Charles J. Aprahamian

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.

Human mesenteric adipose tissue plays unique role versus subcutaneous and omental fat in obesity related diabetes.

Background/Aims: Obesity is a common and rapidly growing health problem today. Obesity is characterized by the increase of body fat and an excess of total body fat and, in particular, visceral fat accumulation, is considered to be a risk factor for type 2 diabetes mellitus. To determine whether the malfunction of the mesenteric adipose tissue plays an important role in the diabetic related metabolic syndrome, in this study, lipolysis and gene expression in the subcutaneous, omental and mesenteric adipose tissue of the diabetic subjects were evaluated. Methods: Lipolysis and real time PCR were utilized to determine adipocyte function. Results: Basal adipose tissue glycerol release is higher in diabetics than that of the non diabetics in all three fat depots. Isoproterenol (ISO) significantly increases glycerol release in subcutaneous, omental and mesenteric adipose tissues of non diabetic subjects but it stimulated glycerol release was significantly impaired in all three fat depots of the diabetic subjects. Gene expression studies indicate that leptin, Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), Fatty acid translocase (FAT/CD36) and 11β-hydroysteroid dehydrogenase (HSD) gene expression were significantly up regulated in the mesenteric adipose tissue of the diabetic patients. Conclusion: Human mesenteric adipose tissue in obese diabetic subjects has high basal glycerol release and impaired isoproterenol stimulated glycerol release. The obesity-related gene expressions in the mesenteric adipose tissue are up regulated, suggesting that the alterations of these genes in mesentery adipose depot may play a critical role in insulin resistance of type 2 diabetes and metabolic syndrome.

Toll-like receptor 2 is protective of ischemia–reperfusion-mediated small-bowel injury in a murine model

Objective: In a murine model of intestinal injury, we hypothesized that Toll-like receptor 2 (TLR2), a recognition molecule for commensal bacteria, plays an important role in the development of mucosal immunity and is protective against ischemia/reperfusion injury via the modulation of both innate and acquired immunity. Design: Interventional laboratory study. Setting: Academic medical research center. Subjects: Four-week-old C57BL/6 wild-type (n = 12) and C57BL/6 TLR2-deficient mice (TLR2−/−) (n = 12). Interventions: Twenty-four mice underwent laparotomy only or laparotomy plus superior mesenteric artery occlusion (n = 6/group) for 60 mins, followed by 90 mins of recovery. Measurements and Main Results: Mid-jejunal sections were taken for histopathology and messenger RNA expression (reverse transcriptase-polymerase chain reaction, normalized to 18s and laparotomy-only controls). Intestinal injury was scored from 0 (no injury) to 4 (transmural necrosis). Statistical analyses were performed using Mann-Whitney U test and Student’s t-test (p < .05 significant). TLR2−/− mice had elevated intestinal injury scores (mean ± sem) after ischemia/reperfusion vs. wild-type (2.17 ± 0.40 vs. 0.67 ± 0.33, p < .05). Intestinal cytokine messenger RNA (mean fold change ± sem) of interferon-γ (0.29 ± 0.12 vs. 3313 ± 1710), interleukin-4 (0.25 ± 0.13 vs. 2.70 ± 1.08), and interleukin-6 (250.63 ± 69.60 vs. 320,300 ± 215,964) in TLR2−/− was significantly decreased (p < .05) after ischemia/reperfusion vs. wild-type. Tumor necrosis factor-α messenger RNA levels were unchanged. Conclusions: TLR2−/− mice have a dysregulated mucosal innate immune response and fail to mount a protective response after ischemia-reperfusion compared with wild-type mice. This murine model of intestinal injury may correlate with the early postnatal course of premature infants who may have decreased TLR2 expression and/or decreased luminal commensal bacteria secondary to antibiotic therapy, thus decreasing TLR2-mediated signaling.

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.

Laparoscopic cholecystectomy for biliary dyskinesia in children provides durable symptom relief.

PURPOSE The purpose of this study was to determine the effectiveness of laparoscopic cholecystectomy in children with biliary dyskinesia. METHODS Reports of children with an abnormal cholecystokinin (CCK)-stimulated HIDA scan between January 2001 and July 2006 who underwent laparoscopic cholecystectomy were reviewed. Postoperatively, a 23-item Likert scale, symptom questionnaire was administered to parents. RESULTS Sixty-four children with chronic abdominal pain and no gallstones on ultrasound had an abnormal CCK-HIDA scan. Twenty-three children (median age, 14 years; 16 girls), with mean (SD) ejection fraction of 17% (8), underwent laparoscopic cholecystectomy and were further analyzed. Preoperatively, these children had right upper quadrant/epigastric pain (78%), nausea (52%), vomiting (43%), and generalized abdominal pain (22%) lasting for a median of 3 months (range, 1 month to 2.5 years). Median postoperative follow-up was 2.7 years. Sixteen (70%) parents completed the questionnaire. Of those who responded, 63% indicated that their children had no abdominal pain, 87% had no vomiting, and 69% had no nausea in the month preceding the questionnaire. Overall, 67% of parents indicated that their childrens symptoms were completely relieved after cholecystectomy, whereas 7% indicated that the symptoms were not relieved. CONCLUSION Laparoscopic cholecystectomy is effective in providing both short-term and long-term improvement of symptoms in children with biliary dyskinesia.

Laparoscopic cholecystectomy for biliary dyskinesia in children: Frequency increasing

PURPOSE The treatment of children with biliary dyskinesia (BD) is controversial. As we recently observed an increasing frequency of referrals for BD in our institution the aim of the study was to re-evaluate the long-term outcome in children with BD. METHODS Children with laparoscopic cholecystectomy (LC) for suspected BD between 8/2006 and 5/2011 were included. A pathologic ejection fraction (EF) was defined as <35%. The long-term effect of cholecystectomy was assessed via a Likert scale symptom questionnaire. RESULTS 82 children (median age 13.5 years, mean BMI 25.8) were included. CCK-HIDA scan was pathologic in 74 children (90.2%). Mean EF was 16.4%. Histology revealed chronic cholecystitis in 48 (58.5%) children and was normal in 30 children (36.5%). The frequency of LC for suspected BD increased by a factor of 4.3 in the last 10 years. Long term follow-up showed that only 23/52 children (44.2%) were symptom-free after LC. Patients with chronic inflammation were more likely to have persistent symptoms (p=0.017). An EF<15% was associated with a resolution of symptoms (p=0.031). CONCLUSION The frequency of LC for suspected BD in our institution has increased significantly during recent years. The long-term efficacy in our cohort was only 44.2%. We believe that laparoscopic cholecystectomy is likely helpful in patients with an EF<15%. However, in children with an EF of 15%-35%, based upon our data, we would highly recommend an appropriately thorough pre-op testing to exclude other gastrointestinal disorders prior to consideration of operative management.

Single-Incision Pediatric Endosurgery for Ovarian Pathology

BACKGROUND AND OBJECTIVES Despite being pioneered by gynecologists, single-incision endosurgery has not been widely reported for the treatment of ovarian and adnexal pathology in neonates, children, and adolescents. We describe our initial experience using single-incision pediatric endosurgery (SIPES) for these indications and discuss advantages and drawbacks. SUBJECTS AND METHODS All children who underwent SIPES with a preoperative diagnosis of ovarian or adnexal pathology were included in the study. Data on age, operative time, complications, length of hospital stay, and outcomes were collected. RESULTS From January 2010 until January 2012, 19 girls (mean age, 11.4 years; range, 6 days-17 years; weight range, 4.0-90 kg) underwent SIPES procedures for ovarian or adnexal diagnoses, including hemorrhagic/follicular/paratubal cysts (n=8), torsion (n=7), tumor (n=3), and parauterine cyst (n=1). The operations included cyst unroofing (n=4), detorsion and oophoropexy (n=7), (salpingo)oophorectomy (n=5), marsupialization of cyst (n=2), and cyst aspiration (n=1). Median operative time was 42 ± 29 minutes; there were no conversions to conventional laparoscopy or open surgery. Fifteen patients (79%) were discharged within 24 hours after the procedure. There were no peri- or postoperative complications. Histopathology showed hemorrhagic/follicular/paratubal cyst (n=7), necrotic/calcified ovarian tissue after torsion (n=6), cystadenofibroma (n=1), granulosa cell tumor (n=1), and mature teratoma (Grade 0) (n=1). CONCLUSIONS SIPES is an excellent alternative to conventional laparoscopy for the treatment of adnexal pathology. Using a single umbilical incision that can be enlarged instead of three smaller trocar sites facilitates the resection and extraction of ovarian masses without compromising cosmesis.

Splenectomy reduces packed red cell transfusion requirement in children with sickle cell disease

PURPOSE The purpose of the study was to measure the effect of splenectomy on packed-cell transfusion requirement in children with sickle cell disease. METHODS Thirty-seven sickle cell children who underwent splenectomies between January 2000 and May 2006 at a childrens hospital were reviewed. Data were collected 6 months preoperatively to 12 months postsplenectomy. Paired t test, analysis of variance, and multivariable regression analyses were performed. RESULTS Of 37 children with median age 11 years (range, 2-18 years), 34 (21 males) had data that allowed analyses. Twenty-six had Hgb-SS, 5 had Hgb-SC, and 3 had Hgb S-Thal. Laparoscopic splenectomy was attempted in 36 and completed successfully in 34 (94% success). The number of units transfused decreased by 38% for 0 to 6 months and by 45% for 6 to 12 months postsplenectomy. Postoperatively, hematocrit levels increased and reticulocytes concurrently decreased with a reduction in transfusion clinic visits. The decrease in transfusion was not influenced by spleen weight, age, or hemoglobin type. Two children had acute chest syndrome (6%), and 1 had severe pneumonia (3%). CONCLUSION Laparoscopic splenectomy can be successfully completed in sickle cell children. Splenectomy significantly reduces the packed red cell transfusion requirement and frequency of clinic visits, in sickle cell children for at least 12 months postoperatively.

Feasibility of Single-Incision Pediatric Endosurgery for Treatment of Appendicitis in 415 Children

INTRODUCTION Single-incision pediatric endosurgery (SIPES) has gained popularity for ablative procedures such as appendectomy in many pediatric surgical centers. This study evaluates the outcome of SIPES for treatment of appendicitis in our institution. PATIENTS AND METHODS After Institutional Review Board approval was obtained, data were prospectively collected on all patients undergoing SIPES appendectomy in our hospital from March 2009 through October 2011. The surgical techniques, operative times, complications, conversion rates, and outcomes were recorded. RESULTS SIPES appendectomy was attempted in 415 children (mean age, 10.9 years; age range, 1.4-17.9 years; 266 males, 149 females; median weight, 43 kg; weight range, 9.8-146 kg). Intraoperatively, acute appendicitis was found in 298 cases and perforated appendicitis in 79 cases. Thirty-eight patients underwent interval appendectomy. Appendectomy was carried out solely as SIPES in 397 cases (96%). Median operative time was 40±16 minutes (37±16 minutes for fellows [n=284] and 46±15 minutes for residents [n=131]). There were three intraoperative complications, which could be handled during the procedure. Pathologic reports revealed inflammatory changes of the appendix (n=386), other pathology (n=11), and no pathologic change (n=18). Overall, 24 patients (5.8%) were readmitted for intra-abdominal abscess (n=14), umbilical wound infection (n=3), and other reasons (n=7). Twelve patients (2.9%) underwent reoperation: drainage of intra-abdominal abscess (n=8) (3 by the surgeon, 5 by the interventional radiologist), wound drainage (n=3), and right hemicolectomy for carcinoid (n=1). In perforated appendicitis the postoperative intra-abdominal abscess rate was 10 of 79 cases (12.7%), which is similar to the previous report with conventional laparoscopic appendectomy from our institution (13.6%). The wound infection rate (5 of 79 cases [6.3%]) was also similar to the previously report (6.8%) with conventional laparoscopic appendectomy for perforated appendicitis. CONCLUSIONS Appendectomy can be accomplished successfully and safely using single-incision endosurgery in children with acceptable operative times without leaving any appreciable scar. Additional trocars are infrequently necessary. So far, the intraoperative and postoperative complication rates are comparable to those of triangulated laparoscopic appendectomy.