Changes in the small bowel of symptomatic kidney transplant recipients converted from mycophenolate mofetil to enteric-coated mycophenolate sodium.

BACKGROUND/AIMS: Gastrointestinal (GI) symptoms in renal transplant recipients may be caused due to mycophenolic acid (MPA) toxicity. Using small bowel capsule endoscopy (SBCE) we examined the impact of conversion from Mycophenolate Mofetil (MMF) to enteric-coated formulation of Mycophenolate Sodium (EC-MPS) given to treat GI mucosal lesions. METHODS: Adult kidney-only recipients at least 30 days after transplant, presenting with GI symptoms while receiving MMF completed a Gastrointestinal Symptom Rating Scale (GSRS) questionnaire, underwent SBCE, and had MMF substituted by EC-MPS. After 30 days, GSRS and SBCE were repeated and findings were compared to baseline values. Patients who were still on EC-MPS 6-24 months post-conversion were contacted for completing a follow-up GSRS questionnaire and SBCE. RESULTS: Eighteen out of 23 subjects completed the first part of the study. Subjects' median ages and post-transplant time were 47.5 years old and 4.5 months, respectively. Tacrolimus, MMF and prednisone was the main regimen (94%), with a median MMF dose of 750 mg BID. The average baseline GSRS was 2.99 ± 0.81; it significantly decreased to 2.19 ± 0.8 at 30 days post-conversion. At baseline, 50 had gastric and 89% had small bowel lesions. At 30 days, 29 and 62% of the SBCE were still showing gastric and small bowel lesions, respectively. Of 5 patients in the study extension, 4 had abnormal SBCE findings but have been reporting improvement in their symptoms. CONCLUSION: Stomach and small bowel mucosal lesions are common in kidney recipients with GI symptoms when treated with MMF. Conversion to EC-MPS for 30 days significantly alleviated the GI symptoms; however, no evident correlation with SBCE findings was found.

Assessment of the stability of citrate-buffered piperacillin/tazobactam for continuous infusion when stored in two commercially available elastomeric devices for outpatient parenteral antimicrobial chemotherapy: a study compliant with the NHS Yellow Cover Document requirements.

OBJECTIVES: To investigate the effect of pH control through the use of a citrate-buffered saline diluent pH 7 on the degradation rate of piperacillin/tazobactam solutions for infusion and to determine if an extended shelf-life of up to 13 days fridge 2°C-8°C plus 24 hours 'in-use' at 32°C in two elastomeric devices: FOLFusor LV10 (Baxter Healthcare, Thetford, UK) and Easypump II (B. Braun Medical Ltd, Sheffield, UK) can be achieved. METHODS: Testing was as per the latest National Health Service (NHS) Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD) requirements.A validated stability indicating high-performance liquid chromatography method was used for assessing the stability of the solutions of piperacillin/tazobactam at a combined concentration of 25 mg/mL and 90 mg/mL respectively. Solutions were tested in two batches in replicate (n=3) at five time points according to the requirements of the YCD. RESULTS: Piperacillin/tazobactam stability was significantly improved when 0.3% w/v citrate-buffered saline pH 7 was used as the diluent, compared with using 0.9% w/v saline as diluent. Greater than 95% of the zero-time concentration of both actives remained following storage at 2°C-8°C for up to 13 days plus 24 hours at 32°C in both devices. The data support extended storage of up to 13 days 2°C-8°C plus 24 hours at 32°C 'in-use' when using FOLFusor LV10 (Baxter) or Easypump II (B. Braun) pump devices. CONCLUSIONS: The enhanced stability complies with UK national standards as stated in the YCD for stability testing of aseptically produced small molecules and supports the storage of piperacillin/tazobactam for up to 13 days 2°C-8°C plus 24 hours at 32°C 'in-use' within two elastomeric pump devices. The extended shelf-life provides a significant advantage over the stability of piperacillin/tazobactam solutions for infusion when reconstituted and diluted in 0.9% w/v saline as diluent. The data open up the possibility of a continuous infusion of piperacillin/tazobactam delivered by elastomeric pump devices over 24 hours in an outpatient parenteral antimicrobial therapy setting.

Integrity performance assessment of a closed system transfer device syringe adaptor lock as a terminal closure for Luer-Lock syringes.

OBJECTIVES: To investigate the container closure integrity of a closed system transfer device syringe adaptor lock in combination with disposable Luer-Lock syringes as the terminal closure device. The UK National Health Service (NHS) Pharmaceutical Quality Assurance Committee (PQAC) requires syringe integrity data for final storage devices of aseptic products such as chemotherapy drugs when prepared in advance and stored before use, as is standard practice for dose banded drugs. The assessment comprised both physical and microbial integrity testing of the combination closed system/Luer-Lock syringe containers at syringe sizes of 1 mL, 20 mL, and 50 mL. METHODS: Integrity testing was performed as described in the NHS Pharmaceutical Quality Assurance Committee yellow cover document, second edition 2013 'Protocols for the Integrity Testing of Syringes', with Chemfort (Simplivia, IL) syringe adaptor lock (SAL) devices as replacement for sterile blind hubs. Microbiological integrity was assessed according to method 1 part 1.4 using Brevundimonas diminuta at 32°C for up to 14 days of contact time. Two positive control devices per syringe size were tested using a blind hub cap as closure which was loosened before the test. Physical integrity was assessed using method 3 of the yellow cover document which is a dye intrusion method. Dye intrusion was assessed both visually and using a validated ultraviolet-visible spectrophotometer method. For each size/batch of test articles a positive control device (n=1) was assessed using a wire wrapped around the syringe plunger tip deliberately compromising integrity. Negative controls for each size (n=1) consisted of devices not immersed in methylene blue dye. RESULTS: Chemfort syringe adaptor lock/Luer-Lock syringe combinations were shown to be: (1) free of microbiological contamination after 14 days of contact time (n=60); and (2) free of dye intrusion at all syringe sizes tested (n=61 in total). The data demonstrate 100% closure integrity of the final container system when the Chemfort syringe adaptor lock replaces the syringe hub as the terminal closure device. All positive control devices demonstrated system suitability as container integrity was compromised in all positive control tests. All negative controls were negative for microbial and dye intrusion. CONCLUSIONS: Syringe adaptor lock components complied with the NHS Pharmaceutical Quality Assurance Committee yellow cover document syringe integrity requirements when used as the terminal closure of Luer-Lock disposable syringes from 1 mL up to 50 mL. Therefore, syringe adaptor lock (Chemfort) can be used as the terminal closure system for pre-filled syringes of chemotherapeutic drug products prepared in advance in UK NHS pharmacy technical services.

Assessment of ceftolozane/tazobactam stability in elastomeric devices and suitability for continuous infusion via outpatient parenteral antimicrobial therapy.

OBJECTIVES: To investigate the stability of ceftolozane/tazobactam 5 mg/mL and 20 mg/mL solutions for infusion in two elastomeric devices: FOLFusor LV10 (Baxter Healthcare) and Easypump® II (B. Braun Medical Ltd) and determine if an extended shelf life of up to 8 days storage at 2-8°C plus 24 h 'in use' at 32°C was achievable. METHODS: Testing was as per the latest NHS Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD) requirements. A stability-indicating LC method was used for assessing the stability of solutions of ceftolozane/tazobactam at 5 mg/mL and 20 mg/mL (combined concentration of both actives) respectively, tested in two batches in triplicate (n = 3) at five timepoints according to the requirements of the YCD. RESULTS: Ceftolozane/tazobactam, diluted in 0.9% w/v sodium chloride at 5 mg/mL and 20 mg/mL, degraded during in-use storage at 32°C with <95% remaining after 18 h for some device/concentration combinations and all device/concentration combinations at 24 h, respectively. The data does support extended storage of up to 8 days at 2-8°C plus 12 h at 32°C 'in-use' when using either FOLFusor LV10 or Easypump® II devices and is compliant with YCD. CONCLUSIONS: Solutions of ceftolozane/tazobactam can be administered in outpatient parenteral antimicrobial therapy (OPAT) services following refrigerated storage for up to 8 days, when limited to a 12 h infusion at in-use temperature of 32°C. For UK OPAT services where twice daily dosing is feasible, our data provides another treatment option for challenging infections. In countries where a 10% loss of ceftolozane/tazobactam is acceptable, a 24 h infusion is supported by the data.

Investigation of meropenem stability after reconstitution: the influence of buffering and challenges to meet the NHS Yellow Cover Document compliance for continuous infusions in an outpatient setting.

Objectives: To determine the influence of different buffers, pH and meropenem concentrations on the degradation rates of meropenem in aqueous solution during storage at 32°C, with the aim of developing a formulation suitable for 24-hour infusion in an ambulatory elastomeric device, compliant with the latest National Health Service Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD) requirements. Methods: Meropenem was diluted to 6.25 mg/mL and 25 mg/mL in aqueous solutions adjusted to various pH with phosphate or citrate buffer and assessed for stability. Meropenem concentrations were determined using a validated stability-indicating high-performance liquid chromatography method at time 0 and following storage for up to 24 hours at 32°C as per the YCD requirements. Results: Degradation was observed to be slowest in citrate buffer around pH 7 and at a meropenem concentration of 6.25 mg/mL; however, losses exceeded 10% after storage for 24 hours at 32°C in all of the diluents tested in the study. Conclusions: Meropenem at concentrations between 6.25 mg/mL and 25 mg/mL as tested is not sufficiently stable to administer as a 24-hour infusion in ambulatory device reservoirs. If the YCD 95% minimum content limit is applied, the infusion period must be reduced to less than 6 hours for body-worn devices, especially at the higher concentration studied (25 mg/mL). This limits the possibility of using elastomeric devices to deliver continuous infusions of meropenem as part of a wider outpatient parenteral antimicrobial therapy service.

Assessment of the stability of citrate-buffered flucloxacillin for injection when stored in two commercially available ambulatory elastomeric devices: INfusor LV (Baxter) and Accufuser (Woo Young Medical): a study compliant with the NHS Yellow Cover Document (YCD) requirements.

Objectives: To investigate the effect of pH and buffers on the degradation rate of flucloxacillin and to determine if flucloxacillin can be stabilised using a buffered diluent for up to 14 days when stored at 2°C-8°C including a 24-hour infusion period at 32°C in two elastomeric devices (Accufuser and INfusor LV) filled to 240 mL. Testing as per the NHS Pharmaceutical Quality Assurance Committee Yellow Cover Document (YCD) requirements. Methods: A validated stability indicating high-performance liquid chromatography method was used for assessing the stability of flucloxacillin diluted in 0.3% w/v citrate-buffered saline pH 7.0 when stored at 2°C-8°C in two ambulatory devices (Accufuser and INfusor LV). Flucloxacillin at 10 and 50 mg/mL diluted in 0.3% w/v citrate-buffered saline pH 7.0 to a final volume of 240 mL and stored at 2°C-8°C, including 24 hours at 32°C, was tested from two batches in replicate (n=3) at five time points for up to 14 days according to the requirements of the YCD. Results: Greater than 95% of the zero-time concentration of flucloxacillin at 10 and 50 mg/mL remained when stored at 2°C-8°C after 14 days including 24 hours at 32°C in both Accufuser and INfusor LV devices. Conclusions: Flucloxacillin sodium stability was improved, and complied with UK national standards, by using a diluent of 0.3% w/v citrate-buffered saline pH 7 in both Accufuser and INfusor LV ambulatory devices when filled to 240 mL. The data support assigning a shelf-life of up to 14 days (13 days stored at 2°C-8°C and 24 hours at 32°C). Flucloxacillin may now be used appropriately as a continuous 24-hour infusion in outpatient parenteral antimicrobial therapy services, providing further opportunity to avoid or shorten patient hospital stays, as well as support ideal antimicrobial stewardship principles.

Management of renal dysfunction in the liver transplant recipient.

PURPOSE OF REVIEW: Acute and chronic kidney injury following orthotopic liver transplantation (OLT) is associated with increased morbidity and mortality. With the increasing longevity of liver transplant recipients, chronic kidney disease (CKD) has become an increasingly prevalent complication among long-term survivors. This article provides an overview of the literature on suggested risk factors for acute and CKD following OLT and a discussion of an approach to their medical management. RECENT FINDINGS: In OLT candidates with pretransplant renal dysfunction, the use of interleukin-2 receptor blockers or antithymocyte globulin induction therapy in conjunction with delayed introduction of calcineurin inhibitors may preserve early renal function. In long-term stable OLT recipients with established calcineurin inhibitor nephrotoxicity, calcineurin inhibitor minimization or withdrawal protocols may halt or ameliorate renal dysfunction without compromising patient and graft survival. However, large-scale, multicenter, randomized controlled trials are still needed. SUMMARY: The occurrence of acute kidney injury is common immediately after OLT, whereas the incidence of CKD and end-stage renal disease increases with time. Identifying patients at risk for acute kidney injury and CKD following OLT and early implementation of measures to preserve, halt, or ameliorate the progression of renal dysfunction should be an integral part in the management of OLT recipients.

Outcomes of dual adult kidney transplants in the United States: an analysis of the OPTN/UNOS database.

BACKGROUND: The organ shortage has resulted in increased use of kidneys from expanded criteria donors (ECD). For ECD kidneys unsuitable for single use, dual kidney transplants (DKT) may be possible. There are limited data comparing outcomes of DKT to single kidney ECD transplants, making it unclear where DKT fits in the current allocation scheme. Our purpose was to compare outcomes of DKT and ECD transplants in the United States. METHODS: From 2000 to 2005, a total of 625 DKT, 7686 single kidney ECD, and 6,044 SCD transplants from donors aged>or=50 years were identified from the Organ Procurement and Transplantation Network/United Network for Organ Sharing data. Allograft survival was the primary outcome. RESULTS: DKT comprised 4% of kidney transplants from donors aged>or=50 years. Compared to the ECD donor group, the DKT donor group was older (mean age 64.6+/-7.7 years vs. 59.9+/-6.2 years) and consisted of more African Americans (13.1% vs. 9.9%), and more diabetic donors (16.3% vs. 10.4%; P<0.001). Mean cold ischemic time was longer in DKT (22.2+/-9.7 hr), but rates of delayed graft function were lower (29.3%) compared to ECD transplants (33.6%, P=0.03). Three-year overall graft survival was 79.8% for DKT and 78.3% for ECD transplants. CONCLUSION: DKT were infrequent and had outcomes comparable to ECD transplants, despite the use of organs from higher risk donors. With a more upfront approach to DKT by offering this option to patients at the time of wait-listing as part of an ECD algorithm, we may be able to further optimize outcomes of DKT and minimize discard of potential organs.

Performance testing protocol for closed-system transfer devices used during pharmacy compounding and administration of hazardous drugs.

OBJECTIVES: The United States National Institute for Occupational Safety and Health (NIOSH) is developing a protocol to assess the containment performance of closed system transfer devices (CSTDs) when used for drug preparation (task 1) and administration (task 2) and published a draft protocol in September 2016. Nine possible surrogates were proposed by NIOSH for use in the testing. The objectives of this study were to: (A) select the most appropriate surrogate; (B) validate the NIOSH protocol using this surrogate; and (C) determine the containment performance of four commercial CSTDs as compared with an open system of needle and syringe using the validated NIOSH protocol. METHODS: 2-Phenoxyethanol (2-POE) was selected as a surrogate based on its water solubility, Henry's volatility constant, detectability by mass spectrometry, and non-toxicity. Standard analytical validation methods including system suitability, limit of detection (LOD), and limit of quantitation (LOQ) as well as system cleaning validation were performed. The amount of 2-POE released when the CSTDs were manipulated according to two tasks defined by NIOSH was determined using mass spectrometry coupled to thermal desorption and gas chromatography. This approach allows sensitivity of detection below 1 part per billion (ppb). Equashield, Tevadaptor (OnGuard), PhaSeal, and ChemoClave were assessed according to manufacturers' instructions for use. RESULTS: 2-POE was tested and validated for suitability of use within the NIOSH protocol. A simple and efficient cleaning protocol achieved consistently low background values, with an average value, based on 85 measurements, of 0.12 ppb with a 95% confidence interval (CI) of ±0.16 ppb. This gives an LOD for the tests of 0.35 ppb and an LOQ of 0.88 ppb. The Equashield, Tevadaptor (OnGuard), and PhaSeal devices all showed average releases, based on 10 measurements from five tests, that were less than the LOQ (i.e. < 0.88 ppb), while the ChemoClave Vial Shield with Spinning Spiros showed average releases of 2.9±2.3 ppb and 7.5±17.9 ppb for NIOSH tasks 1 and 2 respectively at the 95% confidence level. The open system of needle and syringe showed releases, based on two measurements from a single test, of 4.2±2.2 ppb and 5.1±1.7 ppb for NIOSH tasks 1 and 2 respectively at the 95% confidence level. CONCLUSIONS: 2-POE proved to be an ideal surrogate for testing of CSTDs using the NIOSH protocol. We propose that a CSTD can be qualified using the NIOSH testing approach if the experimental LOQ is less than 1 ppb and the release values are below the LOQ. Equashield, Tevadaptor (OnGuard), and PhaSeal meet these acceptance criteria and can therefore all be qualified as CSTDs, but the ChemoClave system does not and so would not qualify as a CSTD.

Donor biopsy and kidney transplant outcomes: an analysis using the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) database.

BACKGROUND: Although the degree of glomerulosclerosis on pretransplant donor biopsy is one criterion used in the decision to accept a deceased donor kidney, its relationship with graft survival remains controversial. This study compared graft survival with the degree of glomerulosclerosis found on donor biopsy. We also examined the agreement in degree of glomerulosclerosis between paired kidneys. METHODS: Biopsy results from 12,129 adult deceased donor transplants between January 1, 2000 and December 31, 2005 were identified in the Organ Procurement and Transplantation Network/United Network for Organ Sharing data, as of September 11, 2006. Of these, 2696 donors had both kidneys biopsied and subsequently transplanted. RESULTS: Among the groups with greater than 5% glomerulosclerosis, there was no statistically significant difference in graft survival rates (log-rank, P=0.44). The overall graft survival rates of the 0-5% group were significantly superior to those of the >5% groups (1-, 3-, and 5-year rates: 85.9%, 72.4%, and 59.0% for 0-5% group vs. 81.6%, 68.1%, and 53.6% for >5% group, log-rank P<0.001). Agreement between paired kidneys from the same donor was highest for the 0-5% glomerulosclerosis groups (90.6% for pairs with 0-5% glomerulosclerosis in the left kidney vs. 42.5% for pairs with >5% glomerulosclerosis in the left kidney). CONCLUSION: Donor kidneys with less than 6% glomerulosclerosis were associated with better graft outcomes and intrapair agreement in the degree of glomerulosclerosis. Among kidneys with greater than 5% glomerulosclerosis, the degree of glomerulosclerosis did not help predict graft outcomes. Sampling error may contribute to the lack of outcome differences seen among these kidneys, given the low intrapair agreement.

Pancreas-kidney and pancreas transplantation for the treatment of diabetes mellitus.

Kidney transplantation is the treatment of choice for end-stage diabetic nephropathy, but the ultimate treatment today for type 1 diabetes mellitus is the whole vascularized pancreas transplant. Although its use is increasing, pancreas transplantation remains an uncommonly used therapeutic option that normalizes glucose levels and results in stabilization or improvement in secondary complications far better than any other strategy available for treatment of type 1 diabetes. These documented benefits of a simultaneous kidney and pancreas transplant are the basis for its acceptance as an appropriate therapy for patients who have type 1 diabetes mellitus and end-stage renal disease.

New onset diabetes mellitus after solid organ transplantation.

This article presents an overview of the literature on the current diagnostic criteria for new onset diabetes mellitus after transplantation (NODAT) and discusses suggested risk factors for the development of NODAT, its potential pathogenic mechanisms, and its impact on post-transplant outcomes after solid organ transplantation. Suggested guidelines for early identification and management of NODAT are also discussed.

Renal function outcomes following liver transplantation and combined liver-kidney transplantation.

Acute renal failure (ARF) is common immediately after orthotopic liver transplantation (OLT), whereas the incidences of chronic kidney disease (CKD) and end-stage renal disease increase with time. Introduction of the Model for End-stage Liver Disease (MELD) score-intended to prioritize patients with more-severe pretransplantation liver disease in general, and worse pretransplantation renal function in particular-for the allocation of liver grafts led to concerns about compromised patient and allograft survival and increased incidence of postoperative ARF and CKD. Nonetheless, it has been suggested that early OLT of candidates with baseline renal dysfunction improves post-transplantation renal outcomes. For OLT candidates with mild to moderate chronic renal impairment or recent-onset ARF, the decision of whether to perform OLT alone or combined liver-kidney transplantation (CLKT) can be challenging because no single factor has been shown to be predictive of the degree of renal function recovery or CKD progression following successful OLT. In this article, we provide an overview of the literature on renal function outcomes following OLT and CLKT, share our perspectives on the potential predictors of renal dysfunction or nonrecovery of renal function after OLT, and present United Network for Organ Sharing data on patient and allograft outcomes in CLKT recipients in the pre-MELD and post-MELD eras. Mechanisms that might underlie immunological protection of kidney grafts by liver allografts are also discussed.

Noninvasive diagnosis of cellular and antibody-mediated rejection by perforin and granzyme B in renal allografts.

A major milestone in transplantation would be the use of biomarkers to monitor rejection. We examined the association between perforin and granzyme-B gene expression detected in the peripheral blood of renal allograft recipients with cellular and antibody-mediated rejection. Furthermore, we judged the appropriateness of assigning negative rejection statuses to persons without a biopsy whose grafts were functioning well clinically. Of the 46 patients who completed the study, recipients with cellular rejection had higher perforin and granzyme-B levels compared with nonrejectors (p = 0.006). Interestingly, recipients with antibody-mediated rejection also had higher perforin and granzyme-B levels compared with nonrejectors (p = 0.04). Patients with high levels of granzyme B had a probability of rejecting that was 26.7 times greater than those patients with low levels of granzyme B. Perforin and granzyme B had sensitivities of 50% and specificities of 95% in predicting rejection (cutoff value = 140). Assigning negative rejection statuses to recipients without a biopsy whose grafts were functioning well did not have a major effect on the direction or significance of covariate values. This study suggests that perforin and granzyme-B gene expressions in peripheral blood are accurate in detecting both cellular and antibody-mediated rejection.

Long-term analysis of combined liver and kidney transplantation at a single center.

OBJECTIVE: To analyze use of combined liver and kidney transplantation (CLKT) for patients with chronic primary diseases of both organs and for patients with hepatorenal syndrome. DESIGN: Retrospective case series. SETTING: Multiorgan transplantation service in a large university medical center. PATIENTS: A total of 98 patients underwent 99 CLKTs during a 16-year period; 76 had primary renal diseases, and 22 had hepatorenal syndrome. Patients receiving isolated liver and kidney transplants were analyzed for comparison. MAIN OUTCOME MEASURES: Patient and graft survival, rejection rates, and need for hemodialysis before and after transplantation. RESULTS: Overall patient survival was 76%, 72%, and 70% at 1, 3, and 5 years, respectively; liver graft survival was 70%, 65%, and 65%; and kidney graft survival was 76%, 72%, and 70%. No risk factors analyzed for recipients or donors were associated significantly with early posttransplantation mortality or graft loss. In 28 patients who received monoclonal antibody induction therapy with interleukin 2 blockers, there were significantly fewer episodes of acute liver rejection. For patients with hepatorenal syndrome, CLKT did not confer a survival advantage over liver-only transplantation (1-year patient survival was 72% vs 66%; P = .88). The 1-year acute kidney rejection rate in the adult CLKT group was 14% vs 23% in a 5-year cadaveric renal transplantation cohort (P<.01). CONCLUSIONS: First, CLKT is indicated in patients with dual organ disease and achieves excellent results. Second, CLKT for hepatorenal syndrome is indicated in patients receiving hemodialysis for longer than 8 weeks and confers advantages in patient survival and use of hospital resources. Third, the liver is immunoprotective for the kidney.

Laser induced disruption of bacterial spores on a microchip.

We report on the development of a laser based spore disruption method. Bacillus globigii spores were mixed with a laser light absorbing matrix and co-crystallized into 200-microm-wide and 20-microm-deep nanovials formed in a polydimethylsiloxane (PDMS) target plate. Surface tension effects were exploited to effect up to 125-fold spore enrichment. When the target zones were illuminated at atmospheric pressure with pulsed UV-laser light at fluences below 20 mJ cm(-2) a change in spore morphology was observed within seconds. Post illumination PCR analysis suggests the release of endogenous DNA indicative of spore disruption. For laser fluences above 20 mJ cm(-2), desorption of spores and fragments was also observed even without a matrix being employed. Desorbed material was collected in a PDMS flowcell attached to the target plate during laser illumination. This opens up a route towards the direct extraction of released DNA in an integrated spore disruption-PCR amplification microchip device.

Development of posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early graft dysfunction.

BACKGROUND: The goal of this study was to determine whether the production of posttransplant antibodies directed against donor HLA mismatches (donor specific antibody; DSA) is associated with renal allograft rejection and early graft dysfunction. METHODS: Forty-nine adult renal allograft recipients with increased risk of rejection were enrolled during the period of October 2001 through May 2003 and were prospectively monitored for the development of anti-HLA antibodies. RESULTS: Of 49 patients, eight (16.3 %) patients were diagnosed with acute humoral rejection (AHR) and 11/49 (22.4%) patients were diagnosed with acute cellular rejection (ACR). A strong association between pretransplant HLA sensitization and AHR was found (P=0.005). Of the eight patients diagnosed with AHR, the majority developed DSA before or concomitant with episodes of rejection (P<0.001). Only 3 of 41 patients (7.3%) without AHR developed DSA. The pathogenic role of alloantibodies was further substantiated by analyzing their association with graft function as measured by serum creatinine levels. The average serum creatinine after the third month posttransplantation in DSA producers was 2.24+/-1.01 mg/dL, while in non-DSA patients the average serum creatinine was 1.41+/-0.37 mg/dL (P<0.01). CONCLUSION: This study reveals a strong association between the production of DSA, AHR, and early graft dysfunction. Our findings indicate that prospective monitoring for anti-HLA antibodies following transplantation is a useful test for the diagnosis and classification of AHR for identifying patients at risk of early graft dysfunction.

A novel flow assay for the detection of cytokine secreting alloreactive T cells: application to immune monitoring.

The direct and indirect allorecognition pathways play an important role in graft rejection. We hypothesized that the presence of alloreactive memory T cells in the recipient's circulation increases the risk of rejection after transplantation. The objective of this study was to develop a noninvasive, immune monitoring tool that simultaneously measures donor-specific responses via both the direct and indirect recognition pathways. Our laboratory developed a whole blood flow cytometric cytokine secretion assay to identify interferon (IFN)-gamma secreting memory T cells in whole blood of renal transplant patients. The assay readily detected IFN-gamma producing CD3+ T cells in response to recall antigens tetanus toxoid, purified protein derivative, and alloantigens in whole blood from healthy controls. Analysis of sequential posttransplant blood samples from 19 renal allograft recipients showed that alloimmune responses were higher in transplant recipients who had undergone acute rejection than in those without acute rejection episodes. In addition, patients showing increased creatinine levels 3 months after transplantation were more likely to exhibit alloimmune responses than recipients with stable graft function. The flow cytokine secretion assay provides a reliable and simple method for identification of patients at risk of acute rejection and early graft dysfunction.

Randomized double-blind study of immunoprophylaxis with basiliximab, a chimeric anti-interleukin-2 receptor monoclonal antibody, in combination with mycophenolate mofetil-containing triple therapy in renal transplantation.

BACKGROUND: Acute rejection remains a major problem in renal transplantation. Immunoprophylaxis with basiliximab (Simulect) has achieved significant reductions in acute rejection episodes in renal allograft recipients receiving dual immunosuppression. This study explored the tolerability and cumulative benefit of combining basiliximab with triple-drug therapy-cyclosporine (USP Modified, Neoral), mycophenolate mofetil, and steroids. METHODS: In a randomized, double-blind, placebo-controlled, multicenter study, 123 kidney transplant recipients received either basiliximab at 20 mg before transplantation (day 0) and 20 mg on day 4 (n=59), or placebo (n=64). All received triple-drug immunosuppression and were followed for 6 months. RESULTS: Tolerability of basiliximab was equivalent to placebo, with no increase in serious adverse events, infection, malignancy, or posttransplant lymphoproliferative disorder. At 6 months, there were trends in favor of basiliximab over placebo in the incidences of first biopsy-confirmed acute rejection (15.3% vs. 26.6%, P=NS) and of acute rejection treated with antibody (5.1% vs. 15.6%, P=NS). Kaplan-Meier estimates at 4 weeks and 6 months were significantly in favor of basiliximab treatment for first acute rejection, biopsy-confirmed rejection, rejection episodes treated with antibody therapy, and treatment failure. Renal function improved more rapidly in the basiliximab group, with mean creatinine clearance at week 2 being 54.7 mL/min versus 43.2 mL/min for placebo (P=0.034). At 12 months, patient survival was 100% in both groups; graft survival was 94.9% with basiliximab and 92.2% with placebo. CONCLUSIONS: Basiliximab immunoprophylaxis is safe, well tolerated, and shows a trend toward reduction in number of acute rejection episodes in renal transplant patients receiving cyclosporine, mycophenolate mofetil, and steroids.

Sirolimus-associated pulmonary toxicity.

BACKGROUND: Pulmonary toxicity has recently been recognized as a potentially serious complication associated with sirolimus therapy. We further detail this condition on the basis of our own cases and those reported in the literature. METHODS: We report three cases of suspected sirolimus-induced pulmonary toxicity that occurred in three renal transplant recipients and searched PubMed for all previously reported cases. RESULTS: Including our current cases, 43 patients with sirolimus-induced pulmonary toxicity have now been reported. Clinical data were incomplete in 28 cases. Analysis of available data for 15 patients revealed that the most commonly presenting symptoms were dyspnea on exertion and dry cough followed by fatigue and fever. Chest radiographs and high-resolution computed tomography scans commonly revealed bilateral patchy or diffuse alveolo-interstitial infiltrates. Bronchoalveolar fluid analysis and lung biopsy in selected case reports revealed several distinct histologic features, including lymphocytic alveolitis, lymphocytic interstitial pneumonitis, bronchoalveolar obliterans organizing pneumonia, focal fibrosis, pulmonary alveolar hemorrhage, or a combination thereof. The diagnosis of sirolimus-associated pulmonary toxicity was made after an exhaustive work-up to exclude infectious causes and other pulmonary disease. Sirolimus discontinuation or dose reduction resulted in clinical and radiologic improvement in all 15 patients within 3 weeks. CONCLUSION: The temporal relationship between sirolimus exposure and onset of pulmonary symptoms in the absence of infectious causes and other alternative pulmonary disease and the associated clinical and radiologic improvement after its cessation suggests a causal relationship. Because the use of sirolimus in organ transplantation has become more widespread, clinicians must remain vigilant to its potential pulmonary complication.