David R. Kelly

Evidence for in vivo peroxynitrite production in human acute lung injury.

Oxidant-mediated toxicity resulting from acute pulmonary inflammation has been demonstrated in acute lung injury. A potent biological oxidant, peroxynitrite, is formed by the near diffusion-limited reaction of nitric oxide with superoxide. In addition to having hydroxyl radical-like oxidative reactivity, peroxynitrite is capable of nitrating phenolic rings, including protein-associated tyrosine residues. Nitric oxide does not directly nitrate tyrosine residues, therefore, demonstration of tissue nitrotyrosine residues infers the action of peroxynitrite or related nitrogen-centered oxidants. Lung tissue was obtained from formalin-fixed, paraffin-embedded autopsy specimens, and specific polyclonal and monoclonal antibodies to nitrotyrosine were visualized by diaminobenzidene-peroxidase staining. Acute lung injury resulted in intense staining throughout the lung, including lung interstitium, alveolar epithelium, proteinaceous alveolar exudate, and inflammatory cells. In addition, staining of the vascular endothelium and subendothelial tissues was present in those patients with sepsis-induced acute lung injury. Antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. Reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. In control specimens with no overt pulmonary disease, there was only slight staining of the alveolar septum. These results demonstrate that nitrogen-derived oxidants are formed in human acute lung injury and suggest that peroxynitrite may be an important oxidant in inflammatory lung disease.

Extensive tyrosine nitration in human myocardial inflammation: Evidence for the presence of peroxynitrite

OBJECTIVES Production of nitric oxide via the cytokine-mediated activation of myocardial inducible nitric oxide synthase decreases myocardial contractility. Whether myocardial dysfunction is mediated directly by nitric oxide or indirectly through the formation of secondary reaction products, such as peroxynitrite, has not been established. Peroxynitrite, but not nitric oxide, reacts with the phenolic ring of tyrosine to form the stable product 3-nitro-L-tyrosine. Demonstration of tissue nitrotyrosine residues, therefore, infers the presence of peroxynitrite or related nitrogen-centered oxidants. DESIGN Retrospective analysis of human autopsy specimens. SETTING University pathology and basic science laboratories. PATIENTS Formalin-fixed, paraffin-embedded myocardial tissue samples were obtained from 11 patients with a diagnosis of sepsis, seven patients with a diagnosis of viral myocarditis, and five control patients without clinical or pathologic cardiac disease. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Specific antibodies to nitrotyrosine were utilized to detect nitrotyrosine residues in human autopsy specimens. Cardiac tissue obtained from patients with myocarditis or sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular endothelium and smooth muscle. In contrast, connective tissue elements were without appreciable immunohistochemical staining. Nitrotyrosine antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin, but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. In situ reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. Densitometric analysis of nitrotyrosine immunoreactivity demonstrated significantly higher values for specimens from myocarditis and sepsis patients when compared with control tissue specimens. CONCLUSION These results demonstrate the formation of peroxynitrite within the myocardium during inflammatory disease states, suggesting a role for peroxynitrite in inflammation-associated myocardial dysfunction.

TGF-β2 Suppresses Macrophage Cytokine Production and Mucosal Inflammatory Responses in the Developing Intestine

BACKGROUND & AIMS Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated whether NEC occurs in the preterm intestine due to incomplete noninflammatory differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products. METHODS We compared inflammatory properties of human/murine fetal, neonatal, and adult intestinal macrophages. To investigate gut-specific macrophage differentiation, we next treated monocyte-derived macrophages with conditioned media from explanted human fetal and adult intestinal tissues. Transforming growth factor-β (TGF-β) expression and bioactivity were measured in fetal/adult intestine and in NEC. Finally, we used wild-type and transgenic mice to investigate the effects of deficient TGF-β signaling on NEC-like inflammatory mucosal injury. RESULTS Intestinal macrophages in the human preterm intestine (fetus/premature neonate), but not in full-term neonates and adults, expressed inflammatory cytokines. Macrophage cytokine production was suppressed in the developing intestine by TGF-β, particularly the TGF-β(2) isoform. NEC was associated with decreased tissue expression of TGF-β(2) and decreased TGF-β bioactivity. In mice, disruption of TGF-β signaling worsened NEC-like inflammatory mucosal injury, whereas enteral supplementation with recombinant TGF-β(2) was protective. CONCLUSIONS Intestinal macrophages progressively acquire a noninflammatory profile during gestational development. TGF-β, particularly the TGF-β(2) isoform, suppresses macrophage inflammatory responses in the developing intestine and protects against inflammatory mucosal injury. Enterally administered TGF-β(2) protected mice from experimental NEC-like injury.

Plasma concentrations of inflammatory cytokines rise rapidly during ECMO-related SIRS due to the release of preformed stores in the intestine

Extracorporeal membrane oxygenation (ECMO) is a life-saving support system used in neonates and young children with severe cardiorespiratory failure. Although ECMO has reduced mortality in these critically ill patients, almost all patients treated with ECMO develop a systemic inflammatory response syndrome (SIRS) characterized by a ‘cytokine storm’, leukocyte activation, and multisystem organ dysfunction. We used a neonatal porcine model of ECMO to investigate whether rising plasma concentrations of inflammatory cytokines during ECMO reflect de novo synthesis of these mediators in inflamed tissues, and therefore, can be used to assess the severity of ECMO-related SIRS. Previously healthy piglets (3-week-old) were subjected to venoarterial ECMO for up to 8 h. SIRS was assessed by histopathological analysis, measurement of neutrophil activation (flow cytometry), plasma cytokine concentrations (enzyme immunoassays), and tissue expression of inflammatory genes (PCR/western blots). Mast cell degranulation was investigated by measurement of plasma tryptase activity. Porcine neonatal ECMO was associated with systemic inflammatory changes similar to those seen in human neonates. Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) concentrations rose rapidly during the first 2 h of ECMO, faster than the tissue expression of these cytokines. ECMO was associated with increased plasma mast cell tryptase activity, indicating that increased plasma concentrations of inflammatory cytokines during ECMO may result from mast cell degranulation and associated release of preformed cytokines stored in mast cells. TNF-α and IL-8 concentrations rose faster in plasma than in the peripheral tissues during ECMO, indicating that rising plasma levels of these cytokines immediately after the initiation of ECMO may not reflect increasing tissue synthesis of these cytokines. Mobilization of preformed cellular stores of inflammatory cytokines such as in mucosal mast cells may have an important pathophysiological role in ECMO-related SIRS.

Desmin Positivity in Primitive Neuroectodermal Tumors of Childhood

In this report, we describe two rosette-forming primitive neuroectodermal tumors that were found to contain desmin by both immunohistochemistry and Western blotting. Electron microscopy on both cases was consistent with primitive neuroectodermal tumors and revealed that the tumor cells contained cytoplasmic bundles of intermediate filaments. In both cases, studies for MyoDl protein using immunohistochemistry and Western blotting were negative. Thus, the detection of desmin in a pediatric neoplasm does not absolutely exclude the diagnosis of primitive neuroectodermal tumor and should not be considered as prima facie evidence that a small-cell tumor is a rhabdomyosarcoma.

Childhood liposarcoma. Report of a case and review of the literature

A case of liposarcoma presenting in an adolescent black male is described. Features unique to this case include a mediastinal primary site (the fourth to be documented in a pediatric patient), a demonstrable response to radiotherapy and chemotherapy allowing complete surgical excision of an initially inoperable tumor, and the apparent histologic maturation of the tumor following multimodal therapy. A review of previously published accounts of pediatric liposarcoma revealed the following: (1) peak incidences during infancy and in early adolescene; (2) the extremities to be the most common site of origin (51%); (3) a predominance of myxoid histology (76%); and (4) a lower overall recurrence rate when compared with adult cases (37% versus 72%, respectively). The influence of histology, location of the tumor, and completeness of surgical excision on the prognosis of adult liposarcoma was confirmed in this limited pediatric experience. While the use of radiotherapy and chemotherapy cannot be advocated in all instances of liposarcoma, our experience and that from other reports would support the incorporation of these modalities of treatment in selected patients where the tumor is surgically inaccessible due to size.

Epithelial cells in fetal intestine produce chemerin to recruit macrophages

Macrophages are first seen in the fetal intestine at 11-12 wk and rapidly increase in number during the 12- to 22-wk period of gestation. The development of macrophage populations in the fetal intestine precedes the appearance of lymphocytes and neutrophils and does not require the presence of dietary or microbial antigens. In this study, we investigated the role of chemerin, a recently discovered, relatively selective chemoattractant for macrophages, in the recruitment of macrophage precursors to the fetal intestine. Chemerin mRNA/protein expression was measured in jejunoileal tissue from 10- to 24-wk human fetuses, neonates operated for intestinal obstruction, and adults undergoing bariatric surgery. The expression of chemerin in intestinal epithelial cells (IECs) was confirmed by using cultured primary IECs and IEC-like cell lines in vitro. The regulatory mechanisms involved in chemerin expression were investigated by in silico and immunolocalization techniques. IECs in the fetal, but not mature, intestine express chemerin. Chemerin expression peaked in the fetal intestine at 20-24 wk and then decreased to original low levels by full term. During the 10- to 24-wk period, chemerin accounted for most of the macrophage chemotactic activity of cultured fetal IECs. The maturational changes in chemerin expression correlated with the expression of retinoic acid receptor-beta in the intestine. Chemerin is an important mediator of epithelial-macrophage cross talk in the fetal/premature, but not in the mature, intestine. Understanding the regulation of the gut macrophage pool is an important step in development of novel strategies to boost mucosal immunity in premature infants and other patient populations at risk of microbial translocation.

Older age at diagnosis of Hirschsprung disease decreases risk of postoperative enterocolitis, but resection of additional ganglionated bowel does not.

PURPOSE This study was conducted to determine the effect of age at diagnosis and length of ganglionated bowel resected on postoperative Hirschsprung-associated enterocolitis (HAEC). METHODS Children who underwent endorectal pull-through (ERPT) between January 1993 and December 2004 were retrospectively reviewed. t Test, analysis of variance, Kaplan-Meier, and Coxs proportional hazards analyses were performed. RESULTS Fifty-two children with Hirschsprung disease (median age, 25 days; range, 2 days-16 years) were included. Nineteen (37%) had admissions for HAEC. Proportional hazards regression showed that HAEC admissions decreased by 30% with each doubling of age at diagnosis (P = .03) and increased 9-fold when postoperative stricture was present (P < .01), after controlling for type of ERPT, trisomy 21, transition zone level, and preoperative enterocolitis. Thirty-six children, with age at initial operation less than 6 months, were grouped based on length of ganglionated bowel excised (A [5 cm] and B [>5 cm]). No significant difference in the number of HAEC admissions during initial 2 years post-ERPT was seen between groups A (n = 18) and B (n = 18). The study had a power of 0.8 to detect a difference of 1 admission over 2 years. CONCLUSIONS Children diagnosed with Hirschsprung disease at younger ages are at a greater risk for postoperative enterocolitis. Excising a longer margin of ganglionated bowel (>5 cm) does not seem to be beneficial in decreasing HAEC admissions.

Myofibroblast differentiation and enhanced TGF-B signaling in cystic fibrosis lung disease.

Rationale TGF-β, a mediator of pulmonary fibrosis, is a genetic modifier of CF respiratory deterioration. The mechanistic relationship between TGF-β signaling and CF lung disease has not been determined. Objective To investigate myofibroblast differentiation in CF lung tissue as a novel pathway by which TGF-β signaling may contribute to pulmonary decline, airway remodeling and tissue fibrosis. Methods Lung samples from CF and non-CF subjects were analyzed morphometrically for total TGF-β1, TGF-β signaling (Smad2 phosphorylation), myofibroblast differentiation (α-smooth muscle actin), and collagen deposition (Masson trichrome stain). Results TGF-β signaling and fibrosis are markedly increased in CF (p<0.01), and the presence of myofibroblasts is four-fold higher in CF vs. normal lung tissue (p<0.005). In lung tissue with prominent TGF-β signaling, both myofibroblast differentiation and tissue fibrosis are significantly augmented (p<0.005). Conclusions These studies establish for the first time that a pathogenic mechanism described previously in pulmonary fibrosis is also prominent in cystic fibrosis lung disease. The presence of TGF-β dependent signaling in areas of prominent myofibroblast proliferation and fibrosis in CF suggests that strategies under development for other pro-fibrotic lung conditions may also be evaluated for use in CF.

A morphologic study of childhood lymphoma of the lymphoblastic type: the pediatric oncology group experience

For this study 227 non‐Hodgkins lymphomas, registered through the Pediatric Oncology Group clinical studies between 1976 and 1982, were morphologically subclassified into major histologic types and subtypes, and their histopathologic and clinical features were compared. These lymphomas were distributed primarily into only three of the recognized major histologic types: lymphoblastic (LB), 106 (47%) undifferentiated (DU), 49 (21%); and diffuse histiocytic (DH), 72 (32%). These patient groups were found to differ in several ways: (1) the LB lymphomas contained most of the patients under two years of age; (2) the LB lymphomas tended to present in higher clinical stages; (3) the LB lymphomas tended to involve lymph node groups and the bone marrow more often than did the DU and DH lymphomas; and (4) the DU lymphomas had a greater tendency for gastrointestinal tract and other major organ system involvement. The complete remission rate of 96%, for the LB lymphomas was better than for either the DU or the DH lymphomas. The disease‐free survival of the LB lymphomas was significantly better than the DU group, but not the DH group. The LB were histologically divisible into three subtypes: convoluted (C), nonconvoluted (NC), and large cell variant (LCV). The C and NC subtypes preferentially involved the mediastinum and peripheral lymph nodes initially, while the LCV tended to involve the abdomen. However, none of the subtypes differed in clinical stage. The complete remission, and the disease‐free survival rates between these subtypes were not statistically different. Cancer 59:1126‐1131, 1987.