Caroline A. Lindemans

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5+ ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

How I treat adenovirus in hematopoietic stem cell transplant recipients

Adenovirus (AdV) infections are very common in the general pediatric population. The delayed clearance in young persons imposes a threat to immunocompromised patients after hematopoietic stem cell transplantation (HSCT), who can reactivate the virus, resulting in life-threatening disseminated disease. Although a definitive cure requires adequate immune reconstitution, 2 approaches appear to be feasible and effective to improve the outcomes of AdV infections. Strict monitoring with AdV quantitative polymerase chain reaction followed by preemptive treatment with low-dose (1 mg/kg) cidofovir 3 times a week, is effective in most cases to bridge the severely immunocompromised period shortly after HSCT, with acceptable toxicity rates. For centers who have the access, AdV-specific cytotoxic T cells can be the other important cornerstone of anti-AdV therapy with promising results so far. Methods to positively influence the reconstitution of the immune system after HSCT and optimizing new and currently available cellular immunotherapies will make HSCT safer against the threat of AdV infection/reactivation and associated disease.

Gut microbiome–derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease

The effect of alterations in intestinal microbiota on microbial metabolites and on disease processes such as graft-versus-host disease (GVHD) is not known. Here we carried out an unbiased analysis to identify previously unidentified alterations in gastrointestinal microbiota–derived short-chain fatty acids (SCFAs) after allogeneic bone marrow transplant (allo-BMT). Alterations in the amount of only one SCFA, butyrate, were observed only in the intestinal tissue. The reduced butyrate in CD326+ intestinal epithelial cells (IECs) after allo-BMT resulted in decreased histone acetylation, which was restored after local administration of exogenous butyrate. Butyrate restoration improved IEC junctional integrity, decreased apoptosis and mitigated GVHD. Furthermore, alteration of the indigenous microbiota with 17 rationally selected strains of high butyrate–producing Clostridia also decreased GVHD. These data demonstrate a heretofore unrecognized role of microbial metabolites and suggest that local and specific alteration of microbial metabolites has direct salutary effects on GVHD target tissues and can mitigate disease severity.

Impact of thymoglobulin prior to pediatric unrelated umbilical cord blood transplantation on immune reconstitution and clinical outcome

In vivo T-cell depletion might contribute to the delayed immune reconstitution observed after unrelated umbilical cord blood transplantation (UCBT). We studied the impact of early, late, and no antithymocyte globulin (ATG) on immune reconstitution and outcome. One hundred twenty seven children receiving UCBT in London or Utrecht were divided into 3 groups: early ATG (days -9 to -5; n = 33), late ATG (days -5 to 0; n = 48), and no ATG (n = 46). The no-ATG group received mycophenolate mofetile + cyclosporin A as graft-versus-host disease (GVHD) prophylaxis, while the ATG groups received cyclosporin A + prednisone. End points studied were survival, immune recovery, infections, and GVHD. The probability of survival was similar in all groups: no ATG, 71% ± 8%; early ATG, 68% ± 9%; and late ATG, 61% ± 7%. CD3(+), CD4(+), and CD4(+)-naive T-cell counts were significantly higher (P < .001) in the no-ATG group at 1, 2, 3, 6, and 12 months post-UCBT. In the no-ATG group, significantly fewer viral reactivations (P = .021) were noted. A higher probability of severe acute GVHD (aGVHD; 31%) was found in the no-ATG group compared with 18% (P = .018) for early-ATG and 5% (P < .001) for late-ATG groups. This was not associated with more chronic GVHD (cGVHD).

Respiratory Syncytial Virus Inhibits Granulocyte Apoptosis through a Phosphatidylinositol 3-Kinase and NF-κB-Dependent Mechanism

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in children. It is associated with increased neutrophil numbers in the airway. In this study, we assessed whether this ssRNA virus can directly influence granulocyte longevity. By culturing RSV with granulocytes, it was observed that virus delays both constitutive neutrophil and eosinophil apoptosis. Using pharmacological inhibitors, the RSV-induced delay in neutrophil apoptosis was found to be dependent on both PI3K and NF-κB, but not p38 MAPK or MEK1/MEK2 activation. Using blocking Abs and a reporter cell line, we were able to exclude TLR4 as the receptor responsible for mediating RSV-induced delay in neutrophil apoptosis. The antiapoptotic effect was abrogated by preincubation with the lysosomotropic agent chloroquine, indicating the requirement for endolysosomal internalization. Furthermore, addition of ssRNA, a ligand for the intracellular TLR7/TLR8, also inhibited neutrophil apoptosis, suggesting that intracellular TLRs could be involved in induction of the antiapoptotic effect. Using the BioPlex cytokine detection assay (Bio-Rad), we found that IL-6 was present in supernatants from RSV-exposed neutrophils. IL-6 was found to inhibit neutrophil apoptosis, suggesting that there is an autocrine or paracrine antiapoptotic role for IL-6. Finally, RSV treatment of neutrophils resulted in increased expression of the antiapoptotic Bcl-2 protein Mcl-1. Taken together, our findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intracellular TLRs in mediating these effects.

Early cytomegalovirus reactivation remains associated with increased transplant-related mortality in the current era: a CIBMTR analysis

Single-center studies have reported an association between early (before day 100) cytomegalovirus (CMV) reactivation and decreased incidence of relapse for acute myeloid leukemia (AML) following allogeneic hematopoietic cell transplantation. To substantiate these preliminary findings, the Center for International Blood and Marrow Transplant Research (CIBMTR) Database was interrogated to analyze the impact of CMV reactivation on hematologic disease relapse in the current era. Data from 9469 patients transplanted with bone marrow or peripheral blood between 2003 and 2010 were analyzed according to 4 disease categories: AML (n = 5310); acute lymphoblastic leukemia (ALL, n = 1883); chronic myeloid leukemia (CML, n = 1079); and myelodysplastic syndrome (MDS, n = 1197). Median time to initial CMV reactivation was 41 days (range, 1-362 days). CMV reactivation had no preventive effect on hematologic disease relapse irrespective of diagnosis. Moreover, CMV reactivation was associated with higher nonrelapse mortality [relative risk [RR] among disease categories ranged from 1.61 to 1.95 and P values from .0002 to <.0001; 95% confidence interval [CI], 1.14-2.61). As a result, CMV reactivation was associated with lower overall survival for AML (RR = 1.27; 95% CI, 1.17-1.38; P <.0001), ALL (RR = 1.46; 95% CI, 1.25-1.71; P <.0001), CML (RR = 1.49; 95% CI, 1.19-1.88; P = .0005), and MDS (RR = 1.31; 95% CI, 1.09-1.57; P = .003). In conclusion, CMV reactivation continues to remain a risk factor for poor posttransplant outcomes and does not seem to confer protection against hematologic disease relapse.

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.

New autoimmune diseases after cord blood transplantation : a retrospective study of EUROCORD and the Autoimmune Disease Working Party of the European Group for Blood and Marrow Transplantation

To describe the incidence, risk factors, and treatment of autoimmune diseases (ADs) occurring after cord blood transplantation (CBT), we analyzed both CBT recipients reported to EUROCORD who had developed at least 1 new AD and those who had not. Fifty-two of 726 reported patients developed at least 1 AD within 212 days (range, 27-4267) after CBT. Cumulative incidence of ADs after CBT was 5.0% +/- 1% at 1 year and 6.6% +/- 1% at 5 years. Patients developing ADs were younger and had more nonmalignant diseases (P < .001). ADs target hematopoietic (autoimmune hemolytic anemia, n = 20; Evans syndrome, n = 9; autoimmune thrombocytopenia, n = 11; and immune neutropenia, n = 1) and other tissues (thyroiditis, n = 3; psoriasis, n = 2; Graves disease, n 1; membranous glomerulonephritis, n = 2; rheumatoid arthritis, n = 1; ulcerative colitis, n = 1; and systemic lupus erythematosus, n = 1). Four patients developed 2 ADs (3 cases of immune thrombocytopenia followed by autoimmune hemolytic anemia and 1 Evans syndrome with rheumatoid arthritis). By multivariate analysis, the main risk factor for developing an AD was nonmalignant disease as an indication for CBT (P = .0001). Hematologic ADs were most often treated with steroids, rituximab, and cyclosporine. With a median follow-up of 26 months (range, 2-91), 6 of 52 patients died as a consequence of ADs. We conclude that CBT may be followed by potentially life-threatening, mainly hematologic ADs.

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.

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)‐5Rα 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‐5Rα 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.