Search for genomic sequences of microbial agents in atherosclerotic plaques.

Atherosclerosis is a complex, multifactorial disease. Several studies have reported a possible association between infection with microbial agents and atherogenesis. Chlamydia pneumoniae (C. pneumoniae), Herpes Simplex Virus 1 (HSV1), Human Cytomegalovirus (HCMV), and Epstein Barr Virus (EBV) have been widely investigated for their possible role in atherosclerosis development, but the results obtained to date are contradictory. The aim of our study is to search DNA of the aforementioned infectious agents by means of Quantitative Real Time PCR in atherosclerotic plaques from carotid arteries obtained from 17 patients. Genomic sequences of C. pneumoniae, HSV1, HCMV were not found in any atherosclerotic lesion. Therefore, our results do not support the hypothesis of an association between these infectious agents and atherosclerosis. Conversely, three patients were found to be positive for EBV DNA, thus indicating that, at least in a limited number of patients, EBV could play a role in atherogenesis.

Pretransplant Single Antigen Bead-Detected HLA Antibodies in Kidney Transplant Long-term Outcome: A Single-Center Cohort Experience.

Single-antigen bead (SAB) platform permits the identification of antibodies not detectable by complement-dependent lymphocytotoxicity test, but their clinical significance is not completely understood. The aim of this study was to evaluate whether the presence of pretransplant SAB-detected antibodies is associated with the development of allograft failure. This is a single-center cohort study with 10-year follow-up in which 573 kidney recipients with negative pretransplant complement-dependent lymphocytotoxicity crossmatch who received transplants at the Kidney Transplant Center of Policlinico, Milan, from deceased donors between 1996 and 2005 were evaluated. Pretransplant plasma samples were retrospectively analyzed by SAB assay. Survival analyses were performed to assess the risk of allograft failures by SAB-detected antibodies. Pretransplant antibodies were found in 160 (28.0%) recipients, of whom 42 subsequently developed an allograft failure for a survival rate of 70.9% (95% confidence interval [CI), 63.5-78.4). Among those without antibodies, 58 (14.0%) returned to dialysis with a survival rate of 84.7% (95% CI, 81.0-88.4). In Cox regression analyses, patients with SAB-positivity had 2-fold higher risk of allograft failure than those who were SAB-negative (hazard ratio, 2.07; 95% CI, 1.39-2.79). Results did not change after adjustment for putative confounders. In conclusion, in this single-center cohort, 10-year allograft survival rate was significantly influenced by the presence of SAB-detected antibodies.

Pediatric kidney transplantation: a snapshot 10 years later.

Herein we report the outcomes of pediatric kidney recipients who underwent transplantation at least 10 years prior. A cohort of 36 patients (mean age, 26.4+/-6 years) with a mean follow-up time of 14.2+/-4 years was selected for the study. Immunosuppression consisted of cyclosporine and steroids. Actuarial patient and graft survivals 15 years after the transplantation were 97% and 86%, respectively. Only 1 patient died due to a complicated sclerosant peritonitis. Graft function was good with a mean serum creatinine of this selected cohort of 1.5+/-0.6 mg/dL. Eighteen percent were class 1, 33% class 2, and 49% chronic kidney disease. Hypertension was treated in almost 80% of the patients. The majority of patients were smaller than the average population with a final height (between 0 and -2) standard deviation score (HSDS) but only 27% had a severe growth impairment (HSDS>-2). Regarding nutritional status, fewer than 30% were overweight and only 1 patient was obese with a body mass index (BMI) >30. The majority of patients, except 2 mentally retarded individuals, are or have been attending normal school and achieved full-time employment. In conclusion, long-term survivors of a kidney transplant received during childhood reached a high degree of rehabilitation despite a long period of immunosuppression.

Intra-operative Postperfusion Micronephrolithotomy for Renal Allograft Lithiasis: A Case Report.

Increasing demand drives the expansion of criteria for kidney donation, and nephrolithiasis is now considered a relative contraindication. We report for the first time a case of intra-operative, postperfusion kidney allograft micronephrolithotomy. A 64-year-old man with end-stage renal disease secondary to Alport syndrome underwent primary deceased donor kidney transplantation at our center. Pre-operative ultrasound of the donor identified a 7-mm calculus in the anterior, lower pole calyx. The kidney was extra-peritoneally implanted in the right iliac fossa and reperfused homogenously. Stone retrieval with a flexible ureteroscope failed due to the narrow calyceal infundibulum. Instead, the calculus was removed using the micropercutaneous nephrolithotomy system under ultrasonographic guidance. The calyx was punctured using a 4.85 Fr needle and the stone was fragmented to dust using a Holmium laser. No bleeding was observed. The post-operative course was uneventful. Outpatient follow up demonstrated good function of the graft which was stone free on ultrasound. Postperfusion micropercutaneous nephrolithotomy for kidney allograft calculi offers a safe and feasible option when pre-operative or intra-operative retrograde intrarenal surgery fails.

Enzyme-Linked Immunospot Assay as a Complementary Method to Assess and Monitor Cytomegalovirus Infection in Kidney Transplant Recipients on Pre-emptive Antiviral Therapy: A Single-Center Experience.

BACKGROUND: Cytomegalovirus (CMV) disease represents a major cause of post-transplantation morbidity and mortality. To estimate the risk of infection and monitor response to antiviral therapy, current guidelines suggest combination of viral load monitoring with direct assessment of CMV-specific immune response. We used enzyme-linked immunospot (ELISpot) for the evaluation of CMV-specific T-cell response in kidney transplant recipients with CMV viremia and investigated how information gained could help manage CMV infection. METHODS: Seventeen patients on pre-emptive antiviral therapy and CMV quantitative polymerase chain reaction (qPCR) ≥500 copies/mL (first episode after transplantation) were assessed using ELISpot and divided into Weak (9 patients with baseline ELISpot <25 spot-forming colonies [SFCs]/200,000 peripheral blood mononuclear cells [PBMCs]) and Strong Responders (8 patients with baseline ELISpot ≥25 SFCs/200,000 PBMCs). CMV-specific T-cell response, infection severity, viral load, and antiviral therapy were prospectively recorded and compared between groups at 1, 2, and 24 months of follow-up. RESULTS: Demographic and transplant characteristics of Weak and Strong Responders were similar. No episodes of CMV disease were observed. Weak Responders were more likely to experience CMV syndrome (56% vs 36.5%) and late virus reactivation (56% vs 25%) than Strong Responders. Weak Responders showed higher baseline median viral loads (19,700 vs 9265 copies/mL) and needed antiviral therapy for longer (179 vs 59.5 days). T-cell response showed 2 main patterns: early and delayed. CONCLUSIONS: ELISpot provides prognostic information about infection severity, risk of late reactivation, and response to therapy. Randomized trials, evaluating the need for antiviral therapy in kidney transplant recipients with asymptomatic infection and effective virus-specific T-cell immune response, are warranted.

Nosocomial infection in kidney transplant recipients: a retrospective analysis of a single-center experience.

Posttransplant bacterial infections are important because of their influence on patient and graft outcomes. Therefore, prevention of infection as well as prompt diagnosis and appropriate treatment are crucial. In this retrospective analysis, we reviewed all posttransplant bacterial infections occurring during the admission of kidney transplant patients from January 2000 to May 2004. Of our patients, 25% had at least one episode of infection. Patients with immunosuppression based on an mTOR inhibitor showed the highest rate of wound infections compared to those receiving a calcineurin inhibitor (odds ratio 5.6, P < .001). Patients with renal failure caused by a urologic disease revealed a increased risk of a urinary tract infections (odds ratio 5.9, P < .001). Although infection complications are an important cause of morbidity in renal transplantation, the extensive use of antibiotics should be avoided in favor of a strict policy for infection prevention and control.

Value of intraoperative resistive index in kidney transplant.

The value of the resistive index (RI) obtained by echo color doppler evaluation of the transplanted kidney is still not well established. Many authors consider the RI to be nonspecific sign of rejection, acute tubular necrosis, or urinary tract obstruction, but its specificity remains low. In this paper, we report our experience with RI determinations in 34 consecutive kidney transplants at different times namely: perioperatively, at 24 hours, at 3 days, at 6 and at 9 days posttransplant. In all patients intraoperative RI was normal. RI increased significantly after transplantation in 10 patients who eventually developed a complication: delayed function, acute rejection, and spontaneous kidney ruptures. This increment from the baseline value was already significant at 24 hours after the kidney transplant, indicating a possible posttransplant complication (0.62 +/- 0.07 vs 0.76 +/- 0.04; P = .0004). We conclude that the value of RI in the early posttransplant phase should be considered an important aid for the early diagnosis of posttransplant complications.

Mycophenolate mofetil pharmacokinetic monitoring in pediatric kidney transplant recipients.

This open-label, longitudinal, long-term study of de novo pediatric renal transplant recipients was designed to investigate the pharmacokinetics (PK) of mycophenolic acid (MPA) and its possible interaction with cyclosporine (CsA). Thirty-four children on an immunosuppressive regimen of CsA, prednisone, and mycophenolate mofetil (MMF, 300-400 mg/m2 twice daily) were investigated at 6, 30, 180, and 360 days after transplantation. Considerable interindividual variability in the areas under the concentration curve (AUC(0-12)) of MPA was observed during the follow-up, although the dose of MMF remained the same over the same time. Predose levels (C0) increased significantly during the first 6 months after transplantation: C0 at 6 and 180 days after transplantation was 0.8 +/- 0.6 and 1.9 +/- 1.1 microg/mL (P < .0001). A significant time-dependent increase in the AUC of MPA was also observed during the first 6 posttransplant months: AUC(0-12) at 6 and 180 days after transplantation was 23.3 +/- 10.8 and 40 +/- 11.6 mg*h/L (P = .003). MPA concentrations 3 and 4 hours after MMF intake were the individual time points that best correlated with the full MPA AUC (r = 0.8 and 0.79; P < .001). The abbreviated MPA AUC (0-4 hours) correlated reasonably with the full AUC (r = 0.87; P < .001). Finally, a significant reduction in CsA dose during the first 6 posttransplant months (P < .001) matched the significant increases in both MPA C0 and full MPA AUC, thus demonstrating the interaction of the 2 immunosuppressive drugs. These observations suggest the need for therapeutic drug monitoring when adjusting the dose of MMF in children.

The mechanism of unresponsiveness to allografts induced by rapamycin and rapamycin/cyclosporine treatment in rats.

The mechanisms by which rapamycin (RAPA) and/or cyclosporine induce unresponsiveness to allografts were investigated in a rat model. Buffalo (BUF, RT-1b) heart allografts were rejected by Wistar-Furth (WFu, RT-1u) recipients at a mean survival time (MST) of 6.5 +/- 0.5 days. A 14-day course of RAPA (0.8 mg/kg) delivered intravenously by an osmotic pump prolonged BUF allograft survival to 76.1 +/- 23.4 days (P < 0.001). Adoptive transfer of 30-50 x 10(6) spleen and lymph node T cells that had been isolated on day 40 postgrafting from CsA- or RAPA/CsA-treated hosts into lightly irradiated (6 Gray) secondary WFu recipients prolonged BUF graft survival from 9.8 +/- 1.2 to 29.2 +/- 11.0 (P < 0.01) and 58.2 +/- 38.9 days (P < 0.004), respectively. T cells transferred from animals treated with RAPA alone failed to prolong graft survival. In contrast, sera isolated on day 40 postgrafting from WFu primary hosts treated with RAPA alone or with the RAPA/CsA combination, but not with CsA alone, extended the survival of BUF hearts: 3 ml serum from RAPA-treated hosts prolonged BUF heart survival to 76.6 +/- 31.3 days (P < 0.002) and from RAPA/CsA-treated hosts to 47.1 +/- 12.8 days (P < 0.001). The effect of serum was immunologically specific: it did not prolong the survival of third-party outbred Sprague Dawley heart allografts. Although the IgM fraction (0.2 mg) purified from the serum of RAPA-treated recipients was ineffective (10.6 +/- 0.8 days; NS), an equal amount of the IgG fraction significantly (P < 0.002) prolonged BUF heart allograft survival to 26 days (n = 4). Thus, hosts treated with RAPA or a RAPA/CsA combination develop IgG antibodies that mediate the unresponsive state toward allogeneic heart allografts.

Evidence that rapamycin rescue therapy delays rejection of major (MHC) plus minor (non-MHC) histoincompatible heart allografts in rats.

The capacity of delayed onset of rapamycin (RAPA) therapy to block process of destruction was examined in rats undergoing heart allograft rejection. Untreated Wistar Furth (WFu; RT-1u) recipients reject Buffalo (BUF; RT-1b) heart allograft with a mean survival time (MST) of 6.5 +/- 0.5 days. A 14-day i.v.infusion of 0.8 mg/kg RAPA begun on the day of transplantation prolonged the survival to 74.1 +/- 20.2 days (P < 0.001), 0.2 mg/kg to 32.2 +/- 10.0 days (P < 0.001), and 0.08 mg/kg to 36.4 +/- 11.8 days (P < 0.001). When RAPA therapy (0.8 mg/kg) was begun 3 or 4 days after transplantation, the grafts survived 85.2 +/- 31.1 (P < 0.001), and 70.2 +/- 43.3 (P < 0.005) days, respectively. Therapy initiated on day 5 was much less effective; most transplants were rejected within 10 days; one graft survived 32 and two grafts 60 days (MST = 17.6 +/- 20.0, NS). A 0.2 mg/kg RAPA dose prolonged graft survival with initial use on days 3 (31.6 +/- 12.2 days; P < 0.001) or 4 (31.4 +/- 8.1 days; P < 0.001) but not on day 5. The 0.08 mg/kg RAPA prolonged hearts only when started on day 3 (47.2 +/- 2.7 days; P < 0.001) but not on days 4 or 5. WFu recipients treated with a subtherapeutic dose of cyclosporine (1 mg/kg; 9.1 +/- 1.5 days) displayed prolonged heart allograft function when treated subsequently with RAPA (0.8 or 0.08) beginning from days 4, 5, or 6 postgrafting. These in vivo results are supported by in vitro experiments. The frequency of BUF alloreactive elements among normal WFu LN cells (fTc) was 337 +/- 139/10(6) T cells in limiting dilution assay. Addition of RAPA (1 muMol) at the beginning of culture significantly reduced (P < 0.025) the fTc to 17 +/- 6.6/10(6), or alternatively on days 4 or 6 to 37.3 +/- 20.0/10(6) and 58.6 +/- 21.8/10(6), respectively. Thus, both in vivo and in vitro data demonstrate that delayed RAPA therapy may interrupt alloimmune reactions.

Immunosuppressive effects of defibrotide.

The effect of defibrotide (DF) alone or in combination with CsA was examined using in vitro proliferation assays with human PBLs and in vivo heterotopic heart allografts in rats. DF alone (12.5-50 mg/ml) inhibited in vitro PBL proliferation more effectively after PHA (50-56%) or OKT3 (50-95%), than after alloantigenic (25-30%), stimulation. Furthermore, the combination of DF (1-4 mg/ml) with CsA (10-40 ng/ml) caused an 85.2-86.8% reduction in proliferative responses after OKT3 stimulation. Median-effect analysis documented that the combination index for DF and CsA was consistently lower than 0.3 at various concentration ratios of the 2 agents. Combination index values below 1.0 reflect drug synergism; those equal to 1.0 show additive, and above 1.0, antagonistic, interactions. Daily intraperitoneal injections of DF (150 mg/kg) failed to prolong the survival of Buffalo (RT-1b) heart allografts in Wistar-Furth (RT-1u) recipients, namely mean survival times of 7.0 +/- 0.7 days with, vs. 6.5 +/- 0.5 days without, DF treatment. Similarly, intravenous or intra-arterial infusion of DF (280 mg/kg) delivered directly into the heart allograft by a 7-day osmotic pump was ineffective. However, a course of local, but not systemic, DF (280 mg/kg) combined with a 14-day i.v. administration of a subtherapeutic dose of CsA (1 mg/kg) significantly prolonged heart allograft survival to 22.8 +/- 5.0 days (P < 0.001). Thus, in vitro DF is immunosuppressive alone at high concentrations, and in combination with CsA at low concentrations. Continuous infusion of DF into the graft combined with systemic administration of CsA prolonged transplant survival in vivo. These findings suggest that high tissue levels of DF potentiate the immunosuppressive effects of CsA.

Conversion from tacrolimus to cyclosporine for a non-dose-dependent tacrolimus-induced toxicity, a pediatric kidney transplant recipient case report.

Tacrolimus-induced toxicity is considered a dose-related side effect largely due to a direct action of this potent calcineurin inhibitor on its targets including the kidney and the pancreas. This paper describes a case of tacrolimus systemic toxicity that appeared in a pediatric kidney transplant recipient who received a low drug dose. The kidney biopsy was a crucial aid toward the correct diagnosis, which reversed upon conversion to cyclosporine-based immunosuppression. A review of the literature suggests a chance of systemic toxicity even when the patient is maintained on therapeutic levels of tacrolimus. Because idiosyncratic reactions to the drug have not yet been postulated, we conclude that this suspicion may be addressed by a safe conversion to cyclosporine in pediatric patients.

C2 is an age-independent parameter for optimal cyclosporine exposure in long-term kidney transplant recipients.

The argument for therapeutic drug monitoring (TDM) of cyclosporine (Cya) has been discussed for the last two decades. So far, a generalized consensus has been reached for TDM of Cya microemulsion in adult transplant recipients, being Cya blood levels obtained 2 hours after the administration (C2), the most reliable in reflecting the overall Cya exposure. However, clear guidelines are not available for the pediatric population because of the distinct metabolism of the drug in this patient population. Therefore, adult data do not necessarily apply to children. In this retrospective analysis, the authors sought to define a universal parameter for pharmacokinetic clinical monitoring of Cya in long-term kidney transplant recipients, regardless of their age. Lower C2 levels were observed in all patients, adult and pediatric, who eventually developed chronic allograft dysfunction (CRAD) compared with patients who maintained stable kidney function throughout the entire follow-up (pediatric CRAD, 933 +/- 455 ng/mL; vs Stable, 1236 +/- 347 ng/mL, P = .0001; and adult CRAD, 781 +/- 518 ng/mL; vs Stable, 1088 +/- 452 ng/mL, P = .009). On the other hand, the risk of Cya underexposure was not highlighted by trough level monitoring (C0) because all patients have been maintained steadily on therapeutical C0 levels for the entire follow-up. In conclusion, for Cya maintenance therapy, C2 appears to be a superior strategy to C0 monitoring in both adult and pediatric kidney transplant recipients.

Cyclosporine monitoring in stable, long-term, pediatric kidney transplant recipients: the value of C2 determination.

Although a generalized consensus has been reached for therapeutic drug monitoring of cyclosporine microemulsion in adult transplant patients, clear guidelines are recently not available for the pediatric population. In this retrospective analysis of pharmacokinetic data obtained from stable, long-term, pediatric kidney transplant recipients, we sought to define a possible approach to manage cyclosporine therapy in a pediatric setting. The 2-hour postdose cyclosporine blood concentration, C(2), rather than trough levels, was the best single time point predictor of the area under the concentration curve. We concluded that therapeutic drug monitoring of cyclosporine-based immunosuppressive regimens should be tailored based on C(2) determinations for pediatric kidney transplant recipients.

[Renal transplantation. 1st year's experience at a new center]. / Trapianto renale. Primo anno di esperienza di un nuovo centro.

The initial activity in the field of organ transplantation always brings some organizational and medical difficulties. We describe the first year experience in the field of kidney transplantation at the Transplantation Unit of Padua University. Sixteen kidney transplants from cadaver donors have been performed. A double drug therapy (cyclosporine and steroids) as immunosuppression was used in 9 patients and a triple drug protocol (cyclosporine, steroids, azathioprine) was used in 7. Two patients also received monoclonal antibodies because of steroid resistant rejection. The technical problems we observed include one case of ureteral duplication corrected with uretero-ureteral anastomosis and one substenosis of an ureteral cystoanastomosis corrected with endoscopic dilatation. Among the complications related to immunosuppressive therapy we had one case of severe cyclosporine nephrotoxicity. Two patients died after few months with a functional graft because of pulmonary embolism and because of severe hepatitis complicated by pancreatitis. At an average of six months follow-up all the remaining patients have a good functioning graft.

Kinetics of in vitro immune responses of T and B cells during tolerance induction by sirolimus.

OBJECTIVES: The purpose of the study presented herein was to examine immune performances of rat heart allograft recipients immunosuppressed with sirolimus (SRL, rapamycin; Rapamune, Wyeth-Ayerst, Princeton, NJ). METHODS: The immune performances of lymphocytes harvested from SRL-treated Wistar Furth (WF; RT1u) recipients of Buffalo (BUF; RT1b) heart allografts were examined on days 7, 14, and 90 postgrafting. RESULTS: Whether derived from normal WF rats, SRL-treated WF heart recipients, or SRL-untreated WF heart recipients, pan-T cell population purified from the lymph nodes or spleens on day 7 or 14 displayed similar responses to phytohemaglutinin, anti-T cell receptor R73 monoclonal antibody, donor-type BUF, or third-party Brown Norway alloantigenic stimulators. There was no in vitro evidence of suppressor T cells in SRL-treated recipients. The frequencies of anti-BUF-specific cytotoxic T cells, as shown by limiting dilution analysis, were similar in the short- (days 7 or 14) and in the long- (day 90) term surviving recipients. SRL treatment did not affect the expression of interleukin-2 (IL-2) messenger RNA (mRNA) by T helper 1 (Th1) or of IL-4 and IL-10 mRNA by Th2 cells on days 7 and 14 postgrafting, but did induce selective activation of Th2 cells on day 60 postgrafting. Administration of SRL induced the production of non-complement (C')-fixing IgG2c BUF-specific alloantibodies that appeared in the sera of unresponsive recipients on day 14 postgrafting and reached a peak concentration on day 120 postgrafting. In contrast to untreated recipients that rejected BUF heart allografts, all SRL-treated WF recipients failed to produce C'-fixing BUF-specific alloantibodies. CONCLUSIONS: SRL promotes long-term selective activation of Th2 cells and the production of non-C'-fixing IgG2c blocking antibodies.

[Surgical central venous catheterization (SCVC) for total parenteral nutrition (TPN) via the basilic vein]. / Il cateterismo venoso centrale chirurgico (CVCC) per nutrizione parenterale totale (NPT) attraverso la vena basilica.

The central venous catheterization (CVC) by subclavian vein puncture for total parenteral nutrition (TPN) has a high risk of complications in some situations such as neck or supraclavicular diseases, tracheostomies, costoclavicular deformities and coagulopathies. Surgical CVC approach to the basilic vein is indicated in these cases. The aim of this study was to evaluate the usefulness of this technique. Sixteen patients had TPN with this technique, utilizing a silicon catheter (Vygon Nutricath). The main time of TPN was 19 days (min 5-max 41). In 12 cases (75%), there were no complications. In two cases (12.5%) we observed an accidental catheter displacement. The incidence of thrombophlebitis was 12.5% (2 cases); in one of two cases (6.25%), it was correlated with the contamination of the catheter (Staphylococcus epidermidis); in the second case it was aseptic. Our results demonstrated the usefulness of this of these technique in cases in where the CVC by subclavian vein presents a high risk of complications.