Cytomegalovirus infection is associated with thymic dysfunction and chronic graft-versus-host disease after pediatric hematopoietic stem cell transplantation.

Pediatric hematopoietic stem cell transplantation (HSCT) is challenged by chronic graft-versus-host disease (cGvHD) significantly affecting survival and long-term morbidity, but underlying mechanisms including the impact of post-HSCT CMV infection are sparsely studied. We first investigated the impact of CMV infection for development of cGvHD in 322 children undergoing standard myeloablative HSCT between 2000 and 2018. Clinically significant CMV infection (n = 61) was an independent risk factor for chronic GvHD in a multivariable Cox regression analysis (HR = 2.17, 95% CI = 1.18-3.97, P = 0.013). We next explored the underlying mechanisms in a subcohort of 39 children. CMV infection was followed by reduced concentration of recent thymic emigrants (17.5 vs. 51.9 × 106/L, P = 0.048) and naïve CD4+ and CD8+ T cells at 6 months post-HSCT (all P < 0.05). Furthermore, CD25highFOXP3+ Tregs tended to be lower in patients with CMV infection (2.9 vs. 9.6 × 106/L, P = 0.055), including Tregs expressing the naivety markers CD45RA and Helios. CD8+ T-cell numbers rose after CMV infection and was dominated by exhausted PD1-expressing cells (66% vs. 39%, P = 0.023). These findings indicate that post-HSCT CMV infection is a main risk factor for development of chronic GvHD after pediatric HSCT and suggest that this effect is caused by reduced thymic function with a persistently impaired production of naïve and regulatory T cells in combination with increased peripheral T-cell exhaustion.

IL-7 and IL-15 Levels Reflect the Degree of T Cell Depletion during Lymphopenia and Are Associated with an Expansion of Effector Memory T Cells after Pediatric Hematopoietic Stem Cell Transplantation.

Differentially and functionally distinct T cell subsets are involved in the development of complications after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about factors regulating their recovery after HSCT. In this study, we investigated associations between immune-regulating cytokines, T cell differentiation, and clinical outcomes. We included 80 children undergoing allogeneic HSCT for acute leukemia using bone marrow or peripheral blood stem cells grafted from a matched sibling or unrelated donor. Cytokines (IL-7, IL-15, IL-18, SCF, IL-6, IL-2, and TNF-α) and active anti-thymocyte globulin (ATG) levels were longitudinally measured along with extended T cell phenotyping. The cytokine profiles showed a temporary rise in IL-7 and IL-15 during lymphopenia, which was strongly dependent on exposure to active ATG. High levels of IL-7 and IL-15 from graft infusion to day +30 were predictive of slower T cell recovery during the first 2 mo post-HSCT; however, because of a major expansion of memory T cell stages, only naive T cells remained decreased after 3 mo (p < 0.05). No differential effect was seen on polarization of CD4+ T cells into Th1, Th2, or Th17 cells or regulatory T cells. Low levels of IL-7 and IL-15 at day +14 were associated with acute graft-versus-host disease grades II-IV in ATG-treated patients (p = 0.0004 and p = 0.0002, respectively). Children with IL-7 levels comparable to healthy controls at day +14 post-HSCT were less likely to develop EBV reactivation posttransplant. These findings suggest that quantification of IL-7 and IL-15 may be useful as biomarkers in assessing the overall T cell depletion and suggest a potential for predicting complications after HSCT.

Insulin-like growth factor-1 and insulin-like growth factor binding protein-3: impact on early haematopoietic reconstitution following allogeneic haematopoietic stem cell transplantation.

OBJECTIVES: The aim of the study was to investigate whether high endogenous levels of insulin-like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3) were related to a faster reconstitution of different blood cell populations in the early phase after allogeneic myeloablative haematopoietic stem cell transplantation (HSCT). METHODS: We measured IGF-1 and IGFBP-3 by chemiluminescence during the first three weeks after transplantation in 35 adult patients undergoing myeloablative HSCT and calculated area under the curve divided by time (AUC/t) for each patient. RESULTS: Circulating levels of IGF-1 and IGFBP-3 correlated with counts of reticulocytes (rs  = 0.44, p = .011 and r = 0.41, p = .017, respectively) and thrombocytes (rs  = 0.38, p = .030 and rs  = 0.56, p = .0008) three weeks post-transplant. Furthermore, high IGFBP-3 levels correlated with absolute lymphocyte counts 3 weeks post-HSCT (rs  = 0.54, p = .012) and were associated with shorter time to neutrophil engraftment (rs  = -0.35, p = .043). Both IGF-1 and IGFBP-3 levels were associated with the number of circulating natural killer cells one month after HSCT (rs  = 0.42, p = .032 and rs  = 0.57, p = .0026). CONCLUSION: These data indicate that high levels of IGF-1 and IGFBP-3 relate to a faster haematopoietic reconstitution after HSCT and suggest a biological influence of these mediators in haematopoietic homeostasis in these patients.

Thymic stromal lymphopoietin levels after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an immunoregulatory, Th2-polarizing cytokine produced by epithelial cells. We hypothesized that TSLP affects immune reconstitution after hematopoietic stem cell transplantation (HSCT) leading to increased alloreactivity. METHODS: We measured plasma TSLP by ELISA in 38 patients and assessed the immune reconstitution by flow cytometry. RESULTS: TSLP levels rose after initiation of the conditioning to peak at day +21 after HSCT (p = .03), where TSLP levels correlated with counts of neutrophils (rho = 0.36, p = .04), monocytes (rho = 0.58, p = .006), and lymphocytes (rho = 0.59, p = .02). Overall absolute TSLP levels were not associated with acute or chronic graft-vs-host disease (a/cGvHD). However, patients mounting a sustained increase in TSLP levels at day +90 had a higher risk of cGvHD compared to patients who had returned to pre-conditioning levels at that stage (cumulative incidence: 77% vs. 38%, p = .01). CONCLUSION: In conclusion, this study suggests a role of TSLP in immune reconstitution and alloreactivity post-HSCT. lymphopoietin (TSLP) is an immunoregulatory, Th2-polarizing cytokine produced by epithelial cells. We hypothesized that TSLP affects immune reconstitution after hematopoietic stem cell transplantation (HSCT) leading to increased alloreactivity. We measured plasma TSLP by ELISA in 38 patients and assessed the immune reconstitution by flow cytometry.

Glucagon-Like Peptide-1 Is a Marker of Systemic Inflammation in Patients Treated with High-Dose Chemotherapy and Autologous Stem Cell Transplantation.

Autologous stem cell transplantation (ASCT) is challenged by side effects that may be propagated by chemotherapy-induced mucositis, resulting in bacterial translocation and systemic inflammation. Because gastrointestinal damage appears as an early event in this cascade of reactions, we hypothesized that markers reflecting damage to the intestinal barrier could serve as early predictive markers of toxicity. Glucagon-like peptide-1 (GLP-1), a well-known regulator of blood glucose, has been found to promote intestinal growth and repair in animal studies. We investigated fasting GLP-1 plasma levels in 66 adults undergoing ASCT for lymphoma and multiple myeloma. GLP-1 increased significantly after chemotherapy, reaching peak levels at day +7 post-transplant (median, 8 pmol/L [interquartile range, 4 to 12] before conditioning versus 10 pmol/L [interquartile range, 6 to 17] at day +7; P = .007). The magnitude of the GLP-1 increase was related to the intensity of conditioning. GLP-1 at the day of transplantation (day 0) was positively associated with peak C-reactive protein (CRP) levels (46 mg/L per GLP-1 doubling, P < .001) and increase in days with fever (32% per GLP-1 doubling, P = .0058). Patients with GLP-1 above the median at day 0 had higher CRP levels from days +3 to +10 post-transplant than patients with lower GLP-1 (P ≤ .041) with peak values of 238 versus 129 mg/L, respectively. This study, which represents the first clinical investigation of fasting GLP-1 in relation to high-dose chemotherapy, provides evidence that GLP-1 plays a role in regulation of mucosal defenses. Fasting GLP-1 levels may serve as an early predictor of systemic inflammation and fever in patients receiving high-dose chemotherapy.

Reduced Plasma Amino Acid Levels During Allogeneic Hematopoietic Stem Cell Transplantation Are Associated with Systemic Inflammation and Treatment-Related Complications.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are challenged by cytotoxic effects of the conditioning regimen, resulting in tissue damage, systemic inflammation, and increased metabolic demands for amino acids to regenerate damaged tissues, reconstitute hematopoietic cells, and establish antioxidant defenses. To date, few studies have addressed the role of plasma amino acid (PAA) levels during transplantation, and it remains unknown if amino acid deficiency can aggravate treatment-related morbidity. We determined plasma levels of the 23 human amino acids in 80 HSCT recipients (age 1.1 to 55.4 years) before conditioning and on days +7 and +21 post-transplant along with C-reactive protein (CRP) and IL-6 levels on day +7. Significant changes were observed in plasma concentrations of several human amino acids during HSCT. On day +7, numerous amino acids were inversely correlated with both CRP and IL-6, including glutamic acid, serine, alanine, glutamine, arginine, cysteine, glycine, histidine, lysine, tryptophan, threonine, taurine, proline, and methionine (r = -.22 to -.66; all P < .05). Patients who developed sinusoidal obstruction syndrome (SOS) had significantly lower mean total PAA levels compared with patients without SOS (2013 ng/L [95% confidence interval (CI), 1709 to 2318 ng/L] versus 2706 ng/L [95% CI, 2261 to 3150 ng/L]; P = .006), along with lower individual levels of glutamic acid, serine, arginine, glycine, lysine, valine, tryptophan, threonine, and proline on day +7 (all P < .05). Patients with severe acute graft-versus-host disease had a lower mean total PAA level (1922 ng/L [95% CI, 1738 to 2106 ng/L] versus 2649 ng/L [95% CI, 2244 to 3055 ng/L]; P = .014) and lower levels of serine, glutamine, cysteine, glycine, lysine, and threonine on day +7 (all P < .05). These results indicate a relationship between low concentrations of certain amino acids and the risk of treatment-related complications.

Morphine consumption is associated with systemic inflammation in children undergoing allogeneic hematopoietic stem cell transplantation.

Background: The majority of children undergoing allogenic hematopoietic stem cell transplantation (HSCT) experience severe pain due to chemotherapy-induced gastrointestinal toxicity. Inter-individual differences in pain perceived and opioid consumption remain unexplained, limiting the possibility for individualized pain control. The aim of this study was to investigate the associations between opioid consumption and markers of gastrointestinal toxicity (plasma citrulline) and systemic inflammation (plasma CRP and IL-6) in these patients. Methods: We retrospectively included 38 children undergoing HSCT in Denmark in 2010-2012. Opioids doses on days 0-21 post-HSCT were registered as intravenous morphine equivalents (MEs). CRP was measured daily on days 0-21. IL-6 was measured on day 7. Citrulline was measured before conditioning, on days 7 and 21. Results: Out of 38 children, 37 (97%) received opioids during days 0-21. CRP level and ME dose peaked on days 9-10 while citrulline level reached a nadir on day 7 indicating maximum enterocyte loss. CRP was associated with ME dose, with an estimated increase of 0.030 mg/kg (95% CI 0.024-0.035) in ME for a 50% increase in CRP level on the same day (p < .001). IL-6 was correlated with ME on day 7 (rho = 0.55, p = .002). Citrulline did not correlate with ME. Conclusions: Opioid consumption in the early post-HSCT period is associated with the degree of chemotherapy-induced systemic inflammation and not with the extent of enterocyte loss. These findings contribute to our understanding of mucositis-related pain and may be of interest for future studies on therapeutic strategies.

Soluble Interleukin-7 receptor levels and risk of acute graft-versus-host disease after allogeneic haematopoietic stem cell transplantation.

Interleukin-7 is a cytokine essential for T cell homeostasis. IL-7 binds to cellular IL-7 receptors in competition with a soluble form of the receptor (sIL-7Rα). We hypothesized that altered sIL-7Rα levels may cause adverse outcomes in patients undergoing HSCT. In parallel, we investigated the impact of the IL-7Rα SNP rs6897932, which has been associated with release of IL-7R. The sIL-7Rα levels decreased during HSCT (from 114ng/ml before to 48ng/ml at day +14 (P<0.0001)). This pattern was inversely mirrored by IL-7. The IL-7/sIL-7Rα ratio at day +14 was significantly higher in patients developing grades II-IV aGVHD (OR=4.3, P=0.026). Furthermore, donor carriage of the rs6897932 T allele was associated with reduced sIL-7Rα levels, increased risk of grades II-IV aGVHD (OR=2.4, P=0.055) and increased transplant-related mortality (CC=4.5%, CT=21.4% and TT=27.3%, P=0.0037). In conclusion, this study suggests an impact of sIL-7Rα levels and rs6897932 donor genotype on alloreactivity and outcome after HSCT.

Gastrointestinal Toxicity, Systemic Inflammation, and Liver Biochemistry in Allogeneic Hematopoietic Stem Cell Transplantation.

Liver toxicity is frequently seen in relation to allogeneic hematopoietic stem cell transplantation (HSCT), but pathogenesis and the risk factors are poorly understood. The purpose of this study was to investigate associations between liver toxicity, gastrointestinal toxicity, and levels of immune-regulating cytokines during the early post-transplantation period. We prospectively included 81 children and adults undergoing HSCT after myeloablative conditioning. Alanine aminotransferase (ALT), total bilirubin levels, and international normalized ratio were measured longitudinally until 3 months after the transplantation and related to levels of inflammatory markers (C-reactive protein [CRP], IL-6, and IL-10) and to plasma citrulline as a marker of intestinal toxicity during the first 3 weeks after HSCT. The majority of patients experienced ALT levels above the normal range (45 U/L) with significant increases at 3 months after HSCT. Increased levels of total bilirubin were observed in 26% during the 3-month period. Citrulline levels decreased significantly to a nadir at day 7 (B = .23; 95% confidence interval [CI], .12 to .35; P < .0001), but citrulline levels at nadir were not associated with parameters of liver toxicity. However, a faster reconstitution of mucosa with higher citrulline levels at day +21 correlated with lower bilirubin levels 3 months after HSCT (r = -.26, P = .034) and increased overall survival (hazard ratio, .88; 95% CI, .79 to .97; P = .008) . Increased levels of CRP and IL-6 at day 7 after HSCT correlated positively with ALT and bilirubin, and in the multivariate analysis, IL-6 at day 7 appeared to be the only predicting risk factor for increased mean bilirubin during the early post-transplantation phase (B = .01; 95% CI, .01 to .02; P = .001) as well as maximum levels of bilirubin (B = .3; 95% CI, .12 to .48; P= .001) and occurrence of sinusoidal obstruction syndrome during the first 3 months after HSCT (odds ratio, 1.003; 95% CI, 1.001 to 1.005; P = .002). The results of this study indicate that liver toxicity after HSCT is associated with an increased inflammatory response mounted during the phase of maximal gastrointestinal toxicity in the early phase after transplantation.

sVEGF-R1 in acute non-infectious toxicity syndromes after pediatric allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is challenged by acute non-infectious toxicities, including sinusoidal obstruction syndrome (SOS), engraftment syndrome (ES) and capillary leak syndrome (CLS) among others. These complications are thought to be driven by a dysfunctional vascular endothelium, but the pathophysiological mechanisms remain incompletely understood, and the diagnoses are challenged by purely clinical diagnostic criteria that are partly overlapping, limiting the possibilities for progress in this field. There is, however, increasing evidence suggesting that these challenges may be met through the development of diagnostic biomarkers to improve diagnostic accuracy of pathogenetically homogenous entities, improved pre-transplant risk assessment and the early identification of patients with increased need for specific treatment. Soluble vascular endothelial growth factor receptor-1 (sVEGF-R1) is emerging as an important biomarker of endothelial damage in patients with trauma and sepsis but has not been studied in HSCT. OBJECTIVES: To investigate sVEGF-R1 as a marker of endothelial damage in pediatric HSCT patients by exploring associations with SOS, CLS, ES, and acute graft-versus-host disease (aGvHD). METHODS: We prospectively included 113 children undergoing myeloablative HSCT and measured sVEGF-R1 in plasma samples obtained weekly during the early period of transplantation and 3 months post-transplant. RESULTS: All over, sVEGF-R1 levels were significantly increased from day +7 after graft infusion, peaking at day +30, most pronounced in patients receiving busulfan. Patients considered to be at increased risk of SOS and therefore commenced on prophylactic defibrotide had significantly elevated levels of sVEGF-R1 before start of conditioning (446 pg/mL vs. 281 pg/mL, p = 0.0035), and this treatment appeared to stabilize sVEGF-R1 levels compared to patients not treated with defibrotide. Thirteen (11.5%) children meeting the modified Seattle criteria for SOS at median day +8 (1-18), had significantly elevated sVEGF-R1 levels on day +14 (489 pg/mL vs. 327 pg/mL, p = 0.007). In contrast. sVEGF-R1 levels in the much broader group of patients (45.1%) meeting EBMT-SOS criteria, including patients with very mild disease, did not overall differ in sVEGF-R1 levels, but higher sVEGF-R1 levels were seen in EBMT-SOS patients with an increased need for diuretic treatment. Importantly, sVEGF-R1 levels were not associated with ES and CLS but were significantly increased on day +30 in patients with grade III-IV aGvHD (OR = 4.2 pr. quartile, p = 0.023). CONCLUSION: VEGF-R1 levels are found to be increased in pediatric patients developing SOS, reflecting the severity of morbidity. sVEGF-R1 were unassociated with both CLS and ES. The potential of sVEGF-R1 as a clinically useful biomarker for SOS should be further explored to improve pre-transplant SOS-risk assessment, SOS-severity grading, and to guide treatment.

Plasma Levels of MRP-8/14 Associate With Neutrophil Recovery, Bacterial Bloodstream Infections, and Engraftment Syndrome Following Pediatric Allogeneic Hematopoietic Stem Cell Transplantation.

Neutrophil engraftment is essential for the successful outcome after allogeneic hematopoietic stem cell transplantation (HSCT), but neutrophil activation may also induce transplant-related complications. Myeloid-related protein (MRP)-8/14 is expressed in granulocytes during inflammatory conditions and secreted in response to tissue damage along with the release of pro-inflammatory cytokines together with leukocyte recruitment and activation. In this study, we investigated associations between levels of the neutrophil activition marker MRP-8/14, neutrophil recovery and toxicities after pediatric HSCT. We included 73 children undergoing allogeneic HSCT using bone marrow or peripheral blood stem cell grafts from matched sibling or unrelated donors. Plasma levels of MRP-8/14 were measured by enzyme-linked immunosorbent assay from preconditioning until 6 months after transplantation. Overall, MRP-8/14 levels decreased from pre-conditioning to a nadir at day 7 and then rose again until day 28, preceding the reappearance of neutrophils. MRP-8/14 levels were significantly reduced at day 14 in patients with delayed neutrophil engraftment compared with patients who engrafted by day 21 (0.20 versus 0.48 µg/mL, P = .0012) and in patients who developed bacterial bloodstream infections compared to patients without this complication (0.2 versus 0.36 µg/mL, P = .048). Patients developing engraftment syndrome had significantly elevated MRP-8/14 levels at day 7 and 21 compared to patients without engraftment syndrome (0.32 versus 0.2 µg/mL, P = .042 and 1.9 versus 0.80 µg/mL, P = .039, respectively), as well as increased neutrophil counts from day 9 to 25 (P ≤ .016). Similarly, neutrophil counts were increased at day 13 to 17 in patients with acute graft-versus-host disease grade III-IV compared with grade 0-II. This study is the first to monitor neutrophil activation by MRP-8/14 in HSCT patients in relation to infectious, as well as noninfectious post-transplantation complications. Our results provide increased insights into the pathophysiology of these complications, and further studies should explore the potential use of MRP-8/14 as a clinically useful biomarker.

Reduced mitochondrial respiration in peripheral T cells after paediatric heamatopoietic stem cell transplantation.

Background: Recovery and functional differentiation of T-cell subsets are central for the development of immune function and complications after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about the cellular respiration and factors influencing T-cell metabolic fitness during immune maturation after HSCT. Method: We included 20 HSCT patients and analysed mitochondrial oxidative phosphorylation and mitochondrial fitness in peripheral blood mononuclear cell samples collected at days +90 and +180 after HSCT. Results: Phenotypic analysis revealed lower overall T-cell counts, lower CD4+/CD8+ ratio and a skewed distribution of early T-cell subsets at day +90, gradually recovering by day +180. Although ATP turnover in HSCT patients was similar to healthy controls, the spare respiratory capacity (SRC) of T cells, reflecting the available energy reserve, was significantly reduced at day +90 and +180 compared to healthy controls. This reduction in SRC was not correlated with the occurrence of acute graft-versus-host disease (aGVHD), the intensity of conditioning regimens and markers of T-cell exhaustion. Conclusion: We found significantly depressed SRC until six months post-HSCT, but we were not able to identify transplant-related risk factors or associations with the clinical outcome.

Ocular graft-versus-host disease and dry eye disease after paediatric haematopoietic stem cell transplantation - incidence and risk factors.

Ocular graft-versus-host disease (oGVHD) contributes substantially to morbidity after allogeneic haematopoietic stem cell transplantation (HSCT) but is sparsely investigated in children. We assessed incidence and risk factors for oGVHD and dry eye disease (DED) in a nationwide, single-centre study of 484 consecutive children receiving HSCT during the period 1980-2016. Ophthalmological examinations were performed before and annually at least until five years after HSCT. Twenty-five patients had DED before transplantation (5.6%). The cumulative incidence was 1.9% for acute oGVHD, 6.0% for chronic oGVHD, 8.7% for new onset DED, and 12.7% for new onset Corneal Fluorescein Staining (CFS). In adjusted Fine-Gray regression models, the use of Busulfan was a risk factor for developing acute oGVHD (HR 5.01, p = 0.03), and malignant disease was a risk factor for developing CFS (HR 2.00, p = 0.047). Younger recipient age was associated with reduced risk of DED when comparing children aged 0-4 years with 10-16 years (HR 0.33, p = 0.03). These data underscore the need of attention to DED and oGVHD in relation to HSCT leading to our recommendation of performing ophthalmic examinations in all children before HSCT, and after HSCT when needed, in order to secure diagnosis and treatment of these complications.

Insulin-like growth factor-I predicts sinusoidal obstruction syndrome following pediatric hematopoietic stem cell transplantation.

Sinusoidal obstruction syndrome (SOS) is a potentially fatal complication of hematopoietic stem cell transplantation (HSCT) initiated through damage of sinusoidal endothelium and inflammation. Insulin-like growth factor-l (IGF-l) maintains and repairs endothelium and intestinal mucosa. We hypothesized that low IGF-l levels may increase the risk of inflammatory complications, such as SOS, in HSCT-patients. We prospectively measured IGF-l concentrations in 121 pediatric patients before, during, and after allogeneic HSCT. Overall, IGF-l levels were significantly reduced compared with healthy sex- and age-matched children. IGF-I levels pre-HSCT and at day 0 were inversely associated with C-reactive protein levels, hyperbilirubinemia, and number of platelet transfusions within the first 21 days post-transplant. Low levels of IGF-I before conditioning and at day of transplant were associated with increased risk of SOS diagnosed by the modified Seattle criteria (pre-HSCT: OR = 1.7 (95% CI: 1.2-2.6, p = 0.01), and the pediatric EBMT criteria (pre-HSCT: 1.7 (1.2-2.5, p = 0.009) and day 0: 1.7 (1.3-2.5, p = 0.001)/SDS decrease in IGF-1). These data suggest that IGF-I is protective against cytotoxic damage and SOS, most likely through trophic effects on endothelial cells and anti-inflammatory properties, and may prove useful as a predictive biomarker of SOS.

Microbiota long-term dynamics and prediction of acute graft-versus-host disease in pediatric allogeneic stem cell transplantation.

BACKGROUND: Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) exhibit changes in their gut microbiota and are experiencing a range of complications, including acute graft-versus-host disease (aGvHD). It is unknown if, when, and under which conditions a re-establishment of microbial and immunological homeostasis occurs. It is also unclear whether microbiota long-term dynamics occur at other body sites than the gut such as the mouth or nose. Moreover, it is not known whether the patients' microbiota prior to HSCT holds clues to whether the patient would suffer from severe complications subsequent to HSCT. Here, we take a holobiont perspective and performed an integrated host-microbiota analysis of the gut, oral, and nasal microbiota in 29 children undergoing allo-HSCT. RESULTS: The bacterial diversity decreased in the gut, nose, and mouth during the first month and reconstituted again 1-3 months after allo-HSCT. The microbial community composition traversed three phases over 1 year. Distinct taxa discriminated the microbiota temporally at all three body sides, including Enterococcus spp., Lactobacillus spp., and Blautia spp. in the gut. Of note, certain microbial taxa appeared already changed in the patients prior to allo-HSCT as compared with healthy children. Acute GvHD occurring after allo-HSCT could be predicted from the microbiota composition at all three body sites prior to HSCT. The reconstitution of CD4+ T cells, TH17, and B cells was associated with distinct taxa of the gut, oral, and nasal microbiota. CONCLUSIONS: This study reveals for the first time bacteria in the mouth and nose that may predict aGvHD. Monitoring of the microbiota at different body sites in HSCT patients and particularly through involvement of samples prior to transplantation may be of prognostic value and could assist in guiding personalized treatment strategies. The identification of distinct bacteria that have a potential to predict post-transplant aGvHD might provide opportunities for an improved preventive clinical management, including a modulation of microbiomes. The host-microbiota associations shared between several body sites might also support an implementation of more feasible oral and nasal swab sampling-based analyses. Altogether, the findings suggest that the microbiota and host factors together could provide actionable information to guiding precision medicine. Video Abstract.

Glucagon-Like Peptide-1 Is Associated With Systemic Inflammation in Pediatric Patients Treated With Hematopoietic Stem Cell Transplantation.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are challenged with severe side effects, which are propagated by mucosal barrier disruption, and the related microbial translocation and systemic inflammation. Glucagon-like peptide-1 (GLP-1), a well-known incretin hormone, possesses anti-inflammatory properties and promotes regeneration of damaged intestinal epithelium in animal studies. We hypothesized that the immense inter-individual variation in the degree of mucosal damage and systemic inflammation, seen after HSCT is influenced by endogenous GLP-1 and could be related to acute post-transplant complications. In this prospective study we measured serial weekly fasting plasma GLP-1, along with C-reactive protein (CRP), and citrulline in 82 pediatric patients during allogeneic HSCT together with a fasting plasma GLP-1 in sex- and age-matched healthy controls. Overall, GLP-1 levels were increased in the patients during the course of HSCT compared with the controls, but tended to decrease post-transplant, most pronounced in patients receiving high-intensity conditioning regimen. The increase in CRP seen in the early post-transplant phase was significantly lower from day +8 to +13 in patients with GLP-1 above the upper quartile (>10 pmol/L) at day 0 (all P ≤ 0.03). Similar findings were seen for peak CRP levels after adjusting for type of conditioning (-47.0%; 95% CI, -8.1 - -69.4%, P = 0.02). Citrulline declined significantly following the transplantation illustrating a decrease in viable enterocytes, most evident in patients receiving high-intensity conditioning regimen. GLP-1 levels at day 0 associated with the recovery rate of citrulline from day 0 to +21 (34 percentage points (pp)/GLP-1 doubling; 95% CI, 10 - 58pp; P = 0. 008) and day 0 to day +90 (48 pp/GLP-1 doubling; 95% CI, 17 - 79pp; P = 0. 004), also after adjustment for type of conditioning. This translated into a reduced risk of acute graft-versus-host disease (aGvHD) in patients with highest day 0 GLP-1 levels (>10 pmol/L) (cause-specific HR: 0.3; 95% CI, 0.2 - 0.9, P = 0.02). In conclusion, this study strongly suggests that GLP-1 influences regeneration of injured epithelial barriers and ameliorates inflammatory responses in the early post-transplant phase.

Assessment of the proposed EBMT pediatric criteria for diagnosis and severity grading of sinusoidal obstruction syndrome.

Sinusoidal obstruction syndrome (SOS) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). We assessed the proposed pediatric EBMT criteria along with the Baltimore and modified Seattle criteria in a population-based cohort. Eighty-seven children (1.1-17.3 years) undergoing myeloablative HSCT from 2010 to 2017 were consecutively included at the Danish National Transplantation Center. In total, 39 (44.8%) patients fulfilled the EBMT criteria and 30 patients (35%) fulfilled the criteria for severe or very severe SOS. Nine (10.3%) patients fulfilled the modified Seattle criteria while none met the Baltimore criteria. Patients fulfilling the EBMT criteria for SOS had longer primary admission (31 days (23-183) vs. 27 days (17-61), p = 0.001), were treated more intensively with diuretics within the first 3 months (29 days (0-90) vs. 3.5 days (0-90), p < 0.0001), and had a longer time to stable platelet counts >50 × 109/L (32 days (16-183) vs. 23 days (14-101), p < 0.0001). Two patients, fulfilling neither Baltimore nor Seattle criteria, but selectively fulfilling EBMT criteria, died of treatment-related acute inflammatory complications within 1 year post-HSCT. In conclusion, application of the pediatric EBMT diagnostic and severity criteria may be helpful in identifying patients at increased risk of severe treatment-related complications and mortality, although with a risk of over-diagnosing SOS.

[Chimeric antigen receptor T-cell therapy].

Although treatment of haematological cancer has improved significantly during the latest decades, the prognosis is poor in case of relapse or refractory disease. This review describes the chimeric antigen receptor (CAR) T-cell therapy, which has emerged as a new promising treatment principle, in which the patient's own T-cells are genetically modified to recognise cancer cells. The possible side effects are usually only transient. A commercial CD19 CAR T-cell product has recently been approved as treatment for acute lympho-blastic leukaemia in children and young adults in Denmark, and a non-commercial CAR T-cell production is being established.

Specific gut microbiome members are associated with distinct immune markers in pediatric allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Increasing evidence reveals the importance of the microbiome in health and disease and inseparable host-microbial dependencies. Host-microbe interactions are highly relevant in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT), i.e., a replacement of the cellular components of the patients' immune system with that of a foreign donor. HSCT is employed as curative immunotherapy for a number of non-malignant and malignant hematologic conditions, including cancers such as acute lymphoblastic leukemia. The procedure can be accompanied by severe side effects such as infections, acute graft-versus-host disease (aGvHD), and death. Here, we performed a longitudinal analysis of immunological markers, immune reconstitution and gut microbiota composition in relation to clinical outcomes in children undergoing HSCT. Such an analysis could reveal biomarkers, e.g., at the time point prior to HSCT, that in the future could be used to predict which patients are of high risk in relation to side effects and clinical outcomes and guide treatment strategies accordingly. RESULTS: In two multivariate analyses (sparse partial least squares regression and canonical correspondence analysis), we identified three consistent clusters: (1) high concentrations of the antimicrobial peptide human beta-defensin 2 (hBD2) prior to the transplantation in patients with high abundances of Lactobacillaceae, who later developed moderate or severe aGvHD and exhibited high mortality. (2) Rapid reconstitution of NK and B cells in patients with high abundances of obligate anaerobes such as Ruminococcaceae, who developed no or mild aGvHD and exhibited low mortality. (3) High inflammation, indicated by high levels of C-reactive protein, in patients with high abundances of facultative anaerobic bacteria such as Enterobacteriaceae. Furthermore, we observed that antibiotic treatment influenced the bacterial community state. CONCLUSIONS: We identify multivariate associations between specific microbial taxa, host immune markers, immune cell reconstitution, and clinical outcomes in relation to HSCT. Our findings encourage further investigations into establishing longitudinal surveillance of the intestinal microbiome and relevant immune markers, such as hBD2, in HSCT patients. Profiling of the microbiome may prove useful as a prognostic tool that could help identify patients at risk of poor immune reconstitution and adverse outcomes, such as aGvHD and death, upon HSCT, providing actionable information in guiding precision medicine.