culture. Since other drugs that affect the neuronal; system also affect the immune system, we investigated the effects of Milk Thistle on the immune system. MATERIAL/METHODS: Standardized Milk Thistle extract was studied in murine lymphocyte proliferation tests using Concanavalin A (ConA) as mitogen for non-specific stimulation and mixed lymphocyte culture (MLC) as allospecific stimulation. Th1 and Th2 cytokine levels in MLC were assayed by two antibody capture ELISA technique. All tests were performed in triplicate and repeated twice. RESULTS: We found that Milk Thistle is immunostimulatory in vitro. It increased lymphocyte proliferation in both mitogen and MLC assays. These effects of Milk Thistle were associated with an increase in interferon gamma, interleukin (IL)-4 and IL-10 cytokines in the MLC (table). This immunostimulatory effect increased in response to increasing doses of Milk Thistle. CONCLUSIONS: Our study has uncovered a novel effect of milk thistle on the immune system. This immunostimulatory effect may be of benefit in increasing the immunity to infectious diseases.