Assessment of rebound and relapse following ecallantide treatment for acute attacks of hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a rare genetic disease characterized by unpredictable and recurring attacks of angioedema. This study assessed potential attack rebound and relapse following treatment with ecallantide, a plasma kallikrein inhibitor approved for HAE attack treatment. METHODS: Results were integrated from 2 double-blind, placebo-controlled studies of ecallantide treatment for HAE: EDEMA3-DB and EDEMA4. Symptoms were assessed by treatment outcome score (TOS), mean symptom complex severity (MSCS) score, and global response. Patients with improvement at 4 h post-dosing in all three measures followed by any sign of worsening at 24 h were considered to show potential rebound if worsening was beyond baseline or potential relapse if not beyond baseline. Likeliness of rebound or relapse was determined by the number of measures showing worsening and the magnitude of worsening. Patients receiving placebo who met the criteria for rebound/relapse were evaluated for descriptive comparison only. RESULTS: Significantly more ecallantide-treated patients (42 of 70) compared to placebo (26 of 71) showed improvement in three measures at 4 h and were thus eligible for rebound/relapse (P = 0.006). Of the nine ecallantide-treated patients with signs of worsening at 24 h, none were likely rebound, one was assessed as possible rebound, one as likely relapse, and two as possible relapse. No patient with potential rebound/relapse experienced new symptoms after dosing. Medical intervention was required in one ecallantide-treated patient. CONCLUSION: Ecallantide was efficacious for treating acute HAE attacks. Relapse was observed in a small proportion of patients, and there was little evidence of rebound.

On-demand IC351 (Cialis) enhances erectile function in patients with erectile dysfunction.

IC351 (Cialis) is a selective inhibitor of PDE5. The efficacy and safety of on-demand dosing of IC351 in men with erectile dysfunction was assessed in a multicenter, double-blind, placebo-controlled study. One hundred seventy-nine men (mean age: 56 y) were randomized to receive placebo or IC351 at doses of 2, 5, 10 or 25 mg, taken on demand over a 3-week period. The primary endpoints were change from baseline in responses to Questions 3 (Q3) and 4 (Q4) of the International Index of Erectile Function (IIEF). IC351 significantly improved IIEF Q3 scores at all doses vs placebo (P < or =0.003). IC351 also significantly improved IIEF Q4 scores in all but the 2 mg group (P < or =0.0003). No significant changes in laboratory values, ECGs, or blood pressure were observed. The most common adverse events were headache and dyspepsia. The conclusion of this study was that on-demand IC351 at doses up to 25 mg was well tolerated and significantly improved erectile function.

Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa.

Alterations in phenotype and function of intestinal macrophages occur in inflammatory bowel disease (IBD) but it is unclear whether these changes result from the recruitment of circulating monocytes to the intestine or from proliferation of resident intestinal macrophages. We sought to demonstrate the arrival of blood monocytes, the precursors of macrophages, in IBD mucosa. Peripheral blood mononuclear cells were isolated from 23 patients with clinically active intestinal inflammation (13 Crohn's disease, eight ulcerative colitis, two infective colitis), then radiolabelled with 99mtechnetium (Tc)-stannous colloid (n = 13) or 111indium (In)-oxine (n = 10) before re-injection and abdominal scanning. Four patients had demonstrable intestinal monocyte uptake using [99mTc]-stannous colloid, while six [111In]-oxine-labelled monocyte scans were positive. Uptake sites correlated with actively inflamed regions. Patients demonstrating monocyte uptake had been treated with corticosteroids for a significantly (P < 0.02) shorter duration (median 3 vs 20 days) than those with negative scans. There was no significant difference between positive and negative scans for disease category, clinical or histological disease, activity, or radioisotope used. Biopsies of inflamed mucosa from two patients suffering ulcerative colitis who had positive scans showed a high proportion of CD14-positive macrophages, 4-9% of which contained autoradiographic grains. These results demonstrate that blood monocytes are recruited to the mucosa of actively inflamed bowel, and suggest that this process may be inhibited by corticosteroids. Moreover, the phenotype of the recently-arrived monocytes indicates their susceptibility to stimulation by lipopolysaccharide, and suggests a mechanism for the continuing inflammation in the bacterial product-rich milieu of IBD.

IL-2 production by intestinal lamina propria cells in normal inflamed and cancer-bearing colons.

Biologically significant levels of IL-2 activity were produced by isolated lamina propria mononuclear cells (LPMC) from normal intestine (n = 12), cancer-bearing colons (n = 35) and inflammatory bowel disease (IBD) affected tissue (n = 12). The levels of IL-2 produced were similar for all three sources of LPMC (normal 252 +/- 48 U/ml, IBD-affected mucosa 197 +/- 42 U/ml and colon cancer 285 +/- 43 U/ml). These levels were significantly greater than those produced by peripheral blood mononuclear cells (20 +/- 5 U/ml, P less than 0.01) on a per cell basis. In mucosa from cancer-bearing colons the amount of IL-2 produced by LPMC was unaffected by the invasiveness of the colon cancer. LPMC IL-2 production was markedly suppressed by drugs used in IBD therapy. 5-Aminosalicylic acid (5-ASA) reduced activity in a dose-dependent fashion. At a dose equivalent to the faecal therapeutic level of 0.5 mg/ml activity, IL-2 production by LPMC was suppressed to 3.4% of controls. Similarly, exposure of LPMC to cyclosporin A (CyA) and hydrocortisone (HC) at therapeutic levels reduced IL-2 activity to less than 1% of controls. The major producers of IL-2 activity were shown to be CD3+ T lymphocytes and those bearing the activation markers IL-2R and TFR. Suppression of mucosal IL-2 production represents an important therapeutic mechanism of drugs used in the management of IBD including HC, 5-ASA and CyA. These results suggest that mucosal T cells produce appreciable levels of IL-2 activity that may be important in maintaining immune homeostasis in the normal intestine, provide anti-neoplastic cytotoxic activity and contribute to the inflammatory events that characterize the mucosal lesions of IBD.

Cloning and characterization of a gene that regulates cell adhesion.

Molecules of the cadherin and integrin families involved in cell-cell and cell-matrix adhesion have been implicated in epithelial differentiation, carcinogenesis and metastasis. Having observed that a colon cancer cell line bound avidly to collagen type I, inducing integrin-triggered glandular differentiation, we investigated the regulation of integrin function in these cells. We modified a mammalian expression cloning system that used monoclonal antibody selection to clone cell surface molecules. Using attachment to collagen type I to select for adhesive phenotype, we isolated a complementary DNA clone that increases cell adhesion to components of the extracellular matrix. The corresponding gene (cell adhesion regulator, CAR) is located on the long arm of chromosome 16 (16q) and encodes a protein of 142 amino acids, which has an N-terminal myristoylation motif and a consensus tyrosine-kinase phosphorylation site at the C terminus. Removal of this tyrosine residue abolishes enhancement of cell-matrix adhesion. This gene may encode an adhesion signal transduction molecule that functions in the suppression of tumour invasion.

Enhanced mucosal cytokine production in inflammatory bowel disease.

Proliferation, maturation, chemotaxis, and activation of neutrophils and monocytes are mediated largely by cytokines, including colony-stimulating factors and lymphokines. Cytokines produced in the intestinal mucosa contribute to the increased migration of neutrophils and monocytes into the lesion of inflammatory bowel disease and to the activation of these inflammatory cells. Lamina propria mononuclear cells isolated from colon tissue from 14 patients with inflammatory bowel disease (IBD) and from histologically normal controls were studied. Cells from IBD-affected tissue produced significantly more colony-stimulating factor activity (1402 +/- 252 U) per 2 x 10(6) cells than those from normal mucosa (362 +/- 85 U), mainly because of the increased production of granulocyte colony-stimulating factor and interleukin 1. This was accompanied by increases in the amount of specific messenger RNA for these two cytokines in lamina propria mononuclear cells from mucosa of patients with Crohn's disease (CD) compared with normal controls. By contrast, there was a substantial reduction in interleukin 3 production in CD and in ulcerative colitis lamina propria mononuclear cells, and this was reflected in significantly reduced expression of interleukin 3 messenger RNA in CD cells. Of the agents used in the therapy of IBD, hydrocortisone and 5-aminosalicylic acid, but not cyclosporin A, markedly suppressed in vitro production of cytokines by lamina propria mononuclear cells, suggesting that their therapeutic efficacy in vivo may be due in part to down-regulation of cytokine production in the inflamed mucosa.

Rapid assay and identification of human haemopoietic growth factors.

In order to facilitate the identification of human haemopoietic growth factors (HGFs), in complex conditioned media from limited cell sources, we have developed a method for the rapid assay of HGFs using bone marrow stem cells (BMSCs) obtained by Percoll density gradient enrichment of non-adherent cells from long-term human bone marrow culture. These BMSCs provide the basis for a 72 h assay where proliferation is triggered by the presence of GM-CSF, G-CSF and IL-3. The assay is up to 1000 times more sensitive than the conventional colony assay in detecting the presence of HGFs. We also describe methods for the rapid separation and identification of HGFs using fast protein liquid chromatography (FPLC) with phenyl-Superose and Mono Q columns. This enables individual HGFs present in complex conditioned media to be rapidly identified from the elution profile of HGF activity determined in the BMSC assay.

Assessment of inflammatory bowel disease activity by technetium 99m phagocyte scanning.

Autologous technetium 99m-labeled phagocyte scanning has been used to assess disease activity in inflammatory bowel disease in 51 consecutive patients. Strong correlations were found between the 24-h fecal excretion of isotope and the histologic score of mucosal biopsy specimens (rS = 0.84, p less than 0.001, where rS is Spearman's rank correlation coefficient), and between the 24-h fecal excretion of isotope and a clinical inflammatory bowel disease activity index based on the Crohn's disease activity index (rS = 0.87, p less than 0.001). To develop a clinically useful and objective measure of inflammatory bowel disease activity that did not require a 24-h stool collection, the intensity of bowel uptake on scanning was graded visually from 0 to 4, a ratio of count rates for the region of interest to the iliac crest reference region was calculated, and the rapidity of labeled phagocyte uptake into inflamed bowel was measured as the peak uptake time. Visual grading of disease activity on the scans was validated by comparing it with the ratio of count rates from inflamed bowel regions of interest and those from the iliac crest reference region. The ratio of count rates showed a significant correlation with the clinical disease activity index (r = 0.75, p less than 0.001). The visual scan grade also correlated well with the clinical activity index (r = 0.87, p less than 0.001). Count rates from hourly scans were also used to calculate the time of peak uptake of counts for a given region of interest. There was a strong negative correlation between this peak uptake time and the fecal excretion of isotope (rS = -0.81, p less than 0.001), a clinical activity index (r = -0.60, p less than 0.001), and the histologic score of the mucosal biopsy specimens (r = -0.84, p less than 0.001). These results indicate that the technetium 99m-labeled phagocyte scan provides an objective assessment of disease activity in inflammatory bowel disease using the visual scan grade, ratio of count rates for the region of inflamed bowel, or by the peak uptake time, thereby avoiding the problems associated with fecal collections. This scanning test may prove to be of significant value in clinical management and in the assessment of treatment response.

Lysis of colonic epithelial cells by allogeneic mononuclear and lymphokine activated killer cells derived from peripheral blood and intestinal mucosa: evidence against a pathogenic role in inflammatory bowel disease.

A sensitive 4 h 51Cr-release cytotoxicity assay has been developed using as targets colonic epithelial cells obtained by Dispase-collagenase digestion of resected mucosa or colonoscopic biopsies. Peripheral blood mononuclear cells (MNC) from most healthy donors showed low, but significant levels of cytotoxicity for normal epithelial cell target cells of 8.7 (4.4) % (mean (SD] and similar levels were found in 14 ulcerative colitis (6.5 (4.4) %) and 16 Crohn's disease (6.2 (5.2) %) patients. Neither drug therapy nor disease activity influenced the results. The sensitivity of colonic epithelial cells isolated from inflamed and histologically normal mucosa to lysis by peripheral blood MNC from a single donor was not affected by the underlying disease. Anti-epithelial cell activity did not correlate with anti-K562 activity and the cytotoxic cell was plastic non-adherent and Leu-11b-. None of 15 MNC populations isolated from mucosa of normal, tumour bearing, or chronically inflamed intestine exhibited significant lysis of colonic epithelial cells despite killing of K562 target cells in 10. Lymphokine activated killer (LAK) cells, generated by interleukin-2 stimulation in vitro of nine intestinal and seven peripheral blood MNC populations, exhibited high levels of lysis of K562 cells but, on every occasion, failed to lyse colonic epithelial cells. These data indicate that spontaneously cytotoxic or LAK cells are unlikely to play a role in the generation of colonic epithelial cell injury by direct cytotoxicity in inflammatory bowel disease.

Technetium-99m autologous phagocyte scanning: a new imaging technique for inflammatory bowel disease.

A method to determine the extent of active inflammatory bowel disease using selective labelling of autologous neutrophils and monocytes by phagocytosis of a technetium-99m (99mTc) stannous oxide colloid is described. Unlike leucocyte scanning techniques using Indium-III (IIIIn), the 99mTc colloid scan uses a cheap, readily available isotope, which specifically labels phagocytes. Scan results in 20 patients with inflammatory bowel disease were compared with barium examinations and colonoscopic appearances. There was close agreement in 15 of 20 patients as to the extent of mucosal disease. In four cases the scan showed more extensive disease than was suggested by barium examination. The scan showed terminal ileal Crohn's disease in three patients in whom the barium studies of the ileum had been reported as normal. In four patients with inactive disease and normal barium examinations no activity was seen on the scans. The 99mTc phagocyte scan is a sensitive, reliable means of determining the extent of active inflammatory bowel disease and can be used to quantify disease activity.

The role of myonuclei in muscle regeneration: an in vitro study.

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or "freed" as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H3-thymidine and their ultimate fate, either as "free" myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.