Fetal Secretory IgA Delivery via Transamniotic Fetal Immunotherapy (TRAFIT) in a Rodent Model.

PURPOSE: We sought to determine the feasibility and routing kinetics of transamniotic fetal delivery of secretory immunoglobulin-A (SIgA), in a rodent model. METHODS: Fetuses (n = 94) from seven time-dated pregnant dams received intra-amniotic injections on gestational day 17 (E17, term = E21-22) of either saline (n = 15) or a solution of 1 mg/mL of ≥95% homogeneous human SIgA (n = 79). Animals were euthanized daily at E18-E21 for quantification of the IgA component by ELISA at gestational membranes, placenta, and select fetal anatomical sites against saline controls procured at term. Statistical analysis was by Mann-Whitney U-test. RESULTS: None of the saline-injected animals had detectable human IgA. SIgA-injected fetuses showed human IgA in the stomach aspirate, intestinal wall, lungs, liver, and serum at all time points. IgA levels were significantly higher in the gastric aspirate and in the intestine than in all other sites (p < 0.001 for both), with intestinal levels remaining stable through E18-E21 (p = 0.09-0.62 pairwise). Serum and placental levels were consistently low throughout, reaching near zero levels by E21. CONCLUSIONS: The chronology of exogenous secretory-IgA kinetics after intra-amniotic injection is suggestive of fetal uptake by ingestion, leading to consistent levels in the gastrointestinal tract. Transamniotic fetal immunotherapy (TRAFIT) with secretory-IgA may become a novel strategy for enhancing early mucosal immunity. LEVEL OF EVIDENCE: N/A (animal and laboratory study). TYPE OF STUDY: Animal and laboratory study.

Impact of institutional prophylaxis guidelines on rates of pediatric venous thromboembolism following trauma-A multicenter study from the pediatric trauma society research committee.

BACKGROUND: A paucity of data exists with regard to the incidence, management, and outcomes of venous thromboembolism (VTE) in injured children. We sought to determine the impact of institutional chemoprophylaxis guidelines on VTE rates in a pediatric trauma population. METHODS: A retrospective review of injured children (≤15 years) admitted between 2009 and 2018 at 10 pediatric trauma centers was performed. Data were gathered from institutional trauma registries and dedicated chart review. The institutions were surveyed as to whether they had chemoprophylaxis guidelines in place for high-risk pediatric trauma patients, and outcomes were compared based on the presence of guidelines using χ 2 analysis ( p < 0.05). RESULTS: There were 45,202 patients evaluated during the study period. Three institutions (28,359 patients, 63%) had established chemoprophylaxis policies during the study period ("Guidelines"); the other seven centers (16,843 patients, 37%) had no such guidelines ("Standard"). There were significantly lower rates of VTE in the Guidelines group, but these patients also had significantly fewer risk factors. Among critically injured children with similar clinical presentations, there was no difference in VTE rate. Specifically within the Guidelines group, 30 children developed VTE. The majority (17/30) were actually not indicated for chemoprophylaxis based on institutional guidelines. Still, despite protocols only one VTE patient in the guidelines group who was indicated for intervention ended up receiving chemoprophylaxis prior to diagnosis. No consistent ultrasound screening protocol was in place at any institution during the study. CONCLUSION: The presence of an institutional policy to guide chemoprophylaxis for injured children is associated with a decreased overall frequency of VTE, but this disappears when controlling for patient factors. However, the overall efficacy is impacted by a combination of deficits in guideline compliance and structure. Further prospective data are needed to help determine the ideal role for chemoprophylaxis and protocols in pediatric trauma. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

Targeted multi-omic analysis of human skin tissue identifies alterations of conventional and unconventional T cells associated with burn injury.

Burn injuries are a leading cause of unintentional injury, associated with a dysfunctional immune response and an increased risk of infections. Despite this, little is known about the role of T cells in human burn injury. In this study, we compared the activation and function of conventional T cells and unconventional T cell subsets in skin tissue from acute burn (within 7 days from initial injury), late phase burn (beyond 7 days from initial injury), and non-burn patients. We compared T cell functionality by a combination of flow cytometry and a multi-omic single-cell approach with targeted transcriptomics and protein expression. We found a significantly lower proportion of CD8+ T cells in burn skin compared to non-burn skin, with CD4+ T cells making up the bulk of the T cell population. Both conventional and unconventional burn tissue T cells show significantly higher IFN-γ and TNF-α levels after stimulation than non-burn skin T cells. In sorted T cells, clustering showed that burn tissue had significantly higher expression of homing receptors CCR7, S1PR1, and SELL compared to non-burn skin. In unconventional T cells, including mucosal-associated invariant T (MAIT) and γδ T cells, we see significantly higher expression of cytotoxic molecules GZMB, PRF1, and GZMK. Multi-omics analysis of conventional T cells suggests a shift from tissue-resident T cells in non-burn tissue to a circulating T cell phenotype in burn tissue. In conclusion, by examining skin tissue from burn patients, our results suggest that T cells in burn tissue have a pro-inflammatory rather than a homeostatic tissue-resident phenotype, and that unconventional T cells have a higher cytotoxic capacity. Our findings have the potential to inform the development of novel treatment strategies for burns.

Enhancing Mucosal-Associated Invariant T Cell Function and Expansion with Human Selective Serum.

Mucosal-associated invariant T (MAIT) cells are promising innate-like lymphocytes with potential for use in anti-tumor immunotherapy. Existing MAIT cell expansion protocols are associated with potentially decremental phenotypic changes, including increased frequency of CD4+ MAIT cells and higher inhibitory receptor expression. In this study, we compared the effect on expansion of human MAIT cells of a serum replacement, Physiologix XF SR (Phx), with traditional serum FBS for supplementing RPMI 1640 media. Using flow cytometry, we found that Phx supported a significantly higher proliferative capacity for MAIT cells and resulted in a lower frequency of CD4+ MAIT cells, which have been associated with reduced Th1 effector and cytolytic functions. We saw that culturing MAIT cells in Phx led to better survival of MAIT cells and lower frequency of PD-1+ MAIT cells than FBS-supplemented media. Functionally, we saw that Phx supplementation was associated with a higher frequency of IFN-γ+ MAIT cells after stimulation with Escherichia coli than FBS-supplemented RPMI. In conclusion, we show that MAIT cells cultured in Phx have higher proliferative capacity, lower expression of inhibitory receptors, and higher capacity to produce IFN-γ after E. coli stimulation than FBS-supplemented RPMI. This work shows that expanding MAIT cells with Phx compared with FBS-supplemented RPMI results in a more functionally desirable MAIT cell for future anti-tumor immunotherapy.

Transamniotic Fetal Administration of Genetically Modified Hematopoietic Stem Cells Carrying a Human Transgene in a Syngeneic Rat Model.

Hematopoietic stem cell (HSC)-based gene therapy has already reached clinical reality in a few applications. Fetal administration of genetically modified HSCs has only been feasible to date through invasive and morbid methods. It has been recently shown that native donor HSCs can reach the fetal circulation and bone marrow after simple delivery into the amniotic fluid, at least in a syngeneic healthy model. We sought to determine whether the transamniotic route could also be a practical alternative for the fetal administration of genetically modified HSCs in a comparable model. Pregnant Lewis rat dams underwent volume-matched intra-amniotic injections in all their fetuses (n = 47) on gestational day 17 (E17; term = E21-22) of donor HSCs genetically modified using a custom lentiviral vector designed to constitutively express both a firefly luciferase reporter gene and a human adenosine deaminase (ADA) transgene. Donor HSCs consisted of syngeneic cells isolated from the amniotic fluid and phenotyped by flow cytometry. Fetuses were euthanized at term, when seven select sites relevant to HSC-based therapies were screened for either luciferase activity by luminometry or for the presence of human ADA mRNA by digital droplet polymerase chain reaction (ddPCR). Among survivors (30/47; 64%), positive luminescence and positive human ADA expression were detected in the bone marrow (respectively, 33% and 76%), liver (respectively, 11% and 81%), spleen (respectively, 11% and 67%), thymus (respectively, 33% and 67%), lungs (respectively, 44% and 86%), and brain (respectively, 22% and 90%). Nucleated peripheral blood cells were analyzed only by ddPCR, showing positive human ADA expression at 54%. We conclude that genetically modified HSCs can reach the fetal circulation and fetal bone marrow after simple intra-amniotic administration in a syngeneic rat model. Gene therapy by transamniotic HSC delivery may become a practicable, minimally invasive strategy for the prenatal treatment of select hemoglobinopathies, immunodeficiencies, and inherited metabolic disorders.

Early functional analysis on the pulmonary hemodynamic effects of Transamniotic Stem Cell Therapy (TRASCET) in the nitrofen model of congenital diaphragmatic hernia.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) has been shown to impact pulmonary vascular development and remodeling in experimental congenital diaphragmatic hernia (CDH), with secondary structural cardiac effects. We sought to determine whether TRASCET has any functional impact on term fetal pulmonary hemodynamics in the nitrofen model. METHODS: Time-dated pregnant rat dams (n = 13) received nitrofen on gestational day 9 (E9) to induce fetal CDH. Fetuses (n = 155) were divided into three groups: untreated (n = 45), and two groups receiving volume-matched intra-amniotic injections on E17 of either saline (sham; n = 46), or a suspension of amniotic fluid-derived MSCs (afMSCs) (TRASCET; n = 64). Donor afMSCs were syngeneic, phenotyped by flow cytometry, and "primed" by exposure to interferon-gamma and interleukin-1beta prior to administration in vivo. At term (E21), fetuses underwent Doppler flow assessment at the mid-pulmonary artery and 4-chamber echocardiogram. Pulmonary vascular resistance was estimated by pulmonary artery acceleration time (PAAT), max velocity (MaxV) and velocity time integral (VTI). Cardiac function was assessed by global longitudinal strain (GLS) and ejection fraction (EF) using speckle analyses. Healthy fetuses (n = 11) served as additional controls. Statistical analysis was by the Mann-Whitney U test RESULTS: High resolution ultrasound data could be obtained from 8 to 13 fetuses per group. The PAAT and the PAAT normalized to cardiac cycle time were significantly improved by TRASCET compared to both untreated and sham-treated CDH (p = 0.004 to <0.001 in all pairwise comparisons). The flow profile sharpness (MaxV:VTI) was increased in untreated (p = 0.06) and sham (p = 0.01) groups but normalized by TRASCET (p<0.01). There was no difference in GLS between TRASCET and either the untreated or sham groups (p = 0.25 to p = 0.93). CONCLUSION: Transamniotic stem cell therapy improves pulmonary vascular resistance in early term fetuses in the Nitrofen model of congenital diaphragmatic hernia. Further focus on the functional pulmonary hemodynamic impact of this therapy is justified. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Brain protection by transamniotic stem cell therapy (TRASCET) in a model of intrauterine growth restriction (IUGR).

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) has been shown experimentally to reverse some of the effects of intrauterine growth restriction (IUGR), apparently by attenuating placental inflammation. Neurodevelopmental deficits driven by neuroinflammation are major complications of IUGR. We sought to determine whether MSC-based TRASCET also mitigates inflammation in the fetal brain. METHODS: Pregnant Sprague-Dawley dams (n = 8) were exposed to alternating 12-hour hypoxia (10.5% O2) cycles from gestational day 15 (E15) until term (E21). One group remained untreated (n = 28 fetuses). Three groups received volume-matched intra-amniotic injections into all fetuses (n = 72) of either saline (sham; n = 19), or a suspension of amniotic fluid-derived MSCs, either in native state (TRASCET; n = 20), or primed by exposure to interferon-gamma (IFN-γ) and interleukin-1beta (IL-1ß) for 24 h prior to administration in vivo (TRASCET-Primed; n = 29). Donor MSCs were syngeneic Lewis rat cells phenotyped by flow cytometry. Normal fetuses served as controls (n = 20). Multiple analyses were performed at term, including ELISA in fetal brains for the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and IL-1ß. Statistical comparisons were by Wilcox-rank sum test, including Bonferroni-adjusted significance. RESULTS: Overall survival was 75% (88/116). Gross brain weights were significantly decreased from normal in both the untreated and sham groups (both p<0.001) and significantly increased in both TRASCET groups when compared to untreated and sham (p = 0.003 to <0.001). TRASCET-Primed led to significantly lower levels of TNF-α and IL-1ß compared to untreated (both p<0.001) and sham (p = 0.017 and p = 0.011, respectively). Non-primed TRASCET led to significantly lower levels of TNF-α and IL-1ß compared to untreated (p = 0.009 to <0.001), but not sham (p = 0.133 and p = 0.973, respectively). CONCLUSIONS: Transamniotic stem cell therapy with primed mesenchymal stem cells reverses some of the central nervous system effects of intrauterine growth restriction in a rat model, possibly by modulating neuroinflammation. TYPE OF STUDY: Animal and laboratory study. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Venous thromboembolic screening in pediatric trauma: A prospective cohort study of risk-stratified ultrasonography.

BACKGROUND: This prospective observational cohort study evaluates risk-stratified venous thromboembolism (VTE) screening in injured children. While the reported incidence of VTE is 6% to 10% among critically injured children, there is no standard for screening. Venous thromboembolism may have long-term sequelae in children, including postthrombotic syndrome. METHODS: Patients admitted to a level 1 pediatric trauma center were risk stratified for VTE using a validated prediction algorithm. Children at high risk (risk scores ≥523; i.e., ≥1% risk) received screening duplex ultrasonography. Children at moderate risk (risk scores 410-522; i.e., 0.3-0.99% risk) were screened as a comparison/control. RESULTS: Three-hundred fifty-five children were consecutively risk stratified from October 2019 to May 2021. Forty-seven children received screening duplex ultrasounds: 21 from a high-risk cohort and 26 from a moderate-risk cohort. Four children were diagnosed with VTE in the high-risk cohort compared with seven in the moderate-risk cohort ( p = 0.53). Total incidence of VTE among screened children was 23.4% (11 of 47). Asymptomatic VTE accounted for 81.8% of all events (9 of 11). Fifty-four percent (6 of 11) of VTE were central venous catheter associated. Venous thromboembolism in surviving children resolved by 3 to 6 months with no symptoms of postthrombotic syndrome after 1 year. No cases of VTE were identified in unscreened children, yielding an institutional VTE incidence of 3.1% (11 of 355). DISCUSSION: Risk-stratified screening demonstrates a significant incidence of asymptomatic VTE in injured children. These results may guide reevaluation of prediction algorithms developed from symptomatic VTE risk and longitudinal study of the sequelae of asymptomatic VTE. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17ß-estradiol.

Asthmatic women tend to develop severe airway disease in their reproductive years, and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells, or ILC2, are readily detected in allergic conditions, such as rhinosinusitis, in individuals that develop nasal polyps do to allergen exposures, and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33, an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in naïve and ovalbumin (OVA) challenged, female BALB/c mice in comparison to male mice following stimulation with IL-33. Here, we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-κB signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated, and had reduced NF-κB activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2, and Nfkb1 was found among top-ranked regulatory interactions. Together, these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases.

Routing pathway of syngeneic donor hematopoietic stem cells after simple intra-amniotic delivery.

BACKGROUND: We sought to determine the pathway through which syngeneic hematopoietic stem cells (HSCs) delivered into the amniotic fluid can reach the fetal circulation. METHODS: Lewis rat fetuses were divided in two groups based on the content of intra-amniotic injections performed on gestational day 17 (E17; term=E21-22): either a suspension of luciferase-labeled syngeneic HSCs (n = 137), or acellular luciferase (n = 44). Samples from placenta, chorion, amnion, amniotic fluid, umbilical cord, and 8 fetal sites were procured at 5 daily time points thereafter until term for analysis. RESULTS: When controlled by acellular luciferase, donor HSCs were identified in the amnion, chorion, placenta, and amniotic fluid of fetuses receiving cells at all time points (p = 0.033 to <0.001), peaking first at the amnion and subsequently at the chorion and placenta. Cells could be detected in the fetal liver as early as day 1, progressively expanding to all the other fetal sites over time, in parallel to their increased presence in the chorion and placenta. CONCLUSIONS: The chronology of syngeneic donor hematopoietic stem cell trafficking after intra-amniotic injection is suggestive of controlled routing through the gestational membranes and placenta. Hematogenous donor cell routing is a constituent of transamniotic hematopoietic stem cell therapy, significantly expanding its potential applications.

Routing kinetics of human immunoglobulin-G after transamniotic fetal immunotherapy (TRAFIT) in a rodent model.

PURPOSE: The transamniotic route was recently discovered as a minimally invasive means of fetal immunoglobulin administration, however by unclear mechanisms. We sought to examine IgG routing after intra-amniotic delivery. METHODS: Sprague-Dawley fetuses (n = 78) received intra-amniotic injections of 15 mg/mL of human IgG on gestational-day 18 (E18; term=21 and 22 days). Amniotic fluid, amnion, chorion, placenta, fetal serum, liver, and stomach-aspirate samples were procured on E19, E20, and E21 for IgG quantification by ELISA. Statistical analysis was by median regression with Bonferroni-adjusted significance at p < 0.017. RESULTS: Human IgG was detected at all sampled sites across all time points, though at significantly higher levels in the gestational membranes and fetal serum than in the stomach aspirate and liver (p < 0.001 for both). Gestational membranes showed a daily decrease after injection, stabilizing by E20 and E21 (p = 0.792 to < 0.001). Placental levels were significantly lower at E21 than E19 (p = 0.010). Fetal serum showed the highest human IgG levels at term. CONCLUSIONS: The chronology of exogenous IgG kinetics after intra-amniotic injection is suggestive of direct placental transport leading to consistently high fetal serum levels, possibly combined with some fetal ingestion. Transamniotic fetal immunotherapy (TRAFIT) may become a practicable strategy for the prenatal treatment of select alloimmune disorders and infections. LEVEL OF EVIDENCE: N/A (Animal and Laboratory study). TYPE OF STUDY: Animal and Laboratory Study.

Intrauterine Growth Restriction (IUGR) as a potential target for transamniotic stem cell therapy.

BACKGROUND: We sought to determine whether intrauterine growth restriction (IUGR) could be a target for mesenchymal stem cell (MSC)-based transamniotic stem cell therapy (TRASCET). METHODS: Pregnant dams subjected to hypoxia (10.5% O2) cycles had their fetuses divided into four groups: untreated (n = 24) and three groups receiving volume-matched intra-amniotic injections of either saline (sham; n = 16), or suspensions of luciferase-labeled, syngeneic amniotic fluid-derived MSCs that were either native (TRASCET-unprimed; n = 29), or primed by exposure to IFNγ and IL-1ß (TRASCET-primed; n = 31). Normal fetuses served as additional controls (n = 22). Multiple analyses were performed at term. RESULTS: Compared to normal, fetal weights were significantly decreased in all hypoxia groups (p = 0.002 to <0.001), except for TRASCET-primed. Placental efficiency (fetal/placental weight) was significantly decreased in all hypoxia groups (p = 0.002 to <0.001), but normalized in both TRASCET groups. A significant increase in metrial expression of IFNγ in both the untreated and sham groups (p = 0.04 to 0.02) was reversed only in the TRASCET-primed group. Luciferase DNA was present in both TRASCET groups' placentas. CONCLUSIONS: Transamniotic stem cell therapy with primed mesenchymal stem cells reverses some of the effects of intrauterine growth restriction in a rat model. Further study into this novel approach for the treatment of this disease is warranted. LEVEL OF EVIDENCE: N/A (Animal and Laboratory Study).

Passive perinatal immunotherapy via transamniotic antibody delivery.

PURPOSE: We sought to determine whether the amniotic cavity/fluid could be an attainable route of administration of therapeutic antibodies to the fetus/neonate. METHODS: Time-dated pregnant dams (n = 9) received volume-matched intra-amniotic injections of either saline (n = 29), or different concentrations of a human IgG that lacked homology with rodents: 5 mg/mL (n = 28); 10 mg/mL (n = 28); or 15 mg/mL (n = 24). At term, the presence of the IgG was quantified by ELISA in the serum, bone marrow, spleen, thymus, and brain of all neonates, and in the maternal serum. Statistical analysis was by median regression with significance set at Bonferroni-adjusted p<0.008. RESULTS: Overall fetal survival was 83% (90/109), with no difference between the groups. Human IgG was detected in the serum, bone marrow, spleen, thymus, and brain of all fetuses for all three injected concentrations, but not in the saline injected controls (p<0.001). A dose dependent relationship between injection concentration and final IgG load was noted in the bone marrow, spleen, and thymus (p = 0.004 to <0.001). Human IgG was also detected in maternal serum. CONCLUSIONS: IgG antibodies can reach high levels in the fetal/neonatal circulation after simple intra-amniotic administration in a healthy rodent model. Transamniotic fetal immunotherapy (TRAFIT) may become a practicable strategy for the perinatal management of select diseases. LEVEL OF EVIDENCE: N/A (animal and laboratory study) TYPE OF STUDY: Animal and laboratory study.

Reoperation to correct unsuccessful vascular ring and vascular decompression surgery.

OBJECTIVE: Although most children do well after operations to relieve vascular compression of the esophagus and airway, many will have persistent/recurrent symptoms. We review our surgical experience using a customized approach to correct various etiologies of failure after vascular ring/decompression surgery. METHODS: Our institutional database identified children who underwent reoperation for persistent/recurrent symptoms after vascular ring or aberrant arterial decompression surgery between January 2014 and December 2019. Charts were reviewed for operative approaches and clinical data. Findings were analyzed by Fisher exact test for comparison between groups. RESULTS: Twenty-seven children required reoperative surgery. Detailed preoperative workup identified 5 etiologies of failure for a customized approach. Residual scarring was corrected by lysis and rotational esophagoplasty (n = 23/27); fibrotic bands re-creating a ring were divided (n = 11); ongoing vascular compression was addressed by descending aortopexy (n = 19), aberrant subclavian division (n = 7), aortic uncrossing procedure (n = 4), and Kommerell resection (n = 8); anterior aortopexy (n = 6) and anterior tracheopexy (n = 9) corrected cartilage malformation; and tracheobronchomalacia was addressed with posterior airway pexy (n = 26). At available short-term follow-up (median 1 year), 21 of 22 patients (95%) had symptom improvement, and on bronchoscopy, the average number of airway sections with severe tracheobronchomalacia decreased from 2.8 ± 1.7 to 0.5 ± 0.9 (P < .001). CONCLUSIONS: Persistent/recurrent symptoms after release of vascular compression are frequently caused by 5 different etiologies. A multidisciplinary strategy for workup and a customized operative approach can effectively treat these cases and may suggest opportunity at the index surgery to prevent reoperation and achieve optimal outcomes.

Novel Approach for Laparoscopically Placed Chronic Amniotic Fluid Catheters in Sheep.

INTRODUCTION: Several fetal therapies involve repeated amniotic fluid intervention. We hypothesize that a minimally invasive approach can be used to safely implant an intrauterine catheter infusion system in a fetal ovine model for chronic use during pregnancy. METHOD: Five pregnant sheep underwent operation between gestational days 110 and 115 (term 145 days). A Codman® implantable infusion pump was adapted for intrauterine use. The chamber was placed in the maternal flank and the tunneled catheter laparoscopically inserted into the amniotic cavity, secured with a pursestring. Three had an additional uterine anchoring suture. Ewes were sacrificed after natural delivery, and the uterus underwent gross and microscopic analyses. RESULTS: There were no maternal mortalities, abortions, or preterm labor. Pumps were accessed and remained functional throughout gestation. Four ewes delivered healthy term lambs; the other delivered twins with failure to progress and demise. On necropsy, catheters secured with an anchoring suture remained in place, while the other 2 dislodged during labor. There was no chorioamnionitis by culture or histology. CONCLUSION: Laparoscopically placed intra-amniotic infusion catheters were implanted safely and remained functional until delivery in an ovine model. This novel approach has promise in providing safe, durable amniotic fluid access for the potential treatment of fetal disease.

Venous thromboembolic risk stratification in pediatric trauma: A Pediatric Trauma Society Research Committee multicenter analysis.

BACKGROUND: Venous thromboembolism (VTE) in injured children is rare, but its consequences are significant. Several risk stratification algorithms for VTE in pediatric trauma exist with little consensus, and all are hindered in development by relying on registry data with known inaccuracies. We performed a multicenter review to evaluate trauma registry fidelity and confirm the effectiveness of one established algorithm across diverse centers. METHODS: Local trauma registries at 10 institutions were queried for all patients younger than 18 years admitted between 2009 and 2018. Additional chart review was performed on all "VTE" cases and random non-VTE controls to assess registry errors. Corrected data were then applied to our prediction algorithm using 10 real-time variables (Glasgow Coma Scale, age, sex, intensive care unit admission, transfusion, central line placement, lower extremity/pelvic fracture, major surgery) to calculate VTE risk scores. Contingency table classifiers and the area under a receiver operator characteristic curve were calculated. RESULTS: Registries identified 52,524 pediatric trauma patients with 99 episodes of VTE; however, chart review found that 13 cases were misclassified for a corrected total of 86 cases (0.16%). After correction, the algorithm still displayed strong performance in discriminating VTE-fated encounters (sensitivity, 69%; area under the receiver operating characteristic curve, 0.96). Furthermore, despite wide institutional variability in VTE rates (0.04-1.7%), the algorithm maintained a specificity of >91% and a negative predictive value of >99.7% across centers. Chart review also revealed that 54% (n = 45) of VTEs were directly associated with a central line, usually femoral (n = 34, p < 0.001 compared with upper extremity), and that prophylaxis rates were underreported in the registries by about 50%; still, only 19% of the VTE cases had been on prophylaxis before diagnosis. CONCLUSION: The VTE prediction algorithm performed well when applied retrospectively across 10 diverse pediatric centers using corrected registry data. These findings can advance initiatives for VTE screening/prophylaxis guidance following pediatric trauma and warrant prospective study. LEVEL OF EVIDENCE: Clinical decision rule evaluated in a single population, level III.

Transamniotic Stem Cell Therapy for Experimental Congenital Diaphragmatic Hernia: Structural, Transcriptional, and Cell Kinetics Analyses in the Nitrofen Model.

PURPOSE: We examined select pulmonary effects and donor cell kinetics after transamniotic stem cell therapy (TRASCET) in a model of congenital diaphragmatic hernia (CDH). METHODS: Pregnant dams (n = 58) received nitrofen on gestational day 9.5 (E9) to induce fetal CDH. Fetuses (n = 681) were divided into 4 groups: untreated (n = 99) and 3 groups receiving volume-matched intra-amniotic injections on E17 of either saline (n = 142), luciferase-labeled amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 299), or acellular recombinant luciferase (n = 141). Pulmonary morphometry, quantitative gene expression of pulmonary vascular tone mediators, or screening for labeled afMSCs were performed at term (E22). Statistical comparisons were by Mann-Whitney U-test, nested ANOVA, and Wald test. RESULTS: TRASCET led to significant downregulation of endothelial nitric oxide synthase and endothelin receptor-A expressions compared to both untreated and saline groups (both p < 0.001). TRASCET also led to a significant decrease in arteriole wall thickness compared to the untreated group (p < 0.001) but not the saline group (p = 0.180). Donor afMSCs were identified in the bone marrow and umbilical cord (p = 0.035 and 0.015, respectively, vs. plain luciferase controls). CONCLUSIONS: The effects of TRASCET in experimental CDH appear to be centered on the pulmonary vasculature and to derive from circulating donor cells.

Enhancement of transamniotic stem cell therapy for spina bifida by genetic engineering of donor mesenchymal stem cells with an Fgf2 transgene.

BACKGROUND/PURPOSE: We examined whether engineered overexpression of fibroblast growth factor-2 (Fgf2) in donor mesenchymal stem cells (MSCs) could enhance spina bifida coverage induced by transamniotic stem cell therapy (TRASCET). METHODS: Pregnant Sprague-Dawley dams (n = 24) exposed to retinoic acid for induction of fetal spina bifida were divided in three groups. An untreated group had no further manipulations. Two groups received volume-matched intra-amniotic injections into all fetuses (n = 157) of either amniotic fluid-derived MSCs (afMSC; n = 85) or afMSCs transduced with an Fgf2 transgene (Fgf2-afMSC; n = 72) on gestational day 17 (term=21-22 days). Defect coverage was categorized at term by histology and pan-cytokeratin immunohistochemistry. Statistical coverage comparisons were by logistic regression. RESULTS: Among 84 survivors with isolated spina bifida, 71 had definitive histology. Defect coverage rates in both the afMSC (38.5%) and Fgf2-afMSC (73.3%) groups were statistically significantly higher than in the untreated group (10%; p<0.001 for both). There was a significantly higher coverage rate in the Fgf2-afMSC group compared with the afMSC group (p = 0.025). CONCLUSIONS: Fgf2 overexpression in donor mesenchymal stem cells increases defect coverage rates in a rodent model of transamniotic stem cell therapy for spina bifida. Genetic engineering of donor cells is a promising strategy for the enhancement of this emerging therapy.

Fetal hematogenous routing of a donor hematopoietic stem cell line in a healthy syngeneic model of transamniotic stem cell therapy.

BACKGROUND/PURPOSE: In utero administration of hematopoietic stem cells (HSCs) has a variety of actual or potential clinical applications but is hindered by invasive, morbid administration techniques. We sought to determine whether donor HSCs could reach the fetal circulation after simple intra-amniotic delivery in a syngeneic rat model of transamniotic stem cell therapy (TRASCET). METHODS: Pregnant Lewis rat dams underwent volume-matched intra-amniotic injections in all fetuses (n = 90) on gestational day 17 (E17; term=E21-22) of a suspension of commercially available syngeneic Lewis rat HSCs labeled with luciferase (n = 37 fetuses) or an acellular suspension of recombinant luciferase (n = 53). HSC phenotype was confirmed by flow cytometry. Fetuses were euthanized at term for screening of luciferase activity at select anatomical sites. Statistical comparisons were by Fisher's exact test. RESULTS: Among survivors (47/90; 52.2%), donor HSCs were identified selectively in the placenta (p = 0.003), umbilical cord (p < 0.001), bone marrow (p < 0.001), thymus (p = 0.009), bowel (p = 0.003), kidney (p = 0.022), and skin (p < 0.001) when compared with acellular luciferase controls. CONCLUSIONS: Donor hematopoietic stem cells undergo hematogenous routing and can reach the fetal bone marrow after simple intra-amniotic delivery in a syngeneic rat model. Transamniotic stem cell therapy may become a practicable, minimally invasive strategy for the prenatal administration of these cells.

A novel two-component, expandable bioadhesive for exposed defect coverage: Applicability to prenatal procedures.

BACKGROUND/PURPOSE: We sought to test select properties of a novel, expandable bioadhesive composite that allows for enhanced adhesion control in liquid environments. METHODS: Rabbit fetuses (n = 23) underwent surgical creation of spina bifida on gestational day 22-25 (term 32-33 days). Defects were immediately covered with a two-component tough adhesive consisting of a hydrogel made of a double network of ionically crosslinked alginate and covalently crosslinked polyacrylamide linked to a bridging chitosan polymer adhesive. Animals were euthanized prior to term for different analyses, including hydraulic pressure testing. RESULTS: Hydrogels remained adherent in 70% (16/23) of the recovered fetuses and in all of the last 14 fetuses as the procedure was optimized. Adherent hydrogels showed a median two-fold (IQR: 1.7-2.4) increase in area at euthanasia, with defect coverage confirmed by ultrasound and histology. The median maximum pressure to repair failure was 15 mmHg (IQR: 7.8-55.3), exceeding reported neonatal cerebrospinal fluid pressures. CONCLUSIONS: This novel bioadhesive composite allows for selective, stable attachment of an alginate-polyacrylamide hydrogel to specific areas of the spina bifida defect in a fetal rabbit model, while the hydrogel expands with the defect over time. It could become a valuable alternative for the prenatal repair of spina bifida and possibly other congenital anomalies. TYPE OF STUDY: N/A (animal and laboratory study). LEVEL OF EVIDENCE: N/A (animal and laboratory study).