Lack of C-KIT+ mast cells and the development of idiopathic gastric perforation in neonates.

BACKGROUND/PURPOSE: The proto-oncogene c-kit encodes a receptor tyrosine kinase C-KIT. W/Wv mice, which are devoid of C-KIT+ mast cells as a result of mutations in the c-kit gene, develop spontaneous gastric ulceration or perforation after day 7 of life at a high frequency, whereas normal litter mates do not. The authors hypothesized that a lack of C-KIT+ mast cells may be implicated in the development of idiopathic gastric perforation (GP) in neonates. METHODS: Postmortem gastric wall specimens were taken from neonates who died of GP (idiopathic, n = 6; secondary, n = 4), and other causes (controls, n = 6). Specimens were taken at random from various sites in the stomach and labeled with antibody to C-KIT. The number of C-KIT+ mast cells from five random fields per specimen were compared under light microscopy (200x). RESULTS: Overall, the number of C-KIT+ mast cells was significantly lower in gastric wall specimens from cases of idiopathic GP when compared with controls or cases of secondary GP irrespective of the sites of sampling (P<.01, analysis of variance test) with the distribution of cells being uniform and unique for each stomach. CONCLUSION: A lack of C-KIT+ mast cells may underlie the development of idiopathic GP in neonates.

Hyaluronic acid: a specific prognostic indicator of hepatic damage in biliary atresia.

BACKGROUND/PURPOSE: Hepatic fibrosis can progress in biliary atresia (BA) and is associated with capillarization of hepatic sinusoids. The significance of serum hyaluronic acid (HA) as a noninvasive indicator of histological sinusoidal endothelial cell (SEC) damage and hepatic fibrosis in BA, is investigated. METHODS: A total of 28 postoperative BA patients (mean age, 11.0+/-3.7 years) and 20 normal controls (mean age, 10.5+/-2.8 years) were studied. BA patients were divided into group I, good liver function (n = 8); group II, moderate liver dysfunction (n = 10); and group III, severe liver dysfunction (n = 10). Serum HA was determined using a one-step sandwich enzyme immunoassay, and liver histological damage was confirmed immunohistochemically using an antibody against factor VIII-related antigen (FVIIIRAg), which is specific for detecting damaged SEC. RESULTS: Serum HA was significantly higher (P < .0001) in group III (84.6+/-36.5 ng/mL) than in group I (15.9+/-6.9 ng/mL) or group 11 (28.7+/-10.7 ng/mL). Although immunoreactive products of FVIIIRAg were abundant in group III, they were not detected in SEC from group II. CONCLUSION: Serum HA may be of value for monitoring postoperative BA patients as a noninvasive indicator of SEC damage and progressive hepatic fibrosis.

Necrotizing enterocolitis and C-KIT.

BACKGROUND/PURPOSE: In the gut, C-KIT is important for immune system homeostasis, and C-KIT+ cells are known to increase during inflammation. Recently the authors identified that spontaneous intestinal mucosal erosion develops in C-KIT-depleted W/Wv mice after day 14 of life at a high frequency, whereas genotypically normal litter mates do not. The authors hypothesized that a lack of C-KIT may be implicated in the development of necrotizing enterocolitis (NEC). METHODS: Bowel specimens were taken during surgery or postmortem from nine cases of NEC (mean gestational age, 32.0 weeks), six age-matched cases of enteritis, and 10 age-matched controls. Specimens were formalin fixed, paraffin embedded, and labeled with antibody to C-KIT. The number of C-KIT+ cells from five random fields per specimen were compared under light microscopy (200x). Results were expressed as the mean +/- SD and compared using the analysis of variance (ANOVA) test. RESULTS: In enteritis, the number of C-KIT+ cells in the lamina propria and submucosa was significantly higher than in controls (P<.01) indicative of their involvement in inflammation. However, in NEC, the number of C-KIT+ cells in the lamina propria and submucosa was significantly lower than in controls (P<.05) despite histological evidence of inflammation. CONCLUSION: A lack of C-KIT+ cells may exert a causal influence on the development of NEC.

Management of vesicoureteral reflux secondary to neurogenic bladder.

The authors encountered 108 cases of vesicoureteral reflex (VUR) in 231 cases of neurogenic bladder complicating spina bifida. Bladder compliance and percent volume (% vol.) were measured pre- and postoperatively and the patients were divided into four groups retrospectively according to the treatment. Ninety-five percent of low-grade VUR (grades I and II) disappeared spontaneously with conservative therapy or after augmentation cystoplasty without antireflux surgery; 92% of high-grade VUR (grade III or more) required ureteral reimplantation with or without bladder augmentation. Reflux did not recur in any case of ureteral reimplantation with bladder augmentation, however, it did recur in 20.4% of the cases of simple ureteral reimplantation without bladder augmentation. Percent volume and bladder compliance in cases of recurrence following simple ureteral reimplantation were significantly lower than in the successful cases. This study suggests that low-grade VUR can resolve spontaneously with conservative therapy or with a suitable maneuver to improve bladder compliance. High-grade reflux in cases of preserved bladder volume (% vol.>75%) and compliance (>7 ml/cmH2O) can be treated successfully with simple ureteral reimplantation, however, in cases of low volume (% vol.<60%) and low compliance (<4 ml/cmH2O), reimplantation with bladder augmentation is recommended.

Administration of antenatal glucocorticoids prevents pulmonary artery structural changes in nitrofen-induced congenital diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: The aim of this study was to investigate whether maternal administration of dexamethasone has any effect on pulmonary vasculature in nitrofen-induced experimental congenital diaphragmatic hernia (CDH) in a rat model. METHODS: A CDH model was induced in pregnant rats after administration of 100 mg nitrofen on day 9.5 of gestation. Antenatal dexamethasone, 0.25 mg/kg was given intraperitoneally on day 18.5 and 19.5 of gestation. The fetuses were divided into three groups: group I (n = 10), normal controls; group II (n = 10), nitrofen-induced CDH; group III (n = 10), nitrofen-induced CDH with maternal antenatal dexamethasone treatment. The fetuses were killed by cesarean section at term. Victorian blue van Gieson staining and immunostaining with antialpha smooth muscle actin (ASMA) were performed on lung tissue. The degree of adventitial thickness and area, and medial thickness and area were measured in pulmonary arteries by image analyzer and analyzed statistically. RESULTS: There was a significant increase in adventitial thickness and area in group II compared with group I and III (P < .01). There was also a significant increase in medial thickness in group II compared with group I and III (P < .01). The degree of adventitial thickness and area and degree of medial thickness and area were similar in controls and maternal dexamethasone-treated CDH group. CONCLUSION: This study demonstrates that antenatal maternal dexamethasone treatment prevents pulmonary artery structural changes in nitrofen-induced CDH in rats.

Abnormal distribution of intestinal pacemaker (C-KIT-positive) cells in an infant with chronic idiopathic intestinal pseudoobstruction.

BACKGROUND: Chronic idiopathic intestinal pseudoobstruction (CIIPO) is a rare syndrome with an obscure pathogenesis. The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor C-KIT that is critical for the development of the interstitial cells of Cajal, cells that are regarded as being the pacemaker cells of the gut. Thus, C-KIT immunopositive (C-KIT-) cells in the muscle layers of the bowel are considered to be intestinal pacemaker cells. METHODS: In this study, the distribution of intestinal pacemaker cells was examined for the first time using C-KIT immunohistochemistry in an infant with CIIPO. RESULTS: C-KIT+ cells were found lying on either side of the border between the two muscle layers (longitudinal and circular) of the bowel and dispersed unevenly throughout both muscle layers. Myenteric plexuses were not demarcated by C-KIT+ cells. In contrast, in controls, C-KIT+ cells were located distinctly between the two muscle layers of the small bowel and dispersed evenly throughout the muscle layers of the colon. Myenteric plexuses were clearly demarcated by C-KIT+ cells. CONCLUSIONS: This case demonstrates for the first time that there is abnormal distribution of intestinal pacemaker cells in CIIPO and provides new evidence that abnormal c-kit gene expression may be responsible for autonomic gut dysmotility. C-KIT immunohistochemistry may be an indispensable tool for diagnosing CIIPO.

Active collagen synthesis in infantile hypertrophic pyloric stenosis.

M-57 antibody, which is capable of distinguishing newly-synthesized type I procollagen from fully-processed, mature collagen, was used to examine the expression of collagen synthesis in hypertrophic pyloric muscle from patients with infantile hypertrophic pyloric stenosis (IHPS). Seven specimens from IHPS patients were removed at the time of operation; age-matched normal pyloric tissue of 5 post-mortem cases was obtained as controls. Immunohistochemistry was performed using antibody of the amino-terminal end of the procollagen type I propeptide (M-57). Newly-synthesized procollagen (M-57) was strongly detected in both the connective tissue septa between circular muscle bundles, and among the circular-muscle fibers in patients with IHPS. No M-57 staining was observed among the circular-muscle fibers in controls. Our findings show that the hypertrophic circular muscle in IHPS is actively synthesizing collagen, and this may be responsible for the characteristic "firm" nature of the pyloric tumor.

Adventitial changes in pulmonary vasculature in congenital diaphragmatic hernia complicated by pulmonary hypertension.

PURPOSE: The purpose of this study was to characterize structural changes in the pulmonary vasculature in congenital diaphragmatic hernia (CDH) complicated by persistent pulmonary hypertension (PPH) with particular emphasis on adventitial thickness. METHODS: Victorian blue Van Gieson (VVG) staining and immunostaining with antialpha smooth muscle actin (ASMA) were performed on lung tissues obtained at autopsy from 23 patients with CDH complicated by PPH and 11 age-matched control tissues of sudden infant death syndrome patients (SIDS). The degree of medial and adventitial thickening was measured in pulmonary arteries with an external diameter (ED) of less than 75 microm, 75 to 100 microm, 100 to 150 microm, 150 to 250 microm, 250 to 500 microm, and greater than 500 microm by IPS-4.01 image analyzer and compared statistically. The degree of medial thickening and adventitial thickening was also measured in pulmonary veins with an ED of less than 100 microm, 100 to 200 microm, and greater than 200 microm. To determine whether the characteristic structural changes were size related, each was related to ED. The area of adventitia and media of the pulmonary arteries and veins was measured using image analyzer. RESULTS: There was a significant increase in medial and adventitial thickness in arteries of all sizes in CDH patients compared with controls (P < .01). The degree of adventitial area was significantly increased for arteries of all sizes (P < .01) and the degree of medial area was significantly increased only for arteries less than 100 microm size (P < .05) in CDH patients compared with controls. Calculation of the areas of the various components in the wall of each artery showed that for small arteries (<100 microm ED), the area of the lumen was smaller, and the areas of the media and adventitia were larger in CDH patients compared with controls (P < .01). There was a significant increase in adventitial thickness and area in veins of all sizes in CDH patients compared with controls (P < .01). The adventitial thickness of pulmonary veins were ED of less than 100 microm: CDH, 13.5 microm +/- 3.5; control, 9.21 microm +/- 2.0; ED 100 to 200 microm: CDH, 21.3 microm +/- 7.5; control, 13.0 microm +/- 4.8; ED greater than 200 microm: CDH, 34.4 microm +/- 12.5; control, 22.3 microm +/- 4.2. CONCLUSIONS: The present study provides the first quantitative demonstration of structural alterations in pulmonary veins in addition to pulmonary arteries in CDH complicated by PPH. The structural remodeling of the pulmonary vein is perhaps as a result of an increase in transvascular pressure in PPH.

Comparison of the pulmonary vasculature in newborns and stillborns with congenital diaphragmatic hernia.

The purpose of this study was to compare structural changes in the pulmonary vasculature in newborns with congenital diaphragmatic hernia (CDH) complicated by persistent pulmonary hypertension (PPH) and stillborns with CDH. Victorian blue van Gieson (VVG) staining and immunostaining with anti-alpha smooth-muscle actin (ASMA) was performed on lung tissue obtained at autopsy from 23 newborns with CDH complicated by PPH, 7 stillborns with CDH, and 11 age-matched controls with sudden infant death syndrome (SIDS). The degrees of adventitial and medial thickness and area were measured in pulmonary arteries with an external diameter (ED) of <75 micrometers, 75-100 micrometers, 100-150 micrometers, 150-250 micrometers, 250-500 micrometers, and >500 micrometers by image analyzer and compared statistically. The degrees of adventitial and medial thickness and area were measured in pulmonary veins with an ED of <100 micrometers, 100-200 micrometers, and >200 micrometers by image analyzer and compared statistically. In order to determine whether the characteristic structural changes were size-related, each was related to ED. There was a significant increase in adventitial thickness and area in arteries of all sizes in both newborns and stillborns with CDH compared to SIDS patients (P < 0. 05). The degree of medial thickness in newborns and stillborns with CDH was significantly increased compared to SIDS patients (P < 0.01). The degree of medial area was significantly increased for arteries with ED less than 100 micrometers (P < 0.05) in newborns and stillborns with CDH compared with SIDS patients. There was a significant increase in adventitial thickness and area in veins of all sizes in newborns with CDH compared to stillborns with CDH and SIDS (P < 0. 05). The degree of adventitial thickness and area of pulmonary veins were similar in stillborns with CDH and SIDS. There were no significant differences in medial thickness of veins between the three groups. The presence of abnormally thick-walled pulmonary arteries in stillborns with CDH suggests that the intrapulmonary arteries in CDH may become excessively muscularized during fetal life, becoming unable to adapt normally at birth. The absence of structural changes in pulmonary veins in stillborns with CDH suggests that the pulmonary venous changes observed in newborns with CDH complicated by PPH occur after birth as a result of increases in transvascular pressure or a response to release of peptide growth factors.

Active collagen synthesis by pulmonary arteries in pulmonary hypertension complicated by congenital diaphragmatic hernia.

The high mortality rate for patients with congenital diaphragmatic hernia (CDH) has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PPH). The factors that cause vasoconstriction and vascular remodeling in PPH are not fully understood. Immunohistochemistry was performed on lung tissue obtained from postmortem CDH patients with pulmonary hypoplasia and PPH (n = 21) using the following antibodies: alpha smooth muscle-actin (ASMA), transforming growth factor-beta (TGF-beta), isoform specific (TGF-beta 1, -beta 2, -beta 3), and M-57. Normal lung tissues from age-matched sudden infant death syndrome patients (SIDS, n = 8) were obtained as controls. TGF-beta 3 immunoreactivity was observed in the adventitia but not in the media of pulmonary muscular arteries in patients with CDH. TGF-beta 1, -beta 2 immunoreactivity was either absent or faintly expressed in pulmonary arteries in CDH patients. No TGF-beta staining was observed in the pulmonary vasculature of SIDS patients. Newly synthesized procollagen (M-57) was easily detected in the media and adventitia in a large number of pulmonary arteries in all patients with CDH and in the neointima in two patients with long standing PPH. No M-57 staining was seen in the media of pulmonary arteries of the lungs of SIDS patients. These observations suggest a potential role of TGF-beta 3 but not TGF beta 1 or TGF beta 2 in pulmonary vascular remodeling and that smooth muscle cells in muscular pulmonary arteries are actively synthesizing collagen in patients with CDH complicated by PPH.

Pulmonary artery structural changes in pulmonary hypertension complicating congenital diaphragmatic hernia.

Primary pulmonary hypertension is characterized by the presence of smooth muscle cells in nonmuscular compartments or segments of the vessel and the abnormal deposition of collagen in both the small muscular arteries and arterioles and the large elastic arteries. Victorian blue van Gieson staining and Immunostaining with anti-alpha smooth muscle actin (ASMA) were performed on lung tissues obtained during autopsy from 21 patients who had congenital diaphragmatic hernia (CDH) complicated by persistent pulmonary hypertension (PPH) and 10 control patients who died of sudden infant death syndrome (SIDS). The degree of medial thickening and adventitial thickening was measured in pulmonary arteries by image analysis and compared statistically. There was a significant increase in adventitial as well as medial thickness in arteries of all sizes in CDH patients compared with control patients (P < .001). The most striking increase occurred in arteries with an external diameter (ED) of less than 75 microns. Calculation of the areas of the various components in the wall of each vessel showed that for smaller vessels (< 75 microns ED), the area of the lumen was smaller and the area of the media and adventitia was larger in CDH patients compared with control patients (P < < .001). In vessels greater than 75 microns ED, the areas of media in CDH was the same as in controls and the area of adventitia in CDH was significantly larger than controls (P < .001). The present study provides evidence that an increase in adventitial thickness and adventitial area occurs in pulmonary arteries in CDH patients complicated by PPH. The structural changes in the adventitia of the pulmonary arteries may be an important factor in the development of PPH in patients with CDH.

Increased intracellular levels of calcitonin gene-related peptide-like immunoreactivity in pulmonary endocrine cells in an experimental model of congenital diaphragmatic hernia.

A congenital diaphragmatic hernia (CDH) model was induced in pregnant rats following administration of 100 mg nitrofen. The fetuses were stored and fixed in Bouin's solution for 24 h after caesarean section at term. After fixation, the lungs were dissected out. Immunostaining of the CDH lungs and controls with rabbit anti-rat calcitonin gene-related peptide (CGRP) antibody at "optimal" and "supraoptimal" dilution levels was obtained by examining the intensity of staining with a series of dilutions of the antisera from 1: 1,000 to 1: 20,000. Supraoptimal dilution detects variations in antigen concentration that may be masked if the routine optimal dilution is used. Immunostaining of the lung by antisera to platelet-derived growth factor (PDGF) and alpha-smooth-muscle actin (ASMA) was performed to examine vascular remodelling. The number of CGRP-immunoreactive cells was significantly (P <0.001) greater in the lungs of CDH rats (n = 26) (0.74 +-0.19 NEB [neuroepithelial bodies]/mm(2); mean +- SEM) compared with controls (n = 21) (0.30+-0.16 NEB/mm(2)) seen at supraoptimal dilution (1:20,000). Since CGRP is a vasodilator, this could have important implications in the development of pulmonary hypertension. The pattern of ASMA and PDGF immunostaining was similar in CDH lungs and controls, and therefore, vascular remodelling is not a feature of CDH lungs in fetuses delivered by caesarean section and not exposed to hypoxia.

The effect of anti-ICAM-1 monoclonal antibody treatment on the transplantation of the small bowel in rats.

At present, organ transplantation has been conducted in various areas. But the most crucial problem is to find an efficient way to determine whether allograft rejection could be interfered by a novel approach, namely interference of adhesive interaction between cytotoxic cells and target organs with a monoclonal antibody (mAb) to intercellular adhesion molecule 1 (ICAM-1). We have studied the effect of anti-rat-ICAM-1 mAb on small bowel transplantation. Inbred Fischer and Lewis rats weighing 250 g were utilized. In allotransplantation, Fischer donor small bowel was transplanted to Lewis recipient. Group 1 consisted of untreated controls (n = 15). Group 2 was treated with the anti-ICAM-1 mAb (1 mg/kg/d intraperitoneally) for the first 5 days posttransplantation (n = 15). A dramatic inhibitory effect on allograft rejection was observed in the early stage of posttransplantation. On histological studies of grafted small intestine, group 2 showed normal morphological appearance while group 1 showed graft rejection until postoperative 5 days. However, changing around crypt cells and endothelial cells of microvasculature have appeared at 15 days of posttransplantation. These findings suggest that anti-ICAM-1 antibody is quite useful for delaying the occurrence of graft rejection in the early stage. Electron microscopic examination demonstrated the same results as conventional HE staining. Endothelial cells of the vessels and crypt cells may have an important role as target cells on graft rejection. Therefore, anti-ICAM-1 mAb may have potential as an alternative to conventional treatment for prevention of allograft rejection.

Prolongation of rat cardiac allograft survival by a monoclonal antibody: anti-rat intercellular adhesion molecule-1.

A mouse monoclonal antibody 1A29, which binds to the rat intercellular adhesion molecule-1 (ICAM-1), was studied for its effect on cardiac allograft survival. Expression of ICAM-1 was detectable only on vascular endothelium in normal heart, but was induced on myocardium associated with interstitial mononuclear cell infiltration during acute rejection. Treatment with monoclonal antibody 1A29 for 10 days after transplantation in 15 rats significantly prolonged allograft survival (mean(s.d.) 18(2) days; P < 0.001), as compared with 15 isotype-matched control monoclonal antibody (mean(s.d.) 10(1) days) recipients. Five-day treatment with monoclonal antibody 1A29, when started at 5 days after transplantation (the time for which acute rejection is ongoing), also significantly extended the survival (mean(s.d.) 12(1) days; P < 0.01) in seven rats. On histological examination, treatment with monoclonal antibody 1A29 reduced the degree of T-cell infiltration of both CD4+ and CD8+ subsets, and greatly reduced myocardial necrosis, vascular injury and intravenous thrombi. These results indicate that an anti-ICAM-1 monoclonal antibody can be used to prevent or treat acute rejection in the rat cardiac allograft model and suggest that human ICAM-1 is an important target for immunosuppression in clinical organ transplantation.

New immunosuppression with monoclonal antibody to intracellular adhesion molecule 1 (ICAM-1) in rat organ transplantation.

Inbred, male Lewis rats underwent heterotopic heart allografting from F344 donor rats, or streptozocin (STZ)-induced diabetic Lewis rats underwent pancreas allografting with bladder drainage from F344 or ACI donor rats. A monoclonal antibody (MoAb) to intracellular adhesion molecule 1 (ICAM-1) was given i. p. (1.0 mg/kg) for 10 days, and its immunosuppressive potency was evaluated. The mean survival time (MST) of the heart allografts was significantly prolonged in the MoAb-treated group. Both exocrine and endocrine MST of pancreas allografts were also prolonged by MoAb administration across the minor and major histocompatibility barriers. However, complete graft tolerance was not induced. Our study demonstrated that the MoAb to ICAM-1 alone can delay the allograft rejection in rat organ transplantation.