David Wattchow

Distribution and coexistence of peptides in nerve fibers of the external muscle of the human gastrointestinal tract

The nerve fibers that supply the external muscle of the human gastrointestinal tract were examined for their immunoreactivity to the neuropeptides enkephalin, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal peptide, for tyrosine hydroxylase (a catecholamine-synthesizing enzyme), and for coexistence between immunoreactivities in nerve fibers. Studies on coexistence revealed that the majority of reactive nerve fibers could be placed in one of two classes: (a) those fibers with reactivity to enkephalin or substance P, or both, and (b) fibers containing one or both of the peptides neuropeptide Y and vasoactive intestinal peptide. Many fibers immunoreactive for vasoactive intestinal peptide or neuropeptide Y, or both, were found throughout the external smooth muscle of the gastrointestinal tract, but neuropeptide Y-reactive fibers were less common in the small and large intestines than in the stomach and esophagus. Fibers immunoreactive for enkephalin or substance P, or both, were sparse in the esophagus, increased in numbers to reach maximal frequency in the pylorus, and maintained a similar frequency in the small and large intestines. Fibers with somatostatin or tyrosine hydroxylase immunoreactivity were rare. In general, sphincter regions were similar to nonsphincter regions in peptide-immunoreactive fiber numbers and types, except that the internal anal sphincter had no enkephalin-immunoreactive fibers and very few substance P-reactive fibers. Moderate numbers of fibers reactive for neuropeptide Y and vasoactive intestinal peptide were found in the internal anal sphincter. It is suggested that enkephalin and substance P are in excitatory fibers and that vasoactive intestinal peptide and neuropeptide Y are in fibers inhibitory to the external muscle.

Prospective randomized comparison of open versus laparoscopic appendectomy in men.

Abstract. A prospective, randomized trial was performed to compare open appendectomy with laparoscopic appendectomy in men with a clinical diagnosis of acute appendicitis. Sixty-four patients with a median age of 25 years (range 18–84 years) were randomized to open appendectomy (n = 31) or laparoscopic (n = 33) appendectomy. Of the 64 men, 56 (87.5%) had appendicitis (27 open, 29 laparoscopic procedures). The mean operating times were 50.6 ± 3.7 minutes (± SEM) for open and 58.9 ± 4.0 minutes for laparoscopic appendectomy (p = 0.13). Five (15%) patients randomized to laparoscopic appendectomy had an open operation. The mean postoperative hospital stay was significantly longer for open appendectomy (3.8 ± 0.4 days) than for laparoscopic appendectomy (2.9 ± 0.3 days) (t = 2.05,df = 62,p = 0.045). The complication rate after open appendectomy (25.8%) was not significantly different from that after laparoscopic appendectomy (12.1%). There was a single postoperative death due to a pulmonary embolus in the laparoscopic group and a single death due to cardiac and renal failure in the open group. The mean time to return to normal activities was significantly longer following open appendectomy (19.7 ± 2.4 days) than after laparoscopic appendectomy (10.4 ± 0.9 days), (t = 3.75,df = 49,p = 0.001). In conclusion, laparoscopic appendectomy in men has significant advantages in terms of a more rapid recovery compared to open appendectomy. There were no significant disadvantages to laparoscopic appendectomy compared to open appendectomy.

The relationship of obesity to the complications of diverticular disease.

Objective  Diverticular disease is common in our community. Most patients remain asymptomatic and the development of diverticular complications is rare. A common clinical observation is that patients presenting with complications of diverticular disease are obese. The aim of this study was to examine the relationship of obesity to the complications of diverticular disease.

Abnormalities of nerve fibers in the circular muscle of patients with slow transit constipation

Abstract Abnormalities of the enteric nervous system are thought to explain the pathophysiology of motility disorders. Our aim was to determine if particular classes of enteric neurons are affected in slow transit constipation (STC). Specimens were taken from the terminal ileum and ascending, transverse and descending colon of patients undergoing subtotal colectomy for STC. Immunohistochemistry was performed using antisera to neuron-specific enolase, tachykinin, leu-enkephalin, choline acetyltransferase, vasoactive intestinal peptide, nitric oxide synthase, tyrosine hydroxylase and neuropeptide Y. The density of nerve fibres labelled with these antibodies in each layer was compared with age-matched controls. The density of nerve fibres with tachykinin and enkephalin immunoreactivity was reduced in the colonic circular muscle of the 15 patients with STC, whereas innervation of all other layers was normal. This reduction of tachykinin-immunoreactive nerve fibres also occurred in nine of the 12 specimens of terminal ileum examined. No difference was detected in the density or distribution of nerve fibres using the other antisera. Excitatory nerve fibres are present in the circular muscle in STC but they are deficient in tachykinins and enkephalin.

Prospective trial of pelvic floor retraining in patients with fecal incontinence

PURPOSE: Our aim was to prospectively evaluate pelvic floor retraining (PFR) in improving symptomatic fecal incontinence. METHODS: PFR was used to treat 30 patients with fecal incontinence (28 women; age range, 29–85 (median, 68) years). PFR was performed by a physiotherapist in the outpatient department according to a strict protocol and included biofeedback using an anal plug electromyometer. Manometry (24 patients), pudendal nerve terminal motor latency (PNTML, 16 patients), and anal ultrasound (14 patients) were done before commencing therapy. Independent assessment of symptoms was done at the commencement of therapy, at 6 weeks, and at 6 and 12 months posttherapy. RESULTS: Twenty patients (67 percent) had improved incontinence scores, with eight patients (27 percent) being completely or nearly free of symptoms. Of 28 patients followed up longer than six months, 14 achieved a 25 percent or greater improvement at six weeks, which was sustained in all cases. Fourteen had an initial improvement of less than 25 percent, with only four (29 percent) showing later improvement (P<0.0001). There was no relationship between results of the therapy and patient age, initial severity of symptoms, etiology of incontinence, and results of anal manometry, PNTML, and anal ultrasound. CONCLUSIONS: PFR is a physical therapy that should be considered as the initial treatment in patients with fecal incontinence. An improvement can be expected in up to 67 percent of patients. Initial good results can predict overall outcome.

Cholinergic and nitrergic interneurones in the myenteric plexus of the human colon

Background: Myenteric interneurones are involved in the reflexes that control the motility of the human colon. Aims: The distribution of choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity in myenteric interneurones was investigated in this study. Methods: DiI (1,1`- didodecyl 3,3,3`,3`-indocarbocyanine perchlorate) was applied to the myenteric plexus of the human colon followed by organotypic culture. Retrogradely labelled neurones, with projections longer than motor neurones (>10 mm), were studied to exclude motor neurone populations. ChAT and NOS immunoreactivity was then determined in the interneurones. Results: We found that 90% of interneurones projecting orally contained ChAT and none contained NOS. Ninety five per cent of descending interneurones were labelled with ChAT and/or NOS antisera; 46% contained NOS immunoreactivity alone, 20% contained ChAT immunoreactivity alone, and 29% contained both ChAT and NOS. Anally directed interneurones had significantly longer projections than orally projecting interneurones. Conclusions: Nearly all interneurones contain either NOS or ChAT immunoreactivity. Orally projecting interneurones are of two types: 90% contain ChAT alone and the remainder contain immunoreactivity for neither ChAT nor NOS. There are three main types of anally projecting interneurones: the largest, which contains NOS but not ChAT, and the two smaller classes which contain ChAT and NOS, and CHAT alone.

Abnormalities of peptide-containing nerve fibers in infantile hypertrophic pyloric stenosis.

The distributions of nerve cells and fibers with immunoreactivity for the peptides enkephalin, gastrin-releasing peptide, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal peptide were examined in specimens of myenteric plexus and external muscle from the pylorus of 20 infants with hypertrophic pyloric stenosis. These were compared with peptide distributions in pyloric samples from unaffected infants and adults. In the normal pylorus the circular muscle was richly supplied with fibers reactive for enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide. In pyloric stenosis, these immunoreactive fiber bundles were either missing or less than 5% of normal. In contrast, there were reactive cell bodies and nerve fibers in the myenteric plexuses of both normal and affected specimens. In the samples from cases of stenosis, swollen nerve fibers that appeared to be in the process of degeneration were frequently encountered. It is concluded that infantile hypertrophic pyloric stenosis is associated with a loss of peptide immunoreactivity in nerve fibers in the circular muscle, although the same peptides are still revealed in fibers and in nerve cell bodies in the myenteric plexus.

A panel of genes methylated with high frequency in colorectal cancer

BackgroundThe development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests.MethodsCombined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).ResultsCombined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas.ConclusionsThis study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Distributions of neuropeptides in the human esophagus

The distributions of nerve cells and fibers with immunoreactivity for the peptides substance P, somatostatin, enkephalin, vasoactive intestinal peptide, gastrin-releasing peptide, and neuropeptide Y and the enzyme tyrosine hydroxylase were examined in 25 samples of human esophagus. These were compared with samples of stomach and intestine. In the smooth muscle of the muscularis externa, the muscularis mucosae, and beneath the epithelium, the most abundant nerve fibers contained vasoactive intestinal peptide and neuropeptide Y, in contrast to the scarcity of substance P, enkephalin, somatostatin, and gastrin-releasing peptide. Gastric and intestinal samples contained dense populations of fibers containing vasoactive intestinal peptide, neuropeptide Y, substance P, and enkephalin in the equivalent layers, but somatostatin- and gastrin-releasing peptide-immunoreactive fibers were scarce. Complete coexistence of vasoactive intestinal peptide and neuropeptide Y in nerve fibers within the muscle layers was demonstrated in the esophagus, but not in gastric and intestinal samples. The myenteric plexus along the length of the esophagus contained cell bodies and fibers reactive for vasoactive intestinal peptide, neuropeptide Y, enkephalin, and substance P. Somatostatin-immunoreactive cell bodies were very rare in the myenteric plexus, no gastrin-releasing peptide-immunoreactive cell bodies were seen, and both somatostatin and gastrin-releasing peptide-immunoreactive fibers were rare. In the upper esophagus, striated muscle bundles did not contain nerve fibers reactive for these peptides but immunoreactive fibers were seen in the muscularis mucosae and subepithelium. It is concluded that the esophagus has a different pattern of innervation by peptide-containing neurons than the stomach and intestines. Esophageal neurons can be classified into separate classes on the basis of their peptide content.

Projections of nitric oxide synthase and vasoactive intestinal polypeptide-reactive submucosal neurons in the human colon

Background : The submucosal plexus is important in the control of secretomotor and motor function of the intestine. Our aim was to describe the projections of submucosal neurons to the mucosa within the submucosal plexus and to the circular muscle of human colon and to determine whether submucosal neurons that projected to different layers were located at different levels of the submucosa.