Study of Transforming Growth Factor-β1 Gene,mRNA, and Protein in Japanese Renal Transplant Recipients

Background

Transforming growth factor (TGF)-β1 may contribute to chronic allograft nephropathy and graft loss; however, the exact molecular mechanism remains unclear. Therefore, we assess the relationship between TGF-β1 gene polymorphisms, expression, and development of allograft nephropathy.

Methods

We studied 135 renal transplant recipients at our hospital. TGF-β1 gene polymorphisms (codons 10 and 25) were determined from peripheral blood leukocyte DNA. Plasma TGF-β1 mRNA was measured by real-time polymerase chain reaction and TGF-β1 protein levels were assessed by enzyme-linked immunosorbent assay. The relationship between TGF-β1 genotyping, expression, and rejection and results of renal biopsy were evaluated.

Results

The genotype frequency of transplant recipients was 49.6%, 30.4%, and 20.0% for C/T, C/C and T/T at codon 10, 100% for G/G at codon 25, respectively. According to the criteria of Banff ‘97 classification, 24 cases were classified as acute rejection and whose genotypes were 16, 3, and 5 cases for C/T, C/C and T/T at codon 10. Plasma mRNA expression was elevated in 14 cases and decreased in 8 cases after acute rejection. We measured 267 specimens of TGF-β1 protein and there was no relation between amount of TGF-β1 protein and mRNA.

Conclusion

Our results suggest that the relationship between plasma TGF-β1 expression and the development of allograft nephropathy remains uncertain. Frequency of allograft rejection differ with TGF-β1 codon 10 genotypes and the high-risk genotype was different from the reports of other countries.     

Locally increased concentrations of inflammatory cytokines in an experimental intraabdominal adhesion model

Background

Peritoneal adhesions may cause bowel obstruction, infertility, and pain. This study investigated cytokines, proteins and growth factors thought to promote formation of adhesions in an experimental intraabdominal adhesion model.

Methods

Male Sprague-Dawley rats were subjected to laparotomy, cecal abrasion, and construction of a small bowel anastomosis and examined at various time points after surgery. Concentrations of cytokines and growth factors in plasma and peritoneal fluid were analyzed using electrochemoluminescence and quantitative sandwich enzyme immunoassay technique.

Results

Concentrations of interleukin-6 (IL-6), interleukin-1beta (IL-1β), and tumor necrosis factor alpha (TNF-α) increased in peritoneal fluid from 6 h after incision. Plasma concentrations of IL-6 increased at 6 h, but plasma concentrations of IL-1β and TNF-α remained low. Peritoneal fluid concentrations of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor beta1 (TGF-β1), vascular endothelial growth factor (VEGF), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were below detection levels at all time points.

Conclusion

Early elevations of IL-6, IL-1β, and TNF-α concentrations in peritoneal fluid correlated to adhesion formation in this rodent model. Our model is relevant and reproducible, suitable for intervention, and indicates that antiadhesion strategies should be early, local and not systemic.     

Ischemic Reperfusion Injury–Induced Oxidative Stress and Pro-inflammatory Mediators in Liver Transplantation Recipients

Objective

Liver ischemic reperfusion injury is harmful to transplant recipients, and is associated with postoperative morbidity and mortality. Our study was designed to investigate the oxidative stress and pro-inflammatory mediators in liver transplant recipients.

Methods

We prospectively analyzed 14 recipients who underwent liver transplantation by measuring their blood levels of malondialdehyde (MDA) and cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, at nine time points perioperatively. We also evaluated the correlations between oxidative stress (MDA levels) and the characteristics of the recipient or the donated graft.

Results

These parameters significantly increased from 1 minute before reperfusion, and the values peaked within 3 to 30 minutes after reperfusion. On the time point at 5 minutes after reperfusion, the MDA levels which were the highest in the recipients correlated with the values of preoperative direct/and total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), model for end-stage liver disease (MELD) score, international normalized ratio (INR), and surgical blood loss.

Conclusion

The levels of MDA, TNF-α, IL-1β, and IL-6 greatly increased with the ischemic reperfusion insult. Recipients with higher values of preoperative direct/and total bilirubin, AST, ALT, MELD score, INR, and surgical blood loss tended to have higher levels of MDA and may suffer more injury from this insult.     

Serum levels of interleukin (IL)-10, IL-17, transforming growth factor (TGF)-β1, and interferon-γ cytokines and expression levels of IL-10 and TGF-β1 genes in renal allograft recipients after donor bone marrow cell infusion

Background

Cytokine storm generated by an alloimmune response after transplantation can lead to either graft survival or rejection. The aim of this study was to evaluate the serum levels of interleukin (IL)-10, IL-17, transforming growth factor (TGF)-β1, and interferon (IFN)-γ and expression levels of IL-10 and TGF-β1 in renal allograft recipients with or without donor bone marrow cell infusion (DBMI).

Methods

We retrospectively followed 28 living unrelated kidney recipients, including 14 with and 14 without DBMI infusion for 2 years. Also, 14 healthy subjects were included as a normal control group. PBMC gene expression analysis for mRNA levels of IL-10 and TGF-β1 cytokines relative to β-actin as a reference gene was performed using quantitative fluorescence real-time polymerase chain reaction at the end of 2 years posttransplantation. Also, serum levels of IL-10, TGF-β1, IFN-γ, and IL-17 in the 3 groups were measured by enzyme-linked immunosorbent assay at the same time.

Results

Both patient groups showed increased gene expression and serum content of IL-10 compared with normal controls. The expression levels were only significant between control patients and normal subjects (P = .02). Serum levels of IFN-γ and IL-17 were higher in untreated patients compared with normal controls (P = .03 and P = .07, respectively). DBMI patients showed significantly lower levels of serum TGF-β1 and IL-17 compared with normal subjects (P = .05 and P = .06, respectively). Also, infused patients showed a positive correlation between circulating levels of IL-17 and IL-10 (r = 0.692; P = .006), and an inverse correlation between serum creatinine and TGF-β1 levels (r = −0.580; P = .03).

Conclusion

The decreased levels of inflammatory cytokines besides IL-10 with increased TGF-β1 levels and better allograft function with improved clinical outcomes were observed among infused patients, possibly indicating immunomodulatory effects of this approach in kidney allograft patients.     

1,25(OH)2D3 Prolongs Islet Graft Survival by Inflammatory Inhibition

Objective

This study aimed to determine the protective effect of 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) against islet graft loss.

Methods

Proliferation of tumor necrosis factor (TNF)-α-induced macrophages was determined in vitro after treatment with different concentrations of 1,25-(OH)₂D₃. Intraportal islet transplantation (IPIT) was performed with islets harvested from the Sprague-Dawley rats and transplanted to the diabetic rats. The transplanted rats were assigned to receive 1,25-(OH)₂D₃ or propylene glycol (control). Islet graft survival; inflammatory cytokine (TNF-α and interleukin [IL]-1); numbers and percentages of macrophages, CD4⁺, and CD8⁺ T cells in bloods; and expression of nuclear factor (NF)-κB and TNF-α were analyzed. Hematoxylin and eosin staining was performed.

Results

We found 100 mg/mL 1,25-(OH)₂D₃ per day to have the strongest inhibitory effect on macrophages. Survival time of islet grafts significantly increased in the rats receiving 1,25-(OH)₂D₃. There were fewer infiltrated inflammatory cells in both islet graft and adjacent tissue in the drug-treated rats with lower serum IL-1 and TNF-α. Furthermore, percentage of macrophages and expression of p-NF-κB p65 and TNF-α in graft sites were significantly lower in the treated rats.

Conclusion

Our results demonstrated that 1,25(OH)₂D₃ prolongs islet graft survival by decreasing nonspecific inflammation in syngeneic IPIT through inhibiting TNF-α/NF-κB pathway and macrophage infiltration.     

TGF-β1 Inhibits Cx43 Expression and Formation of Functional Syncytia in Cultured Smooth Muscle Cells from Human Detrusor

Background

Human detrusor smooth muscle cells (hBSMCs) are coupled by connexin 43 (Cx43)–positive gap junctions to form functional syncytia. Gap junctional communication likely is necessary for synchronised detrusor contractions and is supposed to be altered in voiding disturbances. Other authors have shown that the pleiotropic cytokine TGF-β1 upregulates Cx43 expression in human aortic smooth muscle cells.

Objective

In this study, we examined the TGF-β1 effects on Cx43 expression in cultured hBSMCs.

Design, Setting, and Participants

hBSMC cultures, established from patients undergoing cystectomy, were treated with recombinant human TGF-β1.

Measurements

Cx43 expression was then examined by Western blotting, real-time PCR, and immunocytochemistry. Dye-injection experiments were used to study the size of functional syncytia.

Results and Limitations

Dye-coupling experiments revealed stable formation of functional syncytia in passaged cell cultures (P1–P4). Stimulation with TGF-β1 led to significant reduction of Cx43 immunoreactivity and coupling. Cx43 protein expression was significantly downregulated and Cx43 mRNA was only 30% of the control level. Interestingly, low phosphorylation species of Cx43 were particularly affected.

Conclusions

Our experiments demonstrated a significant down regulation of connexin 43 by TGF-β1 in cultured hBSMCs. These findings support the view that TGF-β1 is involved in the pathophysiology of urinary bladder dysfunction.     

Plasma and urinary levels of cytokines in patients with idiopathic hypercalciuria

Background

Recent studies suggest that cytokines modulate bone turnover. Idiopathic hypercalciuria (IH) seems to be associated with bone mineral loss. Therefore, the aim of this study was to assess cytokines involved in bone turnover in patients with IH.

Methods

Plasma and spot-urine levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor alpha (TNF-α), transforming growth factor β1 (TGF-β1), and monocyte chemoattractant protein (MCP-1) were measured in 70 children and adolescents with IH and in 37 healthy controls. Patients with IH were subdivided according to their calciuria at the time of sample collection: ≥4 mg/kg/day (persistent IH, n=27) and below 4 mg/kg/day (controlled IH, n=43). Cytokines were determined by enzyme-linked immunoassay.

Results

Plasma and urinary concentrations of IL-1β, IL-6, IL-8, and TNF-α were undetectable in all groups. No differences were found between controlled and persistent hypercalciuria for plasma and urinary levels of MCP-1 and TGF-β1. On the other hand, MCP-1 levels were significantly higher in both subgroups of IH in comparison to healthy controls. Furthermore, urinary MCP-1 levels of IH patients correlated positively with bone mineral content (p=0.013).

Conclusion

Although cytokine measurements did not allow the differentiation between persistent and controlled IH, our findings suggest that MCP-1 might play a role in patients with IH.

     

Anti–Human Leukocyte Antigen Antibodies Are Present in Blood of Blood Donors: Is Therapy With Blood Preparations Safe for Graft Recipients?

Background

Blood products infusions are often administrated to graft recipients. Post-transfusion reactions of anti–human leukocyte antigen antibodies (anti-HLA) are responsible for transfusion-related acute lung injury, but cases of graft rejection after blood product infusions were recently also proven.

Methods

The aim of this study was to assess, with the use of the very sensitive Luminex technology and traditional lymphocytotoxic test, the prevalence and cytotoxic activity of anti-HLA in blood donors with different medical histories to evaluate a potential risk of post-transfusion immune complications. Data were analyzed according to different normalized background cutoffs (1.5, 2.2; and the high cutoffs—10.8 for I class and 6.9 for II class anti-HLA).

Results

We observed that anti-HLA may be present in 36% of donors, and even in up to 73.6% of risk groups. Significant risk factors included female sex (23.9% to 64.2% for different cutoffs) and pregnancy history (30% to 72.5%), regardless of the cutoff used in analysis, whereas sera from female donors showed lower cytotoxicity (panel reactive antibodies). Anti-HLA were also detected in men (3.7% to 37%), in donors after a transfusion (0% to 62.5%), and even with no known risk factors (3.8% to 26.9%).

Conclusions

Luminex technology is a sensitive tool in anti-HLA detection, but consensus in measurement interpretation for blood donors is needed. Selection of blood products on the basis of medical history can be a useful alternative for routine testing of blood donors. The clinical significance of treatment of graft recipients with blood products requires further study; until then, more attention should be paid to possible complications.     

Evaluation of breviscapine on prevention of experimentally induced abdominal adhesions in rats

Background

Adhesion formation, which results from mechanical peritoneal damage, tissue ischemia, or the presence of foreign materials, is a complicated process. The formation of adhesions is associated with inflammatory response and extracellular matrix deposition in response to injury. Although the pathophysiology of adhesion formation is widely understood, an absolute solution to this problem does not exist yet. As a main component of Erigeron breviscapus, breviscapine has exhibited the ability of anti-inflammatory and antifibrosis on many diseases. The purpose of this study was to investigate the effect of breviscapine on the development of postoperative intra-abdominal adhesions in Wistar rats.

Methods

Abdominal adhesions were induced by scraping the cecum in rats. Various dosages of breviscapine drugs were administered for 10 days after surgery. On the 11th day after surgery, the levels of interleukin (IL) 18, IL-6, tumor necrosis factor α in blood serum and transforming growth factor β₁ (TGF-β₁), connective tissue growth factor, tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1) in peritoneal fluid were determined by enzyme linked immunosorbent assay. The expression of Smad7 and TGF-β₁ in rat cecum tissue was evaluated by Western blot analysis. Grades of intestinal adhesion were ranked by macroscopic observation.

Results

The intraperitoneal administration of breviscapine is effective on the prevention of the formation of postoperative adhesions in rats. Breviscapine decreased the levels of IL-18, IL-6, and tumor necrosis factor-α in blood serum and TGF-β₁, connective tissue growth factor, PAI-1 in peritoneal fluid. But the levels of tPA and the ratio of tPA and PAI-1 in peritoneal fluid were increased. In addition, breviscapine significantly inhibited the expression of TGF-β₁ and increased the level of Smad7 in the rat cecum tissue.

Conclusions

These results suggested that intraperitoneal administration of breviscapine was effective in preventing intra-abdominal adhesion formation in rats. Breviscapine appears to have synergetic effects which could decrease fibrosis by inhibiting inflammation, upregulating peritoneal fibrinolytic activity and regulating the TGF and/or Smad signaling pathway. These data indicated a potential new therapeutic use of breviscapine on adhesion prevention.     

Time course of pro- and anti-inflammatory cytokine levels in patients with burns—Prognostic value of interleukin-10

Introduction

Trends and the prognostic value of cytokine responses to severe burns have not been fully examined in humans. Therefore, the aim of this study was to determine the time course and prognostic value of pro- and anti-inflammatory cytokines in the immediate post-burn period.

Patients and methods

Blood samples were taken for measuring IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α concentrations from patients with more than 20% burned surface area on admission and on 5 consecutive days. Development of sepsis was assessed using standard criteria twice a day.

Results

IL-12p70 remained under assay detection levels in the study period. IL-1β and TNF-α could be detected in stimulated blood samples with higher levels in survivors (n = 21). IL-6 on days 4–5 and IL-8 on days 4–6 in non-stimulated plasma showed significant elevation in non-survivors (n = 18) whereas in stimulated blood its levels did not differ significantly. IL-10 levels were significantly higher in non-survivors during the study period in non-stimulated, and except day 6 in stimulated blood. Using the cut-off level of 14 pg ml⁻¹ for IL-10 predicted ICU mortality with 85.4% sensitivity and 84.2% specificity on admission.

Conclusion

Early anti-inflammatory excess had a bad prognosis for patients suffering from severe burns.     

Intestine as Source of Cytokines and Growth Factors

Background

The activation status of intestinal immune system cells is much higher than that of analogous peripheral cells. Increased serum concentrations of proinflammatory cytokines have been reported in various pathologic conditions; however, the source of these mediators has not been elucidated.

Objective

To assess the role of the human intestine and its lymphatic system in production of growth factors and proinflammatory cytokines.

Material and Methods

Twenty liver transplant recipients and 20 donors were included in the study. Blood samples were obtained from the artery supplying the intestine, the portal vein, and a peripheral vein during liver harvesting in donors and after transplantation in recipients. An enzyme-linked immunosorbent assay was used to assess serum concentrations of IL-6, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and hepatocyte growth factor (HGF).

Results

In transplant recipients, IL-6 concentration in arterial blood was lower than that in portal blood (P < .049), whereas in donors, there was no significant difference in these concentrations. Neither recipients nor donors demonstrated significant differences in arterial or portal blood concentrations of TNF-α, TGF-β, or HGF.

Conclusions

In healthy human beings, the intestine is not a substantial source of IL-6, TNF-α, TGF-β, or HGF. However, in patients with liver cirrhosis, the intestine is an important source of IL-6 but not of the other studied growth factors and cytokines.     

Aging aggravates long-term renal ischemia-reperfusion injury in a rat model

Aim

Ischemia-reperfusion injury (IRI) has been considered as the major cause of acute kidney injury and can result in poor long-term graft function. Functional recovery after IRI is impaired in the elderly. In the present study, we aimed to compare kidney morphology, function, oxidative stress, inflammation, and development of renal fibrosis in young and aged rats after renal IRI.

Materials and methods

Rat models of warm renal IRI were established by clamping left pedicles for 45 min after right nephrectomy, then the clamp was removed, and kidneys were reperfused for up to 12 wk. Biochemical and histologic renal damage were assessed at 12 wk after reperfusion. The immunohistochemical staining of monocyte macrophage antigen-1 (ED-1) and transforming growth factor beta 1 (TGF-β1) and messenger RNA level of TGF-β1 in the kidney were analyzed.

Results

Renal IRI caused significant increases of malondialdehyde and 8-hydroxydeoxyguanosine levels and a decrease of superoxide dismutase activity in young and aged IRI rats; however, these changes were more obvious in the aged rats. IRI resulted in severe inflammation and tubulointerstitial fibrosis with decreased creatinine (Cr) clearance and increased histologic damage in aged rats compared with young rats. Moreover, we measured the ratio of Cr clearance between young and aged IRI rats. It demonstrated that aged IRI rats did have poor Cr clearance compared with the young IRI rats. ED-1 and TGF-β1 expression levels in the kidney were significantly higher in aged rats than in young rats after IRI.

Conclusion

Aged rats are more susceptible to IRI-induced renal failure, which may associate with the increased oxidative stress, increased histologic damage, and increased inflammation and tubulointerstitial fibrosis. Targeting oxidative stress and inflammatory response should improve the kidney recovery after IRI.     

Chronic Immune-Mediated Reaction Syndrome as the Cause of Late Graft Mortality in Living-Donor Liver Transplantation for Primary Biliary Cirrhosis

Introduction

Few studies to date have investigated the causes of late graft mortality after living-donor liver transplantation (LDLT) for primary biliary cirrhosis (PBC).

Patients and Methods

Fifty-five LDLTs for PBC were retrospectively reviewed. Factors prognostic of graft survival after LDLT were investigated, and histologic findings in patients with late graft loss were assessed.

Results

The 1-, 5-, and 10-year cumulative graft survival rates were 85.1%, 82.5%, and 66.9%, respectively. Multivariate Cox regression analysis found that male donor and ≥4 HLA mismatches were independently associated with poor graft survival. Among the 13 grafts lost, 5 were lost >1 year after LDLT, including 1 each due to chronic rejection, veno-occlusive disease, and obliterative portal venopathy, and 2 to other causes. Pathologic reviews of the serial biopsy specimens and explanted grafts from these 5 patients, with graft rejections from “chronic immune-mediated reaction syndrome,” showed reciprocal changes over time. No patient died of recurrent PBC.

Conclusions

Male donor and ≥4 HLA mismatches were independent factors associated with poor graft survival. Late graft mortality after LDLT for PBC in some patients was due to chronic immune-mediated reaction syndrome, including chronic rejection, veno-occlusive disease, and obliterative portal venopathy, but not to recurrent PBC.