Postoperative Delirium Screening Characteristics in Pediatric Intestinal, Liver, and Renal Transplant Recipients: Single-Center Retrospective Cohort Study.

OBJECTIVES: To describe and compare the results of delirium screening in the immediate post-transplant PICU admission for pediatric intestinal, liver, and renal transplant recipients. We also examined associations with known and suspected risk factors for pediatric delirium (PD). DESIGN: Retrospective analysis of a single-center cohort, 2016-2022. SETTING: Twenty-four-bed PICU in a high-volume transplant center. PATIENTS: All intestinal, liver, and renal transplant recipients under 23 years old admitted between July 2016 and August 2022. MEASUREMENTS AND MAIN RESULTS: We identified 211 pediatric transplant recipients: intestinal (n = 36), liver (n = 78), and renal (n = 97). Results of the Cornell Assessment for PD during the immediate post-transplant PICU admission were reviewed and patients were categorized into screen positive, screen negative, and unscreened. Corresponding data on known and suspected risk factors for PD were also collected. Data on delirium subtypes were not collected. Screens were available for 156 of 211 patients (74%) who were included in the final analysis. The prevalence of a positive screen by transplant category was: intestine 80% (24/30), liver 75% (47/63), and renal 14% (9/63). A positive screen was associated with younger age, greater duration of mechanical ventilation, and greater PICU length of stay (LOS) in bivariate analysis. In multivariable analysis, age and PICU LOS remained strongly correlated with a positive screen (p < 0.05). Deep sedation and agitation as categorized by the State Behavioral Scale was associated with a positive screen, as was significant iatrogenic withdrawal symptoms (p < 0.05). Most patients screened positive by post-transplant days 2 and 3 (58/80 [72%] and 64/80 [80%], respectively). CONCLUSIONS: In our 2016 to 2022 experience, we found a high prevalence of positive PD screens in pediatric intestinal and liver transplant recipients in the immediate post-transplant PICU admission. A positive screen was associated with younger age and greater PICU LOS.

Should a Stoma Be Used After Intestinal Transplant.

As we all acknowledge benefits of ostomies, they can come with significant morbidity, quality of life issues, and major complications, especially during reversal procedures. In recent years, we have started to observe that similar graft and patient survival can be achieved without ostomies in certain cases. This observation and practice adopted in a few large-volume transplant centers opened a new discussion about the necessity of ostomies in intestinal transplantation. There is still more time and randomized studies will be needed to better understand and analyze the risk/benefits of "No-ostomy" approach in intestinal transplantation.

Minnelide suppresses GVHD and enhances survival while maintaining GVT responses.

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide's GVHD suppression after aHSCT. Importantly, Minnelide's GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti-acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.

Regulatory T Cell Amelioration of Graft-versus-Host Disease following Allogeneic/Xenogeneic Hematopoietic Stem Cell Transplantation Using Mobilized Mouse and Human Peripheral Blood Donors.

The present studies examined experimental transplant outcomes using mobilized peripheral blood from mice and humans together with FoxP3+Treg cells. Donor mice were treated with filgrastim and / or plerixafor and their peripheral blood (PB) displayed significant elevations in hematopoietic stem and progenitor populations. Some of these PB donors were concurrently administered a Treg expansion strategy consisting of a TL1A-Ig fusion protein low dose rIL-2. A significant increase (4-5x) in the frequency Tregs occurred during mobilization. C3H.SW PB was collected from mobilized and Treg unexpanded ("TrUM") or mobilized and Treg expanded ("TrEM") donors and transplanted into MHC-matched B6 (H2b) recipients. Recipients of TrEM, exhibited significantly reduced weight loss and clinical GVHD scores compared to recipients of TrUM. Notably, recipients of TrEM exhibited comparable GVL activity to TrUM recipients against leukemia levels. Next, huTregs (CD4+CD25+CD127lo) from a healthy human PB mobilized donor were expanded ex-vivo prior to transplant into NSG/ NOD-scid IL2Rgammanull mice. We found that treatment with ex-vivo expanded huTregs resulted in significant reduction of lethality and clinical xGVHD scores. Notably, post-transplant, PB huTregs levels remained elevated and the frequency of huCD4+Tconv and CD8+ cells was diminished supporting the improved xGVHD outcomes. These findings demonstrated that the use of mPB containing elevated Treg levels significantly reduced GVHD following "MUD" and MHC-mismatched mouse HSCT without loss of GVL activity. Moreover, utilizing ex-vivo expanded huTregs from a mobilized PB donor and added back to donor PB ameliorated xGVHD. In total, these studies support the notion that in vivo or ex-vivo manipulation of donor Tregs together with mobilized peripheral blood could provide therapeutic approaches to improve aHSCT outcomes.

Recipient Tregs: Can They Be Exploited for Successful Hematopoietic Stem Cell Transplant Outcomes?

Human and mouse CD4+FoxP3+ T cells (Tregs) comprise non-redundant regulatory compartments which maintain self-tolerance and have been found to be of potential therapeutic usefulness in autoimmune disorders and transplants including allogeneic hematopoietic stem cell transplantation (allo-HSCT). There is substantial literature interrogating the application of donor derived Tregs for the prevention of graft versus host disease (GVHD). This Mini-Review will focus on the recipient's Tregs which persist post-transplant. Although treatment in patients with low dose IL-2 months post-HSCT are encouraging, manipulating Tregs in recipients early post-transplant is challenging, in part likely an indirect consequence of damage to the microenvironment required to support Treg expansion of which little is understood. This review will discuss the potential for manipulating recipient Tregs in vivo prior to and after HSCT (fusion proteins, mAbs). Strategies that would circumvent donor/recipient peripheral blood harvest, cell culture and ex-vivo Treg expansion will be considered for the translational application of Tregs to improve HSCT outcomes.

Use of Post-transplant Cyclophosphamide Treatment to Build a Tolerance Platform to Prevent Liquid and Solid Organ Allograft Rejection.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after ("post-transplant," PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular "immune privilege." PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

Superior immune reconstitution using Treg-expanded donor cells versus PTCy treatment in preclinical HSCT models.

Posttransplant cyclophosphamide (PTCy) has been found to be effective in ameliorating acute graft-versus-host disease (GVHD) in patients following allogeneic hematopoietic stem cell transplantation (aHSCT). Adoptive transfer of high numbers of donor Tregs in experimental aHSCT has shown promise as a therapeutic modality for GVHD regulation. We recently described a strategy for in vivo Treg expansion targeting two receptors: TNFRSF25 and CD25. To date, there have been no direct comparisons between the use of PTCy and Tregs regarding outcome and immune reconstitution within identical groups of transplanted mice. Here, we assessed these two strategies and found both decreased clinical GVHD and improved survival long term. However, recipients transplanted with Treg-expanded donor cells (TrED) exhibited less weight loss early after HSCT. Additionally, TrED recipients demonstrated less thymic damage, significantly more recent thymic emigrants, and more rapid lymphoid engraftment. Three months after HSCT, PTCy-treated and TrED recipients showed tolerance to F1 skin allografts and comparable immune function. Overall, TrED was found superior to PTCy with regard to weight loss early after transplant and initial lymphoid engraftment. Based on these findings, we speculate that morbidity and mortality after transplant could be diminished following TrED transplant into aHSCT recipients, and, therefore, that TrED could provide a promising clinical strategy for GVHD prophylaxis.

Marked in Vivo Donor Regulatory T Cell Expansion via Interleukin-2 and TL1A-Ig Stimulation Ameliorates Graft-versus-Host Disease but Preserves Graft-versus-Leukemia in Recipients after Hematopoietic Stem Cell Transplantation.

Regulatory T cells (Tregs) are critical for self-tolerance. Although adoptive transfer of expanded Tregs limits graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), ex vivo generation of large numbers of functional Tregs remains difficult. Here, we demonstrate that in vivo targeting of the TNF superfamily receptor TNFRSF25 using the TL1A-Ig fusion protein, along with IL-2, resulted in transient but massive Treg expansion in donor mice, which peaked within days and was nontoxic. Tregs increased in multiple compartments, including blood, lymph nodes, spleen, and colon (GVHD target tissue). Tregs did not expand in bone marrow, a critical site for graft-versus-malignancy responses. Adoptive transfer of in vivo-expanded Tregs in the setting of MHC-mismatched or MHC-matched allogeneic HSCT significantly ameliorated GVHD. Critically, transplantation of Treg-expanded donor cells facilitated transplant tolerance without GVHD, with complete sparing of graft-versus-malignancy. This approach may prove valuable as a therapeutic strategy promoting transplantation tolerance.

Therapeutic plasma exchange in the treatment of exertional heat stroke and multiorgan failure.

BACKGROUND: Exertional heat stroke (EHS) results in a constellation of systemic inflammatory responses resulting in multiorgan failure and an extremely high mortality. CASE DIAGNOSIS AND TREATMENTS: We present the case of an 11-year-old obese male who suffered EHS with rhabdomyolysis and concurrent renal, pulmonary, and hepatic failure. Conventional therapies including continuous veno-venous hemodiafiltration (CVVHDF) were ineffective in preventing ongoing deterioration in clinical status. Liver biopsy was reported as "extensive hepatocyte ballooning" and liver-kidney transplantation was tentatively planned. CONCLUSIONS: The addition of therapeutic plasma exchange using the Prismaflex® system (Gambro, Lakewood, CO, USA) resulted in a reversal of the inflammatory process and recovery from multiorgan failure. Liver biopsy was not a reliable indicator of irreversible hepatic injury.

Murine model of the Ehlers-Danlos syndrome. col5a1 haploinsufficiency disrupts collagen fibril assembly at multiple stages.

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.

Alpha 2(I) collagen deficient oim mice have altered biomechanical integrity, collagen content, and collagen crosslinking of their thoracic aorta.

Collagen and elastin are the primary determinants of vascular integrity, with elastin hypothesized to be the major contributor to aortic compliance and type I collagen the major contributor to aortic strength and stiffness. Type I collagen is normally heterotrimeric composed of two alpha1(I) and one alpha2(I) collagen chains, alpha1(I)(2)alpha2(I). Recent investigations have reported that patients with recessively inherited forms of Ehlers Danlos syndrome that fail to synthesize proalpha2(I) chains have increased risks of cardiovascular complications. To assess the role of alpha2(I) collagen in aortic integrity, we used the osteogenesis imperfecta model (oim) mouse. Oim mice, homozygous for a COL1A2 mutation, synthesize only homotrimeric type I collagen, alpha1(I)3. We evaluated thoracic aortas from 3-month-old oim, heterozygote, and wildtype mice biomechanically for circumferential breaking strength (Fmax) and stiffness (IEM), histologically for morphological differences, and biochemically for collagen content and crosslinking. Circumferential biomechanics of oim and heterozygote descending thoracic aortas demonstrated the anticipated reduced Fmax and IEM relative to wildtype mice. Histological analyses of oim descending aortas demonstrated reduced collagen staining relative to wildtype aortas suggesting decreased collagen content, which hydroxyproline analyses of ascending and descending oim aortas confirmed. These findings suggest the reduced oim thoracic aortic integrity correlates with the absence of the alpha2(I)collagen chains and in part with reduced collagen content. However, oim ascending aortas also demonstrated a significant increase in pyridinoline crosslinks/collagen molecule as compared to wildtype ascending aortas. The role of increased collagen crosslinks is uncertain; increased crosslinking may represent a compensatory mechanism for the decreased integrity.

Novel collagen glomerulopathy in a homotrimeric type I collagen mouse (oim).

BACKGROUND: Oim/oim mice [osteogenesis imperfecta model; homozygous null for the proalpha2(I) collagen gene] synthesize exclusively the homotrimeric type I collagen isotype, alpha1(I)3, and are unable to synthesize the normal heterotrimeric type I collagen isotype, alpha1(I)2alpha2(I). Previous studies of the oim/oim mouse have focused on the musculoskeletal system, with no systematic evaluation of other organ systems. METHODS: Multiple tissues from oim/oim, oim/+ (heterozygous) and +/+ (wild-type) mice were examined for gross and histological abnormalities. Tissues were stained with (1) hematoxylin and eosin (to assess lesion formation), (2) picrosirius red (collagen content), and (3) periodic acid methenamine silver (basement membrane). Kidneys were further evaluated ultrastructurally by electron microscopy and immunohistochemically with anti-alpha1(I) and anti-alpha1(III) collagen antibodies. RESULTS: Histological analyses revealed accumulations of picrosirius red-positive material, consistent with collagen, in glomeruli of 28/29 oim/oim mice, with no evidence of mesangial cell proliferation. Only the most severely affected animals had evidence of increased capillary basement membrane thickening or mild inflammation around the affected glomeruli. Electron microscopy confirmed the presence of fibrillar collagen. Immunohistochemistry with anti-alpha1(I) collagen antibodies confirmed accumulation of type I collagen in the oim/oim glomeruli. The +/+ and oim/+ kidneys had normal mesangium with no evidence of infiltration of collagenous material. CONCLUSIONS: This study demonstrates the first evidence, to our knowledge, of abnormal glomerular collagen deposition associated with a type I collagen defect. Further in vivo and in vitro studies are necessary to elucidate the mechanistic, functional, and pathological significance of the oim/oim collagen glomerulopathy.