Mesenchymal stromal/stem cells promote intestinal epithelium regeneration after chemotherapy-induced damage.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for leukemia and a range of non-malignant disorders. The success of the therapy is hampered by occurrence of acute graft-versus-host disease (aGvHD); an inflammatory response damaging recipient organs, with gut, liver, and skin being the most susceptible. Intestinal GvHD injury is often a life-threatening complication in patients unresponsive to steroid treatment. Allogeneic mesenchymal stromal/stem cell (MSC) infusions are a promising potential treatment for steroid-resistant aGvHD. Data from our institution and others demonstrate rescue of approximately 40-50% of aGvHD patients with MSCs in Phase I, II studies and minor side effects. Although promising, better understanding of MSC mode of action and patient response to MSC-based therapy is essential to improve this lifesaving treatment. METHODS: Single cell human small intestine organoids were embedded in Matrigel, grown for 5 days and treated with busulfan for 48 h. Organoids damaged by treatment with busulfan or control organoids were co-cultured with 5000, 10,000, and 50,000 MSCs for 24 h, 48 h or 7 days and the analyses such as surface area determination, proliferation and apoptosis assessment, RNA sequencing and proteomics were performed. RESULTS: Here, we developed a 3D co-culture model of human small intestinal organoids and MSCs, which allows to study the regenerative effects of MSCs on intestinal epithelium in a more physiologically relevant setting than existing in vitro systems. Using this model we mimicked chemotherapy-mediated damage of the intestinal epithelium. The treatment with busulfan, the chemotherapeutic commonly used as conditioning regiment before the HSCT, affected pathways regulating epithelial to mesenchymal transition, proliferation, and apoptosis in small intestinal organoids, as shown by transcriptomic and proteomic analysis. The co-culture of busulfan-treated intestinal organoids with MSCs reversed the effects of busulfan on the transcriptome and proteome of intestinal epithelium, which we also confirmed by functional evaluation of proliferation and apoptosis. CONCLUSIONS: Collectively, we demonstrate that our in vitro co-culture system is a new valuable tool to facilitate the investigation of the molecular mechanisms behind the therapeutic effects of MSCs on damaged intestinal epithelium. This could benefit further optimization of the use of MSCs in HSCT patients.

Busulfan target exposure attainment in children undergoing allogeneic hematopoietic cell transplantation: a single day versus a multiday therapeutic drug monitoring regimen.

Busulfan exposure has previously been linked to clinical outcomes, hence the need for therapeutic drug monitoring (TDM). Study objective was to evaluate the effect of day 1 TDM-guided dosing (regimen d1) versus days 1 + 2 TDM-guided dosing (regimen d1 + 2) on attaining adequate busulfan exposure. In this observational study, we included all children receiving busulfan-based allogeneic hematopoietic cell transplantation. Primary outcome was the percentage of patients achieving busulfan target attainment in both TDM regimens. Secondary outcomes were the variance in busulfan exposure and day-4 clearance (Clday4) estimates between both TDM regimens and dosing day 1 and 2. In regimen d1, 84.3% (n = 91/108) attained a therapeutic busulfan exposure, while in regimen d1 + 2 a proportion of 90.9% was found (n = 30/33, not-significant). Variance of Clday4 estimate based on busulfan day 2 concentrations was significantly smaller than the variance of Clday4 estimates based on day 1 concentrations (p < 0.001). Therefore, day 1-guided TDM (pharmacometric model-based) of busulfan may be sufficient for attaining optimal target exposure, provided that subsequent TDM is carried out if required. However, performing TDM on subsequent days may be beneficial, as measurements on day 2 seemed to reduce the variance in the estimated clearance as compared to day 1 sampling.

Therapeutic Drug Monitoring of Anti-Thymocyte Globulin in Allogeneic Stem Cell Transplantation: Proof of Concept.

Anti-thymocyte globulin (ATG), a polyclonal antibody, is used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft-vs.-host-disease (GvHD) and graft failure (GF). Overexposure to ATG leads to poor early T-cell recovery, which is associated with viral infections and poor survival. Patients with severe inflammation are at high risk for GF and GvHD, and may have active infections warranting swift T-cell recovery. As ATG exposure may be critical in these patients, individualized dosing combined with therapeutic drug monitoring (TDM) may improve outcomes. We describe the individualized dosing approach, an optimal sampling scheme, the assay to measure the active fraction of ATG, and the workflow to perform TDM. Using a previously published population pharmacokinetic (PK) model, we determine the dose to reach optimal exposures associated with low GvHD and rejection, and at the same time promote T-cell recovery. Based on an optimal sampling scheme, peak and trough samples are taken during the first 3 days of once-daily dosing. The fraction of ATG able to bind to T-cells (active ATG) is analyzed using a bio-assay in which Jurkat cells are co-cultured with patient's plasma and the binding is quantified using flow cytometry. TDM is performed based on these ATG concentrations on the third day of dosing; subsequent doses can be adjusted based on the expected area under the curve. We show that individualized ATG dosing with TDM is feasible. This approach is unique in the setting of antibody treatment and may result in better immune reconstitution post-HCT and subsequently better survival chances.

B-cell depletion abrogates immune mediated cytopenia and rejection of cord blood transplantation in Hurler syndrome.

Umbilical cord blood is the preferred donor cell source for children with Inherited Metabolic disorders undergoing Hematopoietic Cell Transplant (HCT), and its use has been associated with improved "engrafted survival" and higher donor chimerism compared to other cell sources. However, as in other pediatric cord blood transplants for non-malignant disease, immune-mediated cytopenia and primary graft failure limit its use, and the latter remains the commonest cause of death following cord blood transplant for non-malignant disease. We have previously shown an association between immune-mediated cytopenia and graft failure in inherited metabolic diseases suggesting that both immune-mediated cytopenia and graft failure could be mediated by antibodies from the residual recipient B cells. Since rituximab is effective in depletion of B cells and management of refractory immune-mediated cytopenia following HCT, we have added rituximab to the conditioning regimen. We studied 57 patients in 2 centers who received myeloablative conditioning for cord blood transplant in Hurler syndrome, and report a significant improvement in event-free survival with reduced incidence of graft failure and without any evidence of immune-mediated cytopenia in those patients that had received rituximab.

Cross-cutting view of current challenges in paediatric solid organ and haematopoietic stem cell transplantation in Europe: the European Reference Network TransplantChild.

The low prevalence of European paediatric transplanted patients and scarcity of resources and expertise led to the need for a multidisciplinary network able to improve the quality of life of paediatric patients and families requiring a solid organ or haematopoietic stem cell transplantation. The European Reference Network (ERN) TransplantChild is one of the 24 ERNs established in a European legal framework to improve the care of patients with rare diseases. ERN TransplantChild is the only ERN focused on both solid organ and haematopoietic stem cell paediatric transplantation, based on the understanding of paediatric transplantation as a complex and highly specialised process where specific complications appear regardless the organ involved, thus linking the skills and knowledge of different organ disciplines. Gathering European centres of expertise in paediatric transplantation will give access to a correct and timely diagnosis, share expertise and knowledge and collect a critical mass of patients and data that increases the speed and value of clinical research outcomes. Therefore, the ERN TransplantChild aims for a paediatric Pan-European, Pan-transplant approach.

T cell-derived interferon-γ programs stem cell death in immune-mediated intestinal damage.

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.

Fludarabine exposure in the conditioning prior to allogeneic hematopoietic cell transplantation predicts outcomes.

Fludarabine is the most frequently used agent in conditioning regimens for allogeneic hematopoietic cell transplantation (HCT). Body surface area-based dosing leads to highly variable fludarabine exposure. We studied the relation between fludarabine exposure and clinical outcomes. A retrospective, pharmacokinetic-pharmacodynamic analysis was conducted with data from patients undergoing HCT with fludarabine (160 mg/m2) as part of a myeloablative conditioning (busulfan targeted to an area under the plasma-concentration-time curve [AUC] of 90 mg*h/L) and rabbit antithymocyte globulin (6-10 mg/kg; from day -9/-12) between 2010 and 2016. Fludarabine exposure as AUC was calculated for each patient using a previously published population pharmacokinetic model and related to 2-year event-free survival (EFS) by means of (parametric) time-to-event models. Relapse, nonrelapse mortality (NRM), and graft failure were considered events. One hundred ninety-two patients were included (68 benign and 124 malignant disorders). The optimal fludarabine exposure was determined as an AUC of 20 mg*h/L. In the overexposed group, EFS was lower (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.1-3.5; P = .02), due to higher NRM (HR, 3.4; 95% CI, 1.6-6.9; P <001) associated with impaired immune reconstitution (HR, 0.43; 95% CI, 0.26-0.70; P <001). The risks of NRM and graft failure were increased in the underexposed group (HR, 3.3; 95% CI, 1.2-9.4; P = .02; HR, 4.8; 95% CI, 1.2-19; P = .02, respectively). No relationship with relapse was found. Fludarabine exposure is a strong predictor of survival after HCT, stressing the importance of optimum fludarabine dosing. Individualized dosing, based on weight and "renal function" or "therapeutic drug monitoring," to achieve optimal fludarabine exposure might improve survival.

Pre-existing invasive fungal infection is not a contraindication for allogeneic HSCT for patients with hematologic malignancies: a CIBMTR study.

Patients with prior invasive fungal infection (IFI) increasingly proceed to allogeneic hematopoietic cell transplantation (HSCT). However, little is known about the impact of prior IFI on survival. Patients with pre-transplant IFI (cases; n=825) were compared with controls (n=10247). A subset analysis assessed outcomes in leukemia patients pre- and post 2001. Cases were older with lower performance status (KPS), more advanced disease, higher likelihood of AML and having received cord blood, reduced intensity conditioning, mold-active fungal prophylaxis and more recently transplanted. Aspergillus spp. and Candida spp. were the most commonly identified pathogens. 68% of patients had primarily pulmonary involvement. Univariate and multivariable analysis demonstrated inferior PFS and overall survival (OS) for cases. At 2 years, cases had higher mortality and shorter PFS with significant increases in non-relapse mortality (NRM) but no difference in relapse. One year probability of post-HSCT IFI was 24% (cases) and 17% (control, P<0.001). The predominant cause of death was underlying malignancy; infectious death was higher in cases (13% vs 9%). In the subset analysis, patients transplanted before 2001 had increased NRM with inferior OS and PFS compared with later cases. Pre-transplant IFI is associated with lower PFS and OS after allogeneic HSCT but significant survivorship was observed. Consequently, pre-transplant IFI should not be a contraindication to allogeneic HSCT in otherwise suitable candidates. Documented pre-transplant IFI is associated with lower PFS and OS after allogeneic HSCT. However, mortality post transplant is more influenced by advanced disease status than previous IFI. Pre-transplant IFI does not appear to be a contraindication to allogeneic HSCT.

Impact of early CMV reactivation in cord blood stem cell recipients in the current era.

Several studies have reported an association between CMV reactivation and a decreased incidence of relapse for AML after adult donor allogeneic hematopoietic cell transplantation (HCT). Limited data, however, are available on the impact of CMV reactivation on relapse after cord blood (CB) stem cell transplantation. The unique combination of higher incidence of CMV reactivation in the seropositive recipient and lower incidence of graft versus host disease (GvHD) in CB HCT permits a valuable design to analyze the impact of CMV reactivation. Data from 1684 patients transplanted with CB between 2003 and 2010 for AML and ALL were analyzed. The median time to CMV reactivation was 34 days (range: 2-287). CMV reactivation and positive CMV serology were associated with increased non-relapse mortality (NRM) among both AML and ALL CB recipients (reactivation, AML: relative risk (RR) 1.41 (1.07-1.85); ALL: 1.60 (1.14-2.23); Serology, AML: RR 1.39 (1.05-1.85), ALL: RR 1.61 (1.18-2.19)). For patients with ALL, but not those with AML, this yielded inferior overall survival (P<0.005). Risk of relapse was not influenced by CMV reactivation or positive CMV serostatus for either disease.

The incidence, mortality and timing of Pneumocystis jiroveci pneumonia after hematopoietic cell transplantation: a CIBMTR analysis.

Pneumocystis jiroveci pneumonia (PJP) is associated with high morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Little is known about PJP infections after HSCT because of the rarity of disease given routine prophylaxis. We report the results of a Center for International Blood and Marrow Transplant Research study evaluating the incidence, timing, prophylaxis agents, risk factors and mortality of PJP after autologous (auto) and allogeneic (allo) HSCT. Between 1995 and 2005, 0.63% allo recipients and 0.28% auto recipients of first HSCT developed PJP. Cases occurred as early as 30 days to beyond a year after allo HSCT. A nested case cohort analysis with supplemental data (n=68 allo cases, n=111 allo controls) revealed that risk factors for PJP infection included lymphopenia and mismatch after HSCT. After allo or auto HSCT, overall survival was significantly poorer among cases vs controls (P=0.0004). After controlling for significant variables, the proportional hazards model revealed that PJP cases were 6.87 times more likely to die vs matched controls (P<0.0001). We conclude PJP infection is rare after HSCT but is associated with high mortality. Factors associated with GVHD and with poor immune reconstitution are among the risk factors for PJP and suggest that protracted prophylaxis for PJP in high-risk HSCT recipients may improve outcomes.

Biomarker profiling of steroid-resistant acute GVHD in patients after infusion of mesenchymal stromal cells.

We performed a prospective phase II study to evaluate clinical safety and outcome in 48 patients with steroid-refractory grade II-IV acute graft-versus-host disease (aGVHD) treated with mesenchymal stromal cells (MSCs). Clinical outcomes were correlated to comprehensive analyses of soluble and cellular biomarkers. Complete resolution (CR) of aGVHD at day 28 (CR-28) occurred in 12 (25%) patients, CR lasting >1 month (CR-B) occurred in 24 (50%) patients. One-year overall survival was significantly improved in CR-28 (75 versus 33%, P=0.020) and CR-B (79 versus 8%, P<0.001) versus non-CR patients. A six soluble biomarker-panel was predictive for mortality (HR 2.924; CI 1.485-5.758) when measured before MSC-administration. Suppression of tumorigenicity 2 (ST2) was only predictive for mortality 2 weeks after but not before MSC-administration (HR 2.389; CI 1.144-4.989). In addition, an increase in immature myeloid dendritic cells associated with decreased mortality (HR 0.554, CI 0.389-0.790). Patients had persisting T-cell responses against defined virus- and leukemia-associated antigens. In conclusion, our data emphasize the need to carefully assess biomarkers in cohorts with homogeneous GVHD treatments. Biomarkers might become an additional valuable component of composite end points for the rapid and efficient testing of novel compounds to decrease lifecycle of clinical testing and improve the success rate of phase II/III trials.

Predictors of invasive fungal infection in pediatric allogeneic hematopoietic SCT recipients.

This study was aimed at finding predictors of invasive fungal infection (IFI) after pediatric allogeneic hematopoietic SCT (HSCT). All children who received allogeneic HSCT in the Wilhelmina Children's Hospital Utrecht between 2004 and 2012 were included. HSCT data were prospectively collected. Patients were retrospectively classified into high- or low-risk groups for developing IFI using criteria based on available literature. Predictors for the occurrence of IFI were analyzed using Cox regression models. We used logistic regression models to analyze the association between other HSCT-related complications and IFI. Secondary outcomes were overall survival and treatment-related mortality (TRM). Two-hundred nine patients were included in the analysis; median age was 6.6 years. The cumulative incidence of IFI was 12%. In patients classified as 'low risk' (n=75), only 5.3% developed IFI (odds ratio (OR): 0.325; P=0.047). In multivariate analysis, a predictor for the occurrence of IFI was an a priori determined HSCT TRM risk >20% (based on EBMT-risk score). Post-HSCT, the administration of high-dose steroids was associated with IFI (OR: 4.458; P=0.010). Patients who developed IFI showed an increased risk of TRM (OR: 3.773; P=0.004). These results confirm that risk group stratification should guide intensity of monitoring for IFI and use of antifungal prophylaxis.

Fludarabine and exposure-targeted busulfan compares favorably with busulfan/cyclophosphamide-based regimens in pediatric hematopoietic cell transplantation: maintaining efficacy with less toxicity.

Busulfan (Bu) is used as a myeloablative agent in conditioning regimens before allogeneic hematopoietic cell transplantation (allo-HCT). In line with strategies explored in adults, patient outcomes may be optimized by replacing cyclophosphamide (Cy) with or without melphalan (Mel) with fludarabine (Flu). We compared outcomes in 2 consecutive cohorts of HCT recipients with a nonmalignant HCT indication, a myeloid malignancy, or a lymphoid malignancy with a contraindication for total body irradiation (TBI). Between 2009 and 2012, 64 children received Flu + Bu at a target dose of 80-95 mg·h/L, and between 2005 and 2008, 50 children received Bu targeted to 74-80 mg·h/L + Cy. In the latter group, Mel was added for patients with myeloid malignancy (n = 12). Possible confounding effects of calendar time were studied in 69 patients receiving a myeloablative dose of TBI between 2005 and 2012. Estimated 2-year survival and event-free survival were 82% and 78%, respectively, in the FluBu arm and 78% and 72%, respectively, in the BuCy (Mel) arm (P = not significant). Compared with the BuCy (Mel) arm, less toxicity was noted in the FluBu arm, with lower rates of acute (noninfectious) lung injury (16% versus 36%; P = .007), veno-occlusive disease (3% versus 28%; P = .003), chronic graft-versus-host disease (9% versus 26%; P = .047), adenovirus infection (3% versus 32%; P = .001), and human herpesvirus 6 infection reactivation (21% versus 44%; P = .005). Furthermore, the median duration of neutropenia was shorter in the FluBu arm (11 days versus 22 days; P < .001), and the patients in this arm required fewer transfusions. Our data indicate that Flu (160 mg/m(2)) with targeted myeloablative Bu (90 mg·h/L) is less toxic than and equally effective as BuCy (Mel) in patients with similar indications for allo-HCT.

γδT cells elicited by CMV reactivation after allo-SCT cross-recognize CMV and leukemia.

Human cytomegalovirus (CMV) infections and relapse of disease remain major problems after allogeneic stem cell transplantation (allo-SCT), in particular in combination with CMV-negative donors or cordblood transplantations. Recent data suggest a paradoxical association between CMV reactivation after allo-SCT and reduced leukemic relapse. Given the potential of Vδ2-negative γδT cells to recognize CMV-infected cells and tumor cells, the molecular biology of distinct γδT-cell subsets expanding during CMV reactivation after allo-SCT was investigated. Vδ2(neg) γδT-cell expansions after CMV reactivation were observed not only with conventional but also cordblood donors. Expanded γδT cells were capable of recognizing both CMV-infected cells and primary leukemic blasts. CMV and leukemia reactivity were restricted to the same clonal population, whereas other Vδ2(neg) T cells interact with dendritic cells (DCs). Cloned Vδ1 T-cell receptors (TCRs) mediated leukemia reactivity and DC interactions, but surprisingly not CMV reactivity. Interestingly, CD8αα expression appeared to be a signature of γδT cells after CMV exposure. However, functionally, CD8αα was primarily important in combination with selected leukemia-reactive Vδ1 TCRs, demonstrating for the first time a co-stimulatory role of CD8αα for distinct γδTCRs. Based on these observations, we advocate the exploration of adoptive transfer of unmodified Vδ2(neg) γδT cells after allo-SCT to tackle CMV reactivation and residual leukemic blasts, as well as application of leukemia-reactive Vδ1 TCR-engineered T cells as alternative therapeutic tools.

Pulmonary consolidation and inflammation: inflammatory pseudotumour in young children.

Here we report two children with a pulmonary inflammatory pseudotumour; a rare entity in children, that often initially presents as a pneumonia, but with the possibility of serious consequences if unrecognised and untreated. One of the children presented is 6 months which is extremely young for this tumour. Difficulties in presentation, management strategies and prognosis are described. Certainly, this is a condition that should be considered even in a very young child with an inflammatory condition presenting as a solid lesion in the lung which does not resolve or even progresses.

Systemic eosinophil response induced by respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease (LRTD) in infants. Eosinophils have been suggested to play a role in the disease pathogenesis of LRTD. Inflammation can induce functional and morphological alterations of peripheral blood granulocytes. In patients with RSV LRTD, we aimed to investigate the eosinophil activation status by analysing surface markers. In vitro stimulation of eosinophils with cytokines leads to up-regulation of CD11b and priming markers recognized by the recently developed priming markers A17 and A27, whereas interleukin (IL)-5Ralpha is being down-regulated. In 51 patients and 10 controls we examined the expression of these surface markers on eosinophils in moderate to severe RSV-induced LRTD patients at the time of admission and 6 weeks later during the convalescence phase. RSV-patients were characterized by a higher eosinophil CD11b expression compared to controls. Although basal A17 and A27 expression was not increased, we observed a significantly higher expression of these priming epitopes on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated cells of RSV patients compared with cells of controls, indicative of prior in vivo priming. Furthermore, IL-5Ralpha expression was down-regulated on peripheral blood eosinophils of these patients. Follow-up blood samples showed normalization of all markers but CD11b, which was persistently increased. Utilizing cellular markers, we observed that peripheral blood eosinophils from infants with RSV LRTD are in a more activated state compared to eosinophils of controls, which normalizes only partially during convalescence.

Regulation of granulocyte apoptosis by phosphatidylinositol 3-kinase.

Granulocytes are critical components of the innate immune system whose lifespan is limited by an intrinsic, constitutive, apoptotic pathway. However, the lifespan of these cells can be extended at an inflammatory locus through interaction with survival factors. Although a wide variety of factors can modulate granulocyte survival, they often utilize a common subset of intracellular signal transduction pathways. Over the last decade, evidence has accumulated that the PI3K (phosphatidylinositol 3-kinase) family of lipid kinases may be critical in regulating the ability of granulocytes to survive at inflammatory loci. Studies utilizing both pharmacological inhibitors of PI3K and isoform-specific knockout mice have demonstrated that this enzyme is needed for the anti-apoptotic effects of granulocyte survival factors. More recently, a serine/threonine protein kinase, termed protein kinase B (also known as c-akt), has been demonstrated to be important in modulating the prosurvival effects of PI3K activation. This can occur through modulation of the expression or phosphorylation of members of the Bcl-2 (B-cell lymphocytic-leukaemia proto-oncogene 2) family of apoptosis regulators. This review summarizes recent results that have implicated a role for PI3K in regulating granulocyte survival.