Automated ribotyping to distinguish the different non Sau/ non Sep staphylococcal emerging pathogens in orthopedic implant infections.

Several species belonging to Staphylococcus genus, other than Staphylococcus aureus and Staphylococcus epidermidis (non Sau/ non Sep species), exhibit increasing abilities as opportunistic pathogens in the colonisation of periprosthetic tissues. Consequently, the availability of means for accurate identification is crucial to assess the pathogenic characteristics and to clarify clinical relevance of the individual species. Here, 146 clinical staphylococcal isolates belonging to non Sau/ non Sep species from prosthesis-associated orthopedic infections were analyzed by conventional enzymatic galleries and by automated ribotyping. Twelve different species were recognised: S. capitis, S. caprae, S. cohnii, S. equorum, S. haemolyticus, S. hominis, S. lugdunensis, S. pasteuri, S. sciuri, S. simulans, S. warneri, S. xylosus. Ribotype identifications were compared with the phenotypes obtained by the Api 20 Staph system and/or ID 32 Staph system. ID 32 Staph profiles were more consistent with ribotyping results than Api Staph profiles. Across the different staphylococcal species investigated, correct identifications with Api Staph were 45%, while with ID 32 Staph they were 59%. It has, however, to be mentioned that ID 32 Staph was mostly applied to discriminate unmatched ribotyping and Api Staph identifications, thus to a subpopulation of strains with ""atypical"" metabolic profile. Automated ribotyping provided a correct identification for 91% of the isolates. These results confirm automated ribotyping as a convenient rapid technique, still subject to improvements, which will accurately and rapidly recognise the newly emerging staphylococcal pathogens in implant-related orthopedic infections.

Biliary sludge: the sluggish gallbladder.

Biliary sludge is a mixture of particulate matter which has precipitated from bile. It generally consists of cholesterol monohydrate crystals, calcium bilirubinate or other calcium salts. In a clinical setting, biliary sludge is almost always an ultrasonographic diagnosis. Although it is less clinically applicable, direct microscopic examination of gallbladder bile is far more sensitive than ultrasonography into sludge detection, and has to be regarded as the diagnostic gold standard. The overall prevalence of sludge in the general population is relatively low. However, several clinical conditions are associated with a particularly high prevalence of biliary sludge, including pregnancy, rapid weight loss, total parenteral nutrition, octreotide therapy, bone marrow or solid organ transplantation. The clinical course of biliary sludge varies, and complete resolution, a waxing and waning course, and progression to gallstones are all possible outcomes. It may cause complications usually associated with gallstones, such as biliary colic, acute cholecystitis, and acute pancreatitis. The main pathogenic mechanism involved in sludge formation is probably gallbladder dismotility, and in selected patients measures aimed to maintain adequate gallbladder contractions has been shown to effectively prevent sludge development.

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.

[Acute liver failure in chronic hepatic disease. Clinico-therapeutic evaluation]. / Insufficienza epatica acuta in corso di epatopatia cronica. Inquadramento clinico-terapeutico.

Chronic liver diseases are potentially evolving clinical situations which, independently by the etiology, could proceed towards progressive liver structural and functional impairments. The only efficient treatment is orthotopic liver transplantation. Chronic liver diseases, and up to 40% of liver cirrhosis, are initially asymptomatic, but cirrhosis is the most frequent cause of death among non-neoplastic digestive diseases. Important elements complicating a decompensated liver cirrhosis are ascites, hepatic encephalopathy, digestive bleeding and jaundice. Acute liver failure (ALF) is the expression of a clinical state, that is common to many conditions sharing severe liver structural and functional impairments. In patients affected by decompensated liver cirrhosis, ALF could be triggered by several factors, while the death is caused by bleeding episodes, hepato-renal syndrome, spontaneous bacterial peritonitis or hepatocarcinoma. In patients affected by chronic liver diseases, the diagnosis of ALF is based on progressively increasing jaundice, encephalopathy and coagulopathy. Recent clinical trials have evaluated the efficacy of extrahepatic liver support systems, either artificial or bio-artificial, in treating episodes of ALF in chronic liver patients. The preliminary results indicate a potential use of such systems in blood detoxification, but they also showed limits in increasing patient survival.

Review article: gall-bladder motor function in diabetes mellitus.

Although some controversy exists, diabetic patients generally are thought to have a two- to threefold increased risk of cholesterol gallstones. From previous studies there is no convincing evidence for a supersaturated bile in diabetics, whereas several reports indicate that impaired gall-bladder emptying could be one of the important factors in the increased incidence of gallstones in diabetics. However, studies of gall-bladder motility in diabetics have yielded conflicting results, probably because of substantial heterogeneity in the patients studied, emptying stimulus and technique used to assess gall-bladder motor function. The mechanism of the gall-bladder emptying abnormality in diabetics is not completely understood, although it has been proposed that it could represent a manifestation of denervation caused by visceral neuropathy. Based on normal post-prandial cholecystokinin release, it can be ruled out that impaired cholecystokinin release is the mechanism responsible for reduced gall-bladder emptying in diabetics. Other possible explanations for impaired gall-bladder contraction in diabetics include a decreased sensitivity of the smooth muscle of the gall-bladder to plasma cholecystokinin, and/or decreased cholecystokinin receptors on the gall-bladder wall.

Influence of polyethylene terephthalate on the release of growth factors by human endothelial cells.

The influence of polyethylene terephthalate (PET) on the release of platelet derived growth factor AB (PDGF-AB) and basic fibroblast growth factor (bFGF) by in vitro cultured human endothelial cells was assessed by enzyme immunoassay. No significant differences were observed in the production of PDGF-AB with respect to the negative control cultures. A significant increase was observed in the production of bFGF after 48 and 72 h with respect to the negative control cultures. It can be concluded that PET may induce an increase in the production of basic FGF in endothelial cells.

Short-term low-dose pantoprazole-based triple therapy for cure of Helicobacter pylori infection in duodenal ulcer patients.

BACKGROUND: The eradication of Helicobacter pylori infection has been achieved using various therapy regimens, but the efficacy of the proton-pump inhibitor pantoprazole as part of these regimens has not yet been widely tested. AIM: To evaluate the efficacy and tolerability of a 1-week low-dose pantoprazole-based triple therapy in patients with H. pylori-positive duodenal ulcer. METHODS: In an open single-centre prospective study, 71 patients with endoscopically proven active duodenal ulcer and H. pylori infection received pantoprazole 40 mg o.m. for 4 weeks, and during the first week a combination antimicrobial treatment comprising tinidazole 500 mg b.d. plus clarithromycin 250 mg b.d. H. pylori eradication was defined as concordant negative histology and rapid urease test performed at endoscopy 4-6 weeks after the end of treatment, confirmed 4 weeks later by 13C-urea breath test. RESULTS: Sixty-six patients (93%) completed the trial and five patients were lost to follow-up. H. pylori infection was cured in 61 out of the 66 patients who completed the trial (per-protocol analysis: 92.4%, 95% CI: 83.2-97.5%; intention-to-treat analysis: 85.9%, 95% CI: 75.7-93.0%). At final endoscopy, 65 out of 66 patients had healed ulcer (98.5%). Mild adverse events occurred in six patients (9.1%). CONCLUSIONS: One-week low-dose pantoprazole-based triple therapy is a simple, effective and well-tolerated regimen for ulcer healing and H. pylori eradication in patients with duodenal ulcer.

Adhesive protein expression on human endothelial cells after in vitro contact with woven Dacron.

In this research adhesive proteins are studied in order to evaluate the interference of woven Dacron in the endothelialization process and in the ability of endothelial cells to bind circulating leucocytes. Endothelial cells from human umbilical vein (HUVEC) were put in contact with woven Dacron for 24 h. PECAM-1, ELAM-1, ICAM-1 and VCAM-1 expression was then evaluated by flow cytometry, using indirect immunofluorescence reaction with monoclonal antibodies. The study of adhesive proteins was completed with the quantitative determination of surface antigens expressed as the antibody binding capacity (ABC). Antigenic density was calculated by the DAKO QFIT calibration system for indirect immunofluorescence. After contact with woven Dacron no significant change was observed in the percentage of positive cells or in the fluorescence intensity of the adhesins. No significant variation was also noted by calculating the surface antigen density by means of calibration fluorospheres. It can be concluded that the material examined does not significantly affect leucocyte adhesion to the endothelium.

Flow-cytometric analysis of leukocyte activation induced by polyethylene-terephthalate with and without pyrolytic carbon coating.

Leukocyte activation is one test for the evaluation of blood-materials interaction. The expression of adhesion molecules analyzed by flow cytometry provides a simple method to evaluate leukocyte activation by biomaterials: any change in these molecules can be predictive of the inflammatory activity of the materials. In this study the contact between leukocytes and uncoated polyethylene terephthalate or pyrolytic carbon-coated polyethylene terephthalate (PET and PET-PC, respectively) was inspected by analyzing whether the expression of some adhesion molecules involved in leukocyte activation, namely LFA-1 (CD11a/ CD18), Mac-1/CR3 (CD11b/CD18), and LECAM-1 (CD62L) can be modified. By flow cytometry expression of the adhesion molecules can be studied separately on lymphocytes and myeloid cells. The materials tested reduced the total numbers of both leukocytes and neutrophils, although not significantly. Neither PET nor PET-PC changed the expression of the adhesion molecules in lymphocytes: this suggests that no specific immune response is stimulated. On the contrary, statistically significant changes were observed for monocytes and granulocytes: the percentage of cells expressing Mac-1 and the density of such antigens on cell membranes increased while the percentage of LECAM-1 positive cells decreased. Similar changes were observed when the cells underwent the inflammatory stimulus provided by an in vitro challenge with bacterial endotoxin. Our results demonstrated that polyethylene terephthalate activates leukocytes by modifying the expression in neutrophils of the molecules involved in the early phase of the inflammatory response. Even after coating PET with pyrolytic carbon, the ability of this material to activate circulating leukocytes was maintained.

Cell death induced by metal ions: necrosis or apoptosis?

We have evaluated if the cytotoxic effects of metals released from implants are due to necrosis or apoptosis. Peripheral blood mononuclear cells were exposed to different concentrations of chromium, nickel and cobalt extracts and the characteristics of both apoptosis and necrosis were evaluated by flow-cytometry at different culture endpoints. In order to define the prevalence of apoptosis or necrosis, the ratio cell death/apoptosis was calculated. A ratio of 1 indicates the acute toxicity of the tested substance (necrosis). The extracts of chromium, cobalt and nickel had a cytotoxic effect on the mononuclear cells; high concentrations of cobalt and nickel produced cell necrosis, whereas by lowering the extract concentration apoptotic phenomena were observed. High chromium concentrations can induce cell death by apoptosis. Our data suggest that when large amounts of nickel and cobalt are released from implanted metal devices, necrosis is produced and consequently a strong inflammatory tissue reaction is likely to occur. The release of either chromium or limited amounts of nickel and cobalt induces toxicity characterized by apoptotic phenomena, which allows an adaptation of the tissue to the implant.

CD62, thromboxane B2, and beta-thromboglobulin: a comparison between different markers of platelet activation after contact with biomaterials.

The authors examined the modifications of some markers of platelet activation after contact with biomaterials. Glycoprotein GMP-140 (CD62) was evaluated by flow cytometry; beta-thromboglobulin (beta-TG) and thromboxane B2 (TXB2) were determined by radioimmunoassay. Polyethylene terephthalate (PET) induced a remarkable platelet adhesion and a significant increase in beta-TG and TXB2, with no increase in CD62 on the nonadherent platelets. Pyrolytic carbon-coated PET (PC) did not induce platelet adhesion after 15 min of contact, but a significant increase in CD62 was detected. After 30 min a significant increase in platelet adhesion as well as the release of beta-TG and TXB2 were noted. The increase was lower than that observed for uncoated PET, and after 30 min of contact with PC the increase no longer was observed.

[In vitro production of endothelin-1 and prostacyclin by cultured endothelial cells in the presence of polymers]. / Produzione in vitro di endotelina-1 e di prostaciclina da parte di cellule endoteliali coltivate in presenza di polimeri.

The aim of this study was to evaluate endothelin-1 and prostacyclin production by human endothelial cells cultured in the presence of polyethylene terephthalate and collagen-coated PET. Cell counting and the assay of endothelin-1 and 6-keto-prostaglandin F1 alpha, stable metabolite of prostacyclin, were carried out after 48 hour contact of the cells with the examined materials. Endothelial cell contact with uncoated PET caused a significant reduction in cell number, a significant increase in the production of endothelin-1 and a not significant increase in 6-keto-prostaglandin F1 alpha. The endothelial cell contact with collagen-coated PET caused a highly significant decrease in cell number and a not significant decrease in endothelin-1 and 6-keto-prostaglandin F1 alpha. It was concluded that PET causes both a decrease in cell number and a remarkable increase in endothelin-1. On the contrary, collagen-coated PET determines a decrease in cell number and a slight reduction of endothelin-1 and 6-keto-prostaglandin F1 alpha.

Expression of adhesion molecules on endothelial cells after contact with knitted Dacron.

The aim of this study is to evaluate the expression of some adhesion molecules on the surface of endothelial cells cultured in contact with knitted Dacron. These molecules, as mediators of cell adhesion, could play a role in the modulation of adhesion on the biomaterials, therefore conditioning the response of tissues to implant. Twenty different cultures of human umbilical vein endothelial cells (HUVECs) were cultured in contact with knitted Dacron. Both HUVECs grown without the material and HUVECs incubated with endotoxin were used as control. After 24 h, the cell adhesion molecules PECAM-1, ELAM-1, ICAM-1 and VCAM-1 were evaluated on the cells by monoclonal antibodies and flow cytometry. After 24 h of contact with knitted Dacron, a significant decrease in the proportion of cells expressing PECAM-1 was observed, as well as a significant increase in the proportion of cells expressing ELAM-1. The contact with knitted Dacron did not induce significant variations of ICAM-1 and VACM-1. The incubation with endotoxin determined a significant increase in the proportion of ELAM-1-positive cells, a significant increase in ICAM-1 fluorescence intensity, and a significant increase both in fluorescence intensity and in the proportion of VCAM-1-positive cells. The results obtained with the endotoxin are in agreement with those reported in the literature. The ELAM-1 increase, observed after contact with knitted Dacron, could favour leucocyte adhesion, while the decrease in PECAM-1 expression could result from an inhibiting effect on the endothelial cell adhesion so as to hinder the mechanisms involved in the endothelialization of the material. The variations were interpreted as inhibiting endothelialization and favouring the leucocyte adhesion effect by knitted Dacron.

In vitro complement activation after contact with pyrolytic carbon-coated and uncoated polyethylene terephthalate.

This study was undertaken to evaluate whether the pyrolytic carbon coating of polyethylene terephthalate induces complement activation. Complement activation induced by pyrolytic carbon-coated polyethylene terephthalate (PET+PC) in comparison with uncoated polyethylene terephthalate (PET) was assessed on whole blood collected with heparin. The activation of the classic pathway was evaluated by C4d fragment enzyme immunoassay. The activation of the alternative pathway was evaluated with Bb fragment enzyme immunoassay. The results show that uncoated PET activates the alternative pathway, but not the classic one. PET+PC does not induce complement activation, not even through the alternative pathway. Pyrolytic carbon coating therefore contributes to improving blood compatibility.

Cytokine production and adhesive protein expression by endothelial cells after contact with polyethylene terephthalate.

The authors have evaluated adhesive protein expression and cytokine production by human umbilical vein endothelial cells cultured in contact with polyethylene terephthalate (PET). ELAM-1, ICAM-1 and VCAM-1 expression was determined by flow cytometry; the concentration of interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in the supernatant was determined by enzyme immunoassay. The contact with PET determined a significant increase in ELAM-1 expression and insignificant increase in cytokine production, demonstrating that PET had a limited capability to stimulate endothelial cells in a pro-inflammatory sense.

In vitro assessment of phagocytosis of bovine collagen by human monocytes/macrophages using a spectrophotometric method.

The use of a wound dressing with covering and haemostatic properties significantly improves wound healing. In this study, a lyophilized bovine collagen sponge used for the treatment of wounds and ulcerae has been tested in a cell culture system. Phagocytosis of collagen fragments by human blood monocytes/macrophages has been investigated. For the assessment of collagen ingestion by mononuclear phagocytes, a picrosirius dye specific for collagen molecules has been used. By adapting this histochemical technique to microplate cell culture system, replicate monocyte cultures are assayed. Collagen content is determined by evaluating spectrophotometrically at 540 nm the absorbance of a sirius red/picric acid solution. Using this simple and sensitive method, the phagocytosis of bovine collagen by LPS-stimulated monocytes/macrophages has been ascertained.

Activation of the plasma coagulation system induced by some biomaterials.

The ability of some biomaterials to activate plasma coagulation system was examined in vitro. After contact of platelet-rich plasma with biomaterials, some markers of the thrombin formation, i.e., fragment 1 + 2 and fibrinopeptide A, and some inhibitors of the blood coagulation mechanism were tested. Fragment 1 + 2 and fibrinopeptide A were found to be increased by all of the materials, though to a different extent. In particular, fragment 1 + 2 and fibrinopeptide A were significantly increased upon contact with polybutylene terephthalate and with collagen coated polyethylene terephthalate, respectively. Also antithrombin III was shown to decrease following exposure to biomaterials, but statistical significance was found only for polyethylene terephthalate and polyvinylacetate. As a results of this wide range of variability in the parameters, it is advisable to explore the plasma coagulation system with a multiparametric approach in which thrombin formation and coagulation inhibitors are thoroughly investigated.