Lack of Mucosal Cholinergic Innervation Is Associated With Increased Risk of Enterocolitis in Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown. METHODS: We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation. RESULTS: The presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis. CONCLUSIONS: The mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options.

Cholinergic Signaling Attenuates Pro-Inflammatory Interleukin-8 Response in Colonic Epithelial Cells.

Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-ß1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion via the alpha 7 nicotinic acetylcholine receptor (a7nAChR). In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment.

Sacral nerve stimulation is a valuable diagnostic tool in the management of anorectal and pelvic malformations.

BACKGROUND: We hypothesize that the current practice of sphincter assessment in anorectal malformations (ARMs) by direct muscle stimulation can be improved by sacral nerve stimulation (SNS). Focusing on the specific adjustments for infants, we highlight the anatomical and neurophysiologic basis of SNS and its further diagnostic and therapeutic implications. PATIENTS AND METHODS: We examined 20 patients: 12 patients with ARM, 3 with Hirschsprung disease, 3 with sacrococcygeal teratoma, and 2 with cloacal exstrophies. Under general anesthesia and ultrasound guidance, percutaneous needle electrodes were placed within the sacral neural foramina. Electrical stimulation was applied to assess the presence, pattern, and extent of the neuromuscular response of the external anal sphincter. RESULTS: We successfully modified the traditional method used in adults replacing radiographic controls by ultrasound guidance. In 20 SNS procedures with no complications, we found a muscle response on stimulation of the third and/or fourth sacral nerves in 18 of 20 patients (anal sphincter contraction and/or ipsilateral plantar flexion). CONCLUSION: We demonstrated the feasibility of SNS for intraoperative sphincter mapping and detection of primary innervation abnormalities in ARM as well as for the assessment of secondary deficits in postoperative follow-up. Sacral nerve stimulation broadens the spectrum of intraoperative information concerning the sacral innervation pattern by a direct assessment of the sacral nerves. Furthermore, electrophysiologic data may allow prospective criteria for sphincter function to be established. These may improve the accuracy of the present classification-based prognosis as well as the understanding of sphincter physiology in general.

Self- and social perception of physical appearance in chest wall deformity.

This study analyzes self- and social perception of physical appearance in patients with chest wall deformity (CWD), including both pectus carinatum and pectus excavatum. Self-perception of appearance in 76 patients with CWD and social perception of patients' appearance by 20 adult and 20 adolescent raters was assessed using the Appearance Rating Scale (Stangier et al., 2000) and evaluated for agreement and multivariate correlates. Results indicate no agreement between self- and social rating. Based on mean scores patients rated their appearance significantly more negatively than adult raters but only slightly different than adolescent raters. Adolescent raters' judgment of the patients' appearance was related to CWD characteristics, while self-rating rather seems to be related to psychosocial factors. Because adolescents are a relevant peer group for adolescent patients with CWD their evaluation of the appearance might influence patients' self-image and might affect their psychological functioning. Effective interventions focusing on social interactions are needed.

Pectus excavatum and pectus carinatum patients suffer from lower quality of life and impaired body image: a control group comparison of psychological characteristics prior to surgical correction.

OBJECTIVE: The aim of this study was to evaluate the effects of anterior chest-wall deformities on disease-specific and health-related quality of life, body image, and psychiatric comorbidity prior to surgical correction. METHODS: A total of 90 patients (71 with pectus excavatum, 19 with pectus carinatum) presenting themselves for pectus repair and 82 control subjects were recruited for this study. The objective severity of the deformity was determined through the funnel-chest index by Hümmer and the Haller index. Disease-specific quality of life was measured with the Nuss Questionnaire modified for Adults (NQ-mA) and health-related quality of life was determined by the Short-Form-36 Health Survey (SF-36). Body image was assessed via the Body Image Questionnaire (FKB-20), the Dysmorphic Concern Questionnaire (DCQ), and a self-evaluation of the subjective impairment of the appearance. The Diagnostic Interview for Mental Disorders - Short Version (Mini-DIPS), the General Depression Scale (Allgemeine Depressionsskala, ADS), and a self-rating of self-esteem were used to evaluate general psychological impairment. RESULTS: Compared with control group results, physical quality of life was reduced in patients with pectus excavatum, while mental quality of life was decreased in patients with pectus carinatum (p<0.05). Body image was highly disturbed in all the patients and differed significantly from the control group (p<0.01). Patients with pectus carinatum appeared to be less satisfied with their appearance than those with pectus excavatum (p=0.07). Body image distress was multivariately associated with both reduced mental quality of life and low self-esteem (p<0.001). Body image did not influence physical quality of life. Patients displayed no elevated rates of mental disorders according to Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria. CONCLUSION: Since self-perception is a major contributor to therapeutic decision making, a systematic evaluation of body image should be included in the assessment of patients with chest deformities. Body image concerns may be even more relevant to the decision-making process than physical restrictions. Exaggerated dysmorphic concerns should be prospectively investigated in their ability to influence the extent of satisfaction with the surgical outcome.

Aesthetic and functional correction of female, asymmetric funnel chest - a combined approach.

There is still sparse information published about the surgical correction of female funnel chest deformity. Women with severe asymmetric funnel chest deformity often present with asymmetric, hypoplastic breasts. These patients frequently complain of physiological limitations in connection with gross aesthetic impairment. To correct these two features a combined approach is presented in this study. 10 women were operated with correction of the thoracic wall deformity by open retrosternal mobilisation and metal plate fixation. Either during the same procedure or in a secondary operation, submuscular breast augmentation was performed to correct breast hypoplasia and asymmetry. All patients tolerated the operation very well without any complications. Aesthetic outcome was rated good to excellent in secondary breast augmentation, whereas simultaneous implant positioning was prone to cause symmastia. In conclusion we recommend correction of female asymmetric funnel chest by primary sternal reduction with secondary breast augmentation during metal plate removal 1 year after.

Gastroschisis and omphalocele: treatments and long-term outcomes.

Between February 1994 and April 2004, we treated 40 children with gastroschisis and 26 children with omphalocele. We recorded the course of pregnancy, pre- and post-natal complications, delivery, operation, post-operative therapy, and long-term outcomes. Additionally, we conducted follow-up examinations of 37 of these 66 children (56%). We analysed their abdominal musculature, development, cosmetic result and quality of life. The median duration of follow-up was 6.3 years (range 1-10). In 35/40 children (88%) with gastroschisis and in 18/26 children (69%) with omphalocele, there had been prenatal diagnosis. The average maternal age of 23.9 years in the gastroschisis group was lower than in the omphalocele group (29.9 years). Delivery was by caesarean section in 93% of the gastroschisis group and 65% of the omphalocele group. Outcomes following vaginal delivery were no worse than those after caesarean section. Further, congenital abnormalities were shown in 28% of gastroschisis cases, and were limited to the gastrointestinal tract. Of the omphalocele cases 81% showed further abnormalities. Direct closure of the abdominal wall defect was possible in 31/40 (78%) of the gastroschisis cases and 15/26 (58%) of the omphalocele cases. Mortality in gastroschisis was nil; two children with omphalocele died (8%). Outcomes were better after primary closure than in stepwise reconstruction. Follow-up showed good results in all categories. Developmental delays were rapidly made up after treatment, and 75% of the children had no gastrointestinal problems, or suffered from these rarely. Almost all the children were of normal weight and height, and physical and intellectual development were delayed in only one third of the children. The surgical scar was rated as good or very good in about 80% of the cases. Except for those with severe defects, the children had good ratings for quality of life. Improvements in short-term results of gastroschisis and omphalocele treatment can be attributed to recent developments in prenatal diagnosis and the advancements of centralised perinatal care. Our long-term results clearly demonstrate that initial gastrointestinal problems and developmental delays were made up during the first two years of life. Prenatal counselling can now be more optimistic.

Hyperthermic intraperitoneal chemoperfusion is an option for treatment of peritoneal carcinomatosis in children.

BACKGROUND: Gastrointestinal carcinomas in childhood are rare and frequently present at an advanced stage. Besides lymphatic and distant organ metastasis, peritoneal carcinomatosis may be detected and has a poor prognosis. In addition to surgery and intravenous chemotherapy, hyperthermic intraperitoneal chemoperfusion (HIPEC) may be an option for selected patients. Our aim was to demonstrate the feasibility of the method and to discuss possible indications. METHODS: After treating a series of adult patients, HIPEC for peritoneal carcinomatosis from a signet cell carcinoma of the colon was performed intraoperatively in a 12-year-old boy. We gave mitomycin C at a dose of 30 mg/m2 over 90 minutes at maximum temperature of 41.2 degrees C. We performed intraoperative drug level monitoring and daily postoperative liver and kidney function tests and differential blood counts. RESULTS: Hyperthermic intraperitoneal chemoperfusion was performed according to protocol without complications. Perfusate and venous drug levels were similar to those in an adult case. The patient had an uneventful recovery, and serum chemistry and blood count returned to normal after a week. The boy lived for 36 months after initial presentation. Sixteen months after HIPEC, still with excellent quality of life, an elevated carcinoembryonic antigen (CEA) indicated recurrence. Thirty months after HIPEC, he died of progressive recurrent disease. CONCLUSIONS: Hyperthermic intraperitoneal chemoperfusion as performed in adults may be beneficial to children with peritoneal carcinomatosis and merits further study.

Forces to be overcome in correction of pectus excavatum.

OBJECTIVE: The Erlangen technique of funnel chest correction is carried out through an anterior incision, and an essential step is retrosternal mobilization. After elevation of the funnel, the chest wall is stabilized with a lightweight transsternal metal implant. Forces necessary to elevate the chest wall were measured at defined intervals during the operation to prospectively assess the effect of peristernal and retrosternal dissection. METHODS: Over a 3-year period, systematic tension measurements were carried out on 100 consecutive patients with symmetric funnel chest to assess the effect of individual steps in mobilization of the sternum. RESULTS: Whereas in adolescents the extraction force is about 175 N, in adults it is not possible to elevate the sternum to the desired level without surgical mobilization because the force required is, on average, more than 200 N. Only about 50% of this tension can be eliminated by costal chondrotomy. To reduce the tension further and achieve a stable result without the need for heavy-duty internal fixation, we carry out a retrosternal dissection, including removal of the slips of the diaphragm and the insertions of the transversus thoracis muscle. The mean tension at the end of the procedure is 25 N. CONCLUSIONS: Our measurements show that retrosternal dissection is the decisive step in the Erlangen technique, which might explain the low relapse rate and allow for a less extensive anterolateral mobilization.

Femoral vein occlusion and spontaneous collateral ectasia presenting as recurrent hernia of the groin: a case report.

Femoral vein occlusion is not a common complication even after repeated hernia repair. We describe a case of a 14-year-old boy with a visible and soft, yet irreducible, mass below the inguinal ligament after 3 previous inguinal hernia repairs and heart catheterization in infancy. Further examination showed dilated venous collaterals, bypassing an occluded common femoral vein via the testicular sheaths and across the pelvic floor. We discuss etiology, diagnostic pitfalls, therapeutic options, and possible future complications, with a literature review.

Microarray versus conventional prediction of lymph node metastasis in colorectal carcinoma.

BACKGROUND: The authors investigated whether microarray-based gene expression analysis of primary tumor biopsy material could be used to predict lymph node status in patients with colorectal carcinoma (CRC). Lymphatic metastasis strongly determines treatment algorithms in CRC. Currently, postoperative histology results are needed to determine lymph node status. Reliable preoperative information would be useful to advance treatment strategies. METHODS: In specimens from 66 patients with CRC from the Erlangen Registry of Colorectal Cancer, 41 shock-frozen samples of International Union Against Cancer (UICC) Stage I-II CRC and 25 samples of UICC Stage III CRC were microdissected manually, RNA was isolated, and gene chips (HG-U133A; Affymetrix) were hybridized. Prediction rates for lymphatic metastasis were calculated using conventional clinicopathologic parameters, gene expression data, and a combination of both. Prediction error, specificity, and sensitivity were analyzed using six different statistical classifiers. RESULTS: Analysis of conventional parameters produced a positive prediction rate that ranged between 53% and 61%, sensitivity of 42%, and specificity of 72%. Microarray prediction rates were between 62% and 67% for lymphatic metastasis. Specificity was between 76% and 83%, and sensitivity was between 38% and 48%, depending on the statistical procedure. The conventional estimates were improved by 9-12% when array data were added. CONCLUSIONS: Current data show that the prediction of lymphatic metastasis can be improved by gene expression profiling of the primary tumor biopsy, alone or in combination with conventional parameters. Gene expression profiling may become valuable increasingly in planning treatment for patients with CRC.

Characterization of gene expression profiles of T cells during anti-tumor response.

BACKGROUND AND AIMS: T cells of tumor-bearing mice or cancer patients exhibit an immune dysfunction, enabling the tumor to escape immune surveillance. METHODS: The experiments are based on EL4 thymoma cells that were transfected with costimulatory ligands B7-1, B7-2, or both at the same time. We used oligonucleotide-based DNA chip microarrays to characterize the genomic expression profile of peripheral T cells according to their anti-tumor immune response in vivo. These murine T cells were also characterized by ELISA, FACS analysis, and co-stimulatory assays. RESULTS: Using commonly established methods, such as FACS analysis or the analysis of the cytokine profile by ELISA, it was not possible to determine functional differences in the in vivo activity of T lymphocytes against tumor cells. EL4 tumor cells induced multiple anti-tumor immune responses in vivo depending on their B7 expression. We successfully used microarray analysis to identify genes that were differentially expressed in the dysfunctional T cells, which were unable to reject tumors in vivo. Although Th1 and Th2 cytokine expression was not affected, we observed differential expression of genes involved in the regulation of an innate immune response. CONCLUSION: Our results provide evidence that the anti-tumor response can be identified by the "gene profile" of T cells. Genomic scale analysis offers the opportunity to identify subtle changes in gene expression in T cells reflecting a distinct biological behavior in vivo.

Age and manifestation related symptoms in familial adenomatous polyposis.

BACKGROUND: To identify early symptoms of familial adenomatous polyposis with a view to improve early diagnosis and treatment. Diagnosis on the basis of genetic testing is usually limited to where there is a known family history, so FAP is more usually diagnosed on clinical grounds. Except for those identified via FAP registers, the majority of patients are symptomatic at the time of diagnosis. METHODS: We undertook a retrospective study of 143 FAP patients treated at the Department of Surgery, University of Erlangen between 1971 and 2000. We identified patterns of symptoms, endoscopic findings and extracolonic manifestations in three age groups. RESULTS: FAP was diagnosed clinically on the basis of symptoms in 84% (120/143) of these patients. Most presented with intestinal symptoms such as colonic bleeding (68%) and diarrhea (42%). All but one of the patients between 20 and 40 years old had rectal polyps (98.7%, 75/76), whereas in those over 40 years old the prevalence was 76% (35/46). Non-specific symptoms such as abdominal pain, fatigue and bloating were less frequent and were mainly reported by patients older than 40. CONCLUSION: The commonest presenting features of FAP are alteration of bowel habit and rectal bleeding, but both are found in many other conditions. Patients with these findings need immediate endoscopy to allow prompt diagnosis and prophylactic surgery.

Common denominator genes that distinguish colorectal carcinoma from normal mucosa.

PURPOSE: Microarray technology has been used by a growing number of investigators and several studies have been published that list hundreds of genes differentially expressed by colorectal carcinoma (CRC) and normal mucosa (MC). On the basis of our own and other investigators' microarray data, our goal was to identify a common denominator gene cluster distinguishing CRC from MC. METHODS: Thirty GeneChips (HG-U133A, Affymetrix) were hybridized, 20 with RNA of CRC stages I-IV (UICC) and 10 with MC. Expression signals showing at least a 4-fold difference between CRC and MC (p<0.01) were identified as differentially expressed. In addition, in our integrative data analysis approach only those genes whose expression was altered simultaneously in at least 2 of 5 recently published studies were subjected to an unsupervised hierarchical cluster analysis. RESULTS: We detected 168 up- and 283 down-regulated genes in CRC relative to MC. Twenty-three genes were filtered from the five articles reviewed. An unsupervised hierarchical cluster analysis of these 23 genes confirmed the high specificity of these genes to differentiate between CRC and MC in our microarray data. CONCLUSIONS: Colorectal cancer and mucosa could be clearly separated by 23 genes selected for being differentially expressed more than once in a recent literature review. These genes represent a common denominator gene cluster that can be used to distinguish colorectal MC from CRC.

Tissue preparation for gene expression profiling of colorectal carcinoma: three alternatives to laser microdissection with preamplification.

Colorectal-carcinoma specimens are heterogeneous and include areas of nonmalignant mucosal and connective tissue. For those study designs in which laser microdissection and RNA preamplification are impracticable, the optimal yield of genuine cancer RNA is a key factor in gene-expression analysis. In this study we compared alternative methods of tissue purification. Three contiguous 0.5-cm(3) samples taken from an advanced primary adenocarcinoma of the sigmoid colon were processed immediately after surgery with the use of the following methods: (1) cryotomy after manual dissection (CMD), (2) microscopically assisted manual dissection (MAMD), and (3) tumor-cell isolation with the use of Ber-EP4 antibodies and Dynabeads (Dynal Biotech GmbH, Hamburg, Germany; technique abbreviated as DB). We generated gene-expression profiles with the use of GeneChip technology (Affymetrix, Santa Clara, Calif) and recorded preparation times, costs, and RNA quantity and quality. CMD took 60 minutes, MAMD 180 minutes, and DB 90 minutes to isolate 22, 8, and 23 microg of RNA, respectively. Expenses for materials amounted to 41, 23, and 91 US dollars for CMD, MAMD, and DB, respectively. The 3'/5' ratio, as determined with the GeneChips, for GAPDH/beta-actin was 1.01:1.03 for CMD, 1.13:1.28 for MAMD, 1.43:1.68 for DB, K-ras, APC, smad 2, transforming growth factor-beta, and p53 were marked as present in all cases, with the exception of APC, which was graded as marginal on DB. The correlation values of gene-expression profiles were 91% (CMD/DB), 93% (CMD/MAMD), and 97% (DB/MAMD). All 3 methods provided enough RNA, of sufficient quality, for gene-expression microarray analysis in colorectal carcinoma. Cross-methodologic analyses of array data should not be performed uncritically.

Improved prediction of recurrence after curative resection of colon carcinoma using tree-based risk stratification.

BACKGROUND: Patients who are at high risk of recurrence after undergoing curative (R0) resection for colon carcinoma may benefit most from adjuvant treatment and from intensive follow-up for early detection and treatment of recurrence. However, in light of new clinical evidence, there is a need for continuous improvement in the calculation of the risk of recurrence. METHODS: Six hundred forty-one patients with R0-resected colon carcinoma who underwent surgery between January 1, 1984 and December 31, 1996 were recruited from the Erlangen Registry of Colorectal Carcinoma. The study end point was time until first locoregional or distant recurrence. The factors analyzed were: age, gender, site in colon, International Union Against Cancer (UICC) pathologic tumor classification (pT), UICC pathologic lymph node classification, histologic tumor type, malignancy grade, lymphatic invasion, venous invasion, number of examined lymph nodes, number of lymph node metastases, emergency presentation, intraoperative tumor cell spillage, surgeon, and time period. The resulting prognostic tree was evaluated by means of an independent sample using a measure of predictive accuracy based on the Brier score for censored data. Predictive accuracy was compared with several proposed stage groupings. RESULTS: The prognostic tree contained the following variables: pT, the number of lymph node metastases, venous invasion, and emergency presentation. Predictive accuracy based on the validation sample was 0.230 (95% confidence interval [95% CI], 0.227-0.233) for the prognostic tree and 0.212 (95% CI, 0.209-0.215) for the UICC TNM sixth edition stage grouping. CONCLUSIONS: The prognostic tree showed superior predictive accuracy when it was validated using an independent sample. It is interpreted easily and may be applied under clinical circumstances. Provided that their classification system can be validated successfully in other centers, the authors propose using the prognostic tree as a starting point for studies of adjuvant treatment and follow-up strategies.

Prognostic significance of microsatellite instability in curatively resected adenocarcinoma of the small intestine.

Adenocarcinoma of the small intestine (ACSI) is a rare condition with few studies addressing follow-up and prognosis. Tumors of 35 patients with curative resection of an ACSI were retrospectively analyzed by immunohistochemistry: p53, hMLH1, hMSH2 and hMSH6 and microsatellite instability (MSI): BAT-26, BAX, TGF-beta RII. With a median follow up of 6.1 years, the median cancer-specific survival (CSS) was 36.2 months. Patients who were highly instable (MSI-H) (n=10) had a CSS of 49.6 months in contrast to patients with stable tumors (23.2 months) (P=0.010). Additionally, a low tumor stage according to UICC and MSI-H were shown to be independent factors (P=0.005 and P<0.001) for an increased survival in multivariate analysis. Therefore, it is suggested that analysis of the MSI status might prove useful in discerning prognosis within cancers of the same stage.