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1.
bioRxiv ; 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38370768

RESUMO

To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm.The cellular composition in multilineage organoids resembles that of human fetal organs.Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics.Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.

2.
Cell Stem Cell ; 30(11): 1434-1451.e9, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37922878

RESUMO

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.


Assuntos
Células-Tronco Pluripotentes , Humanos , Camundongos , Animais , Células-Tronco Hematopoéticas , Colo , Organoides , Macrófagos
3.
Development ; 150(9)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37070767

RESUMO

The in vitro differentiation of pluripotent stem cells into human intestinal organoids (HIOs) has served as a powerful means for creating complex three-dimensional intestinal structures. Owing to their diverse cell populations, transplantation into an animal host is supported with this system and allows the temporal formation of fully laminated structures, including crypt-villus architecture and smooth muscle layers that resemble native human intestine. Although the endpoint of HIO engraftment has been well described, here we aim to elucidate the developmental stages of HIO engraftment and establish whether it parallels fetal human intestinal development. We analyzed a time course of transplanted HIOs histologically at 2, 4, 6 and 8 weeks post-transplantation, and demonstrated that HIO maturation closely resembles key stages of fetal human intestinal development. We also utilized single-nuclear RNA sequencing to determine and track the emergence of distinct cell populations over time, and validated our transcriptomic data through in situ protein expression. These observations suggest that transplanted HIOs do indeed recapitulate early intestinal development, solidifying their value as a human intestinal model system.


Assuntos
Intestinos , Células-Tronco Pluripotentes , Animais , Humanos , Mucosa Intestinal/metabolismo , Organoides , Diferenciação Celular
4.
Surg Obes Relat Dis ; 19(5): 512-521, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36567232

RESUMO

BACKGROUND: The postoperative course after pediatric metabolic and bariatric surgery (MBS) cuts across a developmental phase when substance-use behaviors emerge as significant public health concerns. OBJECTIVE: We examined use of marijuana, conventional cigarettes, and alternate tobacco products/devices (e.g., e-cigarettes, hookah, smokeless, dissolvable) in young adults (YA) to 6 years postsurgery. SETTING: Five academic medical centers. METHODS: In a prospective observational cohort series, 139 surgical (Mage = 16.9, Mbody mass index [BMI] = 51.5, 80% female, 66% white) and 83 nonsurgical comparisons (Mage = 16.1, MBMI = 44.9, 82% female, 54% white) completed assessments at presurgery/baseline and postsurgery years 2, 4, and 6 (year 6 [2014-2018]: surgical n = 123 [89%], Mage = 23.0, MBMI = 39.8; nonsurgical n = 63 [76%], Mage = 22.4, MBMI = 53.6). Lifetime and current (past 30 days) use were reported. RESULTS: Consistent with national YA trends (2014-2018), the most commonly used were (1) conventional cigarettes (30% surgical, 41% nonsurgical, nonsignificant [ns]); (2) marijuana (25% surgical, 27% nonsurgical, ns); and (3) e-cigarettes (12% surgical, 10% nonsurgical). A sizable minority (26% surgical, 18% nonsurgical) used one or more alternate tobacco product/device. Many YA reported persistent and/or heavy use (e.g., >50% marijuana at year 6 and year 2 or 4; ≈50% ≥.5 pack/d of cigarettes), suggesting more established (versus intermittent) health risk behaviors. For the surgical group at year 6, current tobacco product/device use was associated with lower BMI (P < .001) and greater percent weight loss (P = .002). CONCLUSIONS: Pediatric MBS demonstrates promise in lowering risks for adult chronic disease, which may be diminished by age-typical health risk behaviors. Developmentally salient and holistic pediatric postoperative care guidelines are needed.


Assuntos
Cirurgia Bariátrica , Cannabis , Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco , Humanos , Feminino , Adulto Jovem , Criança , Adolescente , Adulto , Masculino , Uso de Tabaco/epidemiologia
5.
J Pediatr Adolesc Gynecol ; 36(2): 155-159, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36209999

RESUMO

STUDY OBJECTIVE: Describe the current practice patterns and diagnostic accuracy of frozen section (FS) pathology for children and adolescents with ovarian masses DESIGN: Prospective cohort study from 2018 to 2021 SETTING: Eleven children's hospitals PARTICIPANTS: Females age 6-21 years undergoing surgical management of an ovarian mass INTERVENTIONS: Obtaining intraoperative FS pathology MAIN OUTCOME MEASURE: Diagnostic accuracy of FS pathology RESULTS: Of 691 patients who underwent surgical management of an ovarian mass, FS was performed in 27 (3.9%), of which 9 (33.3%) had a final malignant pathology. Among FS patients, 12 of 27 (44.4%) underwent ovary-sparing surgery, and 15 of 27 (55.5%) underwent oophorectomy with or without other procedures. FS results were disparate from final pathology in 7 of 27 (25.9%) cases. FS had a sensitivity of 44.4% and specificity of 94.4% for identifying malignancy, with a c-statistic of 0.69. Malignant diagnoses missed on FS included serous borderline tumor (n = 1), mucinous borderline tumor (n = 2), mucinous carcinoma (n = 1), and immature teratoma (n = 1). FS did not guide intervention in 10 of 27 (37.0%) patients: 9 with benign FS underwent oophorectomy, and 1 with malignant FS did not undergo oophorectomy. Of the 9 patients who underwent oophorectomy with benign FS, 5 (55.6%) had benign and 4 (44.4%) had malignant final pathology. CONCLUSIONS: FSs are infrequently utilized for pediatric and adolescent ovarian masses and could be inaccurate for predicting malignancy and guiding operative decision-making. We recommend continued assessment and refinement of guidance before any standardization of use of FS to assist with intraoperative decision-making for surgical resection and staging in children and adolescents with ovarian masses.


Assuntos
Adenocarcinoma Mucinoso , Neoplasias Ovarianas , Feminino , Humanos , Adolescente , Criança , Adulto Jovem , Adulto , Secções Congeladas/métodos , Neoplasias Ovarianas/patologia , Estudos Prospectivos , Ovariectomia , Estudos Retrospectivos
6.
J Pediatr Surg ; 58(1): 27-33, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36283849

RESUMO

BACKGROUND/PURPOSE: Controversy persists regarding the ideal surgical approach for repair of esophageal atresia with tracheoesophageal fistula (EA/TEF). We examined complications and outcomes of infants undergoing thoracoscopy and thoracotomy for repair of Type C EA/TEF using propensity score-based overlap weights to minimize the effects of selection bias. METHODS: Secondary analysis of two databases from multicenter retrospective and prospective studies examining outcomes of infants with proximal EA and distal TEF who underwent repair at 11 institutions was performed based on surgical approach. Regression analysis using propensity score-based overlap weights was utilized to evaluate outcomes of patients undergoing thoracotomy or thoracoscopy for Type C EA/TEF repair. RESULTS: Of 504 patients included, 448 (89%) underwent thoracotomy and 56 (11%) thoracoscopy. Patients undergoing thoracoscopy were more likely to be full term (37.9 vs. 36.3 weeks estimated gestational age, p < 0.001), have a higher weight at operative repair (2.9 vs. 2.6 kg, p < 0.001), and less likely to have congenital heart disease (16% vs. 39%, p < 0.001). Postoperative stricture rate did not differ by approach, 29 (52%) thoracoscopy and 198 (44%) thoracotomy (p = 0.42). Similarly, there was no significant difference in time from surgery to stricture formation (p > 0.26). Regression analysis using propensity score-based overlap weighting found no significant difference in the odds of vocal cord paresis or paralysis (OR 1.087 p = 0.885), odds of anastomotic leak (OR 1.683 p = 0.123), the hazard of time to anastomotic stricture (HR 1.204 p = 0.378), or the number of dilations (IRR 1.182 p = 0.519) between thoracoscopy and thoracotomy. CONCLUSION: Infants undergoing thoracoscopic repair of Type C EA/TEF are more commonly full term, with higher weight at repair, and without congenital heart disease as compared to infants repaired via thoracotomy. Utilizing propensity score-based overlap weighting to minimize the effects of selection bias, we found no significant difference in complications based on surgical approach. However, our study may be underpowered to detect such outcome differences owing to the small number of infants undergoing thoracoscopic repair. LEVEL OF EVIDENCE: Level III.


Assuntos
Atresia Esofágica , Fístula Traqueoesofágica , Lactente , Criança , Humanos , Fístula Traqueoesofágica/epidemiologia , Fístula Traqueoesofágica/cirurgia , Fístula Traqueoesofágica/complicações , Atresia Esofágica/cirurgia , Atresia Esofágica/complicações , Estudos Retrospectivos , Constrição Patológica/cirurgia , Toracotomia , Estudos Prospectivos , Resultado do Tratamento , Toracoscopia
7.
JAMA Netw Open ; 5(6): e2219814, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35771571

RESUMO

Importance: The ability of computed tomography (CT) to distinguish between benign congenital lung malformations and malignant cystic pleuropulmonary blastomas (PPBs) is unclear. Objective: To assess whether chest CT can detect malignant tumors among postnatally detected lung lesions in children. Design, Setting, and Participants: This retrospective multicenter case-control study used a consortium database of 521 pathologically confirmed primary lung lesions from January 1, 2009, through December 31, 2015, to assess diagnostic accuracy. Preoperative CT scans of children with cystic PPB (cases) were selected and age-matched with CT scans from patients with postnatally detected congenital lung malformations (controls). Statistical analysis was performed from January 18 to September 6, 2020. Preoperative CT scans were interpreted independently by 9 experienced pediatric radiologists in a blinded fashion and analyzed from January 24, 2019, to September 6, 2020. Main Outcomes and Measures: Accuracy, sensitivity, and specificity of CT in correctly identifying children with malignant tumors. Results: Among 477 CT scans identified (282 boys [59%]; median age at CT, 3.6 months [IQR, 1.2-7.2 months]; median age at resection, 6.9 months [IQR, 4.2-12.8 months]), 40 cases were extensively reviewed; 9 cases (23%) had pathologically confirmed cystic PPB. The median age at CT was 7.3 months (IQR, 2.9-22.4 months), and median age at resection was 8.7 months (IQR, 5.0-24.4 months). The sensitivity of CT for detecting PPB was 58%, and the specificity was 83%. High suspicion for malignancy correlated with PPB pathology (odds ratio, 13.5; 95% CI, 2.7-67.3; P = .002). There was poor interrater reliability (κ = 0.36 [range, 0.06-0.64]; P < .001) and no significant difference in specific imaging characteristics between PPB and benign cystic lesions. The overall accuracy rate for distinguishing benign vs malignant lesions was 81%. Conclusions and Relevance: This study suggests that chest CT, the current criterion standard imaging modality to assess the lung parenchyma, may not accurately and reliably distinguish PPB from benign congenital lung malformations in children. In any cystic lung lesion without a prenatal diagnosis, operative management to confirm pathologic diagnosis is warranted.


Assuntos
Pneumopatias , Neoplasias Pulmonares , Estudos de Casos e Controles , Criança , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Masculino , Gravidez , Blastoma Pulmonar , Reprodutibilidade dos Testes , Tomografia Computadorizada por Raios X
8.
JAMA Netw Open ; 5(5): e229712, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35499827

RESUMO

Importance: The factors associated with the failure of nonoperative management of appendicitis and the differences in patient-reported outcomes between successful and unsuccessful nonoperative management remain unknown. Objectives: To investigate factors associated with the failure of nonoperative management of appendicitis and compare patient-reported outcomes between patients whose treatment succeeded and those whose treatment failed. Design, Setting, and Participants: This study was a planned subgroup secondary analysis conducted in 10 children's hospitals that included 370 children aged 7 to 17 years with uncomplicated appendicitis enrolled in a prospective, nonrandomized clinical trial between May 1, 2015, and October 31, 2018, with 1-year follow-up comparing nonoperative management with antibiotics vs surgery for uncomplicated appendicitis. Statistical analysis was performed from November 1, 2019, to February 12, 2022. Interventions: Nonoperative management with antibiotics vs surgery. Main Outcomes and Measures: Failure of nonoperative management and patient-reported outcomes. The relative risk (RR) of failure based on sociodemographic and clinical characteristics was calculated. Patient-reported outcomes were compared based on the success or failure of nonoperative management. Results: Of 370 patients (34.6% of 1068 total patients; 229 boys [61.9%]; median age, 12.3 years [IQR, 10.0-14.6 years]) enrolled in the nonoperative group, treatment failure occurred for 125 patients (33.8%) at 1 year, with 53 patients (14.3%) undergoing appendectomy during initial hospitalization and 72 patients (19.5%) experiencing delayed treatment failure after hospital discharge. Higher patient-reported pain at presentation was associated with increased risk of in-hospital treatment failure (RR, 2.1 [95% CI, 1.0-4.4]) but not delayed treatment failure (RR, 1.3 [95% CI, 0.7-2.3]) or overall treatment failure at 1 year (RR, 1.5 [95% CI, 1.0-2.2]). Pain duration greater than 24 hours was associated with decreased risk of delayed treatment failure (RR, 0.3 [95% CI, 0.1-1.0]) but not in-hospital treatment failure (RR, 1.2 [95% CI, 0.5-2.7]) or treatment failure at 1 year (RR, 0.7 [95% CI, 0.4-1.2]). There was no increased risk of treatment failure associated with age, white blood cell count, sex, race, ethnicity, primary language, insurance status, transfer status, symptoms at presentation, or imaging results. Health care satisfaction at 30 days and patient-reported, health-related quality of life at 30 days and 1 year were not different. Satisfaction with the decision was higher with successful nonoperative management at 30 days (28.0 vs 27.0; difference, 1.0 [95% CI, 0.01-2.0]) and 1 year (28.1 vs 27.0; difference, 1.1 [95% CI, 0.2-2.0]). Conclusions and Relevance: This analysis suggests that a higher pain level at presentation was associated with a higher risk of initial failure of nonoperative management and that a longer duration of pain was associated with lower risk of delayed treatment failure. Although satisfaction was high in both groups, satisfaction with the treatment decision was higher among patients with successful nonoperative management at 1 year. Trial Registration: ClinicalTrials.gov Identifier: NCT02271932.


Assuntos
Apendicite , Antibacterianos/uso terapêutico , Apendicite/complicações , Apendicite/epidemiologia , Apendicite/terapia , Criança , Feminino , Humanos , Masculino , Dor/tratamento farmacológico , Estudos Prospectivos , Qualidade de Vida
9.
Gastroenterology ; 163(2): 411-425.e4, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35487288

RESUMO

BACKGROUND & AIMS: A subset of myeloid-derived suppressor cells (MDSCs) that express murine Schlafen4 (SLFN4) or its human ortholog SLFN12L polarize in the Helicobacter-inflamed stomach coincident with intestinal or spasmolytic polypeptide-expressing metaplasia. We propose that individuals with a more robust response to damage-activated molecular patterns and increased Toll-like receptor 9 (TLR9) expression are predisposed to the neoplastic complications of Helicobacter infection. METHODS: A mouse or human Transwell co-culture system composed of dendritic cells (DCs), 2-dimensional gastric epithelial monolayers, and Helicobacter were used to dissect the cellular source of interferon-α (IFNα) in the stomach by flow cytometry. Conditioned media from the co-cultures polarized primary myeloid cells. MDSC activity was determined by T-cell suppression assays. In human subjects with intestinal metaplasia or gastric cancer, the rs5743836 TLR9T>C variant was genotyped and linked to TLR9, IFNα, and SLFN12L expression by immunohistochemistry. Nuclear factor-κB binding to the TLR9 C allele was determined by electrophoretic mobility shift assays. RESULTS: Helicobacter infection induced gastric epithelial and plasmacytoid DC expression of TLR9 and IFNα. Co-culturing primary mouse or human cells with DCs and Helicobacter induced TLR9, IFNα secretion, and SLFN+-MDSC polarization. Neutralizing IFNα in vivo mitigated Helicobacter-induced spasmolytic polypeptide-expressing metaplasia. The TLR9 minor C allele creates a nuclear factor-κB binding site associated with higher levels of TLR9, IFNα, and SLFN12L in Helicobacter-infected stomachs that correlated with a greater incidence of metaplasias and cancer. CONCLUSIONS: TLR9 plays an essential role in the production of IFNα and polarization of SLFN+ MDSCs on Helicobacter infection. Subjects carrying the rs5743836 TLR9 minor C allele are predisposed to neoplastic complications if chronically infected.


Assuntos
Infecções por Helicobacter , Células Supressoras Mieloides , Neoplasias Gástricas , Receptor Toll-Like 9 , Animais , Helicobacter , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Humanos , Interferon-alfa , Metaplasia , Camundongos , NF-kappa B/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Receptor 4 Toll-Like , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
10.
J Pediatr Surg ; 57(6): 975-980, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35304025

RESUMO

INTRODUCTION: Anastomotic stricture is the most common complication after esophageal atresia (EA) repair. We sought to determine if postoperative acid suppression is associated with reduced stricture formation. METHODS: A prospective, multi-institutional cohort study of infants undergoing primary EA repair from 2016 to 2020 was performed. Landmark analysis and multivariate Cox regression were used to explore if initial duration of acid suppression was associated with stricture formation at hospital discharge (DC), 3-, 6-, and 9-months postoperatively. RESULTS: Of 156 patients, 79 (51%) developed strictures and 60 (76%) strictures occurred within three months following repair. Acid suppression was used in 141 patients (90%). Landmark analysis showed acid suppression was not associated with reduction in initial stricture formation at DC, 3-, 6- and 9-months, respectively (p = 0.19-0.95). Multivariate regression demonstrated use of a transanastomotic tube was significantly associated with stricture formation at DC (Hazard Ratio (HR) = 2.21 (95% CI 1.24-3.95, p<0.01) and 3-months (HR 5.31, 95% CI 1.65-17.16, p<0.01). There was no association between acid suppression duration and stricture formation. CONCLUSION: No association between the duration of postoperative acid suppression and anastomotic stricture was observed. Transanastomotic tube use increased the risk of anastomotic strictures at hospital discharge and 3 months after repair.


Assuntos
Atresia Esofágica , Estenose Esofágica , Fístula Traqueoesofágica , Anastomose Cirúrgica/efeitos adversos , Estudos de Coortes , Constrição Patológica/etiologia , Constrição Patológica/prevenção & controle , Atresia Esofágica/complicações , Atresia Esofágica/cirurgia , Estenose Esofágica/epidemiologia , Estenose Esofágica/etiologia , Estenose Esofágica/prevenção & controle , Humanos , Lactente , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Estudos Prospectivos , Estudos Retrospectivos , Fístula Traqueoesofágica/complicações , Fístula Traqueoesofágica/cirurgia , Resultado do Tratamento
11.
Ann Surg ; 276(5): e622-e630, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33214447

RESUMO

OBJECTIVE: The aim of this study was to assess current clinical outcomes in children with prenatally diagnosed congenital lung malformations (CLMs) and to identify prenatal characteristics associated with adverse outcomes. SUMMARY BACKGROUND DATA: Despite a wide spectrum of clinical disease, the identification of fetal CLM subgroups at increased risk for hydrops and respiratory compromise at delivery has not been well defined. METHODS: A retrospective cohort study was conducted using an operative database of prenatally diagnosed CLMs managed at 11 children's hospitals from 2009 to 2016. Statistical analyses were performed using nonparametric bivariate or multivariable logistic regression. RESULTS: Three hundred forty-four children were analyzed. Fifteen (5.5%) fetuses were managed with maternal steroids in the setting of hydrops, and prenatal surgical intervention was uncommon (1.7%). Seventy-five (21.8%) had respiratory symptoms at birth, and 34 (10.0%) required neonatal lung resection. Congenital pulmonary airway malformation volume ratio (CVR) measurements were recorded in 169 (49.1%) cases and were significantly associated with perinatal outcome, including hydrops, respiratory distress at birth, need for supplemental oxygen, neonatal ventilator use, and neonatal resection ( P < 0.001). An initial CVR ≤1.4 was significantly correlated with a reduced risk for hydrops [area under the curve (AUC), 0.93; 95% confidence interval (CI), 0.87-1.00]. A maximum CVR <0.9 (AUC, 0.72; 95% CI, 0.67-0.85) was associated with a low risk for respiratory symptoms at birth. CONCLUSIONS: In this large, multi-institutional study, an initial CVR ≤ 1.4 identifies fetuses at very low risk for hydrops, and a maximum CVR < 0.9 is associated with asymptomatic disease at birth. These findings represent an opportunity for standardization and quality improvement for prenatal counseling and delivery planning.


Assuntos
Pneumopatias , Ultrassonografia Pré-Natal , Criança , Edema , Feminino , Humanos , Recém-Nascido , Pulmão/anormalidades , Pneumopatias/cirurgia , Oxigênio , Gravidez , Estudos Retrospectivos , Medição de Risco/métodos , Ultrassonografia Pré-Natal/métodos
12.
Cancers (Basel) ; 13(24)2021 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34944780

RESUMO

(1) Background: The expression of programmed death-ligand 1 (PD-L1), which interacts with programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTLs), enables tumors to escape immunosurveillance. The PD-1/PD-L1 interaction results in the inhibition of CTL proliferation, and effector function, thus promoting tumor cell evasion from immunosurveillance and cancer persistence. Despite 40% of gastric cancer patients exhibiting PD-L1 expression, only a small subset of patients responds to immunotherapy. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several solid tumors, including metastatic gastric cancer. Although half of PD-L1-positive gastric tumors co-express HER2, crosstalk between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined. (2) Methods: Human gastric cancer organoids (huTGOs) were generated from biopsied or resected tissues and co-cultured with CTLs and myeloid-derived suppressor cells (MDSCs). Digital Spatial Profiling (DSP) was performed on FFPE tissue microarrays of numerous gastric cancer patients to examine the protein expression of immune markers. (3) Results: Knockdown of HER2 in PD-L1/HER2-positive huTGOs led to a concomitant decrease in PD-L1 expression. Similarly, in huTGOs/immune cell co-cultures, PD-L1 expression decreased in huTGOs and was correlated with an increase in CTL proliferation which enhanced huTGO death. Treatment with Nivolumab exhibited similar effects. However, a combinatorial treatment with Mubritinib and Nivolumab was unable to inhibit HER2 expression in co-cultures containing MDSCs. (4) Conclusions: Our study suggested that co-expression of HER2 and PD-L1 may contribute to tumor cell immune evasion. In addition, autologous organoid/immune cell co-cultures can be exploited to effectively screen responses to a combination of anti-HER2 and immunotherapy to tailor treatment for gastric cancer patients.

13.
Physiol Genomics ; 53(11): 486-508, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34612061

RESUMO

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.


Assuntos
Técnicas de Cultura de Células/métodos , Colo/metabolismo , Meios de Cultura/farmacologia , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Organoides/metabolismo , Transcriptoma/efeitos dos fármacos , Calcitriol/farmacologia , Colágeno/metabolismo , Colágeno/farmacologia , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Meios de Cultura/química , Combinação de Medicamentos , Escherichia coli , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Laminina/metabolismo , Laminina/farmacologia , Organoides/virologia , Proteoglicanas/metabolismo , Proteoglicanas/farmacologia , RNA-Seq/métodos , Transcriptoma/genética , Viroses/metabolismo , Viroses/virologia , Vírus
14.
BMJ Open ; 11(8): e047766, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389568

RESUMO

INTRODUCTION: The pathophysiology of type 2 diabetes (T2D) in youth differs from adults and conventional medical treatment approaches with lifestyle change, metformin, thiazolidinediones or insulin are inadequate. Metabolic bariatric surgery (MBS) improves multiple health outcomes in adults with T2D. Initial small, uncontrolled studies of Roux-en-Y gastric bypass have also suggested beneficial effects in adolescents. Definitive studies in youth with T2D are lacking, especially with the now more common form of MBS, vertical sleeve gastrectomy (VSG). The surgical or medical treatment for paediatric type 2 diabetes (ST2OMP) clinical trial was designed to test the hypothesis that VSG will more effectively reduce hyperglycaemic and diabetes comorbidities than the best currently available medical treatment incorporating state of the art pharmacotherapies. ST2OMP is also designed to better understand the pancreatic and enterohepatic mechanisms by which MBS improves diabetes and its associated comorbidities. METHODS AND ANALYSIS: ST2OMP is a prospective, open-label, controlled clinical trial that will recruit 90 postpubertal participants, age range 13-19.9 years, with body mass index ≥35 kg/m2 or >120% of 95th percentile and youth-onset T2D. The primary outcome is the per cent of youth achieving haemoglobin A1c <6.0% at 12 months postgroup allocation (post-VSG vs postmedical group allocation). Secondary outcomes include remission of comorbidities and measures of ß-cell and incretin responses at 12 and 24 months post VSG versus AMT. ETHICS AND DISSEMINATION: The ST2OMP protocol was approved by the Cincinnati Children's Hospital Medical Center and the University of Colorado Institutional Review Boards. Written informed consent is obtained prior to study enrolment. Study findings will be widely disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: Clinical Trials.Gov NCT04128995.


Assuntos
Diabetes Mellitus Tipo 2 , Derivação Gástrica , Obesidade Mórbida , Adolescente , Adulto , Criança , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , Adulto Jovem
15.
Cancer Lett ; 518: 59-71, 2021 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-34126195

RESUMO

Tumors evade immune surveillance by expressing Programmed Death-Ligand 1 (PD-L1), subsequently inhibiting CD8+ cytotoxic T lymphocyte function. Response of gastric cancer to immunotherapy is relatively low. Our laboratory has reported that Helicobacter pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Hedgehog (Hh) signaling pathway. The PI3K/AKT/mTOR pathway is activated in gastric cancer and may have immunomodulatory potential. We hypothesize that Hh signaling mediates mTOR-induced PD-L1 expression. Patient-derived organoids (PDOs) were generated from gastric biopsies and resected tumor tissues. Autologous organoid/immune cell co-cultures were used to study the immunosuppressive function of MDSCs. NanoString Digital Spatial Profiling (DSP) of immune-related protein markers using FFPE slide-mounted tissues from gastric cancer patients was performed. DSP analysis showed infiltration of immunosuppressive MDSCs expressing Arg1, CD66b, VISTA and IDO1 within cancer tissues. Orthotopic transplantation of patient derived organoids (PDOs) resulted in the engraftment of organoids and the development of histology similar to that observed in the patient's tumor tissue. PDO/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of PMN-MDSCs within these co-cultures sensitized the organoids to anti-PD-1/PD-L1-induced cancer cell death. Rapamycin decreased phosphorylated S6K, Gli2 and PD-L1 expression in PDO/immune cell co-cultures. Transcriptional regulation of PD-L1 by GLI1 and GLI2 was blocked by rapamycin. In conclusion, the PDO/immune cell co-cultures may be used to study immunosuppressive MDSC function within the gastric tumor microenvironment. The mTOR signaling pathway mediates GLI-induced PD-L1 expression in gastric cancer.


Assuntos
Antígeno B7-H1/genética , Proteínas Hedgehog/genética , Organoides/metabolismo , Neoplasias Gástricas/genética , Serina-Treonina Quinases TOR/genética , Transcrição Gênica/genética , Proteína GLI1 em Dedos de Zinco/genética , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Helicobacter pylori/patogenicidade , Humanos , Imunoterapia/métodos , Transdução de Sinais/genética , Neoplasias Gástricas/microbiologia , Linfócitos T Citotóxicos/metabolismo , Microambiente Tumoral/genética
16.
J Pediatr Surg ; 56(12): 2148-2156, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34030879

RESUMO

PURPOSE: The impact of thoracoscopic surgery on outcomes in children with congenital lung malformations (CLM) remains controversial. The purpose of this study was to determine the effect of operative approach on perioperative outcomes in infants undergoing lobectomy for an asymptomatic CLM. METHODS: After IRB approval, a retrospective cohort study was conducted on 506 children with a CLM resected at one of eleven children's hospitals over a seven-year period. Infants undergoing elective lobectomy were identified, and covariates were balanced based on operative approach using propensity scores with full matching. Outcomes were analyzed based on intention to treat with weighted conditional regression. RESULTS: One hundred seventy-five infants met inclusion criteria. There were 67 (38.3%) open, 89 (50.9%) thoracoscopic, and 19 (10.9%) thoracoscopic-converted-to-open lobectomies. Thoracoscopic lobectomy was associated with significantly longer operative times (26 min, 95% CI 6-47 min, p = 0.012) but used less epidural anesthesia (OR 0.02, 95% CI 0.004-0.11, p<0.001) when compared to open lobectomy. There were no significant differences in intraoperative blood loss, postoperative complications, chest tube duration, or length of stay. CONCLUSIONS: Thoracoscopy has become the most common operative approach for elective lobectomy in infants with asymptomatic CLMs. The non-inferiority of thoracoscopic lobectomy in postoperative outcomes supports its continued use as an alternative to open lobectomy. LEVEL OF EVIDENCE: Treatment study, Level III.


Assuntos
Neoplasias Pulmonares , Pneumonectomia , Criança , Humanos , Lactente , Tempo de Internação , Pulmão/cirurgia , Neoplasias Pulmonares/cirurgia , Pontuação de Propensão , Estudos Retrospectivos , Toracoscopia , Resultado do Tratamento
17.
Pediatrics ; 147(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33762310

RESUMO

BACKGROUND: Pediatric lung lesions are a group of mostly benign pulmonary anomalies with a broad spectrum of clinical disease and histopathology. Our objective was to evaluate the characteristics of children undergoing resection of a primary lung lesion and to identify preoperative risk factors for malignancy. METHODS: A retrospective cohort study was conducted by using an operative database of 521 primary lung lesions managed at 11 children's hospitals in the United States. Multivariable logistic regression was used to examine the relationship between preoperative characteristics and risk of malignancy, including pleuropulmonary blastoma (PPB). RESULTS: None of the 344 prenatally diagnosed lesions had malignant pathology (P < .0001). Among 177 children without a history of prenatal detection, 15 (8.7%) were classified as having a malignant tumor (type 1 PPB, n = 11; other PPB, n = 3; adenocarcinoma, n = 1) at a median age of 20.7 months (interquartile range, 7.9-58.1). Malignancy was associated with the DICER1 mutation in 8 (57%) PPB cases. No malignant lesion had a systemic feeding vessel (P = .0427). The sensitivity of preoperative chest computed tomography (CT) for detecting malignant pathology was 33.3% (95% confidence interval [CI]: 15.2-58.3). Multivariable logistic regression revealed that increased suspicion of malignancy by CT and bilateral disease were significant predictors of malignant pathology (odds ratios of 42.15 [95% CI, 7.43-340.3; P < .0001] and 42.03 [95% CI, 3.51-995.6; P = .0041], respectively). CONCLUSIONS: In pediatric lung masses initially diagnosed after birth, the risk of PPB approached 10%. These results strongly caution against routine nonoperative management in this patient population. DICER1 testing may be helpful given the poor sensitivity of CT for identifying malignant pathology.


Assuntos
Neoplasias Pulmonares/patologia , Blastoma Pulmonar/patologia , Pré-Escolar , Estudos de Coortes , RNA Helicases DEAD-box/genética , Feminino , Humanos , Lactente , Recém-Nascido , Tempo de Internação , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Mutação , Metástase Neoplásica/genética , Gravidez , Diagnóstico Pré-Natal , Blastoma Pulmonar/diagnóstico por imagem , Blastoma Pulmonar/genética , Blastoma Pulmonar/cirurgia , Síndrome do Desconforto Respiratório do Recém-Nascido/etiologia , Estudos Retrospectivos , Ribonuclease III/genética , Tomografia Computadorizada por Raios X
18.
J Pediatr Surg ; 56(1): 47-54, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33131776

RESUMO

BACKGROUND/PURPOSE: This study evaluated compliance with a multi-institutional quality improvement management protocol for Type-C esophageal atresia with distal tracheoesophageal fistula (EA/TEF). METHODS: Compliance and outcomes before and after implementation of a perioperative protocol bundle for infants undergoing Type-C EA/TEF repair were compared across 11 children's hospitals from 1/2016-1/2019. Bundle components included elimination of prosthetic material between tracheal and esophageal suture lines during repair, not leaving a transanastomotic tube at the conclusion of repair (NO-TUBE), obtaining an esophagram by postoperative-day-5, and discontinuing prophylactic antibiotics 24 h postoperatively. RESULTS: One-hundred seventy patients were included, 40% pre-protocol and 60% post-protocol. Bundle compliance increased 2.5-fold pre- to post-protocol from 17.6% to 44.1% (p < 0.001). After stratifying by institutional compliance with all bundle components, 43.5% of patients were treated at low-compliance centers (<20%), 43% at medium-compliance centers (20-80%), and 13.5% at high-compliance centers (>80%). Rates of esophageal leak, anastomotic stricture, and time to full feeds did not differ between pre- and post-protocol cohorts, though there was an inverse correlation between NO-TUBE compliance and stricture rate over time (ρ = -0.75, p = 0.029). CONCLUSIONS: Compliance with our multi-institutional management protocol increased 2.5-fold over the study period without compromising safety or time to feeds and does not support the use of transanastomotic tubes. LEVEL OF EVIDENCE: Level II. TYPE OF STUDY: Treatment Study.


Assuntos
Atresia Esofágica , Fístula Traqueoesofágica , Criança , Atresia Esofágica/complicações , Atresia Esofágica/cirurgia , Humanos , Lactente , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/prevenção & controle , Estudos Retrospectivos , Fístula Traqueoesofágica/complicações , Fístula Traqueoesofágica/cirurgia , Resultado do Tratamento
19.
Nat Commun ; 11(1): 4791, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963229

RESUMO

The ability to absorb ingested nutrients is an essential function of all metazoans and utilizes a wide array of nutrient transporters found on the absorptive enterocytes of the small intestine. A unique population of patients has previously been identified with severe congenital malabsorptive diarrhea upon ingestion of any enteral nutrition. The intestines of these patients are macroscopically normal, but lack enteroendocrine cells (EECs), suggesting an essential role for this rare population of nutrient-sensing cells in regulating macronutrient absorption. Here, we use human and mouse models of EEC deficiency to identify an unappreciated role for the EEC hormone peptide YY in regulating ion-coupled absorption of glucose and dipeptides. We find that peptide YY is required in the small intestine to maintain normal electrophysiology in the presence of vasoactive intestinal polypeptide, a potent stimulator of ion secretion classically produced by enteric neurons. Administration of peptide YY to EEC-deficient mice restores normal electrophysiology, improves glucose and peptide absorption, diminishes diarrhea and rescues postnatal survival. These data suggest that peptide YY is a key regulator of macronutrient absorption in the small intestine and may be a viable therapeutic option to treat patients with electrolyte imbalance and nutrient malabsorption.


Assuntos
Células Enteroendócrinas/metabolismo , Absorção Intestinal/fisiologia , Transporte de Íons/fisiologia , Nutrientes/metabolismo , Animais , Enterócitos , Glucose/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Intestino Delgado , Intestinos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo YY , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Trocador 3 de Sódio-Hidrogênio , Água/metabolismo
20.
PLoS One ; 15(8): e0237885, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853234

RESUMO

Our group has developed two transplantation models for the engraftment of Human Intestinal Organoids (HIOs): the renal subcapsular space (RSS) and the mesentery each with specific benefits for study. While engraftment at both sites generates laminated intestinal structures, a direct comparison between models has not yet been performed. Embryonic stem cells were differentiated into HIOs, as previously described. HIOs from the same batch were transplanted on the same day into either the RSS or mesentery. 10 weeks were allowed for engraftment and differentiation, at which time they were harvested and assessed. Metrics for comparison included: mortality, engraftment rate, gross size, number and grade of lumens, and expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism. Mortality was significantly increased when undergoing mesentery transplantation, however engraftment was significantly higher. Graft sizes were similar between groups. Morphometric parameters were similar between groups, however m-tHIOs presented with significantly fewer lumens than k-tHIO. Transcript and protein level expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism were similar between groups. Transplantation into both sites yields viable tissue of similar quality based on our assessments with enhanced engraftment and a dominant lumen for uniform study benefiting the mesenteric site and survival benefiting RSS.


Assuntos
Intestinos/transplante , Organoides/transplante , Animais , Metabolismo dos Carboidratos , Linhagem da Célula , Células Epiteliais/citologia , Sobrevivência de Enxerto , Humanos , Masculino , Camundongos Endogâmicos NOD , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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