RESUMO
Accurate diagnosis and treatment of hepatocellular neoplasm, not otherwise specified (HCN-NOS), poses significant challenges. Our study aimed to investigate the clinicopathologic and genomic similarities and differences between HCN-NOS and hepatoblastoma (HB) to guide diagnostic and treatment strategies. The clinicopathologic characteristics of 16 patients with HCN-NOS and 23 patients with HB were compared. Molecular studies, including the OncoKids DNA- and RNA-based next-generation sequencing panel, chromosomal microarray, and targeted Sanger sequencing analyses of CTNNB1 and TERT promoters, were employed. We found that patients with HCN-NOS were older (P < .001) and more frequently classified as high risk (P < .01), yet they showed no significant differences in alpha fetoprotein levels or survival outcomes compared with those with HB. HCN-NOS and HB had a comparable frequency of sequence variants, with CTNNB1 mutations being predominant in both groups. Notably, TERT promoter mutations (37.5%) and rare clinically significant variants (BRAF, NRAS, and KMT2D) were exclusive to HCN-NOS. HCN-NOS demonstrated a higher prevalence of gains in 1q, encompassing the MDM4 locus (17/17 vs 11/24; P < .001), as well as loss/loss of heterozygosity (LOH) of 1p (11/17 vs 6/24; P < .05) and chromosome 11 (7/17 vs 1/24; P < .01) when compared with HB. Furthermore, the recurrent loss/LOH of chromosomes 3, 4p, 9, 15q, and Y was only observed in HCN-NOS. However, no significant differences were noted in gains of chromosomes 2, 8, and 20, or loss/LOH of 4q and 11p between the 2 groups. Notably, no clinically significant gene fusions were detected in either group. In conclusion, our study reveals that HCN-NOS exhibits high-risk clinicopathologic features and greater structural complexity compared with HB. However, patients with HCN-NOS exhibit comparable alpha fetoprotein levels at diagnosis, CTNNB1 mutation rates, and survival outcomes when subjected to aggressive treatment, as compared with those with HB. These findings have the potential to enhance diagnostic accuracy and inform more effective treatments for HCN-NOS.
Assuntos
Carcinoma Hepatocelular , Hepatoblastoma , Neoplasias Hepáticas , Humanos , Hepatoblastoma/genética , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , alfa-Fetoproteínas , Genômica , Proteínas Proto-Oncogênicas , Proteínas de Ciclo CelularRESUMO
As the classification of kinase-driven spindle cell tumors continues to evolve, we describe the first series of pediatric mesenchymal tumors harboring FGFR1 gene fusions that share histologic overlap with infantile fibrosarcoma and "NTRK-rearranged" spindle cell neoplasms. Herein, we present three cases of FGFR1-rearranged pediatric mesenchymal tumors, including one case with FGFR1::PARD6B gene fusion and two cases with FGFR1::EBF2 gene fusion. The tumors involved infants ranging from 3 to 9 months in age with a male-to-female ratio of 2:1. All tumors involved the deep soft tissue of the gluteal, pelvic, or perirectal region. Histologically, the tumors comprised a cellular spindle cell neoplasm with primitive stellate cells, focal myxoid stroma, focal epithelioid features, no necrosis, and occasional mitotic figures (2-6 per 10 high-power field). By immunohistochemistry, the neoplastic cells focally expressed CD34 but lacked expression of S100 protein, SMA, desmin, myogenin, MyoD1, pan-TRK, and ALK. These three cases, including a case with long-term clinical follow-up, demonstrate that FGFR1 fusions occur in a subset of newly described pediatric kinase-driven mesenchymal tumors with locally aggressive behavior. Importantly, knowledge of these genetic alterations in this spectrum of pediatric tumors is key for diagnostic and targeted therapeutic purposes.
Assuntos
Fibrossarcoma , Neoplasias de Tecido Conjuntivo e de Tecidos Moles , Neoplasias de Tecidos Moles , Feminino , Humanos , Lactente , Masculino , Biomarcadores Tumorais/genética , Fibrossarcoma/patologia , Fusão Gênica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor trkA/genética , Neoplasias de Tecidos Moles/patologiaRESUMO
BACKGROUND: It can be challenging to recognize undifferentiated/immature ganglion cells, especially single forms. Ganglion cells and glia are derived from enteric neural crest cells (ENCCs), a group of autonomic nervous system (ANS)-lineage neural crest progenitors that PHOX2B regulates. Phox2b is an excellent marker for neoplastic and non-neoplastic ANS cells (eg, peripheral neuroblastic tumors [pNTs]). We hypothesized that Phox2b immunohistochemical staining (IHC) would also be useful for detecting ENCCs. METHODS: Hematoxylin and eosin, calretinin IHC, and Phox2b IHC were reviewed on 21 pull-through specimens and on a cohort of 12 rectal biopsies. RESULTS: Phox2b IHC demonstrated nuclear positivity in all of the ganglion cells across the different phases of differentiation without background staining. The Phox2b result correlated with the morphological findings, calretinin IHC results, and diagnoses based on the routine diagnostic method. The intensity was uniformly strong in the undifferentiated/immature forms and became variable in the mature forms; this pattern was similar to that seen in pNTs. CONCLUSION: Phox2b IHC was highly sensitive and specific for detecting ganglion cells. It worked especially well for immature ganglion cells, seen in premature neonates, and scattered single forms in transition zones. In basic research settings, Phox2b can be a useful marker for early differentiation of ENCCs.
Assuntos
Sistema Nervoso Entérico/química , Doença de Hirschsprung/metabolismo , Proteínas de Homeodomínio/análise , Imuno-Histoquímica , Crista Neural/química , Reto/inervação , Fatores de Transcrição/análise , Biópsia , Criança , Pré-Escolar , Sistema Nervoso Entérico/patologia , Feminino , Doença de Hirschsprung/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Crista Neural/patologiaRESUMO
OBJECTIVES: Benign tumors with skeletal muscle differentiation are rare and their characterization in the literature is limited. We present a series of twelve pediatric benign tumors with rhabdomyomatous differentiation including seven rhabdomyomatous mesenchymal hamartomas, four fetal rhabdomyomas, and one benign triton tumor, analyzing myogenic markers as well as clinicopathologic and molecular features. A review of the literature was also performed with an emphasis on myogenic marker expression and correlation with molecular features. METHODS AND RESULTS: Cases obtained from three tertiary pediatric hospitals were retrospectively reviewed. Eleven of twelve cases expressed myogenin in rare to greater than 15% of cells. Five of nine cases had rare to 70-80% of cells positive for MyoD1. One fetal rhabdomyoma demonstrated homozygous deletions in ZEB2. The benign triton tumor harbored a CTNNB1 mutation. Review of the literature identified 160 pediatric benign tumors with skeletal muscle differentiation of which 9 reported myogenin positivity. CONCLUSIONS: Myogenin and MyoD1 may be variably expressed in benign lesions with skeletal muscle differentiation. Recognition of key morphologic features remains critical to diagnose these lesions and, in rhabdomyoma, to exclude malignancy. Our series expands the knowledge of the relationship between rhabdomyoma and rhabdomyosarcoma (RMS) by identifying a shared molecular alteration in ZEB2.
Assuntos
Miogenina/metabolismo , Neoplasias de Tecido Muscular/patologia , Biomarcadores Tumorais/metabolismo , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Lactente , MasculinoRESUMO
Synovial sarcoma (SS) arising within a knee joint is extremely rare, with 10 reported cases in pediatric and adolescent patients in English literature. Its rarity and nonspecific clinical and radiological features pose a diagnostic challenge. We present two cases of primary intra-articular SS of left knee to enhance awareness of this entity. One patient is a 17-year-old male complained of left knee pain and gait abnormality for 9 years. The other one is a 13-year-old female presented with left knee pain for one year. Both cases were clinically diagnosed as benign joint lesion and underwent biopsies. Histological examination, immunohistochemical staining and molecular study confirmed that both patients had primary intra-articular SS, monophasic spindle cell type. Intraarticular SS should be considered as a potential diagnosis with unexplained long-standing knee pain.
Assuntos
Articulação do Joelho/patologia , Sarcoma Sinovial/diagnóstico , Neoplasias de Tecidos Moles/diagnóstico , Adolescente , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Sarcoma Sinovial/patologia , Neoplasias de Tecidos Moles/patologiaRESUMO
BACKGROUND: Pulmonary cysts and spontaneous pneumothorax are presented in most patients with Birt-Hogg-Dubé (BHD) syndrome, which is caused by loss of function mutations in the folliculin (FLCN) gene. The pathogenic mechanisms underlying the cystic lung disease in BHD are poorly understood. METHODS: Mesenchymal Flcn was specifically deleted in mice or in cultured lung mesenchymal progenitor cells using a Cre/loxP approach. Dynamic changes in lung structure, cellular and molecular phenotypes and signalling were measured by histology, immunofluorescence staining and immunoblotting. RESULTS: Deletion of Flcn in mesoderm-derived mesenchymal cells results in significant reduction of postnatal alveolar growth and subsequent alveolar destruction, leading to cystic lesions. Cell proliferation and alveolar myofibroblast differentiation are inhibited in the Flcn knockout lungs, and expression of the extracellular matrix proteins Col3a1 and elastin are downregulated. Signalling pathways including mTORC1, AMP-activated protein kinase, ERK1/2 and Wnt-ß-catenin are differentially affected at different developmental stages. All the above changes have statistical significance (p<0.05). CONCLUSIONS: Mesenchymal Flcn is an essential regulator during alveolar development and maintenance, through multiple cellular and molecular mechanisms. The mesenchymal Flcn knockout mouse model provides the first in vivo disease model that may recapitulate the stages of cyst development in human BHD. These findings elucidate the developmental origins and mechanisms of lung disease in BHD.
Assuntos
Síndrome de Birt-Hogg-Dubé/metabolismo , Síndrome de Birt-Hogg-Dubé/patologia , Cistos/metabolismo , Cistos/patologia , Pneumopatias/metabolismo , Pneumopatias/patologia , Proteínas Proto-Oncogênicas/metabolismo , Alvéolos Pulmonares/crescimento & desenvolvimento , Proteínas Supressoras de Tumor/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fenótipo , Pneumotórax/metabolismo , Pneumotórax/patologia , Transdução de SinaisRESUMO
BACKGROUND: Pelvic neuroblastomas are rare and often present in children as massive tumors whose surgical resection can be associated with significant morbidity, given sacral nerve root involvement and close proximity to pelvic vascular structures. We sought to examine the characteristics of patients with pelvic neuroblastoma and the effect of extent of surgical resection on survival and surgical outcomes. MATERIALS AND METHODS: After institutional review board approval, a retrospective chart review was performed at Children's Hospital Los Angeles from 2000 to 2018. Collected data included tumor location, size, image-defined risk factors histology, stage and risk classification, amplification of the oncogene MYCN or N-myc, use of preoperative chemotherapy, and extent of surgical resection. Outcome variables included postoperative complications and survival. RESULTS: Ten patients with primary pelvic neuroblastoma tumors were identified. The median age at diagnosis was 4.2 y (3 mo to 11 y). Four patients presented with a localized pelvic tumor (stage I or stage II) and underwent upfront tumor resection. Six patients presented with advanced disease (stage III or stage IV) and underwent neoadjuvant chemotherapy, followed by partial resection (30%-90% debulked). One patient experienced a complication: lower extremity hypotonia after tumor resection. One patient died from extensive metastatic disease for which no resection was attempted. The mean postoperative follow-up was 3.9 y with 90% overall survival. CONCLUSIONS: Our data show that patients who undergo gross total resection for localized pelvic neuroblastoma or neoadjuvant chemotherapy, followed by partial resection for advanced disease have excellent survival. We recommend that small localized pelvic neuroblastoma undergo gross total resection and large unresectable tumors undergo neoadjuvant chemotherapy, followed by partial debulking resection to avoid neurovascular morbidity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Procedimentos Cirúrgicos de Citorredução/métodos , Terapia Neoadjuvante/métodos , Neuroblastoma/terapia , Neoplasias Pélvicas/terapia , Criança , Pré-Escolar , Procedimentos Cirúrgicos de Citorredução/efeitos adversos , Feminino , Seguimentos , Humanos , Lactente , Masculino , Estadiamento de Neoplasias , Neuroblastoma/diagnóstico , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Neoplasias Pélvicas/diagnóstico , Neoplasias Pélvicas/mortalidade , Neoplasias Pélvicas/patologia , Pelve/irrigação sanguínea , Pelve/diagnóstico por imagem , Pelve/cirurgia , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Análise de Sobrevida , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
The tuberous sclerosis complex (TSC) proteins are critical negative regulators of the mammalian/mechanistic target of rapamycin complex 1 pathway. Germline mutations of TSC1 or TSC2 cause TSC, affecting multiple organs, including the kidney and lung, and causing substantial morbidity and mortality. The mechanisms of organ-specific disease in TSC remain incompletely understood, and the impact of TSC inactivation on mesenchymal lineage cells has not been specifically studied. We deleted Tsc2 specifically in mesoderm-derived mesenchymal cells of multiple organs in mice using the Dermo1-Cre driver. The Dermo1-Cre-driven Tsc2 conditional knockout mice had body growth retardation and died approximately 3 weeks after birth. Significant phenotypes were observed in the postnatal kidney and lung. Inactivation of Tsc2 in kidney mesenchyme caused polycystic lesions starting from the second week of age, with increased cell proliferation, tubular epithelial hyperplasia, and epithelial-mesenchymal transition. In contrast, Tsc2 deletion in lung mesenchyme led to decreased cell proliferation, reduced postnatal alveolarization, and decreased differentiation with reduced numbers of alveolar myofibroblast and type II alveolar epithelial cells. Two major findings thus result from this model: inactivation of Tsc2 in mesoderm-derived cells causes increased cell proliferation in the kidneys but reduced proliferation in the lungs, and inactivation of Tsc2 in mesoderm-derived cells causes epithelial-lined renal cysts. Therefore, Tsc2-mTOR signaling in mesenchyme is essential for the maintenance of renal structure and for lung alveolarization.
Assuntos
Doenças Renais Policísticas/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Feminino , Humanos , Rim/metabolismo , Rim/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Mesoderma/patologia , Camundongos , Camundongos Knockout , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Serina-Treonina Quinases TOR/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
AIMS: The primary aim of this study is to characterize hepatocellular malignant neoplasm, NOS (HEMNOS), a new provisional entity describing a subset of paediatric hepatocellular tumours, which have histological features of neither typical hepatoblastoma (HB) nor hepatocellular carcinoma (HCC). METHODS AND RESULTS: The clinicopathological features of 11 patients with HEMNOS were analysed retrospectively. The median age and serum alpha-fetoprotein level at diagnosis was 7 years and 182 000 ng/ml, respectively. Ten patients presented with pretreatment extent of disease (PRETEXT) stages III/IV multifocal tumours, eight with major vascular involvement, three with lung metastases and three with extrahepatic extension. The original pathology diagnoses were: HB in seven patients, HCC in two and HEMNOS in two. Our pathology review of pre-chemotherapy specimens showed that six tumours had equivocal/overlapping histological features of HB and HCC, four had predominant HB histology along with focal HCC-like histology and one had HB histology. Seven of nine post-chemotherapy resection specimens showed predominant HCC-like histology. Beta-catenin, glypican 3 and spalt-like transcription factor 4 immunostaining showed that all the tumours had a mixed HB/HCC immunophenotype. Telomerase reverse transcriptase immunostaining showed nuclear staining in nine of the 11 tumours. All patients received chemotherapy and achieved gross total primary tumour resection. Nine of the 11 patients were treated with established HB chemotherapy regimens. After a median follow-up of 6.1 years (range: 1.2-11.8 years), all patients were in remission. CONCLUSIONS: HEMNOS is a subtype of HB with focal HCC-like histology, a high-risk clinical profile but favourable outcome following chemotherapy and complete tumour resection.
Assuntos
Neoplasias Hepáticas/patologia , Adolescente , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/patologia , Criança , Pré-Escolar , Feminino , Hepatoblastoma/patologia , Humanos , Masculino , Estudos RetrospectivosRESUMO
AIMS: To investigate the expression of spalt-like transcription factor 4 (SALL4), a regulator of embryonal development, in three epithelial components of hepatoblastoma (HB) and the relationship between SALL4 expression levels and patients' clinicopathological features. METHODS AND RESULTS: A total of 115 specimens from 79 patients with HB were selected for immunostaining of SALL4. Nuclear staining was semi-quantified using the immunoreactive score (IS; range: 0-12). SALL4 expression was seen in all embryonal components (mean IS = 8.58) and in 41% of fetal components (mean IS = 0.78). No SALL4 expression was seen in either small cell undifferentiated or mesenchymal components of HB. Neither chemotherapy nor metastasis altered SALL4 expression significantly. High SALL4 expression levels were associated significantly with decreased overall survival (OS) (P = 0.004), event-free survival (EFS) (P = 0.003) and the presence of metastasis (P = 0.049) on univariate analysis. Multivariate analysis identified SALL4 as an independent prognostic predictor for OS (P = 0.029). CONCLUSIONS: SALL4 is useful for subtyping HB, and high SALL4 expression is associated with decreased survival in HB.
Assuntos
Biomarcadores Tumorais/análise , Hepatoblastoma/diagnóstico , Neoplasias Hepáticas/diagnóstico , Fatores de Transcrição/biossíntese , Pré-Escolar , Intervalo Livre de Doença , Feminino , Hepatoblastoma/mortalidade , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Masculino , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Transcrição/análiseRESUMO
An eight-yr-old female with a history of multifocal lymphangioendotheliomatosis and thrombocytopenia presented for MVT. The patient had multiple vascular lesions in the skin and stomach in infancy. Although her cutaneous lesions resolved with vincristine and methylprednisolone, her gastric lesions persisted. Eight yr later, she was diagnosed with portal hypertension and decompensating liver function despite therapy with bevacizumab, propranolol, furosemide, and spironolactone. Upon presentation, she was found to have a Kasabach-Merritt-like coagulopathy in association with multiple lesions in her GI tract and persistent gastric lesions. Although treatment with methylprednisolone and sirolimus normalized her coagulation factors and d-dimer levels, she never developed sustained improvement in her thrombocytopenia. Her liver function continued to deteriorate and she developed hepatorenal syndrome. Given better outcomes after OLT in comparison with MVT, she underwent OLT, with the plan to manage her GI lesions with APC post-transplant. Post-transplant, her liver function and coagulopathy normalized, and GI tract lesions disappeared upon screening with capsule endoscopy. The patient is doing well, without recurrence of either GI lesions or thrombocytopenia, at 18 months after transplantation.
Assuntos
Transplante de Fígado/métodos , Linfangioma/complicações , Linfangioma/cirurgia , Trombocitopenia/complicações , Trombocitopenia/cirurgia , Coagulação Sanguínea , Fatores de Coagulação Sanguínea , Criança , Colestase , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Trato Gastrointestinal/patologia , Síndrome Hepatorrenal/complicações , Humanos , Hipertensão Portal , Metilprednisolona/administração & dosagem , Estômago/patologia , Resultado do Tratamento , Vincristina/administração & dosagemRESUMO
PURPOSE: Complete DiGeorge syndrome (cDGS) describes a subset of patients with DiGeorge syndrome that have thymic aplasia, and thus are at risk for severe opportunistic infections. Patients with cDGS and mycobacterial infection have not previously been described. We present this case to illustrate that patients with cDGS are at risk for nontuberculous mycobacterial infections and to discuss further antimicrobial prophylaxis prior to thymic transplantation. METHODS: A 13-month old male was identified as T cell deficient by the T cell receptor excision circle (TREC) assay on newborn screening, and was subsequently confirmed to have cDGS. He presented with fever and cough, and was treated for chronic aspiration pneumonia as well as Pneumocystis jirovecii infection without significant improvement. It was only after biopsy of mediastinal lymph nodes seen on CT that the diagnosis of disseminated Mycobacterium kansasii was made. We reviewed the literature regarding atypical mycobacterial infections and prophylaxis used in other immunocompromised patients, as well as the current data regarding cDGS detection through TREC newborn screening. RESULTS: Multiple cases of cDGS have been diagnosed via TREC newborn screening, however this is the first patient with cDGS and disseminated mycobacterial infection to be reported in literature. Thymic transplantation is the definitive treatment of choice for cDGS. Prophylaxis with either clarithromycin or azithromycin has been shown to reduce mycobacterial infections in children with advanced human immunodeficiency virus infection. CONCLUSIONS: Children with cDGS should receive thymic transplantion as soon as possible, but prior to this are at risk for nontuberculous mycobacterial infections. Severe, opportunistic infections may require invasive testing for diagnosis in patients with cDGS. Antimicrobial prophylaxis should be considered to prevent disseminated mycobacterial infection in these patients.
Assuntos
Antibioticoprofilaxia , Síndrome de DiGeorge/diagnóstico , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium kansasii , Linfócitos T/imunologia , Timo/transplante , Azitromicina/uso terapêutico , Biópsia , Claritromicina/uso terapêutico , Síndrome de DiGeorge/complicações , Síndrome de DiGeorge/imunologia , Humanos , Lactente , Masculino , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/prevenção & controle , Transplante de Órgãos , Timo/patologia , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVES: Cell death in lymphatic organs, such as the spleen, is in part responsible for immunosuppression and contributes to mortality during sepsis. An early and noninvasive detection of lymphoid cell death could thus have significant clinical implications. Here, we tested in vivo imaging of lymphoid cell death using a near-infrared annexin V (AV-750). DESIGN: Animal study. SETTING: Laboratory investigation. SUBJECTS: C57BL/6J wild-type and toll-like receptor 3 knockout mice. INTERVENTIONS: Mild and severe polymicrobial sepsis was induced with cecum ligation and puncture. Serum cytokines and acute kidney injury markers were tested by immunoassay and quantitative reverse transcription-polymerase chain reaction, respectively. Sepsis-induced lymphoid cell death was detected by fluorescent AV-750 accumulation in the thorax and abdomen (in vivo), in isolated organs (ex vivo), and in isolated cells (flow cytometry). Caspase-3 cleavage/activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were tested for apoptosis. MEASUREMENTS AND MAIN RESULTS: Severe sepsis induced marked apoptosis in the thymus, spleen, and liver as demonstrated by cleaved caspase-3 and an increase in caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. A significant increase in fluorescent AV-750 signal was seen in the thoracic and upper abdominal fields and associated with the severity of sepsis. The in vivo thoracic and abdominal AV-750 fluorescent signal was attributed to the thymus, liver, and spleen as determined by ex vivo imaging and highly correlated with the levels of cell death in thymocytes and splenocytes, respectively, as measured by flow cytometry. Compared with wild-type septic mice, toll-like receptor 3 septic mice had attenuated abdominal AV-750 fluorescent signal, reduced ex vivo fluorescence in the spleen, and decreased splenocyte cell death. CONCLUSIONS: In vivo AV-750 fluorescent imaging provides spatially resolved and organ-specific detection of lymphoid cell death during polymicrobial sepsis. The AV-750 fluorescent intensity in the thoracic and abdominal fields is associated with sepsis severity and well correlated with sepsis-induced cell death in the thymus and spleen, respectively.
Assuntos
Bacteriemia/microbiologia , Morte Celular , Coinfecção/microbiologia , Linfócitos/citologia , Análise de Variância , Animais , Apoptose/fisiologia , Bacteriemia/fisiopatologia , Western Blotting , Caspase 3/metabolismo , Coinfecção/diagnóstico , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas/métodos , Linfócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Estatísticas não ParamétricasRESUMO
Necrotizing enterocolitis (NEC) affects up to 10% of premature infants, has a mortality of 30%, and can leave surviving patients with significant morbidity. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes epithelial cell survival. Thus, this pathway could be protective in diseases such as NEC, in which epithelial cell death is a major pathologic feature. We sought to determine whether NRG4-ErbB4 signaling is protective in experimental NEC. NRG4 was used i) in the newborn rat formula feeding/hypoxia model; ii) in a recently developed model in which 14- to 16-day-old mice are injected with dithizone to induce Paneth cell loss, followed by Klebsiella pneumoniae infection to induce intestinal injury; and iii) in bacterially infected IEC-6 cells in vitro. NRG4 reduced NEC incidence and severity in the formula feed/hypoxia rat model. It also reduced Paneth cell ablation-induced NEC and prevented dithizone-induced Paneth cell loss in mice. In vitro, cultured ErbB4(-/-) ileal epithelial enteroids had reduced Paneth cell markers and were highly sensitive to inflammatory cytokines. Furthermore, NRG4 blocked, through a Src-dependent pathway, Cronobacter muytjensii-induced IEC-6 cell apoptosis. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 are present in human breast milk and developing human intestine, respectively. Thus, NRG4-ErbB4 signaling may be a novel pathway for therapeutic intervention or prevention in NEC.
Assuntos
Enterocolite Necrosante/prevenção & controle , Leite Humano/química , Neurregulinas/metabolismo , Receptor ErbB-4/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Citocinas/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Íleo/metabolismo , Intestinos/patologia , Camundongos , Celulas de Paneth/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
UNLABELLED: Biliary atresia (BA), the most common cause of end-stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long-term transplant-free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus-induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ-1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen-1α1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E-CADHERIN, biliary marker CYTOKERATIN-19, and mesenchymal markers VIMENTIN and alpha-SMOOTH MUSCLE ACTIN (αSMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor-beta (TGFß) signaling. In vitro cotreatment of PROM1-expressing Mat1a-/- hepatic progenitor cells with recombinant human FGF10 and TGFß1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen-1α1, Fibronectin, and α-Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype. CONCLUSION: Expansion of collagen-producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGFß pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA.
Assuntos
Antígenos CD/biossíntese , Atresia Biliar/metabolismo , Glicoproteínas/biossíntese , Cirrose Hepática/metabolismo , Antígeno AC133 , Animais , Atresia Biliar/complicações , Modelos Animais de Doenças , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Infecções por Rotavirus/complicações , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismoRESUMO
AIMS: To identify an immunohistochemical panel for paediatric malignant epithelial liver tumours. METHODS AND RESULTS: Forty-five hepatoblastomas (HBs), 13 paediatric hepatocellular carcinomas (HCCs) and two hepatocellular malignant neoplasms not otherwise specified (NOS) were chosen for immunohistochemical staining of glypican 3 (GPC3), ß-catenin, claudin-1, delta-like protein (DLK), and forkhead box protein G1 (FOXG1). Immunostaining was quantitatively analysed with NIH imagej software coupled with colour deconvolution. Different subtypes of HB and HCC showed distinct staining patterns of GPC3, ß-catenin, and claudin-1. Moreover, GPC3, ß-catenin and claudin-1 all showed higher expression in classic HCC and embryonal HB than in fetal HB; GPC3 showed complete negativity in small-cell undifferentiated (SCU) HB and fibrolamellar HCC (FLC); ß-catenin showed the strongest expression in SCU HB but the weakest expression in FLC. A panel of these three immunomarkers was useful for the diagnosis of hepatocellular malignant neoplasms NOS. The expression of DLK and FOXG1 was inconstant among fetal and embryonal HB and classic HCC. CONCLUSIONS: A panel of GPC3, ß-catenin and claudin-1 is helpful for differentiating HB subtypes and distinguishing HB from HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Claudina-1/metabolismo , Glipicanas/metabolismo , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , beta Catenina/metabolismo , Adolescente , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Criança , Pré-Escolar , Feminino , Hepatoblastoma/patologia , Humanos , Lactente , Fígado/patologia , Neoplasias Hepáticas/patologia , MasculinoRESUMO
Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/isolamento & purificação , Cromatografia/métodos , Imunoglobulina G/imunologia , Humanos , CinéticaRESUMO
Severe sepsis involves massive activation of the innate immune system and leads to high mortality. Previous studies have demonstrated that various types of TLRs mediate a systemic inflammatory response and contribute to organ injury and mortality in animal models of severe sepsis. However, the downstream mechanisms responsible for TLR-mediated septic injury are poorly understood. In this article, we show that activation of TLR2, TLR3, and TLR4 markedly enhanced complement factor B (cfB) synthesis and release by macrophages and cardiac cells. Polymicrobial sepsis, created by cecal ligation and puncture in a mouse model, augmented cfB levels in the serum, peritoneal cavity, and major organs including the kidney and heart. Cecal ligation and puncture also led to the alternative pathway activation, C3 fragment deposition in the kidney and heart, and cfB-dependent C3dg elevation. Bacteria isolated from septic mice activated the serum alternative pathway via a factor D-dependent manner. MyD88 deletion attenuated cfB/C3 upregulation as well as cleavage induced by polymicrobial infection. Importantly, during sepsis, absence of cfB conferred a protective effect with improved survival and cardiac function and markedly attenuated acute kidney injury. cfB deletion also led to increased neutrophil migratory function during the early phase of sepsis, decreased local and systemic bacterial load, attenuated cytokine production, and reduced neutrophil reactive oxygen species production. Together, our data indicate that cfB acts as a downstream effector of TLR signaling and plays a critical role in the pathogenesis of severe bacterial sepsis.
Assuntos
Fator B do Complemento/metabolismo , Macrófagos/imunologia , Miócitos Cardíacos/imunologia , Neutrófilos/imunologia , Sepse/imunologia , Animais , Ceco/cirurgia , Movimento Celular/genética , Células Cultivadas , Complemento C3/metabolismo , Fator B do Complemento/genética , Modelos Animais de Doenças , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Fator 88 de Diferenciação Mieloide/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
The sarcomatous element in pleuropulmonary blastoma (PPB) is often histologically indistinguishable from embryonal rhabdomyosarcoma (ERMS). A diagnosis of PPB is often made after definitive surgical resection based on pathologic features, most notably the presence of hamartomatous pulmonary elements. Samples from seven PPB patients were obtained from the rhabdomyosarcomatous portion of the tumor by macrodissection. Representative ERMS tumor tissue was selected from 21 ERMS patient samples. Formalin-fixed paraffin-embedded tissue scrolls from each sample were analyzed using the Affymetrix Human Exon arrays. All PPB patients and 7 of 21 ERMS patients were 3 years old and younger. Twenty transcripts (10 annotated, 10 noncoding RNAs) were significantly differentially expressed in ERMS when compared with PPB samples. Insulin-like growth factor 2 (IGF2) was uniformly overexpressed in ERMS (19/21>400) but was expressed at low levels in PPB (P<0.001). Two ERMS cases that had low level IGF2 expression were 3 years and younger of age. No other differences between the 2 approached this degree of significance, despite a common rhabdomyogenic phenotype in the sarcomatous areas of PPB. PPB, unlike most ERMS, appears not to be driven by autocrine IGF2 signaling.
Assuntos
Biomarcadores Tumorais/genética , Fator de Crescimento Insulin-Like II/genética , Blastoma Pulmonar/diagnóstico , Rabdomiossarcoma Embrionário/diagnóstico , Biomarcadores Tumorais/metabolismo , Pré-Escolar , Diagnóstico Diferencial , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Lactente , Recém-Nascido , Fator de Crescimento Insulin-Like II/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Prognóstico , Blastoma Pulmonar/genética , Blastoma Pulmonar/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismoRESUMO
Chronic bladder dysfunction due to bladder disease or trauma is detrimental to affected patients as it can lead to increased risk of upper urinary tract dysfunction. Current treatment options include surgical intervention that enlarge the bladder with autologous bowel tissue to alleviate pressure on the upper urinary tract. This highly invasive procedure, termed bladder augmentation enterocystoplasty (BAE), significantly increases risk of patient morbidity and mortality due to the incompatibility between the bowel and bladder tissue. Therefore, patients would significantly benefit from an alternative treatment strategy that can regenerate healthy tissue and restore overall bladder function. Previous research has demonstrated the potential of citrate-based scaffolds co-seeded with bone marrow-derived stem/progenitor cells as an alternative graft for bladder augmentation. Recognizing that contact guidance is known to influence tissue regeneration, we hypothesized that patterned scaffolds would modulate cell responses and improve overall quality of the regenerated bladder tissue. We fabricated microgrooved (MG) scaffolds using citrate-based biomaterial poly(1,8-octamethylene-citrate-co-octanol) (POCO) and co-seeded them with human bone marrow derived mesenchymal stem cells (MSCs) and CD34+ hematopoietic stem/progenitor cells (HSPCs). Microgrooved POCO scaffolds supported MSC and HSPC attachment, and MSC alignment within the microgrooves. All scaffolds were characterized and assessed for bladder tissue regeneration in an established nude rat bladder augmentation model. In all cases, normal physiological function was maintained post-augmentation, even without the presence of stem/progenitor cells. Urodynamic testing at 4-weeks post-augmentation for all experimental groups demonstrated that capacity increased and compliance was normal. Histological evaluation of the regenerated tissue revealed that cell-seeded scaffolds restored normal bladder smooth muscle content and resulted in increased revascularization and peripheral nerve regeneration. The presence of microgrooves on the cell-seeded scaffolds increased microvasculature formation by 20% and urothelium layer thickness by 25% in the regenerating tissue. Thus, this work demonstrates that micropatterning affects bladder regeneration to improve overall anatomical structure and re-establish bladder physiology.