Single-exon approach to non-invasive fetal RHD screening in early pregnancy: An update after 10 years' experience.

BACKGROUND AND OBJECTIVES: Anti-D prophylaxis, administered to RhD-negative women, has significantly reduced the incidence of RhD immunization. Non-invasive fetal RHD screening has been used in Stockholm for more than 10 years to identify women who will benefit from prophylaxis. The method is based on a single-exon approach and is used in early pregnancy. The aim of this study was to update the performance of the method. MATERIALS AND METHODS: The single exon assay from Devyser AB is a multiplex kit detecting both exon 4 of the RHD gene and the housekeeping gene GAPDH. Cell-free DNA was extracted from 1 ml of plasma from EDTA blood taken during early pregnancy, weeks 10-12. The genetic RHD results were compared with serological typing of newborns for a determination of sensitivity and specificity. RESULTS: In total, 4337 pregnancies were included in the study; 44 samples (1%) were inconclusive either due to maternal RHD gene variants (n = 34) or technical reasons (n = 10). Of the remaining 4293 pregnancies, a total number of nine discrepant results were found. False positive results (n = 7) were mainly (n = 4) due to RHD gene variants in the child. False-negative results were found in two cases, of which one was caused by a technical error. None of the false-negative cases was due to RHD gene variants. Overall, the sensitivity of the method was 99.93% and specificity 99.56%. CONCLUSION: The single-exon assay used in this study is correlated with high sensitivity and specificity.

The importance of graft cell composition in outcome after allogeneic stem cell transplantation in patients with malignant disease.

BACKGROUND: Graft-versus-host disease (GVHD) and relapse remain majobstacles ftreatment success in allogeneic hematopoietic stem cell transplantation (HSCT). In the present study, we evaluated the immune cell profile of the graft to outcome after HSCT. STUDY DESIGN AND METHOD: Flow cytometry data of graft cell subsets [CD34+ , CD3+ , CD19+ , CD4+ , CD8+ , CD3-CD56+ CD16+ , CD4+ CD127low CD25high ] from G-CSF primed peripheral blood stem cell (PBSC) donors was collected retrospectively from 299 patients with hematological malignancies undergoing HSCT between 2006 and 2013. The association to overall survival, transplant-related mortality (TRM), GVHD and probability of relapse was analyzed. Patients outcome from HLA-identical sibling (Sib) (n = 97) and unrelated donors (URD) (n = 202) were analyzed separately as all URD patients received anti-thymocyte globulin (ATG). RESULTS: Five-year overall survival was similar in the two cohorts (68% (Sib) vs 65% (URD)). The relapse incidence was significantly lower in the Sib cohort (24% vs 35%, P = 0.04). Multivariate analysis in the URD group revealed an association between a higher CD8+ dose and less relapse (HR, 0.94; 95%CI, 0.90-0.98; P = 0.006) as well as an association between higher CD34+ dose and both higher TRM (HR, 1.09; 95%CI, 1.02-1.20; P = 0.02) and relapse (HR, 1.09; 95%CI, 1.01-1.17; P = 0.025). The Sib analysis showed an association between a higher graft CD19+ dose and more severe acute GVHD (HR, 1,09; 95%CI, 1.03-1.15; P = 0.003) and TRM (HR, 1.09; 95%CI, 1.01-1.17; P = 0.036). In addition, a higher CD4+ graft content was associated to an increased risk for chronic GVHD (HR, 1.02; 95%CI 1.00-1.04; P = 0.06). CONCLUSION: These data indicate an importance of PBSC dongraft composition in patients with a hematological malignancy.

Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report.

BACKGROUND: A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 + 3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. CASE PRESENTATION: We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naïve CD4+ and CD8+ T-cells, increased number of activated effector memory CD8+ T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient's cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. CONCLUSIONS: There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing.

T Cell Receptor Excision Circle (TREC) Monitoring after Allogeneic Stem Cell Transplantation; a Predictive Marker for Complications and Clinical Outcome.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up.

Improved overall survival for pediatric patients undergoing allogeneic hematopoietic stem cell transplantation - A comparison of the last two decades.

Pediatric protocols for allogeneic hematopoietic SCT have been altered during the last two decades. To compare the outcomes in children (<18 yr old), who underwent SCT at our center during 1992-2002 (P1) and 2003-2013 (P2). We retrospectively analyzed 188 patients in P1 and 201 patients in P2. The most significant protocol changes during P2 compared with P1 were a decrease in MAC protocols, particularly those containing TBI, an increase in RIC protocols, and altered GvHD prophylaxis. In addition, P2 had more patients with nonmalignant diagnoses (p = 0.002), more mismatched (MM) donors (p = 0.01), and more umbilical CB grafts (p = 0.03). Mesenchymal or DSCs were used for severe acute GvHD during P2. Three-yr OS in P1 was 58%, and in P2, it was 78% (p < 0.001). Improved OS was seen in both malignant disorders (51% vs. 68%; p = 0.05) and nonmalignant disorders (77% vs. 87%; p = 0.04). Multivariate analysis showed that SCT during P2 was associated with reduced mortality (HR = 0.57; p = 0.005), reduced TRM (HR = 0.57; p = 0.03), unchanged relapse rate, similar rate of GF, less chronic GvHD (HR = 0.49; p = 0.01), and more acute GvHD (HR = 1.77, p = 0.007). During recent years, OS has improved at our center, possibly reflecting the introduction of less toxic conditioning regimens and a number of other methodological developments in SCT.

Long-Term Stable Mixed Chimerism after Hematopoietic Stem Cell Transplantation in Patients with Non-Malignant Disease, Shall We Be Tolerant?

Long-term stable mixed chimerism is a rare and poorly understood phenomenon post hematopoietic stem cell transplantation. This study aims to shed light on whether the two hematopoietic systems in patients with mixed chimerism remain functional. Additionally, we investigate possible immunologic differences in these individuals compared to patients with only donor derived immune cells. Patients with donor and mixed chimerism, at median 10 (5-16) years post-HSCT for non-malignant diseases, were assessed regarding clinical situation and immune system (phenotypical and functional). No difference in long-term outcome was seen in terms of general wellbeing, central phenotypic immune system features (e.g., differentiation status, CD4/CD8 ratio, B and NK-cell frequency) and antibody responses to immunizations. At a median of 10 years post transplantation, patients with mixed chimerism had significantly higher IgG3 and platelet levels. Additionally, these patients had higher NKT-cell levels (CD94+CD8+ and CD56+CD8+) than patients with donor chimerism. In depth phenotypic analysis of patients with mixed chimerism demonstrated recipient-derived fractions in most immune cell lineages (e.g., T-cell, B-cell and NK-cell subsets). Recipient cells were also capable of responding to mitogenic stimulation with production of several cytokines. In conclusion, long-term mixed chimerism did not negatively affect patient wellbeing and long-term outcome. Moreover, recipient-derived immunity may still be functional in these patients, suggesting an active state of tolerance and immunologic dependence on both hematopoietic systems.

Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo.

BACKGROUND AIMS: Human decidual stromal cells (hDSCs) may cure acute graft-versus-host disease (GVHD) in humans. We evaluated immunosuppression by xenogenic hDSCs in mice, both in vitro and in vivo. METHODS: hDSCs inhibited mouse lymphocyte proliferation in allo- and xeno-stimulation assays in mixed lymphocyte culture (MLC) and after mitogenic stimulation. The immunosuppressive effect of hDSCs was dose-dependent and strain-independent. Trans-well experiments showed that hDSCs needed cell-to-cell contact to be immunosuppressive. In a GVHD model, Balb/c mice were transplanted with bone marrow and splenocytes from C57BL/6 a donor. Varying doses of hDSCs (10(5)-10(6) per mouse) were infused at different time points. Recipient mice showed lower GVHD scores than untreated mice, but they did not have consistently improved survival. Histopathological investigation of liver, gastrointestinal tract and skin of animals with GVHD did not show any significant improvement from hDSC infusion. RESULTS: hDSCs were transduced with immunosuppressive genes including those encoding interleukin-10, prostaglandin-E2 receptor, indoleamine dioxygenase, interferon-γ and programmed death ligand-1. Transduced and untransduced hDSCs showed similar effects in vitro and in vivo. At a dose of 10(6)hDSCs per mouse, the majority of recipients died of embolism. CONCLUSIONS: hDSCs inhibit allo-reactivity, xeno-reactivity and mitogen-induced stimulation in mouse lymphocytes. Although the GVHD score was reduced by hDSC infusion, survival and GVHD histopathology were not improved. One reason for failure was fatal embolism.

An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation.

INTRODUCTION: An assay to determine glucocorticoid (GC) responsiveness in humans could be used to monitor GC non-responsiveness in states of GC insufficiency and could provide a tool to adapt GC treatment to individual patients. We propose an ex vivo assay to test GC responsiveness in peripheral leukocytes. The assay was evaluated in a human experimental model of surgery-induced inflammation. PATIENTS AND METHODS: Changes in expression of the GC-regulated genes GILZ, IL1R2, FKBP5, and HLA-DR and glucocorticoid receptor alpha (GRα) were determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral leukocytes from surgical patients and healthy blood donors (total n=60) in response to low (1 nM) and high (1 µM) dexamethasone (DEX). The final selection of a suitable endogenous control gene was based on the studies of stability during DEX treatment and inflammation. Correlations between pre- and postoperative GC-induced gene expression, the postoperative systemic inflammatory and metabolic response (CRP, IL-6, white blood cell count, cytokines, resistin, free fatty acids, glucose, insulin, and adiponectin), and the clinical outcome were analyzed. The length of stay in the intensive care unit (ICU-LOS), the length of stay in the hospital, and postoperative complications were used to measure clinical outcome. RESULTS: When the blood donors were compared to the patients, there were no significant differences in the regulation of the genes in response to DEX, except for GRα. Preoperative, but not postoperative, gene regulation of GILZ and GRα was negatively correlated to ICU-LOS (P<0.05 and P<0.01, respectively). Preoperative GILZ and FKBP5 gene regulation was negatively correlated to postoperative systemic TNFα and MIP-1α levels. CONCLUSION: We suggest that this assay could be used to determine GC responsiveness. An alteration in preoperative GC responsiveness may be related to a patient's ability to recover from surgically induced inflammatory stress.

Transplanted Bone Marrow-Derived Cells Contribute to Human Adipogenesis.

Because human white adipocytes display a high turnover throughout adulthood, a continuous supply of precursor cells is required to maintain adipogenesis. Bone marrow (BM)-derived progenitor cells may contribute to mammalian adipogenesis; however, results in animal models are conflicting. Here we demonstrate in 65 subjects who underwent allogeneic BM or peripheral blood stem cell (PBSC) transplantation that, over the entire lifespan, BM/PBSC-derived progenitor cells contribute ∼10% to the subcutaneous adipocyte population. While this is independent of gender, age, and different transplantation-related parameters, body fat mass exerts a strong influence, with up to 2.5-fold increased donor cell contribution in obese individuals. Exome and whole-genome sequencing of single adipocytes suggests that BM/PBSC-derived progenitors contribute to adipose tissue via both differentiation and cell fusion. Thus, at least in the setting of transplantation, BM serves as a reservoir for adipocyte progenitors, particularly in obese subjects.

Hypogammaglobulinemia in children after allogeneic hematopoietic stem cell transplantation: a cytokine mediated immunoglobulin isotype class switch arrest?

BACKGROUND: Hypogammaglobulinemia (hypo-IgG) is common early post-HSCT in children, occasionally necessitating long-term immunoglobulin (Ig) G replacement therapy. IgG replacement may not reduce mortality, although infectious complications are decreased PROCEDURE: Clinical data and samples from 86 children were analyzed retrospectively with the aim to identify risk factors for developing long-term hypo-IgG (i.e., requiring ≥ 3 months IgG replacement) post-HSCT and studying the underlying biology. Laboratory studies covered serum cytokines, IGHG2 genotyping and routine laboratory investigations. Results were analyzed statistically. RESULTS: Forty-eight percent of the children developed long-term hypo-IgG. These children were younger (<5 years) and had higher acute GvHD incidence, but had better overall survival (88% vs. 69%, P = 0.036). Significantly lower Ig levels post-HSCT but equal immune cell recovery were seen in patients with long-term hypo-IgG compared with those of transient or no hypo-IgG. Pre-HSCT IL-6 and -7 and post-HSCT BAFF and APRIL levels were significantly higher in the long-term hypo-IgG group. CONCLUSIONS: Findings suggests an unfavorable cytokine milieu for graft-derived immune recovery, possibly inducing Ig isotype class switch arrest. Younger age, acute GvHD, and higher pre-HSCT IL-6 levels were identified as significant risk factors for long-term hypo-IgG. Long-term hypo-IgG post-HSCT does not need to be unfavorable and could be an effect of deteriorated cytokine signaling.

Antigen-induced regulation of T-cell motility, interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors.

Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility.

T-cell receptor excision circle levels after allogeneic stem cell transplantation are predictive of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome.

In this retrospective study, 209 patients with malignant disease were analyzed for levels of T-cell receptor excision circles (TRECs) for the first 24 months after allogeneic stem cell transplantation. CD3(+) cells were separated by direct antibody-coupled magnetic beads, followed by DNA extraction according to a standard protocol. The δRec-ψJα signal joint TREC was measured with real-time quantitative PCR. Patients were grouped based on malignant disease: chronic myeloid leukemia, chronic lymphatic leukemia, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and myelodysplastic syndrome (MDS). Patients were further subdivided based on TREC levels below (low-TREC) or above (high-TREC) median at each time point. TREC levels were then correlated to relapse incidence and relapse-free survival (RFS). For patients with AML, low TREC levels 2 months post-transplantation were correlated to high relapse incidence at 5 years (P<0.05). In patients with chronic leukemia, high TREC levels were correlated with improved RFS (P<0.05). For patients with MDS, high TREC levels at 9 months post-transplantation were associated with higher RFS at 5 years (P<0.02) and lower relapse incidence (P<0.02). This study shows the potential use of TREC measurement in blood to predict relapse in patients with AML and MDS after allogeneic hematopoietic stem cell transplantation.

A research study of the association between maternal microchimerism and systemic lupus erythematosus in adults: a comparison between patients and healthy controls based on single-nucleotide polymorphism using quantitative real-time PCR.

BACKGROUND: Naturally acquired microchimerism may arise in the mother and her child during pregnancy when bidirectional trafficking of cells occurs through the placental barrier. The occurrence of maternal microchimerism (maternal cells in the offspring) has been associated with several autoimmune diseases, especially in children. Systemic Lupus erythematosus (SLE) is an autoimmune disorder with a resemblance to graft-versus-host disease. The aim of this study was to investigate the association between maternal microchimerism in the blood and SLE. METHODOLOGY/PRINCIPAL FINDINGS: Thirty-two patients with SLE, 17 healthy brothers of the patients, and an additional 12 unrelated healthy men were the subjects in this study. A single-nucleotide polymorphism unique to each mother was identified, and maternal microchimerism in the study group and in the control group was detected using a quantitative real-time polymerase chain reaction technique. No differences in the frequency or the concentration of maternal cells were apparent in the blood of patients with SLE or in that of the controls. Two patients and one control tested positive for maternal microchimerism, but the positive subjects were all negative at a follow-up 16 years later. The sensitivity of the method was estimated to 1/10.000. CONCLUSIONS/SIGNIFICANCE: These results show no association between SLE and maternal microchimerism. The frequency of maternal microchimerism in the blood of adults overall may be lower than earlier reported.

Fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease.

The placenta protects the fetus from the mother's immune system. We have previously found that fetal membrane cells (FMCs) isolated from term placenta prevent alloreactivity in vitro. FMCs share many features with bone marrow-derived mesenchymal stromal cells (MSCs), which we previously introduced to treat severe acute graft-versus-host disease (GVHD). Here, we tested FMCs for treatment of steroid-refractory acute GVHD. After two passages in culture, approximately 10(9) FMCs were obtained from one single placenta, although not all cells from passage 0 and passage 1 were used for expansion. The FMCs were positive for CD29, CD44, CD73, CD90, CD105, and CD49d but were negative for hematopoietic, endothelial, and epithelial markers. Microsatellite polymorphism analysis showed that FMCs were of maternal origin. All FMCs used showed normal karyotype. Nine patients who had undergone hematopoietic stem cell transplantation (HSCT) and who had developed steroid-refractory grade III-IV acute GVHD were given 0.9-2.8 × 10(6) FMCs per kg at 15 infusions. Median age was 57 years. There was no toxicity from infusion of FMCs in eight patients. One patient had seizures after infusion. Two of eight evaluable patients had a complete response and four had a partial response, giving an overall response rate of 75%. Two patients showed no response at all. Three patients are alive from 6 to 21 months after HSCT. One patient is well and two have chronic GVHD. Thus, FMCs may be successfully used for immune modulation and tissue repair.

Factors with an impact on chimerism development and long-term survival after umbilical cord blood transplantation.

BACKGROUND: Umbilical cord blood transplantation (UCBT) is increasingly used and produces similar results to matched unrelated donor transplantation. METHODS: We performed a retrospective single-center analysis of 50 umbilical cord blood transplantations UCBTs performed from 2001 to 2010, including 37 single and 13 double umbilical cord blood transplantations UCBTs. RESULTS: The rate of engraftment of neutrophils was 88% at a median time of 29 days (range, 3-79). Complete donor chimerism (DC) within the CD19, CD3, and CD33 cell lineages was seen in 74%, 72%, and 76% of the patients, respectively. DC was associated with acute graft-versus-host disease (GVHD) grades II to IV for the CD3 cell lineage (P=0.01) and, in multivariate analysis, with total body irradiation for all lineages (P<0.01). Overall survival (OS) at 1 and 5 years was 55% and 43%. Nonmalignant diseases were associated with better 5-year OS (72%) than malignancies (28%; P=0.026). In multivariate analysis, a negative correlation was seen between OS and age (hazard ratio [HR], 1.04; 95% confidence interval [95% CI], 1.02-1.06; P<0.001), acute GVHD grades III and IV (HR, 3.43; 95% CI, 1.95-6.02; P<0.001), and mesenchymal stem cell treatment (HR, 2.66; 95% CI, 1.11-6.35; P=0.027). Transplant-related mortality at 100 days and 1 year was 16% and 30%. The incidence of acute GVHD grades II to IV was 34%. Acute GVHD grades III and IV was associated with ABO incompatibility (HR, 2.61; P=0.05) and myeloablative conditioning (HR, 4.17; P=0.047). CONCLUSIONS: The outcome in patients with nonmalignant diseases was acceptable, but transplant-related mortality in the whole group remains high. A significantly higher rate of DC was associated with total body irradiation-based conditioning and with acute GVHD grades II and IV.

Rapid salvage treatment with virus-specific T cells for therapy-resistant disease.

BACKGROUND: Viral infections are major complications after allogeneic hematopoietic stem cell transplantation (HSCT). During posttransplant immunosuppression the regular T-cell control is compromised. Even if treatment strategies against infections caused by herpes viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus have improved, the mortality rate is still considerable. If primary antiviral therapy fails or cannot be tolerated, adoptive therapy with virus-specific cytotoxic T cells (CTL) can be utilized. METHODS: In this study, we used virus-specific CTLs to treat 8 patients suffering from severe viral infections after allogeneic HSCT. Using positive selection with HLA multimers and magnetic beads, we isolated CTLs from both frozen donor material as well as third-party donors within hours. RESULTS: At 90 days after CTL infusions 7 out of 8 patients were still living. CTLs infused from third-party donors were detected in 5 of 6 patients up to 76 days after infusion. No graft-versus-host disease associated with CTL infusions was observed. CONCLUSIONS: Our separation approach offers a rapid alternative for adoptive CTL therapy if primary antiviral treatment strategies fail. Because no prolonged expansion steps are needed, this method may be used for early treatment of patients suffering from life-threatening infectious complications.

Thymic function after allogeneic stem cell transplantation is dependent on graft source and predictive of long term survival.

T-cell deficiency after allogeneic stem cell transplantation (ASCT) is common and has major impact on clinical outcome. In this retrospective study 210 patients were analyzed with regards to levels of T-cell receptor excision circles (TRECs) during the first 24 months after transplantation. We could for the first time show a significant correlation between the use of bone marrow grafts and higher TREC levels >6 months post-ASCT (p<0.001). Treatment with anti-thymocyte globulin was correlated with lower TREC levels ≤6 months post-ASCT (p<0.001). Patients with TREC levels above median at 3 months had a superior overall survival, 80% vs. 56% (p=0.002), and lower transplantation-related mortality, 7% vs. 21% (p=0.01). We conclude that graft source and conditioning regimen may have a significant effect on T-cell reconstitution after ASCT and can thus affect outcome. These results strongly support the use of TREC measurement as part of the standard repertoire of immunological monitoring after ASCT.

Mesenchymal stem cells inhibit thymic reconstitution after allogeneic cord blood transplantation.

Cord blood (CB) as a source of stem cells has been a successful addition to the field of allogeneic stem cell transplantation (ASCT). The increased human leukocyte antigen (HLA) permissiveness of CB grafts has made it possible for more patients to undergo treatment. The drawback is that patients suffer from a longer period of compromised immunity. We analyzed T-cell receptor excision circles (TRECs), immunoglobulin G (IgG), and immunoglobulin M (IgM) levels after cord blood transplantation (CBT) in 50 patients transplanted at our center. These immunological parameters were compared retrospectively with clinical factors and complications. We found that TREC levels after CBT were lower in adults, patients with myeloablative conditioning, and in patients with a lower nucleated cell dose in the graft. In addition mesenchymal stem cells (MSC) as co-infusion at the time of CBT had a negative effect on TREC reconstitution. This was found to be associated with decreased overall survival for this patient category. Reduced IgM and IgG levels post-CBT were associated with a major AB0 mismatch, and infusion of MSCs. Our results highlight the importance of close monitoring of the immune reconstitution after CBT. In addition it shows a potentially new suppressive effect of MSCs on the immune system.

Improved survival after allogeneic hematopoietic stem cell transplantation in recent years. A single-center study.

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

Sirolimus and tacrolimus as immune prophylaxis compared to cyclosporine with or without methotrexate in patients undergoing allogeneic haematopoietic stem cell transplantation for non-malignant disorders.

For prevention of graft-versus-host disease (GVHD), treatment of 24 haematopoietic stem cell transplantation (HSCT) patients with sirolimus and tacrolimus was compared to treatment of matched controls with cyclosporine-based regimens. The patients mainly had non-malignant disorders. Two-thirds of the donors were unrelated, and bone marrow was the most common source of stem cells. Rejection occurred in four patients in the sirolimus group and three in the control group. Donor chimerism for CD3, CD19 and CD33 was similar in the two groups. The cumulative incidence of grade II acute GVHD was 22% in the sirolimus patients and 17% in the controls (P=0.78). No patients developed acute GVHD of grades III-IV. The cumulative incidence of chronic GVHD was 25% and 37% in the two groups, respectively (P=0.40). Two patients in the sirolimus group developed Epstein-Barr virus lymphoma, and none in the controls. Side effects and toxicity were similar in the two groups. There was no transplant-related mortality at 5 yr in the sirolimus group, as opposed to 8% in the controls (P=0.47). Survival at 5 yr was 95% and 92%. Thus, sirolimus combined with tacrolimus is a promising immunosuppressive regimen in HSCT, also for non-malignancies, and its efficacy should be confirmed in prospective clinical trials.