Daniel H. Winship

Excessive Production of Reactive Oxygen Metabolites by Inflamed Colon: Analysis by Chemiluminescence Probe

Reactive oxygen metabolites (ROMs) are involved in inflammatory diseases and are postulated to contribute to tissue injury in colitis. To determine whether excessive ROMs are generated by inflamed colonic mucosa and to identify possible sources and type of ROMs, mucosal ROMs were estimated in rats and humans using a chemiluminescence probe. Colitis was induced in rats by intracolonic injection of acetic acid or intraperitoneal injection of mitomycin C. Intact, inflamed colon in rats produced more ultraweak chemiluminescence than normal colon. Inflamed mucosal scrapings from both rat models produced significantly more luminol-enhanced chemiluminescence. Addition of catalase, an H2O2 scavenger, or azide, a myeloperoxidase inhibitor, into the media significantly decreased chemiluminescence from inflamed mucosal scrapings. Indomethacin, an antioxidant cyclo-oxygenase inhibitor, also decreased chemiluminescence, but MK-866, a 5-lipoxygenase inhibitor, had no effect. Colonic biopsy specimens obtained during colonoscopy from patients with ulcerative colitis also produced more catalase-inhibitable chemiluminescence than normal colonic mucosa. These data indicate that excessive ROMs are produced by inflamed colonic mucosa in both humans and rats, which may contribute to tissue injury.

Human upper esophageal sphincter: Response to volume, osmotic, and acid stimuli

Abstract Upper esophageal sphincter responses to stimuli of intraesophageal infusion were studied in 9 normal human subjects. Upper esophageal sphincter pressures were measured utilizing a water-perfused multilumen catheter with radially oriented pressure-recording orifices. The central core of the catheter opened distally for infusion of test solutions. The six test solutions (distilled water, 0.9% NaCl, 1.8% NaCl, 3.6% NaCl, 0.1 n HCl, 0.2 n HCl) and sham infusion were administered in a randomized double blind manner on 3 to 9 different days in each subject. Tracings were read and data calculated before the code was broken. Infusion with all solutions increased upper esophageal sphincter resting pressure above sham. The upper esophageal sphincter thus responds to the presence of intraesophageal fluid, a volume response. Pressures during 0.1 n HCl infusion were greater than those during 0.9% NaCl infusion, which demonstrates that the upper esophageal sphincter responds to an acid stimulus. Any pressure response to solutions of varying osmolalities was indistinguishable from the volume effect alone in 6 subjects studied. Upper esophageal sphincter pressures were increased further by increases in the rate of infusion of 0.1 n HCl in the upper esophagus in 7 subjects studied. In addition, the closer to the upper esophageal sphincter the infusion was administered, the greater was the pressure response. The onset of the upper esophageal sphincter response to the presence of intraluminal fluid occurred at a mean time of 2.7 sec after the initiation of a bolus injection into the esophagus in 4 subjects studied. The mean duration of the upper esophageal sphincter pressure elevation, after cessation of the bolus injection, was 33.5 sec. We suggest that the human upper esophageal sphincter functions as a dynamic barrier to esophageal-pharyngeal reflux and possible subsequent tracheobronchial aspiration.

Increased interleukin-8 (IL-8) in rectal dialysate from patients with ulcerative colitis: evidence for a biological role for IL-8 in inflammation of the colon.

OBJECTIVE:Infiltration of neutrophils and their release of toxic reactive oxygen species (ROS) in the colonic mucosa are associated with tissue damage in ulcerative colitis (UC). This neutrophil migration may be induced by chemoattractants, such as cytokines, in the colonic milieu. One such chemoattractant is interleukin-8 (IL-8), a neutrophil chemokine that is present at high concentrations in inflamed mucosa. However, the functional significance of IL-8 in neutrophil attraction and activation in UC has not been established. We hypothesized that IL-8 in the colonic lumen of patients with UC primes neutrophils, leading to their attraction and activation.METHODS:The colonic milieu was sampled by rectal dialysis. Using a semi-permeable membrane with a molecular weight cut-off of 12 kDa, dialysis solution was placed in the rectum and allowed to equilibrate over a 4-h period with the colonic milieu of controls or of patients with UC. IL-8 concentrations were measured by ELISA. Two functions of healthy neutrophils (PMN) were measured: expression of CD11-b surface adhesion molecules (by flow cytometry), and production of ROS (by both chemiluminescence and cytochrome C reduction assays). Neutrophil functions after exposure to rectal dialysates or n-formyl-methionyl-leucyl-phenylalanine (fMLP) were assessed before and after adding anti–IL-8 antibody or the fMLP blocker BMLP.RESULTS:IL-8 concentrations in dialysates from patients with active UC were significantly higher than in controls and correlated with disease activity. UC dialysates significantly increased ROS production and CD11-b expression by neutrophils and anti–IL-8 antibody partially (50%) inhibited these stimulatory effects of UC dialysates. Preincubation of neutrophils with UC dialysates significantly potentiated the fMLP-induced rise in ROS and anti–IL-8 antibody completely abolished this priming effect.CONCLUSIONS:The colonic milieu, sampled by rectal dialysis, from patients with active UC can both activate and prime neutrophils in vitro. High concentrations of IL-8 in the colonic lumen of UC patients are partially responsible for the activating effects of rectal dialysates, and account for all of its priming effects. These findings provide direct evidence for a role for IL-8 in inflammatory bowel disease.

Gastrointestinal Manifestations of Mixed Connective Tissue Disease

We examined the gastrointestinal tract abnormalities in 61 patients with mixed connective tissue disease. The first 34 were part of a prospective longitudinal study that included manometric and radiographic evaluation of the esophagus. Heartburn (48%) and dysphagia (38%) were by far the most common gastrointestinal symptoms. Seventeen percent of patients undergoing manometry had distal esophageal aperistalsis, and 43% low-amplitude peristalsis (less than 30 mmHg). Studies in 10 patients before and after treatment suggested that esophageal dysfunction in mixed connective tissue disease may be responsive to corticosteroids. Upper esophageal sphincter hypotension was also common. One patient had marked upper esophageal sphincter hypotension and recurrent aspiration, which resolved with corticosteroid therapy. Findings on radiographic studies of the stomach and small bowel in 54 patients and barium enemas in 16 patients were reviewed. Our series included one case each of malabsorption, colonic and small bowel perforations due to vasculitis, chronic active hepatitis, and acute pancreatitis. In conclusion, any area of the gastrointestinal tract may be affected by mixed connective tissue disease, although the esophagus is the most common location. The gastrointestinal aspects of mixed connective tissue disease overlap with those of progressive systemic sclerosis, polymyositis, and systemic lupus erythematosus.

Increased production of luminol enhanced chemiluminescence by the inflamed colonic mucosa in patients with ulcerative colitis.

Reactive oxygen species have been implicated as mediators of inflammation in ulcerative colitis. Chemiluminescence is a reliable means of estimating reactive oxygen species in biological media. Increased reactive oxygen species values in the inflamed colonic mucosa in rats were seen by chemiluminescence. The aims of the study were to find out if chemiluminescence is raised in the colonic mucosa of patients with ulcerative colitis and correlates with disease activity, and to elucidate the sources of the chemiluminescence. It was found that reactive oxygen species, as measured by the chemiluminescence technique, are raised in inflamed colonic mucosa and correlates with symptom score, sigmoidoscopic score, disease activity, and activity of the neutrophil enzyme myeloperoxidase. Chemiluminescence was inhibited by a myeloperoxidase inhibitor (azide) and an H2O2 scavenger (catalase) but not by allopurinol, an inhibitor of the enzyme xanthine oxidase. Chemiluminescence was also inhibited by indomethacin, but this did not seem to be related to inhibition of cyclo-oxygenase. These findings suggest that a likely cellular source of reactive oxygen species in the inflamed colon of patients with ulcerative colitis is the neutrophil and that myeloperoxidase conversion of H2O2 to hypochlorous acid, contributes to the chemiluminescence signal and possibly, to the tissue injury. Neither cyclo-oxygenase nor lipoxygenase seem to play a part as sources for the chemiluminescence.

Acid Loss in the Human Duodenum: Volume change, osmolal loss, and CO2 production in response to acid loads

Abstract We infused isotonic hydrochloric acid solutions of different p H into the proximal duodena of normal males. From samples aspirated at 15 cm and 30 cm downstream, we studied some relationships between acid load, acid loss, and chemical neutralization of acid in the duodenal lumen. At infusions with solutions of p H 1, p H 2, and p H 3, gain of fluid was recognized; the gain in the lumen increased progressively with increase in infused acid load. Duodenal fluid P CO 2 , levels increased markedly during p H 1 and p H 2 infusions; values up to 700 mm Hg were recorded. Osmolality of duodenal fluid decreased as a result of the neutralizing reaction between acid and bicarbonate during p H 2 and p H 1 infusions. The major changes were observed in the proximal duodenal segment; P CO 2 and osmolality reverted toward normal in the distal segment. Level of P CO 2 and degree of decrease in osmolality were similarly related to the amount of acid lost from the duodenum and exhibited a strong mutual correlation, reflecting their common origin in the chemical neutralization of HCl with endogenous bicarbonate. Almost all acid loss occurred in the proximal duodenal segment. If acid entered the distal segment it evoked a further secretion of fluid; otherwise, net absorption occurred in the segment.

Loss and Regeneration of the Esophageal Mucosa in Pemphigoid

Summary Serial esophageal biopsies were studied from a patient with pemphigoid after he had sloughed a cast of the esophageal squamous epithelium. Initial biopsies showed no evidence of esophageal epithelium by conventional histological techniques, whereas jejunal and rectal biopsies were normal. Despite these striking histological findings in the esophagus, clinical symptoms were minimal, although significant abnormalities in esophageal motility could be demonstrated. Histologically detectable epithelial regeneration began during the 4th week following loss of the epithelium, and regeneration appeared to progress at the same rate along the entire length of the esophagus. Esophageal motility reverted toward normal within 2 months after loss of the epithelium. Some residual thinning of the squamous epithelium of the esophagus persisted even after 8 months. No stricture formation has been noted.

Basal and Histamine-Stimulated Human Gastric Acid Secretion: Lack of effect of indomethacin in therapeutic doses

The effects of indomethacin administration on gastric acid secretion were studied in 22 patients with arthritic diseases. Twelve patients had rheumatoid arthritis, 8 had osteoarthritis, and 1 each had Reiters syndrome and ankylosing spondylitis. Augmented histamine tests were performed before and after a 1-month trial of the drug, administered in dosage of 75 to 100 mg per day. Repeated augmented histamine tests also were performed in 11 control subjects after a similar interval of time. No statistically significant changes in basal or histamine-stimulated gastric acid secretion were found in either the patients with arthritis or the control subjects.

Regulation of neutrophils in ulcerative colitis by colonic factors: A possible mechanism of neutrophil activation and tissue damage

The mucosal injury of active ulcerative colitis (UC) could involve enhanced migration and activation of neutrophils (PMNs). Because, in vitro, PMNs from patients with UC appear normal and are not therefore a likely cause for the enhancements, we hypothesized an abnormal colonic milieu. We previously found that factors in the UC colonic milieu markedly increase production of reactive oxygen species (ROS) by control PMNs. We now hypothesize that these factors also regulate PMN surface integrins, that regulation of UC PMNs is different than that of control PMNs, and that the integrin regulation is consistent with the ROS regulation. Using rectal dialysis, we sampled the colonic milieu in patients with active UC, in patients with inactive UC, and in control subjects. We monitored a key PMN adhesion molecule, CD11b. When control PMNs were tested, active UC rectal dialysate was almost as effective (+115%) as N-formyl-methionyl-leucyl-phenylalanine (+132%) in up-regulating CD11b. When inactive UC PMNs were tested, baseline CD11b was 50% higher than that for control PMNs. In contrast, rectal dialysates failed to up-regulate CD11b of inactive UC PMNs and in fact down-regulated CD11b. Preincubating control PMNs with UC rectal dialysates converted their CD11b response to PMN activators from up-regulation to down-regulation, mimicking inactive UC PMNs. Changes in intracellular calcium levels paralleled these changes in CD11b. Rectal dialysate-induced changes in both CD11b and calcium paralleled our previous findings on rectal dialysate-induced changes in ROS production. Thus the net overall effect of factors in the colonic milieu is a consistent and predictable regulation of PMN function--proinflammatory in UC, anti-inflammatory in control subjects. These factors may be a critical part of the pathophysiology of UC.

Are dipyridamole (sensitive) calcium channels present in esophageal smooth muscle

UNLABELLED Calcium (Ca2+) entry from the extra-cellular space into the cytoplasm through voltage-dependent Ca2+ channels, specifically dipyridamole (DHP) sensitive ones (L-type), control a variety of biological processes, including excitation-contraction coupling in vascular and GI muscle cells. It has also been proposed that these channels may control esophageal contractility. However, DHP-sensitive Ca2+ channels in esophagus have not been well characterized biochemically. Thus, it is not known if these channels are similar in number or affinity to those in vascular or neural tissues--organs for which clinical use of calcium channel blockers has been successful. Thus, the purpose of this study was to identify and characterize DHP-sensitive calcium channels in esophagus and compare them to vascular, neural, and other GI tissues. METHODS We carried out in vitro receptor binding assays on lower esophageal muscle homogenates, gastric and intestinal and colonic homogenates, and aortic muscle homogenates from ca; and on brain homogenates from rat. We used a radio-labeled dihydropyridine derivative [3H]nitrendipine, to label these sites and co-administration of unlabeled nimodipine to define specific binding. RESULTS As expected, ligand binding to L-type Ca2+ channels in aortic vascular smooth muscle and brain was readily detectable: brain, Bmax=252 fmol/mg protein, Kd=0.88 nM; aorta, Bmax=326 fmol/mg protein, Kd=0.84 nM. For esophagus (Bmax=97; Kd=0.73) and for other GI tissues, using the same assay conditions, we detected a smaller signal, suggesting that L-type Ca2+ channels are present in lower quantities. CONCLUSION L-type Ca2+ channel are present in esophagus and in other GI muscles, their affinity is similar, but their density is relatively sparse. These findings are consistent with the relatively limited success that has been experienced clinically in the use of calcium channel blockers for treatment of esophageal dysmotility.