Saturday, September 29, 2012

Haploidentical hematopoietic stem cell transplantation for lymphoma with monosomy of chromosome 6 (loss of heterozygosity in the HLA region) - Who should be a donor?

Hematopoietic stem cell transplantation (HSCT) from an HLA haploidentical family donor is an option for patients who do not have a full HLA matched donor and lack the time to find an unrelated one.  Furthermore, it may facilitate a powerful graft versus leukemia/lymphoma (GVL) effect to help combat hematological malignancies by directly targeting the mismatched HLA expressed on leukemia/lymphoma cells. [3] On the contrary, leukemia/lymphoma cells escape from the surveillance of the donor-derived GVL effect by losing the target HLA (mismatched HLA in GVH direction). This mechanism has been called loss of heterozygosity (LOH) in the HLA gene region on chromosome 6. Taking  the above into account, in this case report, we present a case of lymphoma with monosomy 6, which means natural LOH of HLA, and suggest that selection of a haploidentical family donor matched with the missing HLA haplotype seems to be very effective (read more)

Thursday, September 27, 2012

Concurrent Acute Cellular Rejection Is an Independent Risk Factor for Renal Allograft Failure in Patients With C4d-Positive Antibody-Mediated Rejection

imageBackground: Identification of risk factors for renal allograft failure after an episode of acute antibody-mediated rejection (AMR) may help the outcome of this difficult-to-treat complication.
Methods: During December 2003 to February 2011, 833 kidney graft recipients underwent 1120 clinically indicated biopsies at our center. We reviewed the biopsy results and identified 87 biopsy specimens from 87 patients positive for the degradation product of complement component 4 (C4d) and acute AMR. We generated Kaplan-Meier survival curves and performed a multivariable analysis using the Cox proportional hazards regression model to identify risk factors for allograft failure after C4d+ acute AMR.
Results: Among the 87 patients, 26 had a diagnosis of acute AMR according to the Banff ’09 classification schema, 29 had acute AMR and chronic active AMR, 18 had acute AMR and acute T-cell mediated rejection (TCMR), and 14 had acute AMR, chronic active AMR, and acute TCMR. Kaplan-Meier survival estimates showed that concurrent acute TCMR (P=0.001, Mantel-Cox log-rank test), concurrent chronic active AMR (P=0.03), and time to biopsy (P=0.04) are associated with graft survival. The Cox proportional hazards regression analysis identified that concurrent acute TCMR (hazard ratio, 2.59 [95% confidence interval, 1.21–5.55]; P=0.01) and estimated glomerular filtration rate (hazard ratio, 0.65 [95% confidence interval, 0.48–0.88]; P=0.01) are independent risk factors for allograft loss. Concurrent chronic active AMR or time to biopsy was not associated with graft failure by the multivariable Cox analysis.
Conclusions: Our single-center study has elucidated that concurrent acute TCMR in kidney transplant recipients with C4d+ acute AMR is an independent risk factor for graft failure. Level of allograft function at the time of diagnosis was also an independent predictor of graft loss (read more).

Long-Term Tolerance to Kidney Allografts After Induced Rejection of Donor Hematopoietic Chimerism in a Preclinical Canine Model

imageBackground: Allogeneic hematopoietic cell transplantation provides a reliable method for inducing tolerance toward solid organ grafts. However, this procedure can result in graft-versus-host disease, thereby limiting its application. Here, we test the hypothesis that mixed chimerism can be intentionally reverted to host hematopoiesis without rejection of a kidney graft.
Methods: Recipient dogs were given 2-Gy total-body irradiation (TBI) before and a short course of immunosuppression after marrow infusion from dog leukocyte antigen–identical littermates. All dogs achieved stable mixed chimerism. After a mean of 20 weeks, one cohort of dogs received kidney transplants from their respective marrow donors. Subsequently, recipients were reconditioned with 2-Gy TBI and given autologous granulocyte colony-stimulating factor–mobilized leukocytes (recipient leukocyte infusion [RLI]) that had been collected before marrow transplantation.
Results: Dogs receiving a second TBI and RLI without a kidney transplant rejected their donor hematopoietic graft within 3 weeks. Dogs that received kidney grafts, followed by a second TBI and RLI, rejected their marrow graft without rejecting their transplanted kidneys for periods greater than 1 year.
Conclusion: Mixed chimerism may be clinically reverted to 100% recipient without rejection of a kidney allograft. This finding may have application toward minimizing the risk of graft-versus-host disease in solid organ transplantation patients given hematopoietic cell transplantation from human leukocyte antigen–identical donors (read more).

A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross-validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q-PCR analysis of a five gene-set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training-set (n = 47) and validated on independent test-set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non-AR phenotypes with 91% sensitivity and 90% specificity. The 5-gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool (read more).

Everolimus is associated with a reduced incidence of cytomegalovirus infection following de novo cardiac transplantation

Background : Cytomegalovirus (CMV) causes several complications following cardiac transplantation including cardiac allograft vasculopathy. Previous studies suggested that immunosuppressive treatment based on everolimus might reduce CMV infection. Aiming to better characterize the action of everolimus on CMV and its interplay with patient/recipient serology and anti-CMV prophylaxis, we analyzed data from 3 large randomized studies comparing various everolimus regimens with azathioprine (AZA)- and mycophenolate mofetil (MMF)-based regimens.
Methods : CMV data were analyzed from 1009 patients in 3 trials of de novo cardiac transplant recipients who were randomized to everolimus 1.5 mg/day, everolimus 3 mg/day, or AZA 1–3 mg/kg/day, plus standard-dose (SD) cyclosporine (CsA; study B253, n = 634); everolimus 1.5 mg/day plus SD- or reduced-dose (RD)-CsA (study A2403, n = 199); and everolimus 1.5 mg/day plus RD-CsA or MMF plus SD-CsA (study A2411, n = 176).
Results : In study B253, patients allocated to everolimus experienced almost a 70% reduction in odds of experiencing CMV infection compared with AZA (P < 0.001). In study A2403, CMV infection was low in both everolimus arms, irrespective of CsA dosing, and in study A2411, patients allocated to everolimus experienced an 80% reduction in odds of experiencing CMV infection, compared with MMF (P < 0.001). CMV syndrome/disease was rare and less frequent in everolimus-treated patients. Subgroup analyses showed that the benefit everolimus provides, in terms of CMV events, is retained in CMV-naïve recipients and is independent of anti-CMV prophylaxis or preemptive approaches.
Conclusions : Everolimus is associated with a lower incidence of CMV infection compared with AZA and MMF, which combined with its immunosuppressive efficacy and antiproliferative effects may positively impact long-term outcomes (read more).

Monday, September 24, 2012

Enrichment of Regulatory T Cells in Acutely Rejected Human Liver Allografts

Acute cellular rejection (ACR) occurs frequently after liver transplantation and can usually be controlled. Triggering of allospecific immune responses and lack of immunoregulation are currently suggested as a cause of ACR, but there are no investigations of intrahepatic immune responses during ACR. Therefore we prospectively analyzed the intrahepatic T cell infiltration pattern in correlation to the severity of ACR in a cohort of patients with graft hepatitis (n = 151). While CD4+ cells dominated the portal infiltrates in mild–moderate ACR, CD8+ cells prevailed in severe ACR. Furthermore portal CD8+ and not CD4+ infiltration correlated with serum transaminases and with the likelihood of subsequent ACRs. Surprisingly, the rise of portal effector T cells density during ACR was surpassed by the increase in portal infiltration of regulatory T cells by a factor of two. Thus ACRs rather showed an increase and not a lack of regulation, as was suggested by analysis of peripheral blood mononuclear cells. Despite the pattern of enhanced immunoregulation, patients with severe ACR had a higher risk for subsequent rejections and showed a trend to a reduced survival. Thus, patients with severe rejections might need a modification of their immunosuppression to improve prognosis (read more).

HIV and HLA Class I: An Evolving Relationship

Successful vaccine development for infectious diseases has largely been achieved in settings where natural immunity to the pathogen results in clearance in at least some individuals. HIV presents an additional challenge in that natural clearance of infection does not occur, and the correlates of immune protection are still uncertain. However, partial control of viremia and markedly different outcomes of disease are observed in HIV-infected persons. Here, we examine the antiviral mechanisms implicated by one variable that has been consistently associated with extremes of outcome, namely HLA class I alleles, and in particular HLA-B, and examine the mechanisms by which this modulation is likely to occur and the impact of these interactions on evolution of the virus and the host. Studies to date provide evidence for both HLA-dependent and epitope-dependent influences on viral control and viral evolution and have important implications for the continued quest for an effective HIV vaccine (read more)

Poor seroprotection but allosensitization after adjuvanted pandemic influenza H1N1 vaccine in kidney transplant recipients

Background : Seasonal and pandemic influenza virus infections in renal transplant patients are associated with poor outcomes. During the pandemic of 2009–2010, the AS03-adjuvanted monovalent H1N1 influenza vaccine was recommended for transplant recipients, although its immunogenicity in this population was unknown. We sought to determine the safety and immunogenicity of an adjuvant-containing vaccine against pandemic influenza A H1N1 2009 (pH1N1) administered to kidney transplant recipients.
Methods : We prospectively enrolled 124 adult kidney transplant recipients in the fall of 2009 at two transplant centers. Cohort 1 (n = 42) was assessed before and after pH1N1 immunization, while Cohort 2 (n = 82) was only assessed post immunization. Humoral response was measured by the hemagglutination inhibition assay. Vaccine safety was assessed by adverse event reporting, graft function, and human leukocyte antigen (HLA) alloantibody measurements.
Results : Cohort 1 had a low rate of baseline seroprotection to pH1N1 (7%) and a low rate of seroprotection after immunization (31%). No patient <6 months post transplant (n = 5) achieved seroprotection. Seroprotection rate was greater in patients receiving double as compared with triple immunosuppression (80% vs. 24%, P = 0.01). In Cohort 2, post-immunization seroprotection was 35%. In both cohorts, no confirmed cases of pH1N1 infection occurred. No difference was seen in estimated glomerular filtration rate before (54.3 mL/min/1.73 m2) and after (53.8 mL/min/1.73 m2) immunization, and no acute rejections had occurred after immunization at last follow-up. In Cohort 1, 11.9% of patients developed new anti-HLA antibodies.
Conclusion : An adjuvant-containing vaccine to pH1N1 provided poor seroprotection in renal transplant recipients. Receiving triple immunosuppression was associated with a poor seroresponse. Vaccination appeared safe, but some patients developed new anti-HLA antibodies post vaccination. Alternative strategies to improve vaccine responses are necessary (read more).

Tuesday, September 18, 2012

Do mesenchymal stem cells function across species barriers? Relevance for xenotransplantation

Background:  Allogeneic mesenchymal stem (stromal) cells (MSC) are a promising therapy for various pathological conditions. Genetically modified pig MSC have been demonstrated to downregulate the human T-cell response to pig antigens in vitro. Before genetically modified pig MSC can be used clinically, however, evidence needs to be provided to indicate whether they will survive in a human (xenogeneic) host.
Literature Search and Results:  A literature search through the end of 2011 identified 94 reports of the in vivo cross-species administration of MSC in a variety of experimental models. The majority (n = 89) involved the use of human MSC in various other species, with an occasional study using pig, rat, or guinea-pig MSC. When human MSC were used, they were largely derived from the bone marrow, adipose tissue, or umbilical cord blood. The routes of administration were varied, although almost half of the studies utilized the intravenous route. In 88 experiments (93.6%), there was evidence that the MSC engrafted and functioned across the species barrier, and in only six cases (6.4%) was there evidence of failure to function. Importantly, MSC function was confirmed in several different cross-species models. For example, human MSC functioned in no fewer than seven different recipient species.
Conclusions:  The data provided by this literature search strengthen the hypothesis that pig MSC will function satisfactorily in a different species, for example, humans. The data also suggest that our own in vitro observations on the efficacy of pig MSC in downregulating the strength of the human T-cell response to pig antigens will likely be reproduced in vivo in pre-clinical large animal models and in clinical trials (read more)

Saturday, September 15, 2012

The Natural History of Clinical Operational Tolerance After Kidney Transplantation Through Twenty-Seven Cases

We report here on a European cohort of 27 kidney transplant recipients displaying operational tolerance, compared to two cohorts of matched kidney transplant recipients under immunosuppression and patients who stopped immunosuppressive drugs and presented with rejection. We report that a lower proportion of operationally tolerant patients received induction therapy (52% without induction therapy vs. 78.3%[p = 0.0455] and 96.7%[p = 0.0001], respectively), a difference likely due to the higher proportion (18.5%) of HLA matched recipients in the tolerant cohort. These patients were also significantly older at the time of transplantation (p = 0.0211) and immunosuppression withdrawal (p = 0.0002) than recipients who rejected their graft after weaning. Finally, these patients were at lower risk of infectious disease. Among the 27 patients defined as operationally tolerant at the time of inclusion, 19 still display stable graft function (mean 9 ± 4 years after transplantation) whereas 30% presented slow deterioration of graft function. Six of these patients tested positive for pre-graft anti-HLA antibodies. Biopsy histology studies revealed an active immunologically driven mechanism for half of them, associated with DSA in the absence of C4d. This study suggests that operational tolerance can persist as a robust phenomenon, although eventual graft loss does occur in some patients, particularly in the setting of donor-specific alloantibody (read more).

Friday, September 14, 2012

Pairs of Kidneys Transplanted From the Same Donor: Is There Any Difference?

The aim of this study was to compare the evolution of the first kidney in relation to the second kidney transplanted from the same donor, focusing on the impact that a longer cold ischemia time may have as an independent variable.
Material and Methods: The study included 44 pairs of kidneys transplanted from the same donor between February 2008 and October 2010, divided into Groups 1 and 2 according to the graft placement order. The variables analyzed were age, sex, comorbidities, number of transfusions, length of hospital stay, maximum peak PRA, immunologic incompatibility, ischemia time, delayed graft function (DGF), presence of rejection, creatinine clearance at first week, at 3 months and at 1 year, and vascular and tract complications in each group.
Results: The mean cold ischemia time was 15.6 hours in Group 1 and 20.1 hours in Group 2 (P < .001). The average recipient age was 52.79 years in Group 1 and 54.52 years in Group 2, with an equal sex ratio in the two groups; an average of 2.06 PRC were transfused prior to transplantation in Group 1 and 0.93 PRC in Group 2; the average length of stay was similar in the two groups. Major DR incompatibility was only found in Group 2 (P < .03). Creatinine clearance at first week, DGF and acute rejection showed worse results in Group 2, but these differences were not significant. Vascular complications were present in 4.5% and 2.3% of Groups 1 and 2, respectively, and tract complications were 6.8% and 11.4%.
Conclusions: A greater tendency to DGF, early rejection and tract complications were found in the group with longer ischemia time, although the difference was not statistically significant. Larger series will be necessary to confirm our results (read more).

History of Eurotransplant

In 1967, the Dutch immunologist Jon van Rood called Eurotransplant into life. From the beginning it was a non-profit private foundation. Initially it was a loose cooperation, where tissue typing laboratories and transplantation centers joined to achieve a better result for their kidney patients, a longer survival based on better immunological matching from a bigger donor pool. Other centers from the Benelux states, Germany and Austria soon joined the first few cooperating centers. Switzerland was also a member of Eurotransplant, but left the organization in 1978. Based on the pioneering work of the Leiden histocompatibility lab, the allocation system became more and more sophisticated and was extended to other solid organs. Since the 1980s Eurotransplant has allocated donor livers, hearts, and pancreas. Thereafter, the allocation also included lungs and small bowel. From 1996 a new kidney allocation system, the ETKAS, was introduced, and after the Acceptable Mismatch program and the Eurotransplant Senior Program (known unofficially as “old-for-old” program) were introduced. The main principle remains to adapt the allocation rules continuously according to the newest scientific data serving all organs. In 1991 the German reunification centers in the former Eastern Germany became part of Eurotransplant. In 1999, Slovenia, and in 2007 Croatia joined Eurotransplant. For the transplant centers in these two countries, membership meant positive changes and is regarded as a success story. Both donor numbers and transplant possibilities increased and equal chances are assured for their patients on the common Eurotransplant waiting list. Hungary, joining Eurotransplant next year, hopes to experience the same (read more).

Thursday, September 13, 2012

Renal grafts from anti-hepatitis B core-positive donors: a quantitative review of the literature

Organ shortage is a major problem in transplantation. The use of organs from hepatitis B surface antigen (HBsAg)-negative and hepatitis B core antibody (HBcAb)-positive donors could significantly increase the donor pool. However, little information is available about the impact of HBcAb status of renal donors on viral transmission to recipients. To address this issue, the present quantitative review of relevant studies has been performed.
Materials and methods : Electronic databases including Medline, EMBASE, ISI, and Scopus were systematically searched for studies that evaluated risk of hepatitis B virus (HBV) transmission through renal transplantation from HBsAg-/HBcAb+ donors. Eligible studies were identified according to predefined criteria. The final outcome was one of HBV markers seroconversion defined as HBsAg, hepatitis B surface antibody (HBsAb), or HBcAb detection in previously seronegative end-stage renal disease (ESRD) patients after transplantation, and without other identified major sources of infection.
Results : Nine studies with 1385 eligible kidney recipients were included. In total, 45 subjects showed seroconversion of HBV markers as follows: HBsAg (n = 4) (0.28%; 95% confidence interval [CI] 0.006; 0.57), HBcAb (n = 32), HBsAb (n = 5), and either HBcAb or HBsAb (n = 4). The total rate of seroconversion after renal transplantation was calculated to be 3.24% (95% CI: 2.31–4.18).
Conclusion : Our review indicates that the risk of HBV transmission from HBcAb-positive kidney donors is extremely low. Therefore, kidneys from these donors can be transplanted safely into ESRD patients (read more).

Saturday, September 8, 2012

Outcome of Renal Transplantation in Patients With Both ANCA and Anti-GBM Antibodies

Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) and anti–glomerular basement membrane (anti-GBM) antibody disease, common causes of end-stage renal disease (ESRD) are both characterized by the presence of circulating autoantibodies. A subset of patients have serologic evidence of both antibodies, also called “double-positive” disease. Patients with double-positive disease present often with multisystem disease like in AAV but have a much worse renal prognosis than AAV. The outcome of renal transplantation in these double-positive patients is not known. We report for the first time the results of renal transplantation in six patients with double-positive disease. We conclude that renal transplantation is a safe and effective for patients with double-positive disease. The modern immunosuppressive drugs are effective in preventing vasculitis relapses (read more)

Porcine Sialoadhesin: A Newly Identified Xenogeneic Innate Immune Receptor

Extracorporeal porcine liver perfusion is being developed as a bridge to liver allotransplantation for patients with fulminant hepatic failure. This strategy is limited by porcine Kupffer cell destruction of human erythrocytes, mediated by lectin binding of a sialic acid motif in the absence of antibody and complement. Sialoadhesin, a macrophage restricted lectin that binds sialic acid, was originally described as a sheep erythrocyte binding receptor. Given similarities between sialoadhesin and the unidentified macrophage lectin in our model, we hypothesized porcine sialoadhesin contributed to recognition of human erythrocytes. Two additional types of macrophages were identified to bind human erythrocytes—spleen and alveolar. Expression of sialoadhesin was confirmed by immunofluorescence in porcine tissues and by flow cytometry on primary macrophages. A stable transgenic cell line expressing porcine sialoadhesin (pSn CHO) bound human erythrocytes, while a sialoadhesin mutant cell line did not. Porcine macrophage and pSn CHO recognition of human erythrocytes was inhibited approximately 90% by an antiporcine sialoadhesin monoclonal antibody and by human erythrocyte glycoproteins. Furthermore, this binding was substantially reduced by sialidase treatment of erythrocytes. These data support the hypothesis that porcine sialoadhesin is a xenogeneic receptor that mediates porcine macrophage binding of human erythrocytes in a sialic acid-dependent manner (read more).

Safety of Blood Group A2-to-O Liver Transplantation: An Analysis of the United Network of Organ Sharing Database

Background: ABO-incompatible organ transplantation typically induces hyperacute rejection. A2-to-O liver transplantations have been successful. This study compared overall and graft survival in O recipients of A2 and O grafts based on Organ Procurement and Transplantation Network data.
Methods: Scientific Registry of Transplant Recipients data were used. The first A2-to-O liver transplantation was entered on March 11, 1990; all previous transplantations were excluded. Between March 11, 1990, and September 3, 2010, 43,335 O recipients underwent transplanation, of whom 358 received A2 grafts.
Results: There were no significant differences in age, sex, and race between the groups. Recipients of A2 grafts versus O grafts were significantly more likely to be hospitalized at transplantation (45% vs. 38%, P≤0.05) and to have a higher mean (SD) model for end-stage liver disease score (24 [11] vs. 22 [10], P≤0.05). 10% of A2 recipients and 9% of O recipients underwent retransplantation. No significant differences existed in rejection during the transplantation admission and at 12 months: 7% versus 6% and 20% versus 22% for A2 recipients and O recipients, respectively; and there were no significant differences in contributing factors to graft failure or cause of death. At 5 years, overall survival of A2 and O graft recipients was 77% and 74%, respectively (log rank=0.71). At 5 years, graft survival was 66% in both groups (log rank=0.52). Donor blood group was insignificant on Cox regression for overall and graft survival.
Conclusions: Using Organ Procurement and Transplantation Network/Scientific Registry of Transplant Recipients data, we present the largest series of A2-to-O liver transplantations and conclude this mismatch option to be safe with similar overall and graft survival. This opens possibilities to further meet the demands of a shrinking organ supply, especially with regard to expanding living-donor options (read more).

Coexistence of Different Circulating Anti-Podocyte Antibodies in Membranous Nephropathy

Background and objectives : The discovery of different podocyte autoantibodies in membranous nephropathy (MN) raises questions about their pathogenetic and clinical meaning. This study sought to define antibody isotypes and correlations; to compare levels in MN, other glomerulonephritides, and controls; and to determine their association with clinical outcomes.
Design, setting, participants, & measurements : Serum IgG1, IgG3, and IgG4 against aldose reductase (AR), SOD2, and α-enolase (αENO) were measured at diagnosis in 186 consecutive MN patients, in 96 proteinuric controls (36 with FSGS, and 60 with IgA nephropathy), and in 92 healthy people recruited in four Italian nephrology units. Anti-phospholipase A2 receptor (PLA2r) and anti-neutral endopeptidase (NEP) IgG4 were titrated in the same specimens. Association with 1-year follow-up clinical parameters was studied in 120 patients.
Results : IgG4 was the most common isotype for all antibodies; IgG1 and IgG3 were nearly negligible. IgG4 levels were positive in a significant proportion of MN patients (AR, 34%; SOD2, 28%; αENO, 43%). Antibody titers were higher in MN than in healthy and pathologic controls (P<0.005). Anti-NEP IgG4 did not differ from normal controls (P=0.12). Anti-PLA2r IgG4 was detected in 60% of patients and correlated with anti-AR, anti-SOD2, and anti-αENO IgG4 (P<0.001). In MN patients negative for the whole antibody panel (20%), 1-year proteinuria was lower compared with patients with at least one antibody positivity (P<0.05).
Conclusions : Our data suggest that IgG4 is the prevalent isotype for antibodies against cytoplasmic antigens of podocytes (AR, SOD2, αENO). Their levels were higher than in other proteinuric glomerulonephritides and in normal controls and were correlated with anti-PLA2r. Only baseline negativity for all known antibodies predicted lower 1-year proteinuria (read more).

Friday, September 7, 2012

Small molecule antibody targeting HLA class I inhibits myeloma cancer stem cells by repressing pluripotency-associated transcription factors

Cancer stem cells have been proposed to be responsible for tumorigenesis and recurrence in various neoplastic diseases, including multiple myeloma (MM). We have previously reported that MM cells specifically express HLA class I at high levels and that single-chain Fv diabody against this molecule markedly induces MM cell death. Here we investigated the effect of a new diabody (C3B3) on cancer stem cell-like side population (SP) cells. SP fraction of MM cells highly expressed ABCG2 and exhibited resistance to chemotherapeutic agents; however, C3B3 induced cytotoxicity in both SP cells and main population (MP) cells to a similar extent. Moreover, C3B3 suppressed colony formation and tumorigenesis of SP cells in vitro and in vivo. Crosslinking of HLA class I by C3B3 mediated disruption of lipid rafts and actin aggregation, which led to inhibition of gene expression of β-catenin and pluripotency-associated transcription factors such as Sox2, Oct3/4 and Nanog. Conversely, knockdown of Sox2 and Oct3/4 mRNA reduced the proportion of SP cells, suggesting that these factors are essential in maintenance of SP fraction in MM cells. Thus, our findings reveal that immunotherapeutic approach by engineered antibodies can overcome drug resistance, and provide a new basis for development of cancer stem cell-targeted therapy (read more).

Pretransplant HLA mistyping in diagnostic samples of acute myeloid leukemia patients due to acquired uniparental disomy

Although acquired uniparental disomy (aUPD) has been reported in relapse acute myeloid leukemia (AML), pretransplant aUPD involving chromosome 6 is poorly documented. Such events could be of interest because loss of heterozygosity (LOH) resulting from aUPD in leukemic cells may lead to erroneous results if HLA typing for hematopoietic stem cell donor searches is performed on blood samples drawn during blastic crisis. We report here six AML patients whose HLA typing was performed on DNA extracted from peripheral blood obtained at diagnosis. We observed LOH involving the entire HLA region (three patients), HLA-A, B, C (two patients) and HLA-A only (one patient). An array-comparative genomic hybridization showed that copy number was neutral for all loci, thus revealing partial aUPD of chromosome 6p21. When HLA typing was performed on remission blood samples both haplotypes were detected. A 3–4% LOH incidence was estimated in AML patients with high blast counts. Based on DNA mixing experiments, we determined by PCR sequence-specific oligonucleotide hybridization on microbeads arrays a detection threshold for HLA-A, B, DRB1 heterozygosity in blood samples with <80% blasts. Because aUPD may be partial, any homozygous HLA result should be confirmed by a second typing performed on buccal swabs or on blood samples from the patient in remission (read more).

Antibodies to anti-thymocyte globulin in aplastic anemia patients have a negative impact on hematopoietic SCT

Severe aplastic anemia (SAA) is a life threatening BM failure and the preferred therapy for children is allogeneic hematopoietic SCT (HSCT) with an HLA-matched sibling donor. Immunosuppressive therapy with anti-thymocyte globulin (ATG) and CsA is an alternative for patients lacking an HLA-identical donor. For many years, horse ATG (hATG) was the preferred source, but in Europe the production of hATG ended in 2007, and hATG was replaced by rabbit ATG (rATG). Recently, Scheinberg et al. published a randomized trial comparing rATG with hATG in SAA patients and concluded that hematological response and survival using hATG was superior to rATG. Here, we report in a pediatric SAA transplant cohort that the use of rATG as first-line therapy, followed by rATG in the HSCT conditioning regimen, can result in severe transplant-related problems. Sensitization toward ATG, defined as production of anti-ATG Abs, has been observed in SAA patients as well as in solid organ and HSCT recipients. When a second immunosuppressive course is required or in vivo T-cell depletion is needed in the context of HSCT, a switch from hATG to rATG is advisable to lower the risk of adverse reactions due to the potential presence of anti-hATG Abs (read more).

Fecal calprotectin in allogeneic stem cell transplantation for the diagnosis of acute intestinal graft versus host disease

Intestinal symptoms are common after hematopoietic SCT (HSCT). In this setting, differential diagnosis specifically for diarrhea includes infections, conditioning regimen toxicity and intestinal GVHD (IGVHD), among others. The importance of an early and accurate diagnosis lies in the different therapeutic approaches, outcome and prognosis. The gold standard for the diagnosis of acute IGVHD is endoscopy, which sometimes yields non-specific findings and is an invasive procedure, and histological confirmation with the demonstration of intracryptal apoptosis. Calprotectin is a major protein found in the cytosol of inflammatory cells, especially granulocytes. It represents about half of the cytosol of neutrophils and is also present in macrophages.4 The protein has two subunits and belongs to the family of S100 proteins. It is stable at room temperature and can be measured in stool with ELISA test in a few hours. Fecal calprotectin (FC) was introduced in clinical practice for the differential diagnosis between functional gastrointestinal diseases and disorders with mucosal inflammation. Several studies have demonstrated the utility of this simple non-invasive and inexpensive test for these diseases. In the same way, it has been used with encouraging results in patients with small bowel transplantation for the differential diagnosis of intestinal graft rejection. Our aim was to evaluate FC for the diagnosis of acute IGVHD in patients with diarrhea following allogeneic HSCT (read more).

Successful Isolated Intestinal Transplantation in Sensitized Recipients With the Use of Virtual Crossmatching

We evaluated virtual crossmatching (VXM) for organ allocation and immunologic risk reduction in sensitized isolated intestinal transplantation recipients. All isolated intestine transplants performed at our institution from 2008 to 2011 were included in this study. Allograft allocation in sensitized recipients was based on the results of a VXM, in which the donor-specific antibody (DSA) was prospectively evaluated with the use of single-antigen assays. A total of 42 isolated intestine transplants (13 pediatric and 29 adult) were performed during this time period, with a median follow-up of 20 months (6–40 months). A sensitized (PRA ≥ 20%) group (n = 15) was compared to a control (PRA < 20%) group (n = 27) to evaluate the efficacy of VXM. With the use of VXM, 80% (12/15) of the sensitized patients were transplanted with a negative or weakly positive flow-cytometry crossmatch and 86.7% (13/15) with zero or only low-titer (≤1:16) DSA. Outcomes were comparable between sensitized and control recipients, including 1-year freedom from rejection (53.3% and 66.7% respectively, p = 0.367), 1-year patient survival (73.3% and 88.9% respectively, p = 0.197) and 1-year graft survival (66.7% and 85.2% respectively, p = 0.167). In conclusion, a VXM strategy to optimize organ allocation enables sensitized patients to successfully undergo isolated intestinal transplantation with acceptable short-term outcomes (read more).

Preformed and De Novo Donor Specific Antibodies in Visceral Transplantation: Long-Term Outcome with Special Reference to the Liver

Despite improvement in early outcome, rejection particularly chronic allograft enteropathy continues to be a major barrier to long-term visceral engraftment. The potential role of donor specific antibodies (DSA) was examined in 194 primary adult recipients. All underwent complement-dependent lymphocytotoxic crossmatch (CDC-XM) with pre- and posttransplant solid phase HLA–DSA assay in 156 (80%). Grafts were ABO-identical with random HLA-match. Liver was included in 71 (37%) allografts. Immunosuppression was tacrolimus-based with antilymphocyte recipient pretreatment in 150 (77%). CDC-XM was positive in 55 (28%). HLA–DSA was detectable before transplant in 49 (31%) recipients with 19 continuing to have circulating antibodies. Another 19 (18%) developed de novo DSA. Ninety percent of patients with preformed DSA harbored HLA Class-I whereas 74% of recipients with de novo antibodies had Class-II. Gender, age, ABO blood-type, cold ischemia, splenectomy and allograft type were significant DSA predictors. Preformed DSA significantly (p < 0.05) increased risk of acute rejection. Persistent and de novo HLA–DSA significantly (p < 0.001) increased risk of chronic rejection and associated graft loss. Inclusion of the liver was a significant predictor of better outcome (p = 0.004, HR = 0.347) with significant clearance of preformed antibodies (p = 0.04, OR = 56) and lower induction of de novo DSA (p = 0.07, OR = 24). Innovative multifaceted anti-DSA strategies are required to further improve long-term survival particularly of liver-free allografts (read more).

Monday, September 3, 2012

Early findings of prospective anti-HLA donor specific antibodies monitoring study in pancreas transplantation: Indiana University Health Experience

The significance of donor-specific antibodies (DSA) is not well known in the setting of pancreas transplantation. Since December 2009, we prospectively followed pancreas transplant patients with single-antigen-luminex-bead testing at one, two, three, six, and then every six months for the first two yr. Thirty-five of the 92 patients that underwent pancreas transplantation (13 pancreas-alone [PTA], 20 with a kidney [SPK], and two after a kidney [PAK]) agreed to participate in study. Median age at transplant was 45 yr and follow-up was 23 months. Majority were Caucasian (n = 33) and male (n = 18). Rabbit anti-thymocyte globulin induction was used. Median HLA-mismatch was 4.2 ± 1.1. Eight patients (7SPK, 1PAK) developed post-transplant DSA at median follow-up of 76 d (26–119), 1 SPK had pre-formed DSA. Seven patients had both class I and class II DSA, one with class I and one with class II only. Mean peak class I DSA-MFI was 3529 (±1456); class II DSA-MFI was 5734 (±3204) whereas cumulative DSA MFI (CI + CII) was 9264 (±4233). No difference was observed in the patient and donor demographics among patients with and without DSA. One patient in non-DSA group developed acute cellular rejection of pancreas. From our data it appears that post-transplant DSA in pancreas allograft recipients may not impact the early-pancreatic allograft outcomes. The utility of prospective DSA monitoring in pancreatic transplant patients needs further evaluation and long-term follow-up (read more)

Saturday, September 1, 2012

Pregnancy after renal transplantation: a review of registry and single-center practices and outcomes

Registries from North America, Australia and Europe are rich sources of clinical data on pregnancy after kidney transplantation. Single-center reports of pregnancy outcomes are limited by small sample sizes but not by the potential reporting bias that can impact registry data. Despite the differences in data pools, the obstetric and graft outcomes reported by single centers and registries have been similar. The majority of pregnancies are successful in renal transplant patients, but the risk of complications like pre-eclampsia, low birth weight and premature birth is high. Pregnancy has no significant impact on graft function or survival when baseline function is normal (read more).

New players in the pathogenesis of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the most common primary glomerular disorder causing end-stage renal disease. Since the first description of this clinicopathological entity in the early 1930s, various studies have identified numerous underlying pathogenetic mechanisms. Nevertheless, FSGS is still a complex, only partially understood and in its classification sometimes confusing disease. A unifying pathophysiological concept has not been identified and might not even exist. However, research efforts of past decades identified FSGS as a podocytopathy with several podocyte molecules being key players in the development and the course of FSGS. Podocytes are crucially involved in the formation of the glomerular barrier and any assault on their delicate physiological balance and architecture can result in the development of proteinuria. The following review article will introduce most recent examples identifying novel players in the complex pathogenesis of FSGS (read more).

Combination of anti-C1q and anti-dsDNA antibodies is associated with higher renal disease activity and predicts renal prognosis of patients with lupus nephritis

That's why you should be careful about C1qScreen® testing interpretation in recipents with lupus!

Background : Although nephritogenic autoantibodies are considered to play a central role in the initiation of lupus nephritis, whether these autoantibodies are associated with renal clinical and pathological activity or renal outcome is still controversial. Here, we investigated the associations of certain serum autoantibodies with renal disease activity and renal outcome in a large cohort of Chinese patients with lupus nephritis.
Methods : One hundred and thirty-six Chinese patients with biopsy-proven lupus nephritis and with long-term follow up data were studied. Sera at renal biopsy were tested for a panel of autoantibodies, including anti-nuclear antibodies, anti-double-stranded DNA (anti-dsDNA) antibodies, anti-extractable nuclear antigen antibodies, anti-C-reactive protein antibodies, anti-C1q antibodies, anti-cardiolipin antibodies and anti-β2-glycoprotein I antibodies. Associations of these autoantibodies with clinical features, laboratory findings, histopathological data and renal outcomes were further investigated.
Results : Among the various autoantibodies, anti-dsDNA and anti-C1q antibodies were better than other antibodies to evaluate the renal disease activity. Anti-dsDNA antibodies were correlated with higher incidence of leukocyturia (P< 0.05), total pathological activity index (AI) score (P< 0.05), endocapillary hypercellularity (P< 0.05), subendothelial hyaline deposits (P< 0.05) and leukocyte infiltration (P< 0.05). Anti-C1q antibodies were correlated with leukocyturia (P< 0.01), hematuria (P< 0.003) and the majority of the histopathological AIs including total AI score (P< 0.003), endocapillary hypercellularity (P< 0.003), cellular crescents (P< 0.05), karyorrhexis/fibrinoid necrosis (P< 0.003), subendothelial hyaline deposits (P< 0.003) and leukocyte infiltration (P< 0.01). Patients with both anti-dsDNA and anti-C1q antibodies had higher renal disease activity and poorer renal outcome (log-rank test: P= 0.048) compared with those without the two antibodies. In univariate survival analysis of renal prognosis, neither the presence of anti-C1q nor the presence of anti-dsDNA antibodies was a risk factor of renal survival. However, the combination of the two antibodies predicted renal prognosis (hazard ratio 4.40, 95% confidence interval: 1.268–15.269, P= 0.02)
Conclusions : Anti-C1q antibodies are more closely correlated with renal disease activity than the other autoantibodies. The combination of anti-C1q and anti-dsDNA autoantibodies indicates higher renal disease activity and predicts poor renal outcome (read more).

Shipping donor kidneys within Eurotransplant: outcomes after renal transplantation in a single-centre cohort study

Background : Shipment of organs during the allocation process aims to improve human leucocyte antigen (HLA) matching but can also have a detrimental effect by prolonging cold ischaemia. The overall effect of organ exchange on post-transplant outcomes in the Eurotransplant (ET) region has not been investigated.
Methods : This is a retrospective single-centre cohort study to investigate the effect of shipment of renal allografts on cold ischaemia times and the incidence of acute rejection (AR) and graft survival in 661 transplantations of deceased donor kidneys.
Results : Forty-six per cent (N = 301) of the patients received a locally procured and 54% (N = 360) a shipped donor kidney. Locally procured donors tended to be older, more often hypertensive and had less frequently died from trauma. Recipients of shipped kidneys were at higher immunological risk, being younger, more frequently retransplanted and immunized against HLA antigens. Shipped kidneys had a 2.2-h prolongation of cold ischaemia time (18.0 versus 20.2 h; P < 0.0001) but significantly less HLA A, B and DR mismatches (2.20 versus 2.84; P < 0.0001). Recipients of shipped kidneys had an increased incidence of first-year AR [19 versus 13%; odds ratio 1.62 (1.06–2.49); P = 0.026] and death-censored graft loss [hazard ratio 1.6 (1.1–2.4); P = 0.01] that was no longer statistically significant after adjustments for risk factors by multivariable modelling.
Conclusions : Shipment of kidneys in the ET region is associated with a modest increase in cold ischaemia time and significantly better HLA matching. This allows for successful transplantation of higher risk patients with no significant penalty with regard to AR rates or death-censored graft survival (read more).

Autoantibodies Targeting Galactose-Deficient IgA1 Associate with Progression of IgA Nephropathy

Mesangial and circulating IgA1 with aberrantly glycosylated hinge region O-glycans characterize IgA nephropathy (IgAN). Unlike healthy individuals, some IgA1 is galactose deficient in patients with IgAN, leaving terminal N-acetylgalactosamine residues in the hinge region exposed. Circulating autoantibodies that recognize such galactose-deficient IgA1 as an autoantigen, or the levels of the autoantigen itself, may allow prediction of disease progression. Here, we analyzed serum samples obtained at diagnosis for autoantigen and autoantibodies from 97 patients with IgAN selected from our prospective cohort according to their absolute renal risk for progression to dialysis or death (0, very low; 1, low; 2, high; 3, very high). We also analyzed samples from controls comprising 30 healthy volunteers and 30 patients with non-IgAN disease. The mean follow-up was 13.8 years. We found that mean serum levels of total autoantigen, normalized IgG autoantibody, and total IgA autoantibody were significantly higher in patients than in the combined controls (all P≤0.01). Furthermore, increasing levels correlated with worse clinical outcomes. In Cox regression and Kaplan–Meier analyses, IgG autoantibody levels ≥1.33 predicted dialysis or death (both P≤0.01). In conclusion, these data suggest that serum levels of IgG and IgA autoantibodies strongly associate with the progression of IgAN nephropathy (read more).