Effect of Early vs Late Inguinal Hernia Repair on Serious Adverse Event Rates in Preterm Infants: A Randomized Clinical Trial.

Importance: Inguinal hernia repair in preterm infants is common and is associated with considerable morbidity. Whether the inguinal hernia should be repaired prior to or after discharge from the neonatal intensive care unit is controversial. Objective: To evaluate the safety of early vs late surgical repair for preterm infants with an inguinal hernia. Design, Setting, and Participants: A multicenter randomized clinical trial including preterm infants with inguinal hernia diagnosed during initial hospitalization was conducted between September 2013 and April 2021 at 39 US hospitals. Follow-up was completed on January 3, 2023. Interventions: In the early repair strategy, infants underwent inguinal hernia repair before neonatal intensive care unit discharge. In the late repair strategy, hernia repair was planned after discharge from the neonatal intensive care unit and when the infants were older than 55 weeks' postmenstrual age. Main Outcomes and Measures: The primary outcome was occurrence of any prespecified serious adverse event during the 10-month observation period (determined by a blinded adjudication committee). The secondary outcomes included the total number of days in the hospital during the 10-month observation period. Results: Among the 338 randomized infants (172 in the early repair group and 166 in the late repair group), 320 underwent operative repair (86% were male; 2% were Asian, 30% were Black, 16% were Hispanic, 59% were White, and race and ethnicity were unknown in 9% and 4%, respectively; the mean gestational age at birth was 26.6 weeks [SD, 2.8 weeks]; the mean postnatal age at enrollment was 12 weeks [SD, 5 weeks]). Among 308 infants (91%) with complete data (159 in the early repair group and 149 in the late repair group), 44 (28%) in the early repair group vs 27 (18%) in the late repair group had at least 1 serious adverse event (risk difference, -7.9% [95% credible interval, -16.9% to 0%]; 97% bayesian posterior probability of benefit with late repair). The median number of days in the hospital during the 10-month observation period was 19.0 days (IQR, 9.8 to 35.0 days) in the early repair group vs 16.0 days (IQR, 7.0 to 38.0 days) in the late repair group (82% posterior probability of benefit with late repair). In the prespecified subgroup analyses, the probability that late repair reduced the number of infants with at least 1 serious adverse event was higher in infants with a gestational age younger than 28 weeks and in those with bronchopulmonary dysplasia (99% probability of benefit in each subgroup). Conclusions and Relevance: Among preterm infants with inguinal hernia, the late repair strategy resulted in fewer infants having at least 1 serious adverse event. These findings support delaying inguinal hernia repair until after initial discharge from the neonatal intensive care unit. Trial Registration: ClinicalTrials.gov Identifier: NCT01678638.

Randomised trial of azithromycin to eradicate Ureaplasma in preterm infants.

OBJECTIVE: To test whether azithromycin eradicates Ureaplasma from the respiratory tract in preterm infants. DESIGN: Prospective, phase IIb randomised, double-blind, placebo-controlled trial. SETTING: Seven level III-IV US, academic, neonatal intensive care units (NICUs). PATIENTS: Infants 240-286 weeks' gestation (stratified 240-266; 270-286 weeks) randomly assigned within 4 days following birth from July 2013 to August 2016. INTERVENTIONS: Intravenous azithromycin 20 mg/kg or an equal volume of D5W (placebo) every 24 hours for 3 days. MAIN OUTCOME MEASURES: The primary efficacy outcome was Ureaplasma-free survival. Secondary outcomes were all-cause mortality, Ureaplasma clearance, physiological bronchopulmonary dysplasia (BPD) at 36 weeks' postmenstrual age, comorbidities of prematurity and duration of respiratory support. RESULTS: One hundred and twenty-one randomised participants (azithromycin: n=60; placebo: n=61) were included in the intent-to-treat analysis (mean gestational age 26.2±1.4 weeks). Forty-four of 121 participants (36%) were Ureaplasma positive (azithromycin: n=19; placebo: n=25). Ureaplasma-free survival was 55/60 (92% (95% CI 82% to 97%)) for azithromycin compared with 37/61 (61% (95% CI 48% to 73%)) for placebo. Mortality was similar comparing the two treatment groups (5/60 (8%) vs 6/61 (10%)). Azithromycin effectively eradicated Ureaplasma in all azithromycin-assigned colonised infants, but 21/25 (84%) Ureaplasma-colonised participants receiving placebo were culture positive at one or more follow-up timepoints. Most of the neonatal mortality and morbidity was concentrated in 21 infants with lower respiratory tract Ureaplasma colonisation. In a subgroup analysis, physiological BPD-free survival was 5/10 (50%) (95% CI 19% to 81%) among azithromycin-assigned infants with lower respiratory tract Ureaplasma colonisation versus 2/11 (18%) (95% CI 2% to 52%) in placebo-treated infants. CONCLUSION: A 3-day azithromycin regimen effectively eradicated respiratory tract Ureaplasma colonisation in this study. TRIAL REGISTRATION NUMBER: NCT01778634.

Epithelial PIK3R1 (p85) and TP53 Regulate Survivin Expression during Adaptation to Ileocecal Resection.

Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of ß-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome.

Neonatal CD71+ Erythroid Cells Do Not Modify Murine Sepsis Mortality.

Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71(+) erythroid splenocytes. We examined the impact of CD71(+) erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71(+) erythroid (CD235a(+)) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71(+) erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b(+) cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71(+) erythroid splenocytes to CD71(+)-reduced animals did not reduce bacterial clearance. Human CD71(+)CD235a(+) cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71(+) erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71(+) erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization.

CD8αα⁺ innate-type lymphocytes in the intestinal epithelium mediate mucosal immunity.

Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.

Smad7 inhibits autocrine expression of TGF-ß2 in intestinal epithelial cells in baboon necrotizing enterocolitis.

Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-ß 2 (TGF-ß(2)) in the developing intestine. We hypothesized that low epithelial TGF-ß(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-ß(2) in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-ß(2), Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-ß(2) than term intestine. TGF-ß(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-ß(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-ß(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-ß(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.

Predicting posttransplantation diabetes mellitus by regulatory T-cell phenotype: implications for metabolic intervention to modulate alloreactivity.

Chronic inflammation and decreased frequency of regulatory T cells (Tregs) in visceral adipose tissue contribute to the propagation of insulin resistance to diabetes mellitus. We tested the hypothesis that new-onset posttransplantation diabetes mellitus (PTDM) is associated with measurable changes in Treg subsets after allogeneic hematopoietic stem cell transplantation (HSCT). PTDM before day 100 and Treg phenotype at engraftment were determined in 36 HSCT recipients without preceding history of diabetes mellitus. Among patients with new-onset PTDM (N = 24), the frequency of circulating CLA(+) (skin-homing) Tregs was decreased (1.53% vs 3.99%; P = .002) and the percentage of α(4)ß(7)(+) (gut-homing) Tregs was increased (17.9% vs 10.7%; P = .048). In multivariate analysis, patients with PTDM continued to demonstrate elevated ratios of α(4)ß(7)(+) Tregs to CLA(+) Tregs (odds ratio, 18.1; P = .020). PTDM is associated with altered immune regulation after HSCT and could represent a target to modulate alloreactivity.

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

Rotavirus-specific CD5+ B cells in young children exhibit a distinct antibody repertoire compared with CD5- B cells.

Antiviral antibody responses in infants are limited in quality. One reason for this finding could be that the majority of B cells in infants are CD5+ cells, a subset of B cells that is thought to contain cells expressing polyreactive, low-affinity B cell receptors. We analyzed the rotavirus (RV)-specific antibody heavy chain variable region (VH) repertoire in CD5+ and CD5- B cells of four RV-infected children between 10 and 19 months of age. We found that the RV-specific B cell repertoire in CD5+ cells was VH3 family biased, in contrast to the VH1/VH4 dominance seen in CD5- B cells. The immunodominant RV-specific gene segment in CD5- B cells was VH1-46, which is the dominant segment used in RV-specific peripheral blood B cells from infants and adults. In contrast, the immunodominant gene segment was VH3-23 in RV-specific CD5+ B cells, which is the dominant gene segment in randomly selected B cells. Both RV-specific CD5+ and RV-specific CD5- B cells from all children studied demonstrated very low frequencies of somatic mutations. In conclusion, CD5+ B cells in infants responding to RV use an antibody gene repertoire that differs from the virus-specific repertoire of CD5- B cells, and both CD5+ and CD5- RV-specific B cells exhibit a low frequency of somatic mutations.

Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires.

Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (V(H) and V(L)) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the V(H)1 and V(H)4 families, for example, V(H)1-46, V(H)4-31, and V(H)4-61. This gene segment bias differed markedly from the V(H)3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant V(H) segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2-11 mo and those of adults were highly related when compared by V(H), D, J(H), V(L), and J(L) segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.