Pathobiology of familial hypercholesterolemic atherosclerosis

Many factors play a role in the development of atherosclerotic lesions. One of the leading risk factors for development of atherosclerosis is familial hypercholesterolemia (FH). FH is a genetic disease characterized by a deficiency, and/or mutation, of receptors for low density lipoprotein (LDL) on the plasmalemma of endothelial cells (EC), a high level of low density lipoprotein in the plasma, and early, spontaneous development of atherosclerosis and skin xanthoma. In this review we describe Watanabe heritable hyperlipidemic (WHHL) rabbits, which represent such an animal model for human FH. This strain of the rabbits is characterized by a genetic deficiency or mutation of functional LDL receptors and develops severe atherosclerosis, which is pathologically similar to familial homozygous hyperlipidemic patients. The most completely characterized animal model is the Watanabe rabbit, a model of homozygous and heterozygous type IIa hypercholesterolemia related to an LDL receptor deficiency. Additional manipulation such as aortic injury in this rabbit model induces the development of atherosclerotic lesions that are structurally similar to those found in humans. Thus, this model of hypercholesterolemia fulfils the above criteria set, i.e. it is able to provide new insights for a better understanding of the pathogenesis of atherosclerosis and for testing new treatment strategies.     

Guidelines for the appropriate use of genetic tests in infertile couples

Research on genetic causes of male and female infertility rapidly expanded in the last years, following the development of in vitro fertilising techniques. Genetic tests are now available to explore the cause of the infertility and assess the risk of a given couple to transmit its genetic characteristics. This allows at-risk couples to take an informed decision when electing for a medically assisted reproduction. It also allows the professionals to offer a prenatal diagnosis when appropriate. Thus, the genetic work-up of the infertile couple has become good practice for an appropriate diagnosis, treatment and prognostic assessment. The lack of national or international rules for the genetic approach to the infertile couple, prompted the Italian community of professionals in the field of reproductive medicine to join and set up guidelines for the genetic diagnosis of male and female infertility. The group of clinical and research experts is representative of 12 national scientific societies and was supported by external experts from four international societies. We examine the clinically relevant genetic causes of male and female infertility and suggest the category of patients for which each genetic test is recommended or optional, both for an accurate diagnosis and prior to ART.     

Localization of Hepatocyte Growth Factor and Its Receptor met in Endocrine Cells and Related Tumors of the Gut and Pancreas: An Immunohistochemical Study

Hepatocyte growth factor (HGF), a stimulator of angiogenesis and cell migration, regulates the growth of a wide variety of cells by binding to its high-affinity receptor met and is involved in the growth and aggressiveness of several tumors. In this study we investigated the expression of HGF and met in normal endocrine cells and related neoplasms of the gut and pancreas to verify their possible role in tumor pathogenesis, growth, and aggressiveness. Normal tissues and 60 different endocrine tumors were immunostained using specific antibodies directed against HGF, met, and various hormones. HGF immunoreactivity (IR) was found in antroduodenal G cells, rectal enterochromaffin (EC) cells, and pancreatic A and B cells, whereas met IR was detected in antral EC and G cells, and in pancreatic B cells; 46 of 60 tumors examined were positive for HGF, and they were mainly represented by ECL-, EC-, and L-cell neoplasms. met IR was identified in 50/60 tumors of various phenotypes. HGF and met coexpression was found in 42/60 cases, most of which were represented by EC-cell tumors. HGF/met coexpression was significantly more frequent in ileolonic EC-cell tumors, which in the majority of cases were malignant, than in appendiceal EC-cell tumors, which were all benign. Our results demonstrated, for the first time, that HGF and met are specifically distributed in normal gut and pancreatic endocrine cells and, in addition, suggest that HGF and met may be implicated as autocrine/paracrine factors regulating the growth of gastroenteropancreatic endocrine tumors, mainly of ileocolonic EC-cell carcinoids.     

Sodium hypochlorite-, chlorine dioxide- and peracetic acid-induced genotoxicity detected by the Comet assay and Saccharomyces cerevisiae D7 tests

Mutagenicity of drinking water is due not only to industrial,agricultural and urban pollution but also to chlorine disinfectionby-products. Furthermore, residual disinfection is used to providea partial safeguard against low level contamination and bacterialre-growth within the distribution system. The aims of this studywere to further evaluate the genotoxic potential of the worldwide used disinfectants sodium hypochlorite and chlorine dioxidein human leukocytes by the Comet assay and in Saccharomycescerevisiae strain D7 (mitotic gene conversion, point mutationand mitochondrial DNA mutability, with and without endogenousmetabolic activation) and to compare their effects with thoseof peracetic acid, proposed as an alternative disinfectant.All three disinfectants are weakly genotoxic in human leukocytes(lowest effective dose 0.2 p.p.m. for chlorine dioxide, 0.5p.p.m. for sodium hypochlorite and peracetic acid). The resultsin S.cerevisiae show a genotoxic response on the end-pointsconsidered with an effect only at doses higher (5- to 10-fold)than the concentration normally used for water disinfection;sodium hypochlorite and peracetic acid are able to induce genotoxiceffects without endogenous metabolic activation (in stationaryphase cells) whereas chlorine dioxide is effective in growingcells. The Comet assay was more sensitive than the yeast tests,with effective doses in the range normally used for water disinfectionprocesses. The biological effectiveness of the three disinfectantson S.cerevisiae proved to be strictly dependent on cell-specificphysiological/biochemical conditions. All the compounds appearto act on the DNA and peracetic acid shows effectiveness similarto sodium hypochlorite and chlorine dioxide. ¹Author to whom correspondence should be addressed. Tel: +39 0521 905608; Fax: +39 0521 905604; Email: mutgen{at}unipr.it Received on September 22, 2003; revised and accepted on November 27, 2003     

Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones

The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria. We have investigated different effects (i.e. growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (rho degrees ) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH). The data confirmed the importance of oxygen production for doxorubicin toxicity. The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin. Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation. Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants. Epirubicin induced levels of cytotoxicity similar to those of doxorubicin. The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450. GSH did not induce an evident alteration. Neither were genotoxic effects induced. Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants. All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450. Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH. However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation. The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH.     

Evaluation of the BDProbeTec ET system for direct detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary samples: a multicenter study

We evaluated the BDProbeTec ET system (Becton Dickinson, Sparks, Md.), a strand displacement amplification-based technique, for direct detection of Mycobacterium tuberculosis in 867 clinical samples. Of 294 extrapulmonary specimens, 52 had positive results by both BDProbeTec ET and culture and 209 had negative results by both methods; sensitivity and specificity were 76.5 and 95.9%, respectively. After resolution of discrepancies, the sensitivity rose to 77.8%.     

Microsatellite instability and p53 expression in gallbladder carcinomas.

We studied the MSI (microsatellite instability) status and p53 expression in a series of 71 gallbladder cancers (GCs) of different histologic type. All neoplasms were examined combining a microsatellite analysis at mononucleotide locus BAT-26 and an immunohistochemical study for hMSH2, hMLH1, and p53 proteins and markers of gastric and intestinal differentiation. All the 71 GCs were MSS (microsatellite stable). The p53 protein was found in 100% of undifferentiated GCs, 67% of conventional gallbladder adenocarcinomas, 50% of mucinous adenocarcinomas, and 20% GCs with squamous differentiation. All 71 MSS tumors showed presence of immunohistochemical expression of both hMLH1 and hMSH2 gene products. We concluded that microsatellite instability does not play a role in the developing of GC while p53 seems to be the most important alteration found in a large proportion of these cancers, with the only exception of mucinous and squamous gallbladder carcinomas.