Tuula Kiviluoto

Randomised trial of laparoscopic versus open cholecystectomy for acute and gangrenous cholecystitis

BACKGROUND Laparoscopic cholecystectomy (LC) has become the treatment of choice for elective cholecystectomy, but controversy persists over use of this approach in the treatment of acute cholecystitis. We undertook a randomised comparison of the safety and outcome of LC and open cholecystectomy (OC) in patients with acute cholecystitis. METHODS 63 of 68 consecutive patients who met criteria for acute cholecystitis were randomly assigned OC (31 patients) or LC (32 patients). The primary endpoints were hospital mortality and morbidity, length of hospital stay, and length of sick leave from work. Analysis was by intention to treat. Suspected bile-duct stones were investigated by preoperative endoscopic retrograde cholangiography (LC group) or intraoperative cholangiography (OC group). FINDINGS The two randomised groups were similar in demographic, physical, and clinical characteristics. 48% of the patients in the OC group and 59% in the LC group were older than 60 years. 13 patients in each group had gangrene or empyema, and one in each group had perforation of the gallbladder causing diffuse peritonitis. Five (16%) patients in the LC group required conversion to OC, in most because severe inflammation distorted the anatomy of Calots triangle. There were no deaths or bile-duct lesions in either group, but the postoperative complication rate was significantly (p=0.0048) higher in the OC than in the LC group: seven (23%) patients had major and six (19%) minor complications after OC, whereas only one (3%) minor complication occurred after LC. The postoperative hospital stay was significantly shorter in the LC than the OC group (median 4 [IQR 2-5] vs 6 [5-8] days; p=0.0063). Mean length of sick leave was shorter in the LC group (13.9 vs 30.1 days; 95% CI for difference 10.9-21.7). INTERPRETATION Even though LC for acute and gangrenous cholecystitis is technically demanding, in experienced hands it is safe and effective. It does not increase the mortality rate, and the morbidity rate seems to be even lower than that in OC. However, a moderately high conversion rate must be accepted.

Gene expression in human NAFLD

Despite the high prevalence of nonalcoholic fatty liver disease (NAFLD), little is known of its pathogenesis based on study of human liver samples. By the use of Affymetrix GeneChips (17,601 genes), we investigated gene expression in the human liver of subjects with extreme steatosis due to NAFLD without histological signs of inflammation (liver fat 66.0 +/- 6.8%) and in subjects with low liver fat content (6.4 +/- 2.7%). The data were analyzed by using sequence-based reannotation of Affymetrix probes and a robust model-based normalization method. We identified genes involved in hepatic glucose and lipid metabolism, insulin signaling, inflammation, coagulation, and cell adhesion to be significantly associated with liver fat content. In addition, genes involved in ceramide signaling (MAP2K4) and metabolism (UGCG) were found to be positively associated with liver fat content. Genes involved in lipid metabolism (PLIN, ACADM), fatty acid transport (FABP4, CD36), amino acid catabolism (BCAT1), and inflammation (CCL2) were validated by real-time PCR and were found to be upregulated in subjects with high liver fat content. The data show that multiple changes in gene expression characterize simple steatosis.

Genes Involved in Fatty Acid Partitioning and Binding, Lipolysis, Monocyte/Macrophage Recruitment, and Inflammation Are Overexpressed in the Human Fatty Liver of Insulin-Resistant Subjects

OBJECTIVE—The objective of this study is to quantitate expression of genes possibly contributing to insulin resistance and fat deposition in the human liver. RESEARCH DESIGN AND METHODS—A total of 24 subjects who had varying amounts of histologically determined fat in the liver ranging from normal (n = 8) to steatosis due to a nonalcoholic fatty liver (NAFL) (n = 16) were studied. The mRNA concentrations of 21 candidate genes associated with fatty acid metabolism, inflammation, and insulin sensitivity were quantitated in liver biopsies using real-time PCR. In addition, the subjects were characterized with respect to body composition and circulating markers of insulin sensitivity. RESULTS—The following genes were significantly upregulated in NAFL: peroxisome proliferator–activated receptor (PPAR)γ2 (2.8-fold), the monocyte-attracting chemokine CCL2 (monocyte chemoattractant protein [MCP]-1, 1.8-fold), and four genes associated with fatty acid metabolism (acyl-CoA synthetase long-chain family member 4 [ACSL4] [2.8-fold], fatty acid binding protein [FABP]4 [3.9-fold], FABP5 [2.5-fold], and lipoprotein lipase [LPL] [3.6-fold]). PPARγ coactivator 1 (PGC1) was significantly lower in subjects with NAFL than in those without. Genes significantly associated with obesity included nine genes: plasminogen activator inhibitor 1, PPARγ, PPARδ, MCP-1, CCL3 (macrophage inflammatory protein [MIP]-1α), PPARγ2, carnitine palmitoyltransferase (CPT1A), FABP4, and FABP5. The following parameters were associated with liver fat independent of obesity: serum adiponectin, insulin, C-peptide, and HDL cholesterol concentrations and the mRNA concentrations of MCP-1, MIP-1α, ACSL4, FABP4, FABP5, and LPL. CONCLUSIONS—Genes involved in fatty acid partitioning and binding, lipolysis, and monocyte/macrophage recruitment and inflammation are overexpressed in the human fatty liver.

Hepatic Stearoyl-CoA Desaturase (SCD)-1 Activity and Diacylglycerol but Not Ceramide Concentrations Are Increased in the Nonalcoholic Human Fatty Liver

OBJECTIVE—To determine whether 1) hepatic ceramide and diacylglycerol concentrations, 2) SCD1 activity, and 3) hepatic lipogenic index are increased in the human nonalcoholic fatty liver. RESEARCH DESIGN AND METHODS—We studied 16 subjects with (n = 8) and without (n = 8) histologically determined nonalcoholic fatty liver (NAFL+ and NAFL−) matched for age, sex, and BMI. Hepatic concentrations of lipids and fatty acids were quantitated using ultra-performance liquid chromatography coupled to mass spectrometry and gas chromatography. RESULTS—The absolute (nmol/mg) hepatic concentrations of diacylglycerols but not ceramides were increased in the NAFL+ group compared with the NAFL− group. The livers of the NAFL+ group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL− group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r = 0.70, P = 0.003) and the hepatic lipogenic index (r = 0.54, P = 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r = 0.71, P = 0.002) but not ceramides (r = 0.07, NS). CONCLUSIONS—We conclude that diacylglycerols but not ceramides are increased in NAFL. The human fatty liver is also characterized by depletion of long polyunsaturated fatty acids in the liver and increases in hepatic SCD1 and lipogenic activities.

Comparison of Lipid and Fatty Acid Composition of the Liver, Subcutaneous and Intra-abdominal Adipose Tissue, and Serum

Ceramides may mediate saturated fat–induced insulin resistance, but there are no data comparing ceramide concentrations between human tissues. We therefore performed lipidomic analysis of human subcutaneous (SCfat) and intra‐abdominal (IAfat) adipose tissue, the liver, and serum in eight subjects. The liver contained (nmol/mg tissue) significantly more ceramides (1.5–3‐fold), sphingomyelins (7–8‐fold), phosphatidylethanolamines (10–11‐fold), lysophosphatidylcholines (7–12‐fold), less ether‐linked phosphatidylcholines (2–2.5‐fold) but similar amounts of diacylglycerols as compared to SCfat and IAfat. The amounts of ceramides and their synthetic precursors, such as palmitic (16:0) free fatty acids and sphingomyelins, differed considerably between the tissues. The liver contained proportionally more palmitic, stearic (18:0), and long polyunsaturated fatty acids than adipose tissues. Stearoyl‐CoA desaturase 1 (SCD1) activity reflected by serum, estimated from the 16:1/16:0‐ratio, was closely related to that in the liver (r = 0.86, P = 0.024) but not adipose tissues. This was also true for estimated elongase (18:1/16:1, r = 0.89, P = 0.01), and Δ5 (20:4/20:3, r = 0.89, P = 0.012) and Δ6 (18:3[n‐6]/18:2, r = 1.0, P < 0.001) desaturase activities. We conclude that the human liver contains higher concentrations of ceramides and saturated free fatty acids than either SCfat or IAfat.

Integrated gene copy number and expression microarray analysis of gastric cancer highlights potential target genes

We performed an integrated array comparative genomic hybridization (aCGH) and expression microarray analysis of 8 normal gastric tissues and 38 primary tumors, including 25 intestinal and 13 diffuse gastric adenocarcinomas to identify genes whose expression is deregulated in association with copy number alteration. Our aim was also to identify molecular genetic alterations that are specific to particular clinicopathological characteristics of gastric cancer. Distinct molecular genetic profiles were identified for intestinal and diffuse gastric cancers and for tumors obtained from 2 different locations of the stomach. Interestingly, the ERBB2 amplification and gains at 20q13.12‐q13.33 almost exclusively discriminated intestinal cancers from the diffuse type. In addition, the 17q12‐q25 gain was characteristic to cancers located in corpus and the 20q13.12‐q13.13 gain was more common in the antrum. Statistical analysis was performed using integrated copy number and expression data to identify genes showing differential expression associated with a copy number alteration. Genes with the highest statistical significance included ERBB2, MUC1, GRB7, PPP1R1B and PPARBP with concomitant changes in copy number and expression. Immunohistochemical analysis of ERBB2 and MUC1 on a tissue microarray containing 78 independent gastric tissues showed statistically significant differences (p < 0.05 and <0.001) in immunopositivity in the intestinal (31 and 70%) and diffuse subtypes (14 and 41%), respectively. In conclusion, our results demonstrate that intestinal and diffuse type gastric cancers as well as cancers located in different sites of the stomach have distinct molecular profiles which may have clinical value.

Infectivity-Enhanced Adenoviruses Deliver Efficacy in Clinical Samples and Orthotopic Models of Disseminated Gastric Cancer

Purpose: Metastatic gastric cancer remains a common and devastating disease without curative treatment. Recent proof-of-concept clinical trials have validated gene therapy with adenoviruses as an effective and safe modality for the treatment of cancer. However, expression of the primary coxsackie-adenovirus receptor is variable in advanced cancers, and therefore, the use of heterologous receptors could be advantageous. Experimental Design: Here, we used capsid-modified adenoviruses for increasing the transduction and subsequent antitumor efficacy. 5/3 chimeric viruses have a serotype 3 knob which allows binding to a receptor distinct from coxsackie-adenovirus receptor. The fiber of Ad5lucRGD is modified with an integrin-targeted motif. Polylysine motifs, pK7 and pK21, bind to heparan sulfates. Oncolytic adenoviruses replicate in and kill tumor cells selectively. Gastric cancer cell lines and fresh clinical samples from patients were infected with transductionally targeted viruses. Capsid-modified oncolytic adenoviruses were used in cell killing experiments. To test viral transduction and therapeutic efficacy in vivo, we developed orthotopic mouse models featuring i.p. disseminated human gastric cancer, which allowed the evaluation of biodistribution and antitumor efficacy in a system similar to humans. Results: Capsid modifications benefited gene transfer efficiency and cell killing in gastric cancer cell lines and clinical samples in vitro and in vivo. Modified oncolytic adenoviruses significantly increased the survival of mice with orthotopic gastric cancer. Conclusions: These preclinical data set the stage for the clinical evaluation of safety and efficacy in patients with disease refractory to current modalities.

Basement membrane laminin‐5 is deposited in colorectal adenomas and carcinomas and serves as a ligand for α3β1 integrin

Interplay between laminin‐5 (Ln‐5) and its integrin (Int) receptors α2β1, α3β1 and α6β4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immuno‐reactivity for chains of Ln‐5 (α3‐β3‐γ2) and Ln‐10 (α5‐β1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln‐5 appeared to accumulate along the invading edges of carcinomas, while Ln‐10 was mostly absent. Immunoreactivity for Ln α1 chain, a component of Lns‐1 and ‐3, was not seen in adenomas or carcinomas. Immunoreactivity for α2, α6, β1 and β4 Ints was found in all tumors and that for α3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln‐5. Immunoblotting of carcinoma tissues showed that the γ2 chain of Ln‐5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln‐5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function‐blocking MAbs to α3 and β1 Int subunits, but not those to Int α2 or α6 subunits, significantly inhibited the adhesion of cells to Ln‐5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln‐5, produced by carcinoma cells, is a major BM component and the cells use the α3β1 Int complex for adhesion to Ln‐5.

Intracellular pH in isolated Necturus duodenal mucosa exposed to luminal acid

Regulation of intracellular pH in gastric epithelial surface cells exposed to luminal acid was investigated in isolated Necturus antral mucosa using microelectrode technique. Exposure of the mucosa to luminal pH 2 acidified intracellular pH from 7.21 +/- 0.01 to 6.95 +/- 0.04 (N = 50). Removal of Na+ from the perfusates or addition of amiloride (1 mM) to serosal perfusate (containing HCO3-) had no influence on intracellular pH during exposure to pH 2 (N = 6), but removal of HCO3-/CO2 from or addition of 4, acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.5 mM) to the serosal perfusate (containing Na+) acidified intracellular pH from 7.02 +/- 0.03 to 6.45 +/- 0.15 (p less than 0.01, N = 10) and from 6.97 +/- 0.06 to 6.58 +/- 0.26 (p less than 0.01, N = 6), respectively, in 15 min. In tissues exposed to mucosal pH 6, epithelial surface pH was about 1.3 pH units higher than pH of the mucosal bulk solution. Removal of Cl-/HCO3- from the serosal perfusate acidified epithelial surface pH by about 0.5 pH units (p less than 0.01, N = 6), suggesting that serosal HCO3- sustains intracellular pH, at least in part, by generating an alkaline buffer layer at the epithelial surface. In the absence of HCO3-/CO2, a stable intracellular pH was obtained when the tissue was exposed to mucosal pH 2.7, but in this situation intracellular pH was sensitive to Na+ removal or amiloride addition, intracellular pH decreasing from 7.00 +/- 0.07 to 6.48 +/- 0.10 (p less than 0.01, N = 6) and from 6.86 +/- 0.06 to 6.32 +/- 0.01 (p less than 0.01, N = 7), respectively, in 15 min. The data suggest that in gastric epithelium exposed to luminal acid, physiological intracellular pH is primarily maintained by the buffer action of serosal HCO3- transported to the epithelial surface to impede the entry of luminal H+ into mucosal tissue. Removal of the sheltering HCO3- unmasks a second line, Na(+)-dependent and amiloride-sensitive intracellular pH regulatory mechanism, presumably a Na+/H+ antiport.

Cytokeratin profile suggests metaplastic epithelial transformation in Barrett's oesophagus

Cytokeratins are subunit proteins of epithelial cell intermediate filaments, which are genetically determined. Because epithelia have their own characteristic cytokeratin profile, this may reveal the origin of the epithelium. The cytokeratin profile of Barretts oesophagus, complicating severe gastro-oesophageal reflux disease, was determined in 35 consecutive patients and in 10 normal controls in order to provide insight into the origin of Barretts epithelium. Immunostaining of frozen sections showed abundant immunoactivity for cytokeratin (CK) 13, which is characteristic of squamous epithelia, including that of the oesophagus, but is not present in the simple columnar epithelium of the cardia. On the other hand, the latter epithelium expresses mainly CK 8, 18 and 19, also found in Barretts epithelium. The presence of CK 13 in Barretts epithelium may indicate its origin from the squamous oesophageal epithelium and not from the proximal migration of columnar epithelial cells of the gastric cardia.