Donor cell engineering with GSK3 inhibitor-loaded nanoparticles enhances engraftment after in utero transplantation.

Host cell competition is a major barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Here we describe a cell-engineering strategy using glycogen synthase kinase-3 (GSK3) inhibitor-loaded nanoparticles conjugated to the surface of donor hematopoietic cells to enhance their proliferation kinetics and ability to compete against their fetal host equivalents. With this approach, we achieved remarkable levels of stable, long-term hematopoietic engraftment for up to 24 weeks post-IUHCT. We also show that the salutary effects of the nanoparticle-released GSK3 inhibitor are specific to donor progenitor/stem cells and achieved by a pseudoautocrine mechanism. These results establish that IUHCT of hematopoietic cells decorated with GSK3 inhibitor-loaded nanoparticles can produce therapeutic levels of long-term engraftment and could therefore allow single-step prenatal treatment of congenital hematological disorders.

In Utero Hematopoietic Cell Transplantation Does Not Alter the Germ Line.

BACKGROUND: In utero hematopoietic cell transplantation (IUHCT) has been demonstrated to reliably generate chimeric offspring. This technique introduces transplanted cells into a fetus while the immune system is still developing, allowing for engraftment without the need for myeloablation. However, little is known about the effect of engraftment on the gonadal tissue or within the germ line of the resultant chimeras. MATERIALS AND METHODS: BALB/cJ mice pups were injected with B6-green fluorescent protein mononuclear bone marrow (BM) cells at gestational ages E13 or E14. Two female and two male chimeras were then crossbred with untreated mice. The gonadal tissue of the chimeras was evaluated with fluorescent stereomicroscopy and green fluorescent protein histologic staining. The progeny of the cross-bred mice was analyzed using flow cytometric evaluation of both the peripheral blood and BM. RESULTS: Although transplanted cells engrafted within the gonads, no evidence of chimerism was found in oocytes or spermatogonia of female and male mice treated with IUHCT, respectively. Crossbreeding chimeric mice with untreated mice generated progeny without evidence of chimerism in peripheral blood and BM. CONCLUSIONS: IUHCT yields chimeric mice that have engrafted cells within the gonads but not within the germ line itself. Correspondingly, progeny from the unaltered germ line has no detectable chimerism. This has clinical implications as the offspring of future patients treated with IUHCT would carry the disease for which their parents were treated with IUHCT.

Case report of mesenteric strangulation secondary to longstanding ventriculoperitoneal shunt catheter.

BACKGROUND: Ventriculoperitoneal (VP) shunts are the most common treatment for hydrocephalus in both pediatric and adult patients. Complications resulting from the abdominal portion of shunts include tube disconnection, obstruction of the shunt tip, catheter migration, infection, abdominal pseudocysts, and bowel perforation. However, other less common complications can occur. The authors present a unique case of a patient with a longstanding VP shunt presenting with an acute abdomen secondary to knotting of the peritoneal portion of the catheter tubing. CASE DESCRIPTION: A 13-year-old male with past medical history significant for myelomeningocele, requiring ventriculoperitoneal shunt placement at 18 months of age, presented to an outside hospital with chief complaint of abdominal pain. Cross-sectional imaging revealed spontaneous knot formation within the shunt tubing around the base of the small bowel mesentery. He was then transferred to our facility for general and neurosurgical evaluation. His abdominal exam was notable for diffuse distension in addition to tenderness to palpation with guarding and rebound. Given his tenuous clinical status and peritonitis, he was emergently booked for abdominal exploration. He underwent bowel resection, externalization of his shunt, with later re-anastomosis and shunt internalization. He eventually made a full recovery. DISCUSSION: Given the potential for significant bowel loss with this and other shunt-related complications, this case serves as a reminder that even longstanding VP shunts should be considered in the differential diagnosis of abdominal pain in any patient with a shunt.

Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation.

The rationale for in utero hematopoietic cell transplantation (IUHCT) rests on exploitation of normal events during hematopoietic and immunologic ontogeny to allow allogeneic hematopoietic engraftment without myeloablative conditioning.  Host hematopoietic competition is among the primary barriers to engraftment in IUHCT. In the murine model this can be partially overcome by delivery of larger donor cell doses, but volume is limiting. Enrichment of donor hematopoietic stem cells (HSCs) would seem to offer a more efficient approach, but such enriched populations have engrafted poorly in existing models of IUHCT. To increase HSC dose while maintaining the presence of accessory cells, we used a less stringent enrichment protocol of single-step lineage depleted cells alone (lin-) or in combination with whole donor bone marrow mononuclear cells. Our results confirm that increasing doses of HSCs in combination with bone marrow accessory cells can dramatically improve engraftment after IUHCT. This represents a practical and clinically applicable strategy to maximize the engraftment potential of the donor graft without risk of treatment-associated toxicity.

Preclinical Canine Model of Graft-versus-Host Disease after In Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allogeneic engraftment and associated donor-specific tolerance without the need for toxic conditioning, as we have previously demonstrated in the murine and canine models. This strategy holds great promise in the treatment of many hematopoietic disorders, including the hemoglobinopathies. Graft-versus-host disease (GVHD) represents the greatest theoretical risk of IUHCT and has never been characterized in the context of IUHCT. We recently described a preclinical canine model of IUHCT, allowing further study of the technique and its complications. We aimed to establish a threshold T cell dose for IUHCT-induced GVHD in the haploidentical canine model and to define the GVHD phenotype. Using a range of T cell concentrations within the donor inoculum, we were able to characterize the phenotype of IUHCT-induced GVHD and establish a clear threshold for its induction between 3% and 5% graft CD3+ cell content. Given the complete absence of GVHD at CD3 doses of 1% to 3% and the excellent engraftment with the lowest dose, there is a safe therapeutic index for a clinical trial of IUHCT.

Enhanced in utero allogeneic engraftment in mice after mobilizing fetal HSCs by α4ß1/7 inhibition.

In utero hematopoietic cell transplantation (IUHCT) is a novel nonmyeloablative approach that results in donor-specific tolerance and mixed allogeneic chimerism. Clinical application is limited by low levels of donor cell engraftment. Competition from endogenous hematopoietic stem cells (HSCs) for limited "space" in fetal hematopoietic organs remains a significant barrier to successful IUHCT. AMD3100, a CXCR4 inhibitor, and firategrast, an α4ß1 and α4ß7 integrin inhibitor (α4ß1/7), have been shown to disrupt HSC retention in the postnatal hematopoietic niche. We hypothesized that maternal administration of AMD3100 and/or firategrast prior to IUHCT would mobilize endogenous HSCs from the fetal liver (FL) and result in preferential FL homing of donor HSCs and enhanced long-term engraftment following IUHCT in an allogeneic mouse model. We demonstrate that (1) both agents cross the placenta with rapidly detectable fetal serum concentrations following maternal administration; (2) firategrast treatment alone or with AMD3100 mobilizes endogenous HSCs from the FL and results in increased FL homing of donor HSCs following IUHCT; and (3) enhanced donor HSC homing following firategrast treatment translates into increased long-term multilineage donor cell engraftment. This approach highlights the potential of mobilization strategies to overcome barriers to successful engraftment and increase the clinical promise of IUHCT.

Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation.

Evidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days' gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial of IUHCT.

Outcomes of prenatally diagnosed lung lesions in multigestational pregnancies.

BACKGROUND: The outcomes of prenatally diagnosed lung lesions in the context of multigestational pregnancies are unknown. METHODS: Of 960 fetal lung lesion cases evaluated at a single tertiary center over 16 years, 30 occurred in multigestational pregnancies. We reviewed this series to aid in prenatal counseling of affected families and to provide prognostic information for decision making. Pre- and postnatal clinical characteristics were gathered for these pregnancies, and the morbidity and mortality were determined for both affected and normal fetuses, whether twins or triplets. RESULTS: Mortality was found to be 3/30 (10%) for affected fetuses, and morbidity in normal co-twins was consistent with the degree of prematurity. No morbidity was seen in co-twins born at or after 36 weeks of gestation. Median gestational age at delivery was 35 5/7 weeks. CONCLUSIONS: Outcomes for the affected fetus correlate with the size and pathophysiologic consequences of the lesion and are not worse than previously reported outcomes for similar lesions in singleton pregnancies, while morbidity in the normal co-twin is consistent with prematurity related to the fetal age of the multiple gestation at delivery, irrespective of the fetal lung lesion.

In utero hematopoietic cell transplantation--recent progress and the potential for clinical application.

In utero hematopoietic stem cell transplantation (IUHCT) is a potential therapeutic alternative to postnatal hematopoietic stem cell transplantation (HSCT) for congenital hematologic disorders that can be diagnosed early in gestation and can be cured by HSCT. The rationale is to take advantage of normal events during hematopoietic and immunologic ontogeny to facilitate allogeneic hematopoietic engraftment. Although the rationale remains compelling, IUHCT has not yet achieved its clinical potential. This review will discuss recent experimental progress toward overcoming the barriers to allogeneic engraftment and new therapeutic strategies that may hasten clinical application.

Fetal surgical intervention: progress and perspectives.

Building upon over 30 years of experimental and clinical development, fetal surgery can be argued to be a standard of care for selected indications, though application of these techniques remains limited to a small number of highly selected fetuses, and availability to a small number of highly specialized centers. Despite its limited application to date, the field of fetal surgery continues to evolve, spurred both by technological advances allowing earlier and more accurate diagnosis of fetal anomalies as well as improved capability to intervene when appropriate. The efficacy of fetal surgical intervention has now been validated for selected indications by well-designed, randomized controlled trials. In this review, we summarize the evidence or lack thereof supporting the current most common indications for fetal surgical intervention.

False Positive Rate Among Pediatric Appendicitis Patients Transferred to a Tertiary Children's Hospital.

BACKGROUND: Many patients with suspected appendicitis are initially evaluated at outlying hospitals and then transferred to a tertiary care pediatric hospital for surgical management. We sought to evaluate whether diagnosis prior to transfer provides a reliable basis for direct admission to a pediatric surgery service. METHODS: Patients transferred during calendar year 2018 with the principal diagnosis of acute appendicitis were compared based on the service accepting the patient: Emergency Department (ED) or Pediatric Surgery (PS). Data were evaluated using Student's t-tests. RESULTS: Overall patient characteristics were consistent among ED and PS transfers. The number of patients accepted directly to PS underwent significantly more computed tomography (80.2% vs 54.1%, P = .0002). Despite diagnostic "confirmation" with cross-sectional imaging, 14.7% of patients admitted directly to PS were found to be false positives. CONCLUSION: A significant proportion of patients referred to pediatric hospitals for appendicitis do not require admission or operation. A protocol which encourages cross-sectional imaging before PS evaluation may subject children to unnecessary radiation and still result in non-surgical admissions. Routine ED transfer allows PS evaluation, targeted imaging, and discharge for non-surgical patients. This approach decreases costs for the families whose children received a false positive diagnosis at a referring facility, while preserving inpatient bed availability.

Acceptability of In Utero Hematopoietic Cell Transplantation for Sickle Cell Disease.

BACKGROUND: In utero hematopoietic cell transplantation (IUHCT) has curative potential for sickle cell disease (SCD) but carries a risk of fetal demise. METHODS: We assessed the conditions under which parents of children with SCD and young adults with SCD would consider IUHCT in a future pregnancy, given a 5% fixed risk of fetal demise. Participants were randomized to consider a hypothetical cure rate (20%, 40%, or 70%). Subsequently, cure rate was either increased or decreased depending on the previous answer to reveal the lowest acceptable rate. Participants also completed the Pediatric Research Participation Questionnaire (PRPQ) and an omission scale. RESULTS: Overall, 74 of 79 (94%) participants were willing to consider IUHCT, and 52 (66%) participants accepted IUHCT at a cure rate of 40%, the estimated rate of therapeutic mixed chimerism. Participants with higher scores on the PRPQ perceived benefits scale were more likely to participate at lower cure rates (OR 1.08, p=0.007) and participants with a greater degree of omission bias were less likely to participate at lower cure rates (OR 0.83, p=0.04). Demographics and SCD severity were not significantly associated with acceptability of IUHCT. CONCLUSION: This study suggests that the majority of parents >and young adults would consider IUHCT under expected therapeutic conditions.

Corrigendum: An extra-uterine system to physiologically support the extreme premature lamb.

This corrects the article DOI: 10.1038/ncomms15112.

An extra-uterine system to physiologically support the extreme premature lamb.

In the developed world, extreme prematurity is the leading cause of neonatal mortality and morbidity due to a combination of organ immaturity and iatrogenic injury. Until now, efforts to extend gestation using extracorporeal systems have achieved limited success. Here we report the development of a system that incorporates a pumpless oxygenator circuit connected to the fetus of a lamb via an umbilical cord interface that is maintained within a closed 'amniotic fluid' circuit that closely reproduces the environment of the womb. We show that fetal lambs that are developmentally equivalent to the extreme premature human infant can be physiologically supported in this extra-uterine device for up to 4 weeks. Lambs on support maintain stable haemodynamics, have normal blood gas and oxygenation parameters and maintain patency of the fetal circulation. With appropriate nutritional support, lambs on the system demonstrate normal somatic growth, lung maturation and brain growth and myelination.

Fast-track management is safe and effective after bowel resection in children with Crohn's disease.

BACKGROUND: "Fast-track" management (FT) challenges traditional postoperative tenets in order to minimize discomfort and optimize inpatient care. We examined the outcomes of consecutively performed laparoscopic-assisted ileocecectomy for Crohn's disease (CD), with particular focus on FT's effects in patients with underlying bowel inflammation. METHODS: We retrospectively reviewed all patients undergoing isolated laparoscopic-assisted ileocecectomy for CD at our institution between 12/2000 and 12/2010, excluding patients with multiple areas of surgical CD, bladder involvement, or age >18years. RESULTS: Seventy-one patients aged 8-18years underwent isolated laparoscopic-assisted ileocecectomy for CD, of which 45 met FT criteria. Individual practice patterns primarily determined which patients were FT-managed. FT management led to decreased length of stay (LOS), time to first stool, time to full diet, and intravenous narcotic use. No significant difference in complications or disease progression was observed between the two groups during 2-year follow up. CONCLUSIONS: Our results suggest that FT is safe and effective in patients with CD. In a chronically ill population, counseling patients and families to expect early discharge is critical to the success of this strategy. Despite CD-related GI pathology, FT patients realized benefits in terms of LOS, time to bowel function, and narcotic use without any increase in complications.

Use of manipulated stem cells for prenatal therapy.

Prenatal stem cell therapy has broad potential for therapeutic application. "Stem cells" of interest include multipotent adult-derived stem cells, cord blood, amniotic fluid, or fetal stem cells, and embryonic or induced pluripotent stem cells. Potential manipulations of stem cells prior to their administration may include harvest, processing, enrichment, expansion, and genetic transduction. A complete description of the methodology related to all of the above is well beyond the scope of this chapter. In the interest of practical application and proven efficacy, we limit our description to adult-derived hematopoietic stem cells (HSCs) and their application to in utero transplantation with or without HSC-targeted gene transfer.