Characterizing the human intestinal chondroitin sulfate glycosaminoglycan sulfation signature in inflammatory bowel disease.

The intestinal extracellular matrix (ECM) helps maintain appropriate tissue barrier function and regulate host-microbial interactions. Chondroitin sulfate- and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs) are integral components of the intestinal ECM, and alterations in CS/DS-GAGs have been shown to significantly influence biological functions. Although pathologic ECM remodeling is implicated in inflammatory bowel disease (IBD), it is unknown whether changes in the intestinal CS/DS-GAG composition are also linked to IBD in humans. Our aim was to characterize changes in the intestinal ECM CS/DS-GAG composition in intestinal biopsy samples from patients with IBD using mass spectrometry. We characterized intestinal CS/DS-GAGs in 69 pediatric and young adult patients (n = 13 control, n = 32 active IBD, n = 24 IBD in remission) and 6 adult patients. Here, we report that patients with active IBD exhibit a significant decrease in the relative abundance of CS/DS isomers associated with matrix stability (CS-A and DS) compared to controls, while isomers implicated in matrix instability and inflammation (CS-C and CS-E) were significantly increased. This imbalance of intestinal CS/DS isomers was restored among patients in clinical remission. Moreover, the abundance of pro-stabilizing CS/DS isomers negatively correlated with clinical disease activity scores, whereas both pro-inflammatory CS-C and CS-E content positively correlated with disease activity scores. Thus, pediatric patients with active IBD exhibited increased pro-inflammatory and decreased pro-stabilizing CS/DS isomer composition, and future studies are needed to determine whether changes in the CS/DS-GAG composition play a pathogenic role in IBD.

Imaging and endoscopic tools in pediatric inflammatory bowel disease: What's new?

Pediatric inflammatory bowel disease (IBD) is a chronic inflammatory disorder, with increasing incidence and prevalence worldwide. There have been recent advances in imaging and endoscopic technology for disease diagnosis, treatment, and monitoring. Intestinal ultrasound, including transabdominal, transperineal, and endoscopic, has been emerging for the assessment of transmural bowel inflammation and disease complications (e.g., fistula, abscess). Aside from surgery, IBD-related intestinal strictures now have endoscopic treatment options including through-the-scope balloon dilatation, injection, and needle knife stricturotomy and new evaluation tools such as endoscopic functional lumen imaging probe. Unsedated transnasal endoscopy may have a role in patients with upper gastrointestinal Crohn's disease or those with IBD with new upper gastrointestinal symptoms. Improvements to dysplasia screening in pediatric patients with longstanding colonic disease or primary sclerosing cholangitis hold promise with the addition of virtual chromoendoscopy and ongoing research in the field of artificial intelligence-assisted endoscopic detection. Artificial intelligence and machine learning is a rapidly evolving field, with goals of further personalizing IBD diagnosis and treatment selection as well as prognostication. This review summarized these advancements, focusing on pediatric patients with IBD.

Reverse-engineered exclusive enteral nutrition in pediatric Crohn's disease: A pilot trial.

BACKGROUND: In pediatric Crohn's disease (CD), commercial formulas used as exclusive enteral nutrition (EEN) are effective at inducing remission. This study aims to assess the impact of a whole-food blended smoothie as EEN on CD activity and the intestinal microbiome. METHODS: A 4-week prospective trial assessed the impact of EEN with a whole-food smoothie on newly diagnosed mild-to-moderate active pediatric CD. The smoothie with a multivitamin were developed to meet age-appropriate nutritional requirements. Assessment over 4 weeks included Pediatric Crohn's Disease Activity Index (PCDAI), serum laboratories, fecal calprotectin (FCP), and stool collection for metagenomic shotgun sequencing and microbiota composition analysis. Clinical remission was defined as PCDAI ≤ 10 at week 4. RESULTS: Ten participants were enrolled with median age 14.5 years, and 8 completed the trial. Baseline mean PCDAI was 26.3 ± 9.1 and mean FCP 1149 ± 718 µg/g. At week 4, 80% of participants achieved clinical remission. FCP decreased by over half in 60% of participants, with FCP below 250 µg/g in 60% and below 100 µg/g in 40%. Microbiome analysis showed a significant increase in species richness over 4 weeks (p = 0.01). Compared to baseline, the relative abundance at week 2 and at week 4 was significantly increased for Bifidobacterium and Streptococcus and decreased for Blautia (p < 0.05 for all). CONCLUSION: A whole-food blended smoothie was effective for inducing clinical remission and decreasing FCP in pediatric CD similar to commercial EEN formulas. Further research may give insight into data-driven whole-food dietary approaches for CD management. CLINICALTRIALS: gov NCT03508193.

Notch signaling drives intestinal graft-versus-host disease in mice and nonhuman primates.

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Association between iron deficit repletion with ferric carboxymaltose relative to iron sucrose in children with inflammatory bowel disease: A retrospective cohort study.

BACKGROUND: Iron deficiency and iron deficiency anemia are common in pediatric inflammatory bowel disease and often require supplementation with iron. There is a paucity of literature regarding optimal iron formulation. The aim of this study is to compare outcomes in pediatric patients with inflammatory bowel disease receiving either iron sucrose or ferric carboxymaltose during inpatient hospitalizations. METHODS: This was a single-center retrospective study of pediatric patients with inflammatory bowel disease admitted for newly diagnosed disease or flare who received either iron sucrose or ferric carboxymaltose. Linear regression was used to assess differences in iron repletion. Longitudinal linear mixed-effects models and generalized estimating equations compared hematologic and iron outcomes 6 months post-iron repletion. RESULTS: Thirty patients received ferric carboxymaltose. Sixty-nine patients received iron sucrose. Baseline hemoglobin and iron deficits were similar in both groups. A larger percentage of iron deficit was repleted in the ferric carboxymaltose group (81.4%) compared with iron sucrose (25.9%) (P < 0.001) with fewer infusions. Cumulative doses of ferric carboxymaltose administered (18.7 mg/kg) were higher than iron sucrose (6.1 mg/kg) (P < 0.001). Hemoglobin increased more quickly with ferric carboxymaltose compared with iron sucrose (P = 0.04 and P = 0.02, respectively). Total iron binding capacity and red cell distribution width levels decreased more over time with ferric carboxymaltose vs iron sucrose (P < 0.01 and P = 0.01, respectively). No adverse effects were seen. CONCLUSIONS: Hematologic and iron parameters responded more quickly with fewer infusions in patients who received ferric carboxymaltose vs iron sucrose. Patients who received ferric carboxymaltose achieved a higher percentage of iron deficit repleted.

Sirolimus for management of GI bleeding in blue rubber bleb nevus syndrome: A case series.

Blue rubber bleb nevus syndrome (BRBNS) commonly presents with anemia from bleeding gastrointestinal (GI) vascular malformations. Management is highly variable, as no consensus guidelines for medical treatment currently exist. Sirolimus has been used in BRBNS to decrease GI bleeding and seems well tolerated, though questions remain regarding dosing, duration of therapy, and adverse effects. Here, we report our single-center experience of four pediatric patients with BRBNS who were successfully treated with sirolimus and review the existing literature regarding sirolimus for treatment of GI bleeding in BRBNS. Further prospective studies are needed to establish optimal dosage, drug monitoring, and duration.

Risk Factors for Development of Pneumatosis Intestinalis after Pediatric Hematopoietic Stem Cell Transplantation: A Single-Center Case-Control Study.

The significance of pneumatosis intestinalis (PI) in pediatric patients following hematopoietic stem cell transplantation (HSCT) is poorly understood. A knowledge gap remains with respect to the etiology, risk factors, and evidence-based treatment of these patients. As a result, management is frequently based on each center's clinical practice, without standardization across treatment centers. In this single-center trial, we aimed to validate both previously proposed and additional risk factors for the development of PI and to examine our management and outcomes for these patients. We performed a retrospective case-control study examining risk factors for the development of PI in pediatric HSCT patients at a single tertiary referral children's hospital. We used univariate and multivariable conditional logistic regression analysis to explore differences in pharmacologic and other transplantation-specific risk factors. Between 2012 and 2019, PI was diagnosed in 212 patients at our pediatric hospital, of whom 42 were HSCT recipients. The majority of patients (88%; n = 37 of 42) with PI were diagnosed by X-ray. Eighteen patients (43%) were asymptomatic and diagnosed incidentally after imaging was obtained for standard post-transplantation surveillance or other nonrelated indications. All patients with PI were hospitalized and placed on strict bowel rest while receiving parenteral nutrition and antibiotics. Recurrence of PI occurred in 4 patients (10%) following their initial diagnosis. Increased doses of steroid exposure within 30 days of PI diagnosis (odds ratio [OR], 5.7; 95% confidence interval [CI], 2.1 to 15.3; P = .0006), presence of grade II-IV gastrointestinal acute graft-versus-host disease (GVHD) (OR, 5.3; 95% CI, 1.0 to 28.1; P = .05), and receipt of >50% of total daily nutrition by nasogastric (NG) tube feeds (OR, 22.0; 95% CI, 1.3 to 370.2; P = .03) were identified as independent risk factors for the development of PI. Intensity of the conditioning regimen, exposure to total body irradiation, stem cell source, donor type, HLA matching, use of mycophenolate mofetil, and presence of bacterial or viral infection at the time of PI diagnosis were not demonstrably associated with the development of PI in our study. We conclude that development of asymptomatic PI is a benign condition following HSCT, and that the risk for PI is increased in patients with gastrointestinal GVHD, patients receiving steroid therapy, and patients relying on supplemental NG tube feeds for at least one-half of their total daily nutrition.

Linking Genetic Diagnosis to Therapeutic Approach in Very Early Onset Inflammatory Bowel Disease: Pharmacologic Considerations.

Very early onset inflammatory bowel disease (VEO-IBD) is diagnosed in children < 6 years of age, and in rare cases may be due to an identifiable monogenic cause. Recent advances in genetic testing have allowed for more accurate diagnosis, with as many as 100 genes now known to be associated with monogenic inflammatory bowel disease. These genes are involved in many immune pathways and thus may represent potential avenues for targeted precision medicine with pharmacologic treatments aimed at these. This review describes the broad classifications of monogenic disorders known to cause VEO-IBD, as well as empiric and disease-specific medical therapies. These include immune-modulating or immunosuppressant medications, nutritional therapy, surgery, and hematopoietic stem cell transplantation. We aim to provide an overview of the current state of targeted therapy for VEO-IBD.

Adjunctive Dietary Therapy Is Associated With Improved Gastrointestinal Symptoms in Steroid-Refractory Gastrointestinal Graft-Versus-Host Disease: A Case Series.

Acute gastrointestinal graft-versus-host disease (GI GVHD) is a complication after hematopoietic stem cell transplant with high morbidity and mortality. In particular, steroid-refractory GI GVHD can be difficult to treat. Recent investigations have revealed that patients after transplant can experience intestinal dysbiosis contributing to the progression of GVHD. Modulation of the gut microbiome through dietary intake could potentially improve the intestinal dysbiosis in GI GVHD. In this case series, we present 3 patients where dietary therapy was used in conjunction with immunosuppression to achieve clinical remission of GI GVHD.

Spatiotemporal single-cell profiling reveals that invasive and tissue-resident memory donor CD8+ T cells drive gastrointestinal acute graft-versus-host disease.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRM cells during aGVHD in primate transplant recipients.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

CD28 blockade controls T cell activation to prevent graft-versus-host disease in primates.

Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab', in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-γ response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.

Chimeric Antigen Receptor T Cell-Mediated Neurotoxicity in Nonhuman Primates.

Chimeric antigen receptor (CAR) T-cell immunotherapy has revolutionized the treatment of refractory leukemias and lymphomas, but is associated with significant toxicities, namely cytokine release syndrome (CRS) and neurotoxicity. A major barrier to developing therapeutics to prevent CAR T cell-mediated neurotoxicity is the lack of clinically relevant models. Accordingly, we developed a rhesus macaque (RM) model of neurotoxicity via adoptive transfer of autologous CD20-specific CAR T cells. Following cyclophosphamide lymphodepletion, CD20 CAR T cells expand to 272 to 4,450 cells/µL after 7 to 8 days and elicit CRS and neurotoxicity. Toxicities are associated with elevated serum IL6, IL8, IL1RA, MIG, and I-TAC levels, and disproportionately high cerebrospinal fluid (CSF) IL6, IL2, GM-CSF, and VEGF levels. During neurotoxicity, both CD20 CAR and non-CAR T cells accumulate in the CSF and in the brain parenchyma. This RM model demonstrates that CAR T cell-mediated neurotoxicity is associated with proinflammatory CSF cytokines and a pan-T cell encephalitis.Significance: We provide the first immunologically relevant, nonhuman primate model of B cell-directed CAR T-cell therapy-mediated CRS and neurotoxicity. We demonstrate CAR and non-CAR T-cell infiltration in the CSF and in the brain during neurotoxicity resulting in pan-encephalitis, accompanied by increased levels of proinflammatory cytokines in the CSF. Cancer Discov; 8(6); 750-63. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Combined OX40L and mTOR blockade controls effector T cell activation while preserving Treg reconstitution after transplant.

A critical question facing the field of transplantation is how to control effector T cell (Teff) activation while preserving regulatory T cell (Treg) function. Standard calcineurin inhibitor-based strategies can partially control Teffs, but breakthrough activation still occurs, and these agents are antagonistic to Treg function. Conversely, mechanistic target of rapamycin (mTOR) inhibition with sirolimus is more Treg-compatible but is inadequate to fully control Teff activation. In contrast, blockade of OX40L signaling has the capacity to partially control Teff activation despite maintaining Treg function. We used the nonhuman primate graft-versus-host disease (GVHD) model to probe the efficacy of combinatorial immunomodulation with sirolimus and the OX40L-blocking antibody KY1005. Our results demonstrate significant biologic activity of KY1005 alone (prolonging median GVHD-free survival from 8 to 19.5 days), as well as marked, synergistic control of GVHD with KY1005 + sirolimus (median survival time, >100 days; P < 0.01 compared to all other regimens), which was associated with potent control of both TH/TC1 (T helper cell 1/cytotoxic T cell 1) and TH/TC17 activation. Combined administration also maintained Treg reconstitution [resulting in an enhanced Treg/Teff ratio (40% over baseline) in the KY1005/sirolimus cohort compared to a 2.9-fold decrease in the unprophylaxed GVHD cohort]. This unique immunologic signature resulted in transplant recipients that were able to control GVHD for the length of analysis and to down-regulate donor/recipient alloreactivity despite maintaining anti-third-party responses. These data indicate that combined OX40L blockade and sirolimus represents a promising strategy to induce immune balance after transplant and is an important candidate regimen for clinical translation.