This is the second annual report of an international collaborative research group that is examining the cellular impact of laryngopharyngeal reflux (LPR) on laryngeal epithelium. The results of clinical and experimental studies are presented. Carbonic anhydrase (CA), E-cadherin, and MUC gene expression were analyzed in patients with LPR, in controls, and in an in vitro model. In patients with LPR, we found decreased levels of CAIII in vocal fold epithelium and increased levels in posterior commissure epithelium. The experimental studies confirm that laryngeal CAIII is depleted in response to reflux. Also, cell damage does occur well above pH 4.0. In addition, E-cadherin (transmembrane cell surface molecules, which have a key function in epithelial cell adhesion) was not present in 37% of the LPR laryngeal specimens. In conclusion, the laryngeal epithelium lacks defenses comparable to those in esophageal epithelium, and these differences may contribute to the increased susceptibility of laryngeal epithelium to reflux-related injury.

Esophageal epithelium has intrinsic antireflux defenses, including carbonic anhydrases (CAs I to IV) that appear to be protective against gastric reflux. This study aimed to investigate the expression and distribution of CA isoenzymes in laryngeal epithelium. Laryngeal biopsy specimens collected from the vocal fold and interarytenoid regions were analyzed by Western blotting and immunofluorescence. Carbonic anhydrases I and II were expressed by the majority of samples analyzed. In contrast, CA III was differentially expressed in the interarytenoid samples and was not detected in any vocal fold samples. The expression of CA III was increased in esophagitis as compared to normal esophageal tissue. Carbonic anhydrase I and III isoenzymes were distributed cytoplasmically in the basal and lower prickle cell layers. The laryngeal epithelium expresses some CA isoenzymes and has the potential to protect itself against laryngopharyngeal reflux. Laryngeal tissue may be more sensitive to injury due to reflux damage than the esophageal mucosa because of different responses of CA isoenzymes.

SUMMARY The value of serum bile acids (SBA) in the diagnosis of hepatobiliary disease has been investigated. A modified GLC method was used, with an overall coefficient of variation of + 11 % in the control range. Serum was obtained after a 12 hour fast, and two hours after a fatty meal from 73 patients and 14 control subjects. In controls the total fasting SBA of 2 17 + 0-86 ,umol/l increased significantly (P < 0 001) to 3-81 + 1-14 umol/l after a meal. All icteric patients had raised SBA, but in 23 anicteric patients there was no significant difference in the detection of chronic liver disease by fasting SBA, postprandial SBA, AST, or y GTP. Compared with controls, serum in patients contained proportionately less deoxycholic acid (P < 0 001), there was proportionately more cholic acid in extrahepatic obstruction (P < 0O001), and proportionately more chenodeoxycholic acid in patients with cirrhosis, viral hepatitis, and neoplasia (P < 0001). In control subjects, the fasting cholic:chenodeoxycholic acid ratio ranged from 0*5-1*0, and differed significantly (P < 0O001) from patients with extrahepatic obstruction 0-96-3'6, and cirrhosis 0 1-0 5. It is concluded that serum bile acids measured by sensitive methods can provide useful diagnostic information.

Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial‐induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53‐mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA‐induced apoptotic cell death, and can attenuate DCA‐mediated increases in intracellular free calcium. Deletion of the highly conserved EF‐hand calcium‐binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell‐stress protein SEP53 can function as a modifier of the DCA‐mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.

SUMMARY The characteristics of cholesterol uptake by 83 human gall bladders (obtained at cholecystectomy) were studied with a modified Ussing technique. Real and artificial biles labelled with 14C-cholesterol and 3H-dextran (the latter to correct for adherent mucosal bile) were used; all gall bladders absorbed cholesterol (average 3 5 nmol/cm2/minute). Recovery of the absorbed cholesterol from the tissue showed that about 4% was esterified over 60 minutes. In artificial bile the rate of absorption of cholesterol increased as the bile saturation index rose, but became constant once supersaturation was achieved. In contrast, supersaturated real bile permitted greater absorption of cholesterol, possibly due to enhanced cholesterol solubilisation.Preincubation of gall bladder tissue in sodium cyanide (5 mM) caused a 30% reduction in cholesterol uptake indicating that, although absorption is predominantly a "passive" process, there is a partial "active" component. There were no pronounced differences in the rate of cholesterol absorption as gall bladders became more diseased, but there was a reduction in the amount of cholesterol ester formed.Cholesterolosis of the human gall bladder is common1 2 and is characterised by the accumulation of free3 and esterified4 cholesterol and triglycerides5 in the mucosa of the gall bladder wall. The most common symptoms are colicky epigastric pain and nausea, but vomiting and intolerance to specific foods also occur.' The aetiology of this condition is poorly understood but is believed to be associated with absorption of cholesterol from the bile.' 5 -7 Both the guinea pig8 and human gall bladder9 '0 have been shown to absorb small but noticeable amounts of cholesterol from bile, and patients with cholesterolosis have a high biliary cholesterol content.5 7