Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells.

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes.

Colo-rectal anastomotic leakage often masquerades as a cardiac complication.

OBJECTIVE: The aim of this study was to identify the mode of presentation of patients with clinical anastomotic leaks following restorative colorectal resection for carcinoma. PATIENTS AND METHODS: Prospective information was collected on all patients having restorative resection of colorectal cancer. These data were reviewed for a five-year period (1994-1998) to identify all patients who had suffered a clinical anastomotic leak and their notes were retrieved and reviewed. RESULTS: Three hundred and seventy-nine patients underwent restorative resection for colorectal cancer during the study period (178 female, 201 male), mean age 70 years (range 36-94). There were 22 (6%) clinical anastomotic leaks. Seven (32%) patients presented with obvious abdominal peritonitis. The remaining 15 (68%) were initially misdiagnosed. Thirteen (59%) patients were treated for cardiac symptoms, 1 (5%) patient for obstruction and 1 (5%) for ascites. The delay in diagnosis ranged from 0 to 11 days (mean 4 days). For the whole series of 379 there were 30 patients who suffered cardiac symptoms (8%) 13(43%) of whom had an anastomotic leak. CONCLUSION: Patients who develop cardiac symptoms following restorative colorectal resection for carcinoma should have a water soluble enema as there is a 40% chance that they will have an anastomotic leak.

Idiopathic chronic ulcerative enteritis--the role of radical surgical resection.

Idiopathic chronic ulcerative enteritis is uncommon. It is a term that describes ulceration of the small bowel in the absence of a recognisable cause. Patients mainly present with a surgical abdomen and their management often proves to be a therapeutic challenge. Our series describes three such cases: the first patient presented with a tender left iliac fossa mass and rectal bleeding, the second with peritonitis and pneumoperitoneum, the third with severe acute colitis. All three patients needed urgent surgical intervention with further laparotomies due to recurrent ulceration, perforation and fistula formation in addition to intensive supportive measures such as inotropes and total parenteral nutrition. The importance and challenges of idiopathic chronic ulcerative enteritis are therefore discussed.

Endoscopic-assisted intrathoracic oesophagogastrostomy without thoracotomy for tumours of the lower oesophagus and cardia.

AIMS: This study aimed to evaluate the efficacy of a novel technique enabling a trans-hiatal oesophagectomy with intrathoracic anastomosis under direct vision, without thoracotomy. METHODS: Trans-hiatal dissection of the oesophagus was performed using direct and laparoscopic visualization. The oesophagus was transected above the tumour with a linear endo-GIA-2 60 mum stapler. The stomach was transected and a gastric tube fashioned. The anvil of an appropriately sized CEEA circular stapler was modified enabling it to flatten. It was attached to a novel delivery system introduced under direct vision along a guidewire into the stapled oesophagus. The anvil was realigned to its original position in the distal oesophagus, docked with the body of the stapler and an intrathoracic anastomosis performed. RESULTS: Ten patients (female n=3, male n=7) aged from 39--77 years (mean age 65 years), ASA 2--3 with distal third tumours were treated. Duration of procedure ranged from 2--5 hours (mean 4 hours). One patient suffered a post-operative chest infection and an anastomotic leak treated successfully with a self-expanding metal stent. Hospital stay ranged from 6--28 days (mean 17 days). There was no mortality. CONCLUSION: This technique allows a safe intrathoracic anastomosis to be performed trans-hiatally under direct vision, avoiding the need for thoracotomy in patients with high comorbidity.

Transanal endoscopic microsurgery--lessons from a single UK centre series.

OBJECTIVES: Transanal endoscopic microsurgery (TEM), a minimally invasive technique has been employed in the excision of benign and selected malignant rectal tumours since 1983. We present a single surgeon's series of 102 procedures. PATIENTS AND METHODS: A retrospective case note review of 102 procedures performed over a 6-year period between 1996 and 2001. RESULTS: One hundred and two TEM procedures were performed on 100 patients. 68 for adenomas, 19 potentially curative excisions for carcinoma, 13 palliative procedures for advanced carcinoma and 2 for solitary rectal ulcer syndrome (SRUS). Four adenomas recurred and were successfully treated by various procedures. None went on to develop malignancy, or a further recurrence. Of the cancers, six T1 and 10 T2 were excised with curative intent. Three T3 cancers were excised before endorectal ultrasound was available in the unit and went on to have definitive procedures. One T1 and two T2 carcinomas were not completely histologically excised. These patients were offered definitive procedure and there have been no recurrences. 11 patients underwent palliative TEM procedures, 2 went on to have a recurrence of symptoms. Both underwent a successful second TEM procedure. CONCLUSIONS: Although longer term follow up is still required, TEM appears to be an effective method of excising benign tumours and T1 carcinomas of the rectum. The role of TEM in the treatment of T2 carcinomas is, as yet, unclear and needs further evaluation although the results of our series and others are encouraging.

IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component.

The bronchial epithelium is a source of both alpha and beta chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and alpha(2)-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene alpha, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC(50) approximately 0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC.

Nucleoside diphosphate kinase--a component of the [Na(+)]- and [Cl(-)]-sensitive phosphorylation cascade in human and murine airway epithelium.

We have shown that proteins within apically enriched fractions of human nasal respiratory epithelium vary their phosphohistidine content with ambient [Cl(-)] and other anion concentrations. This membrane-delimited phosphorylation cascade includes a multifunctional protein histidine kinase - nucleoside diphosphate kinase (NDPK). NDPK is itself a cascade component in both human and ovine airway, the self-phosphorylation of which is inhibited selectively by [Na(+)] in the presence of ATP (but not GTP). These findings led us to propose the existence of a dual anion-/cation-controlled phosphorylation-based "sensor" bound to the apical membrane. The present study showed that this cascade uses ATP to phosphorylate a group of proteins above 45 kDa (p45-group, identities unknown). Additionally, the Cl(-) dependence of ATP (but not GTP) phosphorylation is conditional on phosphatase activity and that interactions exist between the ATP- and GTP-phosphorylated components of the cascade under Cl(-)-free conditions. As a prelude to studies in cystic fibrosis (CF) mice, we showed in the present study that NDPK is present and functionally active in normal murine airway. Since NDPK is essential for UTP synthesis and regulates fetal gut development, G proteins, K(+) channels, neutrophil-mediated inflammation and pancreatic secretion, the presence of ion-regulated NDPK protein in mouse airway epithelium might aid understanding of the pathogenesis of CF.

Metazoan parasite species richness and genetic variation among freshwater fish species: cause or consequence?

The factors responsible for the maintenance of genetic variation among natural populations remain a mystery. Recent models of host-parasite co-evolution assume that parasites exert frequency-dependent selection on their hosts by favouring rare alleles that may confer resistance against infection. We tested this prediction in a comparative analysis that sought relationships between levels of genetic variation and the number of metazoan parasite species exploiting each host species. We used data on 40 species of North American freshwater fishes. After controlling for sampling effort and phylogenetic influences, we found no relationship between genetic polymorphism and parasite species richness among fish species. However, we found a marginal negative correlation between parasite species richness and heterozygosity. This result goes against the prediction that increased selective pressure by parasites should be associated with higher levels of genetic variation. Instead, it suggests that parasites may be colonising host species showing low levels of genetic variation with greater success than genetically more variable host species.

Na+ and K+ regulate the phosphorylation state of nucleoside diphosphate kinase in human airway epithelium.

We describe how cations, in the presence of ATP, regulate the phosphorylated form of 19- and 21-kDa nucleoside diphosphate kinase (NDPK; EC 2.7.4.6), a kinase controlling K+ channels, G proteins, cell secretion, cellular energy production, and UTP synthesis. In apically enriched human nasal epithelial membranes, 10 mM Na+ inhibits phosphorylation of NDPK relative to other cations. Dose response showed that, whereas K+ induces a fourfold greater phosphate incorporation (EC50 10 mM), Na+ is inhibitory (EC50 10 mM) compared with respective buffer controls. Cation discrimination is nucleotide selective (not seen with [gamma-32P]GTP) and NDPK specific (not seen with p37h, a previously characterized Cl--sensitive phosphoprotein). Na+ does not exert an inhibitory effect on NDPK phosphorylation directly but is likely to act via an okadaic acid-insensitive phosphatase. We speculate that the ability of NDPK to discriminate between physiologically relevant cation concentrations provides a novel example of cross talk within the apical membrane.

Nucleoside diphosphate kinase and Cl(-)-sensitive protein phosphorylation in apical membranes from ovine airway epithelium.

We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl-], and anion species determine the steady-state phosphorylation of apical membrane proteins within human airway epithelium in vitro. We found that a Cl(-)-regulated 37-kD protein (p37) principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from sheep tracheal epithelium also contain a Cl(-)-regulated 37-kD phosphoprotein (p37s) and characterize one of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins with gamma[32P]GTP in the presence of MgCl2 showed that two proteins circa 19 and 21 kD (p19s and p21s) were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either gamma[32P]GTP or gamma[32P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical membrane proteins in the presence of gamma[32P]ATP and guanosine diphosphate (GDP) (but not GDPbetaS) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg2+, Mn2+, and Co2+ (but not Zn2+ or Ca2+). Our data show that ovine trachea is a good model for further characterization of the chloride-dependent cascade in airway epithelium.

A novel chloride-dependent GTP-utilizing protein kinase in plasma membranes from human respiratory epithelium.

The protein kinases that stimulate ion flux across airway epithelium are believed to utilize ATP as phosphate donor. Here we show that a chloride-sensitive protein kinase (in an apically enriched plasma membrane fraction from human nasal respiratory epithelium) uses guanosine 5'-triphosphate in preference to ATP as phosphate donor and is not inhibited by the protein kinase inhibitors staurosporine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, and N-(2-guanodinoethyl)-5-isoquinoline sulfonamide. This kinase phosphorylates a 37-kDa membrane protein (p37), which exhibits a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive phosphorylation peak at 40 mM Cl- (DIDS inhibition constant = 8 microM). p37 is additionally phosphorylated by an N-(2-guanodinoethyl)-5-isoquinoline sulfonamide-inhibitable protein kinase that uses ATP and shows a similar chloride sensitivity. The profile of membrane phosphoproteins generated by both kinases is also dependent on the source of Pi, the species of anion, and the concentration of anion. We propose a molecular mechanism for the transduction of Cl- concentration into a guanosine 5'-triphosphate-selective protein kinase signal and show that anion substitution alters the intensity of phosphorylation of membrane proteins in the absence of exogenously added protein kinases.