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.

Booster immunization of HIV-1 negative volunteers with HGP-30 vaccine induces protection against HIV-1 virus challenge in SCID mice.

Eleven HIV-1 seronegative subjects previously injected with an HIV-1 p17 synthetic peptide vaccine (HGP-30) were given two booster immunizations to evaluate memory cell responses and the ability to boost cellular and humoral immune responses. Five of 11 subjects showed a significant increase in their antibody titres to HGP-30 or p17 and 6/11 had T-cell proliferation responses to either HGP-30 or p17. HIV-1 virus challenge studies in SCID mice demonstrated that 39 of 50 mice (78%) receiving PBMC from 5 of the HGP-30 immunized subjects were protected from infection with a different strain of HIV-1 compared to 4 of 30 mice (13%) that received PBMC from 3 non-immunized subjects (p < 0.001). These studies show that booster immunizations with HGP-30 vaccine are safe and non-toxic and induce protective cell mediated immune responses.

Determination of protein carbonyl groups by immunoblotting.

Free radical-mediated oxidation of proteins results in the formation of carbonyl groups in quantities that reflect the intensity of the oxidative stress. We have developed an immunochemical technique for the quantification of carbonyl groups in protein samples prepared from small tissue samples and cell cultures. Protein samples were slot-blotted onto a polyvinylidene difluoride membrane, which was sequentially treated with 2,4-dinitrophenylhydrazine (DNPH), a primary antibody specific for the 2,4-dinitrophenol group, and a peroxidase-labeled second antibody. After the blots were developed with a chemiluminescent substrate and exposed to X-ray film, the level of immunostaining was quantitated by densitometry. Using oxidized bovine serum albumin as a standard and loading 5 microg of protein per slot, the minimum detectable carbonyl content was approximately 60 pmol carbonyl/mg protein. When necessary, nonspecific staining by noncarbonyl constituents in complex sample matrices was accounted for by using sodium borohydride-treated blanks. Results by the new method were highly correlated (r = 0.932, P < 0.0001) with those of the standard DNPH-based spectrophotometric technique. The coefficient of variation at a carbonyl level of 1.5 nmol/mg protein was 9.7%. The utility of this new method was demonstrated by measuring protein oxidation in cultured human colon cells (SW620) that were briefly exposed to H2O2.

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.

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.

The role of nitric oxide in ethanol-induced gastrointestinal dysfunction.

We recently showed that acute ethanol inhibits contractility of the lower esophageal sphincter (LES) and the lower esophageal body (LEB) both in vivo and in vitro. To evaluate the mechanism of this inhibitory effect of ethanol, we investigated the role of nitric oxide (NO) on contractility of isolated LES and LEB circular muscle strips using inhibitors of NO synthase (NOS), NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine. Ethanol significantly decreased LES basal tone. This effect was not mediated by NO, because inhibition was not prevented by inhibitors of NOS. Electrical field stimulation caused an On-response relaxation from LES strips, and an Off-response contraction from both LES and LEB strips. Inhibitors of NOS prevented the On-response relaxation of LES, but had no significant effect on LES Off-response contraction. Ethanol potentiated the On-response relaxation of the LES Off-response contraction. Ethanol potentiated the On-response relaxation of LES, but had no significant effect on Off-response contraction. Ethanol's potentiating effect of the On-response relaxation is NO-mediated, because it was abolished by NOS inhibitors. Ethanol also inhibited carbachol-induced LES contractility. This inhibitory affect was NO-mediated, because NOS inhibitors abolished it. Ethanol inhibited both the Off-response contraction and carbachol-induced contraction of LEB strips. These effects were not NO-mediated, because they were not affected by NOS inhibitor. These data suggest that NO is not a mediator for the inhibitory effect of ethanol on LEB contractility, and that NO seems to be a mediator of ethanol inhibition of some aspects of LES motor functions.

Restructuring medical schools to better manage their three missions in the face of financial scarcity.

The leaders of schools of medicine face a major financial and management challenge as they approach the end of the twentieth century. The new environment for the practice of medicine is diminishing the clinical income that schools have used for so long to supplement their operations. Deans and their staffs must discover new ways to manage their schools and their schools' finances. In particular, schools that continue to rely on subsidies from their health care operations will very likely face the prospect of having to defend themselves as pressures for more cost-effective operations increase and clinical and other subsidies dwindle. In sum, for deans to remain captains of their own ships, preserve their schools' three missions, and balance their books, it is essential that they lead their schools in change toward better administrative, information, and financial management systems. Stritch School of Medicine at Loyola University of Chicago is reinventing itself in response to this challenge. The organizational and management models being used there have been partially guided by the two approaches to redesigning schools of medicine described in detail in this article, the service line management matrix (SLMM) and the fractal management (FM) method. In turn, the experience of implementing the education and research service lines at Loyola has permitted a refinement of the approaches, reflected in the descriptions in this article. These approaches constitute a new model that provides for the development of three administrative service lines-teaching, research, and clinical service (not implemented at Loyola). Each service line has its own leaders and managers, who are responsible for balancing income against expenses. Each service line is a profit-and-loss center, an approach that highlights how effectively the service line is being managed and leads to greater accountability and productivity of faculty and staff. This new approach makes it possible for the school to answer the question, "Can research and teaching pay for themselves?" If they cannot, any subsidies that are needed from clinical income are identified as such. This businesslike approach supplants the traditional one of commingling all income streams to pay for operations, a strategy that worked well enough when research and clinical income constantly increased but now places medical schools in harm's way.

Human neutrophil functions are inhibited in vitro by clinically relevant ethanol concentrations.

Neutrophils [polymorphonuclear neutrophils (PMNs)] play a pivotal role in host defense in man. These defenses may be compromised, however, in alcohol users and abusers. We therefore evaluated the effect of ethanol levels (12.5 to 500 mg/dl), on key functions of human PMNs-chemotaxis and production of reactive oxygen species-and on changes in cytosolic-free calcium ([Ca2+]i), a pivotal intracellular mechanism of PMN activation. Ethanol significantly inhibited chemotaxis as evaluated by formyl-methionyl-leucyl-phenylalanine (fMLP)-induced upregulation of surface adhesion molecules (CD11b). fMLP-induced PMN elongation was only inhibited by a very high ethanol concentration of 500 mg/dl. Production of reactive oxygen species by normal PMNs was assessed by either chemiluminescence (CL) for hypochlorous acid or ferricytochrome c reduction (FCR) for superoxide anions. For PMN stimulated by fMLP, ethanol inhibited CL but not FCR. For PMNs activated by phorbol myristate acetate, ethanol inhibited both CL and FCR. Ethanol did not alter baseline [Ca2+]i, as assessed by videomicroscopy using the Ca(2+)-sensing fluorescent dye Fura-2-AM, but did significantly potentiate the increase in peak [Ca2+]i levels that occurs in response to stimulation by fMLP. Calcium channel blockers attenuated ethanol's inhibition of CL. Thus, acute in vitro ethanol, at clinically relevant concentrations, can inhibit several critical aspects of PMN functions. But, in PMNs, unlike neural cells, these inhibitory effects do not seem to be mediated by decreases in Ca2+ influx or in [Ca2+]i.

Ethanol inhibits contractility of esophageal smooth muscle strips.

Acute ethanol (EtOH) in vivo decreases both the pressure of the lower esophageal sphincter (LES) and the amplitude of contractions of the smooth muscle of the lower esophageal body (LEB) in both man and cat. However, the mechanism of this inhibitory effect of EtOH is unclear. This inhibitory effect could be caused by a direct effect of EtOH on the esophagus or be secondary to known inhibitory effects of EtOH on the central nervous system. To this end, we evaluated the in vitro effect of EtOH on contractility of smooth muscle strips from both LES and LEB. Circular muscle strips from LES and LEB were isolated from cats. Changes in resting tension of LES strips and changes in stimulant-induced tension of LES or LEB strips were measured in the presence of up to five concentrations of EtOH (12.5- 100 mM). Stimulants included electric field stimulation (EFS) and carbachol. EtOH at 75 mM significantly decreased resting LES tension. EtOH also decreased maximal contractile responses to carbachol in both LES and LEB and increased the EC50 of carbachol for LES, but not LEB. EtOH also modulated EFS-induced esophageal contractility; EtOH potentiated EFS-induced "on-response relaxation" in LES and decreased EFS-induced "off-response contractions" In LEB. EtOH-induced inhibition of esophageal contractility seemed to be reversible. EtOH did not result in muscle fatigue. Thus, EtOH can directly inhibit contractility of the esophagus, and does so reversibly and at pharmacologically relevant concentrations.

Modulation of neutrophil function by novel colonic factors: possible role in the pathophysiology of ulcerative colitis.

Tissue damage in acute ulcerative colitis (UC) may be triggered by neutrophils (PMNs) and their inflammatory mediators such as reactive oxygen species (ROS). Because circulating PMNs appeared normal in subjects with UC, we hypothesized that the critical abnormality that attracts and activates PMNs in UC is a local colonic factor. Accordingly, the colonic milieu was sampled by using in vivo rectal dialysis (mol wt < or = 12 kd). Normal PMNs were exposed in vitro to rectal dialysates (RD) from control subjects (cRD) or subjects with active UC (aRD) or inactive UC (IRD). PMN-derived ROS were measured by chemiluminescence. cRD did not increase ROS production by unstimulated PMNs; aRD significantly and concentration-dependently increased ROS; IRD gave intermediate results. cRD inhibited the PMN-stimulating effects of both the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA). aRD and IRD blunted the effect of fMLP and PMA significantly less than did cRD. Rectal dialysates from 44% of subjects with active UC exaggerated the fMLP effect, whereas potentiation occurred for only 13% of cRDs and 18% of iRDs. cRD preconditioned with either activated or nonactivated PMNs was not significantly different than unconditioned cRD. We thus infer the existence of colonic factors in UC that (1) can trigger PMNs to produce ROS and (2) have a proinflammatory modulatory effect on bacterial peptide-induced, PMN-mediated ROS production, thereby initiating or perpetuating inflammation and eventually causing tissue damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute ethanol inhibits calcium influxes into esophageal smooth but not striated muscle: a possible mechanism for ethanol-induced inhibition of esophageal contractility.

In both humans and cats, EtOH administered in vivo and acutely decreases contractility of smooth muscle of lower esophageal sphincter (LES) and lower esophagus (LE), but not striated muscle of upper esophagus. To see if these effects are associated with perturbation of Ca++ homeostasis, esophageal muscle slices were incubated in vitro with EtOH and then 45Ca++. At steady-state Ca++ uptake, some slices were exposed to 1 microM carbachol (CCH). Although 100 mM EtOH had no effect on Ca++ uptake into resting or stimulated striated muscle of upper esophagus, it significantly inhibited Ca++ uptake into smooth muscle of LES and LE. For unstimulated LE and resting LES, 100 mM EtOH significantly inhibited both initial uptake and steady-state levels, whereas lower doses had no significant effect. EtOH at 100 mM also affected changes in Ca++ content induced by CCH stimulation. CCH increased total exchangeable tissue Ca++ content in LE, whereas it decreased Ca++ content in LES. EtOH at 100 mM blunted these CCH-induced effects in both LES and LE. In contrast to resting muscle, inhibition of CCH-stimulated LE muscle was not limited to 100 mM EtOH, because substantial and significant inhibition was also seen at EtOH doses of 25 and 50 mM, doses which are relevant even in social drinking. Thus, EtOH inhibition of Ca++ influx into esophageal muscle is selective for smooth muscle, can occur at pharmacologically relevant EtOH doses and could be the underlying mechanism for EtOH's inhibition of contractility of esophageal smooth muscle.

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.

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.

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.