Urinary Thromboxane B2 in Cardiac Transplant Patients as a Screening Method of Rejection

Noninvasive methods for regular monitoring of cardiac transplant patients for acute rejection are preferable to the only currently accepted method involving frequent endomyocardial biopsies. Thromboxane A₂ (TXA₂) is synthesized in large amounts by monocytes/macrophages during organ graft rejection. It enhances T-lymphocyte clonal expansion and cytotoxic function as well as upregulating the major histocompatibility class II expression on antigen presenting cells. Experimentally increased urinary excretion of TXA₂ metabolites is associated with cardiac transplant rejection. We therefore compared urinary immunoreactive thromboxane B₂ (i-TXB₂) levels to the rejection score of the endomyocardial biopsies. In addition we graded the degree of activated lymphocytes in peripheral blood. Urinary i-TXB₂ was significantly higher in patients exhibiting medium to severe rejection than in patients without rejection (1236 ± 372 vs. 526 ± 57 pg/mL). The urine i-TXB₂ (704 ± 48 pg/mL) of all patients who participated in this study, whose endomyocardial biopsy indicated rejection, was also significantly higher than in the non-rejecting group. Increased levels of urine i-TXB₂ were associated with increased biopsy scores. Circulating activated lymphocytes was also significantly increased in patients with moderate/severe rejection compared to patients with no rejection (66 ± 11 vs. 39 ± 4 per mm (3)) (p < 0.01). Further, this study shows that urine i-TXB₂ is associated with increased endomyocardial biopsy scores (acute rejection scores) and blood lymphocyte activation. Thus we conclude that urine i-TXB₂ may be of potential value as a diagnostic screening test for helping identify cardiac transplant patients undergoing acute rejection.     

Clinical Evaluation of a Rosette Inhibition Test in Renal Allotransplantation

The formation of spontaneous rosettes by peripheral blood or spleen mononuclear cells when incubated with sheep red blood cells has proved a useful way of assessing the potency of immunosuppressive drugs and antilymphocyte sera in vitro. A test employing the inhibition by antilymphocyte globulin (A.L.G.) of spontaneous rosette formation around peripheral blood mononuclear cells is described. This has been used to assess the degree of immunosuppression in patients with renal allografts and uraemic patients on regular haemodialysis.Twenty-three patients with renal allografts had 21 clinically diagnosed episodes of rejection. In none of these rejection episodes was the minimal inhibitory concentration (M.I.C.) of A.L.G. (that necessary to reduce the spontaneous rosette formation of peripheral cells by 75%) less than 1/50,000. Nineteen patients had no rejection episodes during 57 patient/months of continuous observation while the M.I.C. was at a greater dilution than 1/50,000. The test has therefore been of great value in suggesting when an individual is capable of rejecting his graft, and allows the dose of immunosuppressive drugs to be adjusted to a minimum in a controlled fashion. It has been of use in diagnosing rejection in the anuric patient, when the distinction between rejection, urinary tract obstruction, and infection is particularly difficult.Fifteen patients maintained on regular haemodialysis for more than a year had, as judged by this technique, less reactive lymphocytes than normal healthy controls. The degree of immunosuppression was not as great as in the patients on full immunosuppressive regimens.     

Serious renal transplant rejection and adrenal hypofunction after gradual withdrawal of prednisolone two years after transplantation.

Ten patients with stable renal function two years after transplantation had their sole immunosuppressive treatment (oral prednisolone 10 mg daily) withdrawn by reducing the daily dose by 1 mg at monthly intervals. Plasma prednisolone concentration, cortisol concentration, creatinine clearance, and serum creatinine concentration were measured in all patients, and the adrenal response to corticotrophin was determined in five by measuring plasma cortisol concentrations before and after tetracosactrin injection. No episodes of rejection occurred in patients taking over 7 mg prednisolone daily. Although three patients apparently required only minimal immunosuppressive treatment (less than 5 mg daily) the remainder suffered episodes of rejection at daily doses below 7 mg. There was a tenuous association between rejection and low plasma cortisol concentration, but neither the pattern of plasma prednisolone concentrations nor the response to tetracosactrin were related to episodes of rejection. Reducing the daily dose of oral prednisolone to under 7 mg should not be attempted in patients with renal transplants unless there are extenuating circumstances.     

Pre-Transplant Donor-Specific T-Cell Alloreactivity Is Strongly Associated with Early Acute Cellular Rejection in Kidney Transplant Recipients Not Receiving T-Cell Depleting Induction Therapy

Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90), to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67). We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction therapy.     

Cytometric bead array (CBA) for the measurement of cytokines in urine and plasma of patients undergoing renal rejection

Renal rejection is associated with an active immune response regulated by cytokines and in which immunocompetent cells are involved. Previous studies have measured high levels of cytokines in the urine and plasma in various renal dysfunction states. However, some methods used to measured cytokines hinder their use as a diagnostic tool in renal rejection. In this report, cytokine levels were determined in the plasma and urine of kidney transplant patients, with renal rejection and without it, using a cytometric bead array (CBA) technique. Concentrations of six human cytokines (IL-2, IL-4, IL-5, IL-10, TNF-alpha and INF-gamma) were established. Results show that patients who develop renal rejection presented high levels of IL-10 and IFN-gamma cytokines in plasma and urine compared to patients without renal rejection. The CBA technique displayed greater sensitivity in the determination of cytokines in urine than the conventional ELISA technique. Finally, when standard cytokines in plasma and in urine were compared, it was observed that, in plasma, levels of IL-4, IL-5, IL-10, TNF-alpha and IFN-gamma were detected, whereas in urine the levels detected were of IL-4, IL-5, IL-10 and IFN-gamma. These results indicate that the CBA assay is a sensitive method to measure cytokines in urine. In kidney transplant patients undergoing acute renal rejection, the presence of cytokines in urine reflects renal damage and could be a useful method in the diagnosis of renal rejection.     

Leucocytosis is not a manifestation of rejection.

The leucocyte response to allograft rejection was retrospectively analysed in 80 rejection episodes that occurred in 50 patients. There was no significant change in leucocyte count in 32 rejection episodes. In 27 there was a fall in leucocyte count of more than 20% and in only 21 was there a rise in count of more than 20%. Thus leucocytosis seems not to be a manifestation of rejection; indeed, it is the least common response, a fall in the count being commoner. The results also suggest that the prognosis for the graft is poorer when the leucocyte count falls significantly.     

Viral Infection and Renal Transplant Rejection

The occurrence of an outbreak of influenza in a renal transplant unit is described. Five patients had a proved episode of infection, confirmed by a rise in the complement fixation titre to influenza virus A, and this coincided in three of the patients with episodes of acute rejection. It seems likely that the virus infection was responsible for the rejection, possibly through a stimulating effect of the virus on the host''s immune response.     

Association of genetic variation in co-stimulatory molecule genes with outcome of liver transplant in Iranian patients

CD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS) and programmed cell death 1 are closely-linked genes located on chromosome 2q and encode co-stimulatory molecules, which are T-cell activity regulators. The principal assignment of T-cell mediated immune response in allograft rejection is an interesting topic of multiple studies. Although the variation in these genes may influence the graft survival and the amount of immunosuppression needed, the studies so far have been restricted solely to the CTLA4 gene. In 145 patients who underwent liver allograft transplantation, 10 single nucleotide polymorphisms of CD28, CTLA4, ICOS, and PD.1 genes were defined. To distinguish the polymorphisms of all 10 SNPs, PCR-RFLP method was used and according to the standard criteria, acute rejection episodes were determined. CTLA4-1661, AA genotype was significantly more frequent in the patients with acute rejection and AG genotype was significantly more frequent in the patients without rejection. Frequencies of CTLA4+49 AG A allele and CTLA4-1661AG A allele were significantly higher than those of CTLA4+49 AG and CTLA4-1661AG, G allele in the patients with acute rejection. ICOS+693, GG genotype and G allele were significantly less frequent in the patients with acute rejection and CD28 CT genotype was significantly more in patients with acute rejection. The present results demonstrate that potentially functional genetic variation in T-cell co-stimulatory molecules including ICOS, CTLA4 and CD28 can influence liver transplant outcome.     

Evaluation of Rosette Inhibition Test in Renal Transplantation

Serial rosette inhibition tests were performed on 11 renal transplant patients in an attempt to predict graft rejection. The rosette inhibition titre was higher in immunosuppressed patients than in normal subjects. The test was of predictive value in only two out of 12 rejection episodes, where a fall in titre to normal levels occurred 48 hours and 24 hours, respectively, before biochemical evidence of rejection. In two further rejection episodes the titre fell at the time of rejection. The titre changes in all tests were small and there were frequent inconsistencies in the results of individual tests. A study of the variables was undertaken, with standardization of the technique, and improvements were made in reading the test. Despite these changes the test was still not sufficiently accurate or reliable to be used as the basis of treatment of rejection episodes.     

Participation of coenzyme Q10 in the rejection development of the transplanted heart: a clinical study.

Coenzyme Q10 and alpha-tocopherol concentrations were assessed in 28 endomyocardial biopsies from 22 patients and in 61 blood samples from 31 patients after heart transplantation with histologically confirmed signs of rejection. The values were compared to the group of 14 patients with cardiomyopathies of unclear etiology as candidates for heart transplantation. Blood analyses were also compared with 50 healthy persons. Myocardial and blood coenzyme Q10 concentrations were already significantly decreased in the incipient phase of rejection (degree 0-1) and also in rejection phase 1 and 2. In patients without rejection signs myocardial and blood coenzyme Q10 values were similar to those of cardiomyopathic patients. No significant differences were found in alpha-tocopherol concentrations in relation to signs of rejection. Increased plasma lipid peroxidation quantified as malondialdehyde production was detected in all groups of transplanted patients. The results contribute to the explanation of some pathobiochemical mechanisms participating in the rejection development of the transplanted heart.     

Partial suppression of cell mediated immunity in chromoblastomycosis

The cellular immune response of 8 patients from the Brazilian Amazon region with chromoblastomycosis was analyzed. Primary immunological responses of patients were tested by contact sensitization to 2,4-dinitro-chlorobenzene (DNCB), or rejection of first set skin allografts. 2 of 8 patients were reactive to DNCB after sensitization, and skin allograft rejection occured in an average of 14 days. Capacity of patients to mount recall immunological responses was measured by skin testing with two fungal antigens and three bacterial antigens. Delayed skin reaction to trichophytin and Candida antigens was negative in the majority of the patients. However, reactivity to mycobacterial (tuberculin), and bacterial (staphylococcal, streptococcal) antigen was high, or only slightly diminished respectively. The data suggest that patients with chromoblastomycosis have suppressed nonspecific, cell mediated immunity for some antigens (skin allografts, DNCB, fungal antigens), while reactivity to bacterial and mycobacterial antigens is not impaired.     

Increased Numbers of Circulating CD8 Effector Memory T Cells before Transplantation Enhance the Risk of Acute Rejection in Lung Transplant Recipients

The effector and regulatory T cell subpopulations involved in the development of acute rejection episodes in lung transplantation remain to be elucidated. Twenty-seven lung transplant candidates were prospectively monitored before transplantation and within the first year post-transplantation. Regulatory, Th17, memory and naïve T cells were measured in peripheral blood of lung transplant recipients by flow cytometry. No association of acute rejection with number of peripheral regulatory T cells and Th17 cells was found. However, effector memory subsets in acute rejection patients were increased during the first two months post-transplant. Interestingly, patients waiting for lung transplant with levels of CD8⁺ effector memory T cells over 185 cells/mm³ had a significant increased risk of rejection [OR: 5.62 (95% CI: 1.08-29.37), p=0.04]. In multivariate analysis adjusted for age and gender the odds ratio for rejection was: OR: 5.89 (95% CI: 1.08-32.24), p=0.04. These data suggest a correlation between acute rejection and effector memory T cells in lung transplant recipients. The measurement of peripheral blood CD8⁺ effector memory T cells prior to lung transplant may define patients at high risk of acute lung rejection.     

Liver Transplantation in Man—III,Studies of Liver Function,Histology, and Immunosuppressive Therapy

The experience gained from 13 hepatic transplant operations is described, with particular reference to the findings in nine patients who survived the immediate operative period. A major problem was found to be infection. Fulminant pneumonia caused death in two adults, at a time when liver function was virtually normal. Infection related to bile fistula and sepsis may be overcome by an improved method of biliary drainage by cholecyst-dochostomy, which was carried out in the last two patients. Jaundice in the second week due to rejection was observed in several patients. The striking histological change was centrilobular cholestasis. The jaundice, which was not prevented by administration of antilymphocyte globulin, was rapidly controlled by temporarily increasing die dose of prednisone. One patient who survived for four and a half months and who had a poor tissue match subsequently developed chronic rejection with progressive cholestatic jaundice. Five of the patients were able to go home and at time of publication two are alive and well 14 and 20 weeks after treatment.     

Urinary excretion of factor VIII after renal transplantation.

The urinary excretion of factor-VIII-related antigen (VIIIRAg) was measured in 72 patients with kidney transplants and compared with that of two end-products of fibrin-fibrinogen lysis (fragments D and E) to assess their usefulness in monitoring the onset of rejection episodes. Specific and sensitive radioimmunoassays were used to measure the three proteins. Unconcentrated urine samples of 24-hour collections were obtained from 20 healthy subjects, 48 patients with stable transplants, and 24 patients with recent transplants serially followed up from the day of transplantation. Factor VIIIRAg and fragments E and D were not detectable in the urine from healthy subjects but were present in 39%, 60%, and 100% respectively of samples from patients with stable transplants. During 33 acute rejection episodes in 19 patients with recent transplants factor VIIIRAg and fragments E and D were significantly increased above the values observed in patients with stable transplants in 82%, 73%, and 64% of samples respectively; in patients with recent transplants showing no clinical sign of rejection increased excretion of these proteins was observed in 11%, 26%, and 22% of samples respectively. The presence of factor VIIIRAg in urine from patients with kidney allografts suggests that endothelial cell-factor VIII-platelet interactions might pay a key part in the pathogenesis of acute rejection. The results suggest that the assay of factor VIIIRAg in urine is more useful than assays of fragments D and E as a corroborative index of transplant rejection.     

Bleeding in renal failure: a possible cause.

Increased concentrations of factor VIII-related antigen (VIIIRA), factor VIII-procoagulant activity (VIIC), and decreased factor VII-von Willebrand activity (VIIIVWF) were found in the plasma of patients with chronic renal failure (CRF). This functional abnormality of the factor VII protein may partly explain the prolonged bleeding time commonly found in CRF. It was not improved by dialysis, but it was no longer found in patients with normally functioning grafted kidneys after the sixth month after transplantation. VIIIVWF levels remained decreased when compared with VIIIRA or VIIIC in transplanted patients undergoing acute reversible rejection soon after transplantation. Yet, not only VIIIC and VIIIRA but also VIIIVWF were greatly increased in patients with hyperacute irreversible rejection. Possibly a high VIIIVWF level in these patients is a thrombogenic factor.     

Influence of glutathione<Emphasis Type="Italic"> S</Emphasis>-transferase M1 and T1 polymorphisms with acute rejection in Iranian liver transplant recipients

Glutathione-S-transferase (GST) has a major protective role against free radicals and plays a vital role in phase II of biotransformation of many substances. In liver transplantation, reperfusion injury, calcineurin drug consumption and infection produce free radicals that cause tissue injury and organ damage. Genetic variations of GST may influence individual susceptibility to some diseases associated with the deleterious effects of oxidative metabolism. Although it is well known that the rejection is an immunological process, however, in this study, we have investigated the gene frequency and relationship between human GST gene polymorphism and rejection in liver transplant recipients. We have assessed 51 liver transplant recipients from Shiraz, South of Iran. The GSTT1 and GSTM1 genotypes were identified by polymerase chain reaction (PCR). The gene frequency of GSTM1 and GSTT1 polymorphism were evaluated. We observed that GSTM1 null genotype was present in 68.62% of the liver transplant recipients while GSTT1 null genotype was present in 37.25% of the liver transplant subjects. There was a trend between increasing age and acute rejection episode. No statistically significant correlation was present between GSTM1 null and GSTT1 null genotypes with an acute rejection episode in transplant recipients. No relationship was observed between GST genotypes and acute rejection. It is likely that development and progression of rejection are determined by genes which is involved in immunological pathways rather than genes that is participated in free radicals destruction. However, these findings need to be confirmed in a larger series of patients.     

Kidney graft rejection studies with labeled platelets and lymphocytes

The usefulness of In-111-labelled platelets and lymphocyte scintigraphy in acute kidney graft rejection is evaluated.One hundred fifty-five patients (36 treated with cyclosporine A) were studied with labelled platelets and 27 with labelled lymphocytes.Blood cells were labelled with 100–150 uCi of In-111-oxine and reinjected. Subsequently patients were scanned once daily from 2 hours post-reinjection up to a week. The graft / contralateral area activity ratio was calculated in all scans (Index I).Four groups of patients were established: Functioning grafts (FG); post-operative acute renal failure (p-ARF);acute rejection (AR) and nephrotoxicity (NTX), the last one only in patients under cyclosporine therapy.Results with labelled platelets showed similar index I mean values in FG, p-ARF and NTX patients I = 1.1 ± 0.1 and a significant increase (p <O.001), in acutely rejecting grafts I = 1.9 ± 0.4.Evolving controls showed a decrease of graft activity parallel to rejection resolution while the activity maintains or increases in patients with less or no response to treatment.Overall sensitivity was 97.2 %, specificity 90.2 % and accuracy 92.8 %.Results with labelled lymphocytes were similar to those with platelets. They showed a significant (p 0.001) difference of activity index between rejecting (I = 1.86 ± 0.3) and non rejecting grafts (I = 1.05 ± 0.1) Decrease of graft activity was only seen in patients with good response to treatment.It is concluded that In-111-labelled platelets scintigraphy is nowadys the method of choice for acute kidney graft rejection diagnosis, especially in patients under cyclosporine immunosuppression.     

Analysis of CC chemokine receptor 5 and 2 polymorphisms and renal transplant survival

Chronic rejection is an immune process leading to graft failure. By regulating the trafficking of leukocytes, chemokines and chemokine receptors are thought to be one of the reasons causing acute renal rejection (ARE), which increases the possibility of chronic rejection and organ destruction. This study was designed to investigate, in the Turkish population, an association of chemokine receptor genetic variants, CCR2V641, CCR5-59029-A/G, CCR5-Delta32 and acute renal rejection after renal transplant surgery. We carried out our study in 85 Turkish renal transplant patients (45 men, 40 women; mean age 39 +/- 2 years) by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. We found no significant difference in the incidence of rejection among patients possessing or lacking CCR5-Delta32. For the groups with and without acute renal rejection, we found a significant difference between the groups in A and G allele distribution in both CCR2V641and CCR559029 gene variants (p = 0.003 and p = 0.003, respectively). According to our findings, the risk of acute rejection in renal transplantation may be associated with genetic variation in the chemokine receptor genes CCR5-59029 and CCR2V641 in Turkey, and studies on these gene polymorphisms could be an ideal target for future interventions intended to prevent renal transplant loss.     

组织相容性Ⅰ类相关链A位点基因多态性在预判肾移植物排斥中的意义

目的 研究组织相容性Ⅰ类相关链A位点(MICA)基因多态性和抗MICA特异性抗体在肾移植排斥反应发生中的意义.方法 采用免疫磁珠液相芯片技术对40例肾移植患者在移植术前和移植术后1个月、3个月、6个月、1年和2年动态检测抗MICA抗体的特异性和阳性分值的变化,同时采用SSOP方法分析16对肾移植供受者的MICA基因分型...     

Elevated urinary sVCAM-1, IL6, sIL6R and TNFR1 concentrations indicate acute kidney transplant rejection in the first 2 weeks after transplantation

We tested the hypothesis that increased urinary cytokine concentrations may indicate an acute kidney transplant rejection. Eight patients with an early rejection in their protocol biopsy about 14days after transplantation (group A), 9 patients with a biopsy proven rejection 2-3months after transplantation (group B) and 18 patients without acute rejection in their protocol biopsies both at 14days and 3months (group C, represents the control group) were chosen for this study. At the time of biopsy, the mean urinary concentration of interleukin 6 (IL6), soluble IL6 receptor (sIL6R), tumor necrosis factor receptor 1 (TNFR1), and soluble vascular cell adhesion molecule -1 (sVCAM-1) were significantly higher in patients with an early acute transplant rejection, i.e. in group A compared to patients in the control group (p<0.01). Additionally we found already 14days after transplantation significantly higher concentrations of urinary sIL6R and sVCAM-1 in group B patients who suffered of late acute rejection compared to patients with no acute rejection (group C, p<0.05). No significant correlation could be shown for interleukin 1 receptor antagonist (IL1ra), TNF, and TNFR2. In conclusion, elevated urinary concentrations of IL6, sIL6R, TNFR1 and sVCAM-1 clearly indicate an early acute transplant rejection. Especially sVCAM-1 may also serve as an early marker of an upcoming late rejection. However, further studies are warranted to verify the value of individual cytokine profiles to predict acute rejection episodes.