Early Post-transplant Phase Research Articles

Infectious agents after liver transplant: etiology, timeline and patients' cell-mediated immunity responses.

Infections continue to be one of the leading causes of morbidity and mortality in liver transplant recipients. We retrospectively reviewed the symptomatic infectious episodes that occurred during the first year post-transplant to determine time of onset, causative pathogens and cell-mediated immunity response patterns. Ninety-eight of the 202 (48.5%) recipients enrolled developed at least one infectious episode. The total number of infectious episodes was 135: 77 (57.1%) bacterial, 45 (33.3%) viral and 13 (9.6%) fungal. The most frequently isolated bacteria were Escherichia coli (21 isolates) and Klebsiella pneumoniae (19 isolates). Overall, extended-spectrum beta lactamase-producing and methicillin-resistant organisms were responsible for 29 (29/77; 37.7%) infectious episodes. Members of the herpes virus group, in particular cytomegalovirus (34/45 viral infections, 75.5%), were detected. Candida species (9 isolates) followed by Aspergillus species (4 isolates) were isolated. The majority of infections (63%) occurred during the early post-transplant phase (<1month), whereas only 8/135 episodes (5.9%) were detected after the sixth month (late phase). Significantly lower median ImmuKnow® intracellular ATP values in patients who developed bacterial and fungal infections compared to infection-free patients were observed (P<0.0001 and P=0.0016, respectively), whereas patients who developed a viral infection had a median intracellular ATP level not statistically different compared to uninfected patients (P=0.4). Our findings confirm that bacteria are responsible for the majority of symptomatic infections and occur more frequently during the first month post-transplant. The ImmuKnow® measurements can be a useful tool for identifying patients at high risk of developing infection, particularly of fungal and bacterial etiology.

In Vivo Tracking of Human Hematopoiesis Reveals Patterns of Clonal Dynamics during Early and Steady-State Reconstitution Phases

SummaryHematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.

Influence of combined CYP3A4 and CYP3A5 single-nucleotide polymorphisms on tacrolimus exposure in kidney transplant recipients: a study according to the post-transplant phase.

The present study investigated in Tunisian renal transplant patients, genetic polymorphisms of CYP3A4 -392A>G and CYP3A5 6986A>G and their influence on tacrolimus (Tac) pharmacokinetics during early and late post-transplant (PT) phases and established customized ranges of Tac doses matching the C0 target levels according to CYP3A4 and CYP3A5 genotype combination and the PT phase. We included adult Tunisian patients having received Tac for de novo kidney grafts and undergone a therapeutic drug monitoring of Tac by morning C0 monitoring during early (1 to 90 days) and late (over 90 days) PT phases. The genomic DNA was extracted from peripheral blood mononuclear cells using a salting-out procedure. CYP3A4 promoter (rs2740574; -392A>G) and CYP3A5 (rs776746; 6986A>G) SNP genotyping was analyzed using PCR-RFLP. Fifty-two patients were enrolled in the study. During the early PT phase, the CYP3A5 polymorphism but not that of CYP3A4, correlates significantly with Tac dose-normalized C0 (C0/D ratio). During the late PT phase, the effect of CYP3A4 polymorphism becomes significant and that of CYP3A5 becomes nonsignificant on Tac C0/D Tac. The mean daily doses (mg/kg) matching therapeutic C0, regardless of the CYP3A genotypes, were 0.16 ± 0.05 and 0.10 ± 0.05 during early and late PT phase, respectively. Carriers of the CYP3A4*1B allele require higher doses to maintain the C0 in the therapeutic range during the two PT phases. However, patients carrying the CYP3A5*1 require significant higher Tac doses, only during the early phase. Our data support a critical role of the CYP3A5 6986A>G and CYP3A4 -392A>G polymorphisms on the variation of Tac exposure during the early and the late PT phase, respectively. The establishment of customized Tac doses, according to CYP3A4/CYP3A5 genotype combination and the PT time, may allow preventing graft rejection and improving the safety profile of this drug. Further studies are needed to investigate this issue.

Limited Sampling Strategy for Mycophenolic Acid in Chinese Kidney Transplant Recipients Receiving Enteric-Coated Mycophenolate Sodium and Tacrolimus During the Early Posttransplantation Phase.

Mycophenolic acid (MPA), a potent immunosuppressant, is widely used in solid organ transplantations. This study aimed to investigate the pharmacokinetics of enteric-coated mycophenolate sodium (EC-MPS) in Chinese adult renal allograft recipients and to generate optimal model equations for estimation of the MPA area under the concentration-time curve from 0 to 12 hours (AUC0-12 h), using a limited sampling strategy (LSS). Serial blood samples were collected over 12 hours from 38 recipients of a primary living-related donor kidney graft treated with EC-MPS, tacrolimus, and corticosteroid. MPA concentrations were evaluated using an enzyme-multiplied immunoassay technique. The LSSs were developed and validated by multiple regression analysis using a 2-group method (test group, n = 19; validation group, n = 19). The best algorithms obtained from the test group were the following: 15.09 + 1.05 × C1.5 + 1.8 × C4 + 4.18 × C6 (for 3 time points, r = 0.902) and 10.44 + 0.7 × C1 + 1.22 × C2 + 1.75 × C4 + 4.36 × C6 (for 4 time points, r = 0.941). When these algorithms were tested in the validation group, there were no significant differences in prediction errors. LSSs using time points of 1.5, 4, and 6 hours or 1, 2, 4, and 6 hours after dose provide effective and reliable estimations of the MPA AUC0-12 h in Chinese renal allograft recipients treated concomitantly with EC-MPS and tacrolimus during the early posttransplantation phase.

Nutritional Status in Japanese Renal Transplant Recipients With Long-term Graft Survival

BackgroundNutritional status affects clinical outcomes in patients with chronic renal failure. Glucose intolerance, dyslipidemia, obesity, hypertension, and a calcium–phosphorus–vitamin D imbalance are the major nutritional and metabolic problems that occur in posttransplant patients. In this study, we assessed the daily intake in long-term renal transplant recipients to determine whether they have sufficient nutrients based on the Japanese nutrition recommendations (recommended dietary allowances [RDA] in Japan 2010). Subjects and MethodsThirty-one renal allograft recipients followed for >10 years (median, 16.3) were recruited. The median serum creatinine level was 1.2 g/dL (95% CI, 0.6–3.4). We estimated the intake of nutrients, including protein and salt, using a simple food frequency questionnaire. ResultsThe median body mass index was 20.1 kg/m2. The median total energy intake was 1566 kcal/d (95% CI, 892–2556). The daily intake of protein and salt was 65.1 and 9.1 g/d, respectively. The calcium, iron, vitamin D, and vitamin K intakes were 423 mg, 7.0 mg/d, 9.7 μg/d, and 197 μg/d, respectively. Patients with dyslipidemia displayed greater amounts of lipid and calcium than those with normal lipid levels. DiscussionOur findings suggest that long-term renal transplant recipients in Japan seem to restrict caloric intake, while maintaining appropriate intake of protein, lipids, carbohydrates, and vitamins A, D, and K. However, daily calcium and iron intake were insufficient; salt intake was greater than the recommended dietary allowances in all subjects. In patients with dyslipidemia, calcium intake was lower than those in patients without dyslipidemia, although their intake of lipids was also lower than those without dyslipidemia. ConclusionNutritional guidance beginning during the early posttransplant phase helps to foster a healthy body mass index and nutritional balances for long-term renal transplant recipients. However, greater salt restriction was needed, and additional nutritional guidance aiming to prevent osteoporosis seems to be considered.

γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay.

γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical evaluation.

Decrease in the percentage of peripheral blood CXCR3highCD4+ lymphocytes after renal transplantation

IntroductionPrevious studies suggest that infiltration into the graft of active T cells following kidney transplantation depends on the expression of chemokines and their interaction with their T-cell receptors. However, little is known about the natural history of the expression of these molecules during the early post-transplantation phase. AimTo evaluate the percentage of CXCR3highCD4+ and CCR4highCD4+ cells, as markers of the Th1 and Th2 populations, in peripheral blood from uremic patients before transplantation and six months after maintaining an acceptable kidney graft function. Material and methodsFlow cytometry was used to measure CXCR3highCD4+ and CCR4highCD4+ cells from 44 consecutive patients who received a kidney transplant at our center. Measurements were made at the time of transplantation and six months later. ResultsThere was a significant reduction after transplantation in the CXCR3highCD4+/CCR4highCD4+ balance (10.68±20.28 vs. 2.01±3.15, p=0.001). Separate analysis of each subset showed a significant reduction after transplantation in CXCR3highCD4+ (2.37±2.75 vs. 1.49±2.66, p=0.010) but no difference in CCR4highCD4+ (0.83±1.01 vs. 1.01±1.12, p=0.812). ConclusionPrior to kidney transplantation uremic patients have an immunologic activation with Th1 polarization (studied by analyzing the CXCR3highCD4+ and CCR4highCD4+ populations) that falls after transplantation. This can be monitored with the CXCR3highCD4+ lymphocyte subset. This may help understand the pathologic mechanisms intervening in immunologic dysfunction of kidney grafts.

The Optimal MMF Dose in Tacrolimus Treated Patients

To the Editor: We appreciate the opportunity to respond to the Letter to the Editor of Knotek et al (1Knotek M Galešić Ljubanović D Mihovilović K Maksimović B Tacrolimus or mycophenolate in kidney transplantation—Less, or more?.Am J Transplant. 2014; 14: 1220Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). In their letter they propose a novel immunosuppressive strategy, aimed at treating de novo kidney transplant recipients with (very) low-dose tacrolimus and a higher mycophenolate mofetil (MMF) dose. The hypothesis of their prospective study is that reduced exposure to tacrolimus will result in an improved histology in 1-year protocol biopsies (less interstitial fibrosis and tubular atrophy), while the higher MMF dose (3 g daily) compensates for the lower tacrolimus exposure and will maintain efficacy. We feel that the observations in our analysis of three large, randomized-controlled trials (2Bouamar R Shuker N Hesselink DA et al.Tacrolimus predose concentrations do not predict the risk of acute rejection after renal transplantation: A pooled analysis from three randomized-controlled clinical trials.Am J Transplant. 2013; 13: 1253-1261Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) suggest that the currently targeted tacrolimus predose concentrations may be too high and that a further reduction of the target levels is possible without compromising efficacy. We agree that optimizing MMF exposure is likely to further improve the outcome. What the optimal MMF dose would be has been a matter of debate for some time. In the registration trials, MMF doses of 2 or 3 g per day were compared with placebo or azathioprine treatment. Patients in these studies were on full-dose cyclosporine (CsA) treatment. It may well be that in a setting of low-dose tacrolimus treatment (say target predose concentrations in the range of 3 ng/mL) higher MMF doses would offer a benefit. It is known that in tacrolimus-treated patients an MMF dose of 2 g/day will result in mycophenolic acid (MPA) exposure below the target in 25% of patients in the first month after transplantation (and in CsA co-treated patients this occurs in no less than 50%). Temporary intensified dosing has been suggested to avoid reduced MPA exposure in the early posttransplant phase (3Gourishankar S Houde I Keown PA et al.The CLEAR study: A 5-day, 3-g loading dose of mycophenolate mofetil versus standard 2-g dosing in renal transplantation.Clin J Am Soc Nephrol. 2010; 5: 1282-1289Crossref PubMed Scopus (48) Google Scholar). Knotek et al (1Knotek M Galešić Ljubanović D Mihovilović K Maksimović B Tacrolimus or mycophenolate in kidney transplantation—Less, or more?.Am J Transplant. 2014; 14: 1220Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar) propose a 3 g MMF dose in the first week, and in half of the patients this dose is maintained thereafter. Whether or not a prolonged duration of higher MMF dosing would offer a benefit when very low tacrolimus concentrations are targeted remains to be seen. One could expect more MMF-related toxicity in a substantial proportion of patients, as observed in an older MMF dose finding study in tacrolimus-treated patients (4Squifflet JP Bäckman L Claesson K et al.Dose optimization of mycophenolate mofetil when administered with a low dose of tacrolimus in cadaveric renal transplant recipients.Transplantation. 2001; 72: 63-69Crossref PubMed Scopus (122) Google Scholar). Therapeutic drug monitoring for MPA may identify patients with over-exposure, and in such patients, dose reductions may improve tolerability to the proposed regimen (5van Gelder T Therapeutic drug monitoring for mycophenolic acid is value for (little) money.Clin Pharmacol Ther. 2011; 90: 203-204Crossref PubMed Scopus (15) Google Scholar). The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. Dr. Hesselink has received lecture fees from Astellas Pharma. Dr. van Gelder has received lecture fees from Astellas Pharma, and Roche. Dr. Bernasconi has received consulting fees from F. Hoffmann-La Roche Ltd, Basel, Switzerland. All other authors have no conflicts to declare.

Which is more suitable for kidney transplantation at the early post-transplantation phase in China - low dosing or standard dosing of enteric-coated mycophenolate sodium?

To investigate the pharmacokinetics of enteric-coated mycophenolate sodium (EC-MPS) and the clinical outcome in kidney transplant recipients in the early post-transplantation phase. Then explain which regimen is more suitable for Chinese renal transplant recipients. In total, 60 de novo kidney transplant recipients treated with tacrolimus and steroids were randomised to receive EC-MPS at standard dose (SD; 1440mg/day; n=28) or low dose (LD; 1080mg/day; n=32). Efficacy parameters, safety and tolerability were assessed over a 6-month study period. Full mycophenolic acid (MPA) areas under the curve (AUCs) were completed on days 3 and 5, whereas a three-point limited sampling strategy (LSS) was utilised for MPA AUC assessments at 2weeks and months 1, 3 and 6 (the LSS for three-time-point MPA AUC 0-12h (mgh/l) =15.99+0.87C1h +0.68C2h +7.85C4h ; r(2) =0.8670. The mean AUC levels at day 3 and day 5 in the SD group were significantly higher than in the LD group (57.4mg·h/l vs. 38.2mg·h/l and 59.3mg·h/l vs. 44.8mg·h/l, respectively, p<0.01). There was a trend for fewer clinically diagnosed acute rejections in the SD group vs. the LD group at 6months (7.1% vs. 12.5%). This trend was also present when acute rejection was analysed as biopsy-proven cases. There were significantly more acute rejections (all definitions) in patients with MPA AUC levels <30mg·h/l compared with those with MPA AUC levels ≥30mg·h/l within 6months (p<0.05). Renal function, incidence of infection and haematological disorders were not significantly different in either study group. Early adequate MPA exposure in renal transplant recipients can be achieved with a higher starting dose. In addition, a SD regimen was as well-tolerated as a LD regimen. Furthermore, early adequate MPA exposure significantly lowered the rate of acute rejection without compromising safety and tolerability.

Cyclosporine A and tacrolimus combined with enteric-coated mycophenolate sodium influence the plasma mycophenolic acid concentration - a randomised controlled trial in Chinese live related donor kidney transplant recipients

To observe the different concentrations of mycophenolic acid (MPA) during the early post-transplant phase for various combinations of cyclosporine A (CsA) and tacrolimus (Tac) with enteric-coated mycophenolate sodium (EC-MPS). A total of 42 Chinese adults receiving live related donor kidney transplants were studied. All received a triple immunosuppressive regimen of EC-MPS, CsA/Tac and corticosteroids and were divided randomly into CsA (n=21) and Tac (n=21) combination groups. The dosage of EC-MPS was the same (1440mg/day) in the two groups. The MPA concentration was evaluated with an enzyme-multiplied immunoassay technique (EMIT) and the pharmacokinetic characteristics were investigated in both groups. The mean maximum plasma concentrations (Cmax ) of MPA in the CsA and Tac groups were 11.365±9.522 and 9.748±7.523μg/ml, respectively (p=0.137). The maximum times to Cmax (Tmax ) were 2.54±1.53 and 2.67±1.08h, respectively (p=0.341). The mean MPA 12-h areas under the curve (MPA-AUC0-12h ) were 59.463±16.252 and 77.535±33.615μgh/ml (p=0.003) and the mean residence times (MRT) were 3.71±0.829 and 3.928±0.923h (p=0.038). Combined with the same EC-MPS dosage (1440mg/day), the MPA-AUC0-12 of the Tac group was higher than that of the CsA group, and the Tac group had a longer MRT after kidney transplantation. Our data indicate that the concentration of MPA should be monitored in clinical therapy when EC-MPS is combined with different calcineurin inhibitors to reduce acute allograft rejection and avoid adverse events.

Anthocyanins from Chinese Bayberry Extract Activate Transcription Factor Nrf2 in β Cells and Negatively Regulate Oxidative Stress-Induced Autophagy

Islet replacement is a promising cure for insulin-dependent diabetes but is limited by a massive early cell death following transplantation. Overburden oxidative stress is one of the major factors causing cell damage. We have shown previously that anthocyanins in Chinese bayberry extract protected β cells (INS-1) from hydrogen peroxide (H₂O₂)-induced apoptosis and decreased grafts' apoptosis after transplantation partially through heme oxygenase-1 (HO-1) up-regulation. In the present study, we observed that H₂O₂ stimulation induced autophagy in β cells. Inhibition of autophagy increased cell viability and decreased cell death. Anthocyanin pretreatment attenuated oxidative stress-mediated autophagic cell death. Anthocyanins activated antioxidant transcription factor Nrf2 in INS-1 cells, and Nrf2/HO-1 negatively regulated autophagy process. Furthermore, we here demonstrate that autophagy also took place in β cell grafts during the early post-transplantation phase. β Cells pretreated with anthocyanins displayed decreased extent of autophagy after transplantation. Taken together, these findings further supported the conclusion that anthocyanins could serve as a protective agent of β cells and suggested that autophagy might play a role in β cells during transplantation.

Urinary proteomic shotgun approach for identification of potential acute rejection biomarkers in renal transplant recipients

BackgroundAcute rejection (AR) episodes in renal transplant recipients are suspected when plasma creatinine is elevated and other potential causes out ruled. Graft biopsies are however needed for definite diagnosis. Non-invasive AR-biomarkers is an unmet clinical need. The urinary proteome is an interesting source in the search for such a biomarker in this population.MethodsIn this proof of principle study, serial urine samples in the early post transplant phase from 6 patients with biopsy verified acute rejections and 6 age-matched controls without clinical signs of rejection were analyzed by shotgun proteomics.ResultsEleven proteins fulfilled predefined criteria for regulation in association with AR. They presented detectable regulation already several days before clinical suspicion of AR (increased plasma creatinine). The regulated proteins could be grouped by their biological function; proteins related to growth and proteins related to immune response. Growth-related proteins (IGFBP7, Vasorin, EGF and Galectin-3-binding protein) were significantly up-regulated in association with AR (P = 0.03) while proteins related to immune response (MASP2, C3, CD59, Ceruloplasmin, PiGR and CD74) tended to be up-regulated ( P = 0.13).ConclusionThe use of shotgun proteomics provides a robust and sensitive method for identification of potentially predictive urinary biomarkers of AR. Further validation of the current findings is needed to establish their potential clinical role with regards to clinical AR diagnosis.Trial registrationClinicalTrials.gov number NCT00139009

Lactic Acidosis After Cardiac Transplantation: Foe or Common Innocent Bystander?

Lactic acidosis (LA) can occur in the early post heart transplant (HTx) phase. It is hypothesized to be related to high-dose inotropic support post HTx and/or metabolic derangements from chronic advanced heart failure. As such, restoring hemodynamic stability with drugs and/or mechanical circulatory support (MCS) prior to HTX should help mitigate this problem. Our aim was to evaluate the incidence of LA and associated outcomes post HTx.

A case of BK virus nephropathy and cytomegalovirus infection concurrent with plasma cell–rich acute rejection

The BK virus is a double-stranded DNA virus to which 90% of adults have been exposed. BK virus infections typically result in an oral or respiratory infection; however, BK virus reactivation is an infectious disease of concern in kidney transplant recipients. The prevalence of BK virus nephropathy (BKN) in kidney transplant recipients is approximately 5%, and most cases occur within one yr after kidney transplantation. Graft survival of BKN is reported to be 30-60%, and the standard treatment strategy for BKN is reducing immunosuppressive therapy and close monitoring for rejection. Viral infection is most common in the early post-transplantation phase, and BKN or acute rejection is one of the major factors involved in graft loss. However, in this report, we describe the successful management of BKN and cytomegalovirus infection concurrent with plasma cell-rich acute rejection.

Functional assessment of automatically sorted pancreatic islets using large particle flow cytometry

The size composition of human islet preparations has been attributed to functional potency, islet survival and transplantation outcomes. In the early post-transplantation phase islets are supplied with oxygen by diffusion only and are at risk of critical hypoxia. The high rate of early islet graft dysfunction is in part attributed to this condition. It has been presumed that islets with smaller diameter, and therefore smaller diffusion distance, are superior to large islets regarding early survival rate and graft function. In this study we aimed to evaluate Complex Object Parametric Analysis and Sorting (COPAS) as a device for automated sorting of human islets. The use of COPAS was validated for accuracy and sensitivity using polystyrene beads of known diameters. Based on time of flight relative to particle isolated islets were then automatically sorted and analyzed for viability and function using handpicked islets as control. Our results suggest that COPAS enables the automated and accurate sorting of islets with no negative impact on their integrity and viability. Thus, COPAS is an adequate tool for size-specific analysis of pancreatic islets and may be considered as part of a platform for automated high-throughput screening of pancreatic islets.

Prospective evaluation of biliary reconstruction with duct-to-duct continuous suture in adult live donor liver transplantation

PurposeBiliary reconstruction remains the Achilles’ heel of adult live donor liver transplantation (LDLT). The study aims to investigate the feasibility of duct-to-duct hepaticocholedochostomy in LDLT.MethodsPerioperative data from 30 consecutive LDLT aiming at duct-to-duct reconstruction of the biliary tract using a continuous suture technique were prospectively collected. Nineteen recipients (63.3%) had one graft bile duct. Eleven recipients (36.7%) had two or three graft bile ducts. The median follow-up was 50 months.ResultsThe overall biliary complication rate was 23.3%. Two recipients developed biliary stricture (6.7%), and two recipients (6.7%) presented with biliary leakage in early posttransplant phase (<90 days). One recipient suffered from bilioma (3.3%), and two recipients (6.7%) presented with biliary stricture in later posttransplant phase (>90 days). No correlation was found between the number of graft bile ducts and the incidence of biliary complications. No biliary complication-associated necessity for re-transplantation or mortality was observed. On multivariate analysis, no single risk factor associated with biliary complication could be identified. All biliary complications were successfully treated with Roux-en-hepaticojejunostomy and/or with endoscopic interventions.ConclusionDuct-to-duct hepaticocholedochostomy with continues suture represents a safe and feasible procedure for biliary reconstruction in LDLT. Recipients may benefit from aggressive management of biliary complications with Roux-en-hepaticojejunostomy as compared with repeated endoscopic interventions in early posttransplant phase.

SDF1 Plasma Levels and CD34+ Cells Recirculation After Autologous Peripheral Blood Stem Cell Transplant.

Abstract 4571The chemokine stromal-cell derived factor-1alpha (SDF1) is the major chemotactic stimulus for human hematopoietic stem cell (HSC) and through its receptor CXCR4 is critically involved in directional migration and homing of hematopoietic progenitors and stem cells. Usually HSC mobilization is achieved after a proteolytic disruption of SDF1-CXCR4 axis after G-CSF administration. Several report showed HSC circulating during early post transplant phase but the mechanism of recirculation is not clear. Based on these premises we studied the role of SDF1 during autologous peripheral blood stem cell transplant (aPBSCT) and its relations with recirculating HSC and G-CSF administration. From January 2009 to July 2009 we enrolled 12 patients undergoing aPBSCT for hematologic malignancies or solid tumors: 7 males, 5 females, with a median age of 60 years, affected by multiple myeloma (6 patients), Non Hodgkin's lymphoma (5 patients) and Ewing's sarcoma (1 patient). After conditioning regimen (6 Busulfan and Melphalan, 6 High Dose Melphalan) they received a median of 7.56 ×106/kg cell CD34+ on day 0 and, subsequently early (day +1) or delayed (day +6) G-CSF administration. SDF1 was assayed in plasma samples collected at baseline, on day 0,2,6,8,10,12 after aPBSCT using an immunoenzymatic technique. Characteristics of patients of two groups are shown in table 1. There was no statistical difference in CD34+ cell dose infused.All patients achieved engraftment: neutrophil recovery (PMN count >500 cells/microL) was achieved after a median time of 11 days; platelet recovery (Plt>50×103/microL) after a median time of 14 days; patients were discharged after a median time of 20.5 days. CD34+ cells after transplant were detectable on day +10 (9.06 cells/microL) achieving peak level on day +12 (21.69 cells/microL) confirming timing and magnitude of this phenomenon.SDF1 assay showed a decrease in concentration from the baseline to hematopoietic engraftment: 2278.3 pg/ml (range 1799.1-3619.6) at baseline and 1850.1 pg/ml on day +12 (range 564.4-2551.7). This decrease in concentration was statistically significant (p=0.0094) and was chronologically related to engraftment ad CD34+ recirculation. We found a statistically significant negative correlation between SDF1 plasma levels and CD34+ cell count after aPBSCT: r=-0.957, p0.043. Interestingly we found a negative correlation between SDF1 plasma levels and white blood cell count starting from day +6 to day +12 (r=-0.91, p=0.03). This finding was confirmed by a positive correlation between SDF1 plasma levels on day +6 and time to achieve a PMN count >500 cells/microL: r=0.67, p=0.01 (Figure 1). The correlations found between SDF1 plasma levels and kinetics of engraftment suggested us to compare engraftment of patients on the base of SDF1 plasma levels. Interestingly patients showing SDF1 plasma levels <2300 pg/ml on day +6 (8 patients) compared to patients with higher levels (4 patients) had a faster PMN recovery in terms of days to achieve a PMN count >500 cells/microL (10.5 days vs 16.5 days, p=0.043), days to achieve a PMN count >1000 cells/microL (11 days vs 18 days, p=0.044) and days with a PMN count <100 cell/microL (5 days vs 12 days, p=0.02). Interestingly taking in to account G-CSF administration schedule, the significant decrease of SDF1 plasma levels observed in whole group of patients was noticed only in patients receiving delayed G-CSF. Patients receiving early G-CSF administration showed small variation of chemochine concentration confirming the key role of G-CSF in perturbing SDF1-CXCR4 axis.In conclusion our data confirm the involvement of SDF1-CXCR4 axis in post transplant CD34+ recirculation. SDF1 decrease with subsequent CD34+ release may represent a key mechanism to explain repopulation of hematopoietic stem cell niche after myeloablation induced by conditioning regimen. Furthermore there was a clear relation between SDF1 plasma levels and kinetic of engraftment.Table 1:Characteristics of patientsNumber of patients12sex7 male; 5 femaleage60 years (range 26-65)diagnosisA Group 5 NHL 1 ESB Group 6 MMconditioning regimenBuMelHdMelG-CSF starting day+1+6CD34+x106/kg cells infused7.56 (range 3.1-16.6)Legend: NHL Non Hodgkin's Lymphoma; ES: Ewing's Sarcoma;MM: Multiple Myeloma; BuMel: Busulfan and Melphalan;HdMel:High Dose Melphalan [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Successful High-Dose Ribavirin Treatment of Respiratory Syncytial Virus-Induced Tracheobronchitis and Pneumonia Occuring Pre-Engraftment in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

Abstract 1163Poster Board I-185 IntroductionRespiratory syncytial viruses (RSV) frequently cause severe respiratory disease in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Within the first post-transplant month, progression of upper respiratory tract infection to life-threatening bronchiolitis and pneumonia is common and mortality rates of up to 80% have been reported if left untreated. Aerosolized ribavirin plus/minus immunoglobulins is considered the treatment of choice for RSV pneumonia. With this regimen, the 30-day all cause mortality is still 40% in immunosuppressed HSCT patients. In addition, nebulizing ribavirin may induce toxic side effects on patients and staff. Patients and treatmentConcurrent with an outbreak in the community, RSV infection was diagnosed in 10 out of 29 patients (34%) undergoing allogeneic HSCT in the winter season 2008. Diagnosis was based on RSV-specific PCR of routinely collected throat swaps. Intravenous ribavirin was dosed according to Schleuning et al., 2003 (day 1: 33 mg/kg, day 2-5: 4×16 mg/kg, day 6-10: 3×8 mg/kg) for 8-10 days, followed by oral ribavirin (1800 mg per day) until recovery from symptoms. Airflow obstruction (AFO) was defined as a FEV1/FVC ratio <0.7 or a drop in FEV1 >20% from baseline. All RSV-infected patients (median age 60 years) had undergone reduced-intensity conditioning HSCT for high-risk acute myeloid leukemia (1st CR: n=2; subsequent CR: n=3; untreated or refractory relapse: n=5). GvHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil with (n=8) or without (n=2) antithymocyte globulin. Donors were HLA-idential siblings (n=1), matched (n=8) or mismatched unrelated donors (n=1). ResultsMedian time from HSCT to detection of RSV was 15 (range, 1-40) days. In 7 out of 10 patients, RSV infection was diagnosed pre-engraftment during neutropenia. Four patients had bronchiolitis or pneumonia and 6 patients suffered from tracheobronchitis. High-dose intravenous ribavirin was administered to all patients with pneumonia and/or diagnosis of RSV infection within 7 days after transplantation (n=5). These patients had hypoxia and required oxygen supplementation. Treatment with oral ribavirin was initiated in the other five patients with tracheobronchitis diagnosed on days 12-40 after HSCT. With a median follow up of 6 months, 9 out of 10 patients are alive and in complete remission of their AML. All 9 patients became RSV-negative in throat swabs after a median time of 22 days from start of therapy. Severe AFO caused by RSV pneumonia occurred in 2 patients and improved after treatment. Despite severe lymphopenia, no patient treated for tracheobronchitis progressed to RSV pneumonia. Neutrophil recovery was delayed in the three patients diagnosed with RSV pneumonia pre-engraftment until days 26, 28+ and 111 after HSCT, due to high-dose intravenous ribavirin and/or RSV infection itself. One of these patients died of septic shock associated with pseudomonas aeruginosa-induced pneumonia on day 28 after HSCT in prolonged neutropenia. No other ribavirin-associated toxicity such as hepatotoxicity or clinically relevant hemolysis was observed. Three patients developed acute intestinal GvHD, 2 of them recovering upon steroid treatment and one proceeding to chronic disease despite salvage therapy. One additional patient had steroid-responsive acute GvHD of the skin. ConclusionsHigh-dose intravenous or oral ribavirin therapy appears to be safe and effective even if administered to neutropenic allogeneic HSCT recipients. None of these patients with RSV infection in the early post-transplant period developed bronchiolitis obliterans or persistent AFO. Based on our favourable results, further studies are required. Meanwhile, we suggest to treat all patients with symptomatic RSV infections in the early post-transplant phase with oral or intravenous ribavirin. DisclosuresOff Label Use: Ribavirin is approved for inhalative treatment of RSV infections but not for systemic therapy..

Early Lymphocyte Recovery Predicts Superior Survival after Unmanipulated HLA-Mismatched/Haploidentical Blood and Marrow Transplantation for Patients with Hematological Malignancies.

Background: The crucial role of repopulating lymphocytes following allogeneic hematopoietic stem cell transplantation (allo-HSCT), including the prevention of infections and erudiating residual tumor cells in the early post-transplant phase, has been demonstrated. Currently, unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation with T cell repletion has been an established treatment for patients without HLA-matched related or unrelated donors. However, the prognostic significance of early lymphocyte recovery in this transplant settings is not defined.Methods: We retrospectively analyzed the relationship between day 30 absolute lymphocyte count (ALC30) and transplant outcomes in 217 paients with hematological malignancies receiving T-cell-repleted transplantation from an HLA-mismatched/haploidentical related donors.Results: At the time of this analysis, 44 of the cohort patients had died. The choice of ALC ≥ 235 cells/μL as the cut-off value point was supported by the data because it yielded the greatest differential in survival, based on the chi-square (×2=23.448, P<0.0001) analysed at different cut-off points between the 25% and 75% quartiles (190–460 cells/μL) from the log-rank test. The median follow-up was 543 days (range: 150–1873 days). Patients with an ALC ≥ 235 cells/μL (n=136) experienced a superior median OS compared to patients with an ALC < 235 cells/μL (n=81) (median OS, 582 days vs 510 days, respectively, P<0.0001). Two groups were identified based on an ALC of ≥ 235 cells/μL and <235 cells/μL at days 30 post HSCT. There were no statistical differences in patient's characteristics between the two groups at days 30 taking into accout a P-value of 0.05. One patients (0.46%) failed to neutrophil engraft, 12 patients (5.53%) failed to platelet engraft. The median time to neutrophil and platelet recovery was 13 days (range, 9–49 days) and 16 days (range, 7–356 days), respectively. Eighty-two patients developed grade II to IV acute GVHD with a cumulative incidence of 49%±3.80%. 113 of 160 evaluable patients surviving more than 100 days developed chronic GVHD (limited 82 [51.25%]; extensive 31 [19.38%]) with a cumulative incidence of 63.8%±3.90%. Forty-four of 217 (20.28%) patients died from nonrelapse causes, with a cumulative incidence of TRM of 23.6%±3.40%. In multivariate analysis the ALC30 above the cutoff value 235 cells/μL was associated with improved overall survival (OS) (HR 0.331, 95% CI 0.189–0.578; P<0.0001), leukemia free survival (LFS) (HR 0.299, 95% CI 0.179–0.501; P<0.0001), less relapse (HR 0.345 (95% CI 0.147–0.808); P=0.014), and less transplant-related mortality (HR=0.284; 95%CI 0.148–0.544; P<0.0001). The beneficial effects of a higher ALC30 (≥235 cells/μL) influenced outcomes in patients with both standard and high-risk disease but did not affect relapse in these two groups. The higher ALC30 was also correlated with improved OS and LFS in patients with AML, ALL, and CML, respectively.Conclusion: Our results suggest that the day 30 ALC would appear to be a useful and simple measurement to predict those patients with superior survival after unmanipulated HLA-mismatched/haploidentical transplantation.

Inflammatory Cytokine Levels in the Peri-Stem Cell Transplantation Period Correlate with Fluctuations in Peripheral Blood Counts but Not with Inflammation.

The period from the conditioning regimen to engraftment of allogeneic stem cells is characterized by regimen-induced tissue damage, major fluctuations in blood counts and periods of fever associated with infection and graft-versus-host disease. To understand accompanying changes in cytokine in the post-graft period we studied 11 patients receiving T cell depleted (TCD) (5) or selectively T cell depleted SCT (6). Plasma was stored for every 3 days (total 126 samples) from first day of conditioning with fludarabine, cyclophosphamide and total body irradiation up to day 42 post-transplant. Cytokines measured were:lymphocyte- (TNFa; IFNg, IL-1, IL-6, IL-17), monocyte/macrophage- (IL-1, IL-6; TNFa; IL-12p70; IL-18) or somatic cell- (IL-6, IL-17) derived inflammatory cytokines,anti-inflammatory cytokines (IL-4, IL-5; IL-10, IL-13; IL- 1ra), (c) cytokines involved in leukocyte trafficking (CCL2; CCL4; CCL5; CXCL10), (d) cytokines involved with hematopoietic cell recovery (SCF, IL-7, IL-15, G-CSF), and tissue repair and remodeling following injury (PDGFbb, VEGF, HGF).Cyclosporine was given as GvHD prophylaxis. Three TCD patients developed limited, grade I-II skin GvHD between 21–24 days post-SCT. Four cytokine patterns were observedrarely detected or detected but never elevated: IL-1, IL-2, IL-4, IL-5, IL-10, IL-13 were detectable in some samples, but at a median level of below 10 pg/ml (Fig. A).detectable in >90% of samples in normal levels without significant fluctuations: SCF, SCGF, HGF, and GM-CSF (Fig. B)Inverse correlation of levels with neutrophil counts: G-CSF, MCP-1/CCL2, and IL-6 (FFig. C); lymphocyte counts: IL- 15 (r = −0.44; p < 0.05) (Fig. D), or monocyte counts MCP-1/CCL2 levels (r = −0.61; p < 0.05).Direct correlation with lymphocyte counts: RANTES, IFNg, IL-17, and TNFa, IL-7, LIF, IL-18, and IP-10/CXCL10. RANTES significantly correlated with IL-1ra (monocyte, neutrophil-derived; r=0.79), and with IFNg (r=0.82) levels, as were IFNg (T cell/NK cell-derived) levels with IL-1ra (r=0.89), suggesting that both were regulated through exposure to pathogen-derived Toll-like receptor ligands.To examine the effect of inflammation, we compared body temperature and C-reactive protein (CRP) with cytokine levels. A significant positive correlation was found between these parameters and IL-6 (r=0.33 for temperature; r=0.42 for CRP). Furthermore, IL-6 correlated significantly with G-CSF (r=0.75) and MCP-1/CCL2 (r=0.53), suggesting a common regulatory mechanism and/or cellular source of their synthesis and secretion. These data indicate that fluctuations in many cytokines around the time of SCT are governed by homeostatic mechanisms, or reflect the cell count. Notably during the conditioning regimen cytokine levels tended to fall, and we did not identify cytokine characteristic of the cytokine storm. Inflammatory cytokines were mainly detected in the early post-transplant phase associated with fever and raised CRP but not with GVHD. [Display omitted]