Effect of temperature on the shift of Pseudomonas fluorescens from an environmental microorganism to a potential human pathogen.

Pseudomonas fluorescens is a Gram-negative bacterium generally considered of scarce clinical significance. However, in the last few years, the isolation of P. fluorescens as the causative agent of nosocomial infections has rapidly increased. P. fluorescens is a psychrophile microorganism which grows at an optimal temperature of 25-30 degrees Celcius. In spite of this constraint, it has recently been reported that the human physiological temperature does not appear to be a barrier for this microorganism. In this study we examined the ability of P. fluorescens, grown at 28 degrees C or at 37 degrees C, to adhere to cultured human A549 pulmonary cells and to form biofilm. The ability of P. fluorescens to induce expression of proinflammatory cytokines, beta-defensin 2 and the intercellular adhesion molecule-1 was also investigated. Our results clearly indicate that inflammatory mediators are induced when the microorganism is grown at a lower temperature, while biofilm is formed only at 37 degrees C. The results presented are consistent with previous reports indicating P. fluorescens as an opportunistic pathogen and underscore the urgent need for further studies to better characterize the virulence of this microorganism.

Effects of toluidine blue-mediated photodynamic therapy on periopathogens and periodontal biofilm: in vitro evaluation.

Photodynamic therapy (PDT) is a selective modality of killing targeted cells, mostly known for its application in neoplasms. PDT can be considered to be an alternative method for the elimination of periodontal bacteria from the pocket without harms for the resident tissues. Therefore, PDT may replace systemic antibiotics and enhance the effect of mechanical treatments of periodontal defects. This effort focused on the in vitro sensitization of periopathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia ) Toluidine Blue mediated and on the use of a Diode laser emitting source. The objective of this research was to evaluate the bactericidal in vitro effect of laser diodes 830 nm (as the light source) after photosensitization with Toluidine Blue (TBO) on the following periopathogenic bacteria: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia. After evaluating the effect on the single bacterial strain, the ability of Diode Laser to disrupt the structure of biofilms produced by A. actinomycetemcomitans after photosensitization with TBO was also analyzed. The study suggests that the association of TBO and diode laser light 830 nm is effective for the killing of bacteria strains and determines the photoinactivation of Aggregatibacter biofilms. In summary, photodynamic therapy has effectively shown its capabilities and, therefore, it can be considered a valid alternative approach to antimicrobial therapy of periodontitis.

The Helicobacter pylori's protein VacA has direct effects on the regulation of cell cycle and apoptosis in gastric epithelial cells.

In this study, we have evaluated the effects on cell cycle regulation of VacA alone and in combination with other two Helicobacter pylori proteins, cytotoxin-associated protein (CagA) and HspB, using the human gastric epithelial cells (AGS). Our results indicate that VacA alone was able to inhibit the G1 to S progression of the cell cycle. The VacA capacity of inhibiting cell progression from G1 to S phase was also observed when cells were co-transfected with CagA or HspB. Moreover, VacA over-expression caused apoptosis in AGS cells through activation of caspase 8 and even more of caspase 9, thus indicating an involvement of both the receptor-mediated and the mitochondrial pathways of apoptosis. Indeed, the two pathways probably can co-operate to execute cell death with a prevalence of the mitochondrial pathways. Our data taken together provide additional information to further enhance our understanding of the molecular mechanism by which H. pylori proteins alter the growth status of human gastric epithelial cells.

Inhibitory effect of SV-IV, a major protein secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes.

The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical correlates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 X 10(-7) M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.

Porins from Salmonella typhimurium accelerate human blood coagulation in vitro by selective stimulation of thrombin activity: implications in septic shock DIC pathogenesis.

The effect of porins, major hydrophobic outer membrane proteins purified from Salmonella typhimurium, on human blood coagulation was investigated. It was found that micromolar concentrations of porins accelerated markedly human blood coagulation in vitro. Using appropriate experiments, data were obtained showing that the main target of the porin-induced procoagulant effect was thrombin. A possible binding of porins with thrombin has been suggested to be the basis of this effect. The implications of this finding in the pathogenesis of the disseminated intravascular coagulation syndrome (DIC) occurring during the Gram-negative septic shock is discussed.

3-O-methylfunicone, a secondary metabolite produced by Penicillium pinophilum, induces growth arrest and apoptosis in HeLa cells.

3-O-Methylfunicone (OMF) is a secondary metabolite produced by the soil fungus Penicillium pinophilum which has cytostatic properties. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to any anti-proliferative and apoptotic potential, on HeLa cells. OMF treatment caused about 44% inhibition of cell growth after 24 h, and modifications in the tubulin fibre organization. In addition, a significant increase in p21 mRNA expression and a decrease in cyclin D1 and Cdk4 mRNA expression resulted at the same time. Apoptosis induction was demonstrated by the annexin V assay, cytofluorimetric analysis of the DNA content of the sub-G1 fraction and DNA laddering. Taken together, our data showed that the compound inhibits proliferation of HeLa cells by several mechanisms, such as disruption of tubulin fibres, cell cycle arrest and apoptosis induction. The capacity of the compound to affect the cell cycle and to modulate apoptosis is indicative of a potential for the development of a new agent for cancer chemotherapy.

UVA irradiation induces L-isoaspartyl formation in melanoma cell proteins.

It has been reported that UVA effects are partly mediated by production of reactive oxygen species. Moreover, oxidative stress increases protein damage, involving the occurrence of isoaspartyl residues, a product of protein deamidation/isomerization reactions. This work was undertaken in order to study the effects of UVA irradiation, mediated by oxidation, on sensitive protein targets. Melanoma cells exposed to UVA rays have been chosen as a model for monitoring the occurrence of L-isoaspartyl sites. A dramatic increase of these abnormal residues, specifically recognized and methylated by the enzyme L-isoaspartate(D-aspartate) O-methyltransferase (PCMT; EC 2.1.1.77), can be detected after exposure of M14 cells to raising doses of UVA. The effect of UVA on NO and TBARS accumulation, as well as on DNA fragmentation, has also been investigated. NO formation parallels the increase in isoaspartyl formation, while lipid peroxidation occurs only at the highest UVA doses. No DNA fragmentation has been detected under the employed experimental conditions. These results, as a whole, indicate that protein damages are one of the early events on UVA-induced cell injury. The endogenous activity of PCMT remains remarkably stable under UVA treatment, suggesting that this enzyme might play a crucial role in the repair and/or disposal of damaged proteins in UVA-irradiated cells.

Properties of Yersinia enterocolitica porins: interference with biological functions of phagocytes, nitric oxide production and selective cytokine release.

We have extracted and purified Yersinia enterocolitica ATCC 9610 porins that have molecular weights of 36-38 kDa. They inhibited phagocytosis and phagosome-lysosome fusion (30%) in human monocytes and caused enhanced nitrite production. Preincubation of polymorphonuclear neutrophils with porins (1-10 micrograms/ml/10(6) cells) induced a reduction in chemotaxis, adherence to nylon wool and chemiluminescence. Human lymphomonocytes treated with Y. enterocolitica porins showed a distinctive cytokine profile. Interleukin-1 alpha, interleukin-6 and tumour necrosis factor alpha were released within 3-6 h, while interleukin-8, gamma interferon and granulocyte-macrophage colony stimulating factor were released after 18 h. Interleukin-3 and interleukin-4 were not detected at up to 48 h of incubation. In conclusion, these immunomodulating and histotropic properties may account for Y. enterocolitica infection and its sequelae.

Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells. Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV. The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed.

Protein A and other surface components of Staphylococcus aureus stimulate production of IL-1 alpha, IL-4, IL-6, TNF and IFN-gamma.

Studies were carried out on the ability of protein A (PA) and of muramic acid (MA) from S. aureus to induce the release of cytokines both from monocytes and lymphocytes in vitro. Results show that protein A induces the greatest activity, compared to the activity already known for the theicoic acid (TA) and for muramyl dipeptide (MDP). At concentration of 10 micrograms/ml; PA induces roughly +180% release of TNF with respect to controls, while release of IL-1 alpha is about 500% control values, and is higher than those obtained when cells are treated with TA and MDP; IL-6 release is higher than that stimulated by Con A, used as standard challenge. At PA concentrations of 5 micrograms/ml, IL-4 release is about five times higher than that induced by Con A. Release of IFN-gamma showed similar dose-dependent stimulations. Muramic acid (MA) is particularly active in inducing the release of cytokines from target cells, inducing TNF release of about +75% with respect to the controls. This increase is less than that obtained with PA. Also IL-4 and IFN-gamma are released by PA in quantities higher than those induced by TA and MDP. Our results lead us to believe that during infections by Gram-positive bacteria, their surface components are able to induce a series of chain reactions ranging from the inflammatory to the immunologic responses which are also conditioned by release of cytokines.

Isolated autoimmune response: definition, analysis of the prevalence in an outpatient rheumatology clinic, relationship to pre-clinical autoimmune disease and infections by hepatotropic viruses.

OBJECTIVE: To define the clinical significance of non-organ specific autoantibody positivity in patients in whom routine clinical and laboratory examinations did not detect any disease that might have caused the serological finding. METHODS: Out of 1,120 patients consecutively admitted to an outpatient rheumatology clinic, 28 were referred for the evaluation of an autoantibody positivity unrelated to the clinical status. These patients and 28 sex- and age-matched controls underwent a specific work-up with the aim of detecting any underlying infection or autoimmune disease. RESULTS: Eight of the 28 patients (28.5%) were found to be affected by a previously undetected disease: 3 chronic hepatitis C, 3 Sjögren's syndrome, and 2 autoimmune thyroiditis. The remaining 20 did not show any autoimmune or hepatic disease, although 4 of them showed active infection by HBV (n = 1) or HGV (n = 3) and 15 had had a previous infection by hepatotropic viruses (HBV, CMV or EBV). After a follow-up lasting 6-54 months, none of the last 20 patients developed any autoimmune or chronic hepatic disease. CONCLUSIONS: A diagnostic work-up is necessary in patients presenting with unexpected autoantibody positivity in order to detect an underlying pre-clinical autoimmune disease and/or unexpected hepatic infection. Patients in whom such a work-up fails to point out any condition should be further followed in order to make an early diagnosis of autoimmune disease.

Ketoconazole inhibits lipopolysaccharide-induced activation of the nitric oxide synthase gene in the murine macrophage cell line J774.

The aim of this study was to determine whether ketoconazole can affect the expression of the nitric oxide (NO) synthase gene in the murine macrophage cell line J774. The inducible enzyme (i-NOS) is activated in murine macrophages by LPS and cytokines. Exposure of the J774 cell line to ketoconazole for 24 h did not induce any NO release. Cells preincubated with ketoconazole and treated with LPS showed a significant decrease in nitrite levels in the culture medium, compared with controls (cells treated with LPS alone). The addition of 1 mM N-monomethyl-L-arginine (L-NMMA), a structural analogue of arginine, reduced nitrite levels by about 88+/-9.2% in cells treated with LPS alone, whereas in those treated with ketoconazole + LPS, the levels were comparable to the baseline values detected in control cells. Northern blotting, used to assess i-NOS mRNA expression in the J774 cells, showed that ketoconazole reduced the LPS-induced increase in i-NOS mRNA activation by about 50%. These results support another mechanism for the antiinflammatory effect of ketoconazole (i.e. reduction in i-NOS gene expression and consequently inhibition of reactive radical NO production), that may explain the antierythema and antiedema action of this compound, besides its antimycotic effects.

Bilateral femoral osteomyelitis with knee arthritis due to Salmonella enteritidis in a patient with systemic lupus erythematosus.

A bilateral knee septic arthritis due to Salmonella enteritidis developed in a female patient affected by long-standing systemic lupus erythematosus (SLE) with cardiac and renal involvement treated with immunosuppressants and corticosteroids. Because avascular necrosis and multiple osteomyelitic areas were detected at the same time in both right and left femoral condyles, an early localisation of Salmonella into the bone was assumed. Involvement of the joints was regarded as consequence of local dissemination of infection. Ampicilline (0.2 g/kg body weight daily for 2 months) plus ciprofloxacin (1.5 g daily for 12 months) and withdrawal of immunosuppressants appeared to be effective in preventing complications of infection.

Pulmonary disposition of lomefloxacin in patients with acute exacerbation of chronic obstructive pulmonary disease. A multiple-dose study.

In this study we have measured the concentrations of lomefloxacin at steady state in serum and in the intrapulmonary region at specified intervals for 24 h following administration of the last dose of drug in patients suffering from acute exacerbation of chronic obstructive pulmonary disease (COPD). Twenty subjects were enrolled. They received lomefloxacin 400 mg orally once-daily for 5 consecutive days. All patients were divided into five groups, with 4 subjects in each group, according to sampling times (2, 4, 8, 12, and 24 h after the last dose). At bronchoscopy, bronchial biopsies and bronchoalveolar lavage (BAL) were performed. At 12 h after the last dose, serum concentration of lomefloxacin was >1.0 microg/mL and at 24 h it was still detectable, but, at all times, the concentrations in bronchial secretion, bronchial mucosa, and epithelial lining fluid (ELF) were greater than the concentrations in serum [bronchial secretions (pg/mL) = 2.5+/-1.2; 2.2+/-1.0: 2.0+/-1.1; 1.8+/-1.1; 0.6+/-0.3. bronchial mucosa (microg/g) = 5.9+/-2.1; 6.2+/-1.8; 2.6+/-2.2; 1.9+/-1.5; 1.0+/-0.9. ELF (microg/mL) = 6.9+/-2.8; 5.9+/-2.6; 3.1+/-1.9; 2.2+/-1.0; 0.8+/-1.3. serum (microg/mL) = 3.2+/-1.4; 2.8+/-0.9: 2.1+/-1.5; 1.2+/-1.1; 0.4+/-0.81. We must stress that we observed a large inter-individual variability in concentrations. Our data show that lomefloxacin once-daily induces high and sustained concentrations in the various potential sites of pulmonary infection and clearly indicate that the pharmacokinetic behavior of this fluoroquinolone permits once-daily administration in patients with acute exacerbations of COPD.

[The role of anaerobe adhesion in the colonization of the vaginal mucosa]. / L'aderenza degli anaerobi nella colonizzazione della mucosa vaginale.

Some our previous research works on bacterial adhesion to vaginal cells in the different phases of the menstruum showed that adhesion changes depending on changing environmental conditions. We therefore considered interesting to extend our investigations to anaerobic flora, in the light of recent observations intended to attribute an important role to anaerobic flora in the pathogenesis of vaginitis. The results obtained so far indicate that the maximum adhesion capability is found in the middle of the menstruum. The very low adhesion of bacteria belonging to the Leptothrix genus remains substantially unaltered throughout the menstruum. Low adhesion is also found in sporogenic bacteria, whereas the coccoid ones have a stronger adhesion, particularly about the middle of the menstruum. With lower pH values adhesion of the anaerobic flora is enhanced, whereas in the final phase of the menstruum, with higher pH values, adhesion is reduced. Competition tests evidence a stronger adhesion of coccoid as compared to bacillar types.

[Neutrophil function and microbial associations in rapidly progressing periodontitis and chronic periodontitis in the adult]. / Funzionalità dei neutrofili e associazioni microbiche nella parodontite rapidamente progressiva e nella parodontite cronica dell'adulto.

Our study was focused on the functional characteristics of neutrophil leukocytes (PMN) and the subgingival microflora in two different forms of periodontal disease: 1) adult periodontitis (AP); 2) rapidly progressive periodontitis (RPP). Our study dealt with the functional characteristics of neutrophil leukocytes in the gingival fluid and in the peripheral blood. These were found markedly reduced in the RPP group, while, in the AP group, they were comparable to those of a healthy control group. No difference between local and systemic values was detected. Moreover, some samples of subgingival plaque were taken from two groups of patients, affected by AP and RPP respectively. The above samples showed a predominance of Gram-negative flora over Gram-positive flora, and of anaerobic flora over the aerobic one, and the predominance of specific pathogens in each of the two forms of periodontal disease. The subgingival plaque samples taken at the end of the periodontal treatment from five out of ten patients affected by RPP showed inverse ratios, as well as the absence of the previously detected pathogens. The findings underline the relevance of tests of leukocytes functionality and that of microbiological analysis to allow correct diagnosis of dubious forms of periodontal disease and the checking of the posttreatment results.

Modification of osteopontin and MMP-9 levels in patients with psoriasis on anti-TNF-α therapy.

Psoriasis is a chronic skin inflammatory disease in which a pleiotropic cytokine, tumor necrosis factor alpha (TNF-α), plays a central role, as demonstrated by the clinical success of anti-TNF-α therapy. Among the multiple effects of TNF-α on keratinocytes, the induction of matrix metalloproteinase-9 (MMP-9), a collagenase implicated in joint inflammation, might be one of the key mechanisms in psoriasis pathogenesis. Interestingly, MMP-9 expression can be enhanced also by osteopontin (OPN), a glycosylated protein whose levels are increased in skin and peripheral blood mononuclear cells (PBMC) of psoriasis patients. The aim of the current study is to investigate the relationship between OPN, MMP-9 and TNF-α in psoriasis. Our survey identified high levels of both OPN and MMP-9 in PBMC as well as skin of psoriatic patients with respect to healthy controls. Significant reduction of OPN and MMP-9 levels in PBMC, plasma and lesional skin of psoriasis patients was observed after 24 weeks of anti-TNF-α therapy. Moreover, OPN and MMP-9 were enhanced by TNF-α and down-regulated by anti-TNF-α treatment in healthy PBMC. These findings may suggest that OPN and MMP-9 may be regulated by TNF-α, indicating a possible role in the pathogenesis of psoriasis.

3-O-methylfunicone, from Penicillium pinophilum, is a selective inhibitor of breast cancer stem cells.

OBJECTIVES: Cancer stem cells make up a subpopulation of cells within tumours that drive tumour initiation, growth and recurrence. They are resistant to many current types of cancer treatment, causing failure of such therapeutic approaches, including chemotherapy and radiotherapy. In the study described here, anti-proliferative effects of 3-O-methylfunicone (OMF), a metabolite from Penicillium pinophilum, were investigated on human breast cancer MCF-7 cells and cancer stem cells selected as mammospheres derived from MCF-7s. MATERIALS AND METHODS: Stemness markers were analysed on isolated mammospheres showing positive expression of CD24, CD29, CD44, CD133, CD184 and CD338. Cell proliferation and apoptosis were analysed by flow cytometry and RT-PCR. Cell colony formation assays were performed to evaluate colony formation of mammospheres. RESULTS AND CONCLUSION: OMF treatment affected both MCF-7 and mammosphere growth, inducing apoptosis. In addition, OMF strongly reduced stemness markers and survivin, hTERT and Nanog-1 gene expression. Growth of colonies in soft-agar was significantly affected by OMF treatment, too. Lastly, we tested ability of MCF-7 cells to form mammospheres after treatment with OMF or cisplatin, demonstrating that OMF treatment resulted in drastic reduction in number of mammospheres. These results introduce OMF as an effective molecule in suppressing breast cancer stem cells.

Hepatocyte growth factor (HGF) modulates Leydig cell extracellular matrix components.

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays multiple roles during mammalian development. We previously demonstrated that in the postnatal testes, the HGF receptor, c-met, is expressed by Leydig cells and HGF increases the steroidogenetic activity of the cells. In the present article, we report that HGF modifies the composition of the extracellular matrix of cultured Leydig cells. We show that HGF increases the metabolic activity of isolated Leydig cells; in particular, the factor increases urokinase plasminogen activator and matrix metalloproteinase 2 secretion. We have also shown that the levels of active transforming growth factor beta are increased by HGF. On the contrary, using the Western blotting technique, a strong reduction in the amount of fibronectin present in the culture medium of cells cultured in the presence of HGF has been detected. The presented data demonstrate that HGF modulates several functional activities of Leydig cells, further supporting the hypothesis that this factor has a relevant role in the regulation of mammalian spermatogenesis.