Epidemiology and prevention of surgical site infections after cardiac surgery.

Deep sternal wound infection is the major infectious complication in patients undergoing cardiac surgery, associated with a high morbidity and mortality rate, and a longer hospital stay. The most common causative pathogen involved is Staphylococcus spp. The management of post sternotomy mediastinitis associates surgical revision and antimicrobial therapy with bactericidal activity in blood, soft tissues, and the sternum. The pre-, per-, and postoperative prevention strategies associate controlling the patient's risk factors (diabetes, obesity, respiratory insufficiency), preparing the patient's skin (body hair, preoperative showering, operating site antiseptic treatment), antimicrobial prophylaxis, environmental control of the operating room and medical devices, indications and adequacy of surgical techniques. Recently published scientific data prove the significant impact of decolonization in patients carrying nasal Staphylococcus aureus, on surgical site infection rate, after cardiac surgery.

Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor.

Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture.

BACKGROUND: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts. METHODS: Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis. RESULTS: In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml. CONCLUSIONS: These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.

HIP/PAP gene, encoding a C-type lectin overexpressed in primary liver cancer, is expressed in nervous system as well as in intestine and pancreas of the postimplantation mouse embryo.

We originally isolated the HIP/PAP gene in a differential screen of a human hepatocellular carcinoma cDNA library. This gene is expressed at high levels in 25% of primary liver cancers but not in nontumorous liver. HIP/PAP belongs to the family of C-type lectins and acts as an adhesion molecule for hepatocytes. In normal adult human tissues, HIP/PAP expression is found in pancreas (exocrine and endocrine cells) and small intestine (Paneth and neuroendocrine cells). In order to gain insight into the possible role of HIP/PAP in vivo, we have investigated the pattern of HIP/PAP expression in the developing postimplantation mouse embryo by in situ hybridization. Detailed analysis of developing mouse embryos revealed that HIP/PAP gene exhibits a restricted expression pattern during development. Thus, HIP/PAP transcripts are first observed within the nervous system from day 14.5 onwards in trigeminal ganglia, dorsal root ganglia, and spinal cord where it appears to be an early specific marker of a subpopulation of motor neurons. At laster stages, HIP/PAP transcripts were detected in intestine and pancreas at day 16.5 but not in embryonic liver. This highly restricted expression pattern suggests that HIP/PAP might participate in neuronal as well as intestinal and pancreatic cell development.

Modifications of the antioxidant enzymes in relation to chromosome imbalances in human melanoma cell lines.

Five human melanoma cell lines were investigated for their antioxidant activities. These metabolic data were correlated with cytogenetic analysis giving the relative numbers of chromosomes or chromosomal segments carrying the gene encoding for each enzyme. Particular attention was focused on the expression of superoxide dismutase 2 (SOD2), whose gene, located on the long arm of chromosome 6 (6q), has been proposed as a tumour suppressor gene. The activity of glutathione peroxidase (GPX), glutathione reductase (GSR) and catalase appeared to be unrelated to the relative number of 3q, 8p and 11p arms which, respectively, carry their encoding genes. GPX activity paralleled that of total SOD activity, and GSR variations followed those of GPX, suggesting possible metabolic regulation. Both the activity and the amount of SOD1 immunoreactive protein correlated with the number of chromosomes 21, suggesting a gene dosage effect. The three cell lines with deletions of the 6q arm had lower SOD2 activity and less immunoreactive protein than the two cell lines without 6q deletion. In addition, they demonstrated high thymidine kinase and thymidylate synthetase activities, which are directly linked to the cell proliferation rate. These results strengthen the hypothesis that SOD2 has a function as a tumour suppressor gene, but also suggest that the expression of other antioxidant enzymes might be altered in human melanomas.

Strong decrease in biotin content may correlate with metabolic alterations in colorectal adenocarcinoma.

Short-chain fatty acids are an important source of energy for colonocytes. One of these is propionate, which is metabolized through carboxylation by propionyl-CoA carboxylase (PCC), an enzyme encoded by 2 genes, PCCA and PCCB. The co-factor of this reaction is biotin, a product of intestinal bacterial metabolism, as is propionate. Despite detailed knowledge about the metabolic effects and physiology of biotin, the relative amounts of this vitamin in normal colonic mucosae and in tumour tissue remains quite unknown. The biotin content in normal and cancerous cells from the distal digestive tract was examined on 10 pairs of tissue specimens of colorectal cancer and adjacent normal mucosae using reflectance in situ hybridization (RISH). Having observed a high biotin content in colon mucosae and a low content in colorectal-cancer cells, we then studied the transcription levels of PCCA and PCCB genes in 9 colorectal cancers and the corresponding mucosae. In all cases, the levels of mRNA were lower in colorectal cancers than in normal mucosae, the decrease being always more marked for PCCB than for PCCA. In normal mucosae and in adenocarcinoma cancer cells, PCCA and PCCB transcription levels were strongly related to the amount of biotin detected, but not to the number of chromosomes 13 (which carries PCCA) or 3 (which carries PCCB).

Suppression of apoptosis by overexpression of Bcl-2 or Bcl-xL promotes survival and mutagenesis after oxidative damage.

Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, and its close relative Bcl-xL, are both effective inhibitors of apoptosis induced by a wide variety of stimuli in many different cell types. In a previous study, we reported that suppression of apoptosis by Bcl-2 or Bcl-xL, markedly elevates the levels of radiation-induced mutations at the specific locus thymidine kinase. We investigated the effect of the Bcl-2 or Bcl-xL overproduction on hydrogen peroxide-induced mutagenesis. Oxidative DNA damage has been implicated in biological processes such as mutagenesis, carcinogenesis and aging. Overexpression of either Bcl-2 or Bcl-xL enhances oxidative stress mutagenesis in cells with wild type p53 as well as with mutated p53 protein. These results support the hypothesis that apoptosis plays a crucial role in maintaining genomic integrity by selectively eliminating highly mutated cells from the population.

HIP/PAP is an adhesive protein expressed in hepatocarcinoma, normal Paneth, and pancreatic cells.

Human hepatocarcinoma-intestine-pancreas (HIP) cDNA, isolated from a hepatocellular carcinoma, encodes a C-type lectin. According to published cDNA sequences, HIP protein is identical to human pancreatitis-associated protein (PAP). In these sequences, a putative signal peptide and the carbohydrate recognition domain (CRD) can be recognized. In the present study, we established transgenic mice to drive the production of soluble recombinant HIP/PAP protein in the milk of lactating animals; using this model, we showed that HIP/PAP protein was secreted after suitable cleavage of the potential signal peptide. Moreover, we also produced HIP/PAP protein by Escherichia coli cultures performed to generate specific antibodies. These antibodies enabled the detection of HIP/PAP protein in normal intestine and pancreas (both in endocrine and exocrine cells), e.g., intestinal neuroendocrine and Paneth cells, pancreatic islets of Langerhans, and acinar cells. HIP/PAP protein was also identified in the cytoplasm of tumoral hepatocytes but not in nontumoral hepatocytes. Finally, HIP/PAP protein activity was tested and we showed that HIP/PAP induced the adhesion of rat hepatocytes and bound strongly to extracellular matrix proteins (laminin-1, fibronectin), less strongly to type I and IV collagen, and not at all to heparan sulfate proteoglycan. In conclusion, these results showed that HIP/PAP protein was matured on secretion. We also demonstrated that HIP/PAP protein was specifically expressed in hepatocarcinoma cells and interacted with rat hepatocytes and the extracellular matrix. Taken overall, these results suggest that HIP/PAP protein may be of potential importance to liver cell differentiation/proliferation.

Suppression of apoptosis by Bcl-2 or Bcl-xL promotes susceptibility to mutagenesis.

Bcl-2 appears to contribute to neoplasia primarily by promoting cell survival, rather than by stimulating cellular proliferation. Bcl-2, and the related protein Bcl-xL, each suppress apoptosis induced by a wide variety of stimuli in many different cell types. Here we report that suppression of apoptosis by Bcl-2 or Bcl-xL markedly elevates the levels of radiation-induced mutations. This enhanced mutagenesis is the result of an increase in mutation frequency (mutations per survivor) together with a moderate increase in viability. Ectopic expression of either Bcl-2 or Bcl-xL enhances radiation mutagenesis in cells with wtp53. Surprisingly, we found that ectopic expression of Bcl-xL also promotes mutagenesis in p53- cells. These results support the hypothesis that apoptosis plays a crucial role in maintaining genomic integrity by selectively eliminating highly mutated cells from the population.

Structural organization and chromosomal localization of a human gene (HIP/PAP) encoding a C-type lectin overexpressed in primary liver cancer.

We previously identified, through differential screening of a human primary liver cancer library, a novel gene (named HIP) the expression of which is markedly increased in 25% of human primary liver cancers. HIP mRNA expression is tissue specific since it is restricted to pancreas and small intestine. HIP protein consists in a signal peptide linked to a carbohydrate-recognition domain (CRD), typical of C-type lectins without other binding domains. We have proposed that HIP and related proteins belong to a new family of C-type lectins. Drickamer [Drickamer, K. (1993) Curr. Opin. Struct. Biol. 3,393-400] included this group of proteins in his classification of C-type lectins as the free CRD (group VII) lectins. In the present report we describe the genomic organization and the chromosomal localization of HIP. We have shown that HIP is in fact the pancreatitis-associated protein (PAP) and provided a phylogenetic analysis of the free CRD lectins. Furthermore, the analysis of HIP/PAP gene indicates that the HIP/PAP CRD is encoded by four exons, a pattern shared with all members of this group of proteins. This common intron-exon organization indicates an ancient divergence of the free CRD-lectin group from other groups of C-type lectins. We provide evidence for the localization of HIP/PAP on chromosome 2, suggesting previous duplication of HIP/PAP and the related reg I alpha and reg I beta genes from the same ancestral gene. Finally, the sequence of the 5' upstream region of the HIP gene shows several potential regulatory elements which might account for the enhanced expression of the gene during pancreatic inflammation and liver carcinogenesis.

Overexpression of glutamine synthetase in human primary liver cancer.

BACKGROUND/AIMS: We have identified several clones specifically expressed during malignant cell proliferation by screening a complementary DNA library constructed from a human primary liver cancer with subtractive probes. One clone was identified as the glutamine synthetase (GS) transcript. Its expression is tightly regulated during development, especially in the hepatic lobule. Because this enzyme is involved in nitrogen homeostasis, it might contribute to tumor development/progression in primary liver cancer. METHODS: We compared the expression of GS messenger RNA (mRNA) and protein in tumorous and nontumorous liver from 34 patients with primary liver cancers, using a combination of Northern blot, dot blot, western blot, and determination of GS enzyme activity. RESULTS: GS mRNA was higher in tumors versus nontumors in 23 of 34 primary liver cancer samples. GS activity was higher in 6 of 8 selected primary liver cancer samples with high RNA levels. GS protein levels were proportional to enzyme activity. A major GS transcript of 2.8 kilobase was detected by Northern blotting and sequencing. This comprised the minor 1.8-kb transcript and a long 3' untranslated region; the latter contained an AT-rich zone, fully conserved in the chicken, mouse, and rat, which might be important for stability. CONCLUSIONS: Our results show an overexpression of GS in human primary liver cancers and, thus, point to its potential involvement in hepatocyte transformation.

Gene dosage and expression, and enzyme activity of thymidine kinase and thymidylate synthase in xenografted colorectal adenocarcinomas.

Cytogenetic studies performed on human colorectal tumors have revealed 2 specific patterns of chromosomal anomalies. The major pattern, known as the monosomic type (MT), is characterized by the loss or deletion of chromosomes 18, 17 (short arm 17p) and, less frequently, 1p, 4, 15, 5 (long arm 5q) and 21. The other one, known as the trisomic type (TT), is characterized by the gain of several chromosomes: 7, 12, X, 5 and 8. Losses of chromosome 18 and of the 17p arm never coexist in TT tumors. It was observed that many chromosome losses or deletions involved genes encoding for enzymes of the de novo pathways of nucleotide synthesis. In contrast, gains involved genes encoding for enzymes of the salvage pathways of the same metabolism. This led to the hypothesis that chromosome imbalances corresponded to those of nucleotide synthesis in tumor cells. Such an interrelation was confirmed by the dosage of thymidylate synthase (TS) and thymidine kinase (TK) activities in a series of colorectal grafted tumors. This study has been expanded to a larger series of xenografted tumors (23 cases) in which both TS and TK activities were studied, in parallel with an analysis of mRNA, by Northern blotting. The amount of mRNA was found to correlate with the number of gene copies calculated from cytogenetic data, indicating a direct gene-dosage effect. It also correlated with enzyme activities, but less strongly. This suggests the existence of an efficient post-transcriptional regulation, in particular for TS, whose level of expression varies over a wide range. Such variations may explain the diversity of responses to chemotherapy.

The human HIP gene, overexpressed in primary liver cancer encodes for a C-type carbohydrate binding protein with lactose binding activity.

HIP was originally identified as a gene expression in primary liver cancers, and in normal tissues such as pancreas and small intestine. Based on gene data base homologies, the HIP protein should consist of a signal peptide linked to a single carbohydrate recognition domain. To test this hypothesis HIP and the putative carbohydrate recognition domain encoded by the last 138 C-terminal amino acids, were expressed as glutathione-S-transferase proteins (GST-HIP and GST-HIP-142, respectively). Both recombinant proteins were purified by a single affinity purification step from bacterial lysates and their ability to bind saccharides coupled to trisacryl GF 2000M were tested. Our results show that HIP and HIP-142 proteins bind to lactose, moreover the binding requires divalent cations. Thus the HIP protein is a lactose-binding lectin with the characteristics of a C-type carbohydrate recognition domain of 138 amino acids in the C-terminal region.

Expression of thymidylate synthase gene in human colorectal carcinomas by northern blotting and reflectance in-situ hybridization (rish) - correlation with the number of chromosome-18.

The thymidylate synthase (TYMS) gene expression was analysed by Northern-blotting and by reflectance in situ hybridization (RISH) in human histological colorectal cancer. Scattering reflectance signals from 1-nm colloidal-gold particles after in situ hybridization, using digoxigenin-labeled probe, were quantified by confocal scanning laser microscopy. The importance of the RISH method is demonstrated by results obtained on colorectal cancer specimens. This study demonstrates a quantitative difference between cancer cells and normal colon epithelial cells in situ. In addition, TYMS expression varies from turner to tumor, in relation with karyotypic patterns. When the number of copies of chromosome 18, which carries the TYMS gene, are considered, there is a significant correlation with TYMS mRNA amounts, suggesting the existence of a gene dosage effect in colorectal cancer cells.

A novel gene (HIP) activated in human primary liver cancer.

Differential screening of a human hepatocellular carcinoma complementary DNA library using subtracted probes allowed us to identify a novel gene named HIP whose expression at the transcriptional level was elevated in liver tumors. The protein potentially encoded by the complementary DNA showed 68.5% identity with the bovine pancreatic thread protein and 49% identity with the human reg protein, which has been proposed as a pancreatic islet cell regenerating factor and is identical to the pancreatic stone or pancreatic thread protein. Sequence analysis suggests that the bovine pancreatic thread protein encoding gene is, in fact, the bovine homologue of the HIP gene. Furthermore, data base searches revealed a significant similarity of the HIP and pancreatic stone protein/pancreatic thread protein/reg sequences with the C-type lectin superfamily. The HIP sequence, like pancreatic stone protein/pancreatic thread protein/reg protein, consists of a single carbohydrate recognition domain linked to a signal peptide which would be involved in secretion of the protein. HIP mRNA was expressed at a high level in the tumors of seven of 29 hepatocellular carcinomas. In contrast, HIP mRNA was not detected in nontumorous adjacent areas or in normal adult and fetal liver, suggesting that HIP could be involved in liver cell proliferation or differentiation. HIP mRNA expression is tissue specific, since it is present in the normal small intestine and pancreas, while it could not be evidenced in colon, brain, kidney, or lung. In summary, our results show the existence of a novel family within the superfamily of C-type lectin which may be involved in liver, pancreatic, and intestinal cell proliferation or differentiation.

Insulin-like growth factor II (IGF-II) mRNA expression during hepatocarcinogenesis in transgenic mice.

Insulin-like growth factor II (IGF-II) mRNA expression is developmentally regulated in liver tissue. We previously observed the reexpression of fetal IGF-II mRNAs in human primary liver cancer and in surrounding cirrhotic tissue. In order to determine the steps of liver cancer progression where the activation of IGF-II fetal mRNAs occurs, we analyzed IGF-II mRNA expression during hepatocarinogenesis in transgenic mice carrying an antithrombin III-SV40 early region hybrid gene. The comparative analysis of mRNAs encoding IGF-II and other differentiation-associated proteins, as well as histological analysis, indicate that the reexpression of fetal IGF-II mRNAs takes place in specific steps of liver cancer progression, both in early pretumorous lesions and in well-differentiated hepatocellular carcinomas.

Expression of insulin-like growth factor II, alpha-fetoprotein and hepatitis B virus transcripts in human primary liver cancer.

Insulin-like growth factor II is a fetal growth factor structurally and functionally related to insulin and insulin-like growth factor I. Its mRNA expression is developmentally regulated in human liver, the reexpression of insulin-like growth factor II fetal transcripts being often observed in primary liver cancer. Insulin-like growth factor II and alpha-fetoprotein mRNAs were studied in 16 human primary liver cancers, most of which were highly differentiated. Hepatitis B virus transcripts were also analyzed in the tumors from hepatitis B virus chronic carriers. alpha-Fetoprotein mRNA was detected in only four tumors and in one nontumorous cirrhotic tissue; all these samples also displayed insulin-like growth factor II fetal transcripts. Furthermore, fetal insulin-like growth factor II mRNAs were observed in five tumors and six nontumorous cirrhotic areas not expressing alpha-fetoprotein mRNA. The presence of hepatitis B virus RNA was only observed in tissues not expressing alpha-fetoprotein or fetal insulin-like growth factor II mRNA. In conclusion, fetal insulin-like growth factor II transcripts are more frequently observed than alpha-fetoprotein mRNA in highly differentiated liver cancers and in surrounding cirrhotic areas. The reexpression of fetal insulin-like growth factor II transcripts might then be a marker of early steps of liver cell transformation.

Expression of insulin-like growth factor II (IGF-II) in human primary liver cancer: mRNA and protein analysis.

Insulin-like growth factor II (IGF-II) is a polypeptide growth factor thought to be involved in fetal tissue development. We previously showed an increased expression of IGF-II mRNA in human primary liver cancer. The present investigation was undertaken to characterize the overexpressed IGF-II transcripts and to determine whether they are translated into protein. Two cDNAs with distinct 5' untranslated regions, corresponding to IGF-II transcripts expressed in fetal liver, were isolated from a primary liver cancer. Complete nucleotide sequence analysis showed an identical open reading frame of 540 bp, encoding a predicted polypeptide identical to the IGF-II isolated from serum. An increased synthesis of IGF-II protein was demonstrated by a protein-binding assay in tumorous liver samples, the highest levels being found in primary liver cancers with the highest IGF-II steady state level. By contrast, serum IGF-II content was low in most of primary liver cancer cases analyzed. Altogether, the results indicate reexpression of IGF-II both at the mRNA and protein levels in primary liver cancer. This finding is consistent with IGF-II being a marker of liver cell differentiation. In addition, this growth factor might be involved in liver cancer progression by an autocrine and/or paracrine mechanism.

Differential expression of insulin-like growth factor II mRNA in human primary liver cancers, benign liver tumors, and liver cirrhosis.

We investigated insulin-like growth factor II (IGF-II) mRNA in three groups of human liver samples including primary liver cancers, benign liver tumors and cirrhosis; indeed these pathological conditions would allow us to distinguish between different steps in liver carcinogenesis. A 40- to 100-fold increase in IGF-II mRNA was shown in 9/40 of the liver cancer samples as compared to normal adult liver. RNA blot analysis using both IGF-II cDNA and oligonucleotide probes showed the reexpression of two fetal (6 and 5 kilobases) IGF-II transcripts in primary liver cancers and in some cirrhotic adjacent tissues; these included all the samples with enhanced IGF-II expression. By contrast the adult (5.3 kilobases) IGF-II transcript was identified in most of the benign liver tumors and liver cirrhosis; in addition, in some of these samples, the 5-kilobase fetal transcript was also detected. The increase of IGF-II mRNA in some liver cancers is consistent with an autocrine mechanism conferring a selective growth advantage to tumorous liver cells. Furthermore, these results indicate a differential expression of IGF-II transcripts in nonmalignant hepatocyte proliferation (benign liver tumors and cirrhosis) as compared to liver cancer. Finally this study suggests that, in liver cirrhosis and in some benign liver tumors, premalignant proliferative states might be identified by the presence of IGF-II fetal transcripts.