Proteomic peptide scan of porphyromonas gingivalis fima type ii for searching potential b-cell epitopes.

PURPOSE: To identify potential antigenic targets for Porphyromonas gingivalis vaccine development. MATERIALS AND METHODS: In the present study, we analyzed the Porphyromonas gingivalis, fimA type II primary amino acid sequence and characterized the similarity to the human proteome at the pentapeptide level. RESULTS: We found that exact peptide-peptide profiling of the fimbrial antigen versus the human proteome shows that only 19 out of 344 fimA type II pentapeptides are uniquely owned by the bacterial protein. CONCLUSIONS: The concept that protein immunogenicity is allocated in rare peptide sequences and the search the Porphyromonas gingivalis fimA type II sequence for peptides unique to the bacterial protein and absent in the human host, might be used in new therapeutical approaches as a significant adjunct to current periodontal therapies.

Assessment of host defence mechanisms induced by Candida species.

Some species of Candida are opportunistic pathogens that can cause disease in a host immunocompromised by underlying local or systemic pathological processes. C. albicans is the species most often associated with oral lesions, but other species of Candida, including C. glabrata, C. tropicalis and C. parapsilosis, have also been isolated in the saliva of subjects with and without candidiasis. In the present study we evaluated the host defence mechanisms induced by Candida albicans and other Candida species in monocytes and oral epithelial cells in order to establish the existence of a species-specific cellular response. Our results indicated that, during Candida species infection, the epithelial cells actively participate in the host defence by producing antimicrobial peptides and proinflammatory cytokines. Moreover, in infections caused by Candida tropicalis and Candida glabrata, the host defence may be strengthened by the release of perforin and granzyme by polymorphonuclear leukocytes recruited at the site of infection.

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.

Inhibition of HSV-1 replication by laser diode-irradiation: possible mechanism of action.

Herpes labialis are the most frequent clinical manifestations of HSV-1 infection. Epithelial cells are able to respond to HSV-1 presence inducing the expression of IL-6, IL-1, TNF-α and IL-8. These proinflammatory cytokines have a function in the acute-phase response mediation, chemotaxis, inflammatory cell activation and antigen-presenting cells. In the human epithelial cell models, it has been demonstrated that, after an early induction of proinflammatory host response, HSV-1 down-modulates the proinflammatory cytokine production through the accumulation of two viral proteins, ICP4 and ICP27, whose transcription is induced by tegument protein VP16. These viral proteins, through the decreasing of stabilizing the mRNAs of proinflammatory genes, delay cytokine production to an extent that allows the virus to replicate. Moreover, viral transactivating proteins, ICP-0 and VP-16 induce IL-10 expression. The conventional treatment of herpes labialis involves the topical and systemic use of antiviral drugs but it is necessary to find new therapies that can act in a selective and non-cytotoxic manner in viral infection. Laser diode therapy has been considered as a non-invasive alternative treatment to the conventional treatment of herpes labialis in pain therapy, in modulation of inflammation and in wound healing. This study aims to report a possible mechanism of action of laser diode irradiation in prevention and reduction of severity of labial manifestations of herpes labialis virus. We investigated, in an in vitro model of epithelial cells HaCat, the laser-effect on HSV-1 replication and we evaluated the modulation of expression of certain proinflammatory cytokines (TNF-α, IL-1ß and IL-6), antimicrobial peptide HBD2, chemokine IL-8 and the immunosuppressive cytokine, IL-10. Our results lead us to hypothesize that LD-irradiation acts in the final stage of HSV-1 replication by limiting viral spread from cell to cell and that laser therapy acts also on the host immune response unblocking the suppression of proinflammatory mediators induced by accumulation of progeny virus in infected epithelial cells.

[Resistant hypertension in non-dialysis chronic kidney disease]. / Ipertensione resistente nella malattia renale cronica non-dialitica.

Resistant hypertension is defined as blood pressure that remains above the target of <140/90 mm Hg in the general population and <130/80 mm Hg in people with diabetes mellitus or chronic kidney disease (CKD) in spite of the use of at least three full-dose antihypertensive drugs including a diuretic, or as blood pressure that reaches the target by means of four or more drugs. Hypertension is a frequent complication in CKD and a determining factor in the progression of renal damage, especially in proteinuric and diabetic patients, as well as contributing to a high cardiovascular risk. Clinical practice guidelines recommend blood pressure levels below 130/80 mm Hg in all CKD patients, but the target is reached in only a small proportion (10-20%), both in nephrology and non-nephrology settings. The resistance to antihypertensive treatment may be considered one of the causes of the poor achievement of blood pressure targets in CKD patients.

The PEA15 gene is overexpressed and related to insulin resistance in healthy first-degree relatives of patients with type 2 diabetes.

AIMS/HYPOTHESIS: Overexpression of the gene encoding phosphoprotein enriched in astrocytes 15 (PEA15), also known as phosphoprotein enriched in diabetes (PED), causes insulin resistance and diabetes in transgenic mice and has been observed in type 2 diabetic individuals. The aim of this study was to investigate whether PEA15 overexpression occurs in individuals at high risk of diabetes and whether it is associated with specific type 2 diabetes subphenotypes. SUBJECTS AND METHODS: We analysed PEA15 expression in euglycaemic first-degree relatives (FDR) of type 2 diabetic subjects. RESULTS: The expression of PEA15 in peripheral blood leucocytes (PBLs) paralleled that in fat and skeletal muscle tissues. In PBLs from the FDR, PEA15 expression was two-fold higher than in euglycaemic individuals with no family history of diabetes (control subjects), both at the protein and the mRNA level (p < 0.001). The expression of PEA15 was comparable in FDR and type 2 diabetic subjects and in each group close to one-third of the subjects expressed PEA15 levels more than 2 SD higher than the mean of control subjects. Subjects with IFG with at least one type 2 diabetes-affected FDR also overexpressed PEA15 (p < 0.05). In all the groups analysed, PEA15 expression was independent of sex and unrelated to age, BMI, waist circumference, systolic and diastolic BP, and fasting cholesterol, triacylglycerol and glucose levels. However, in euglycaemic FDR of type 2 diabetic subjects, PEA15 expression was inversely correlated with insulin sensitivity (r = -557, p = 0.01). CONCLUSIONS/INTERPRETATION: We conclude that PEA15 overexpression represents a common defect in FDR of patients with type 2 diabetes and is correlated with reduced insulin sensitivity in these individuals.

Immunomodulatory effects of peptide T on human keratinocyte cells.

BACKGROUND: Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. PT is from the V2 region of HIV-1 gp120, an exterior envelope glycoprotein that is a target for host immune responses. The anti-inflammatory mechanisms of PT are not well understood. OBJECTIVES: We studied the immunomodulatory effects of PT on the human keratinocyte cells. METHODS: Cultured human keratinocytes were treated with PT, proteins extracted and analysed by Western blotting and reverse transcriptase polymerase chain reaction. RESULTS: Our findings show reduced expression of intercellular adhesion molecule 1 and an increase in transforming growth factor (TGF)-beta and heat shock protein (HSP)-70 in human keratinocyte cells treated with PT. The HSP-70 increase is modulated by TGF-beta. In fact, we demonstrated that anti-TGF-beta antibodies reduce HSP-70 overexpression. In addition, we show a modulation of alphav integrins after 4 hours of treatment with PT. These receptors favour keratinocyte migration and epidermal regeneration. It has been reported that overexpression of HSP results in dramatic changes to intermediate filaments. These proteins act on keratin intermediate filaments and determine their retraction. The consequence is cell-cell contact detachment and inhibition of cellular hyperproliferation. CONCLUSIONS: Our results support the beneficial effect of PT found in vivo, suggesting, moreover, the primary role of keratinocytes upon which PT acts directly by stimulating the anti-inflammatory function and favouring the regeneration of tissue.

A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning.

A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the N-terminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.

Increased expression of periplasmic Cu,Zn superoxide dismutase enhances survival of Escherichia coli invasive strains within nonphagocytic cells.

We have studied the influence of periplasmic Cu,Zn superoxide dismutase on the intracellular survival of Escherichia coli strains able to invade epithelial cells by the expression of the inv gene from Yersinia pseudotuberculosis but unable to multiply intracellularly. Intracellular viability assays, confirmed by electron microscopy observations, showed that invasive strains of E. coli engineered to increase Cu,Zn superoxide dismutase production are much more resistant to intracellular killing than strains containing only the chromosomal sodC copy. However, we have found only a slight difference in survival within HeLa cells between a sodC-null mutant and its isogenic wild-type strain. Such a small difference in survival correlates with the very low expression of this enzyme in the wild-type strain. We have also observed that acid- and oxidative stress-sensitive E. coli HB101(pRI203) is more rapidly killed in epithelial cells than E. coli GC4468(pRI203). The high mortality of E. coli HB101(pRI203), independent of the acidification of the endosome, is abolished by the overexpression of sodC. Our data suggest that oxyradicals are involved in the mechanisms of bacterial killing within epithelial cells and that high-level production of periplasmic Cu,Zn superoxide dismutase provides bacteria with an effective protection against oxidative damage. We propose that Cu,Zn superoxide dismutase could offer an important selective advantage in survival within host cells to bacteria expressing high levels of this enzyme.

New strategies in dandruff treatment: growth control of Malassezia ovalis.

BACKGROUND: Cutaneous infections induced by Malassezia ovalis (Pityrosporum ovale) represent a therapeutic problem due to the high rate of recurrence. OBJECTIVE: We studied feasible strategies to control the growth of M. ovalis, compatible with topical use in cosmetic formulations. Studies were performed on the effects of pH, ionic strength, cinnamic acid and related compounds on mycotic growth. METHODS: M. ovalis was cultivated in modified Sabouraud agar. The effects of pH, ionic strength and cinnamic acid and related compounds on mycotic growth were studied by the membrane filter method. RESULTS: In vitro growth of M. ovalis is strongly affected by pH and ionic strength. pH 4.5 induced a growth inhibition of about 95% and 1 M NaCl, at the optimal growth pH, reduced cell growth by over 90%. Cinnamic acid showed an inhibitory effect of 50% at 0.005 g/dl; 30 min incubation with cinnamic acid 0.5 g/dl had a mycocidic effect. CONCLUSION: These results suggest the use of cosmetic compositions containing cinnamic acid or buffered acidic lotions and shampoos in the treatment of M. ovalis infections of the scalp, eventually in addition or alternative to antimycotic drugs or in maintenance therapy. Cosmetic formulations with high ionic strength or skin irritant derivatives such as cinnamaldehyde cannot be proposed for practical use.

TNF-alpha expression and herpes simplex infection in human monocytes treated with growth hormone, prolactin and insulin.

We evaluated the in vitro effect of growth hormone (GH), prolactin (PRL) and insulin treatment of human monocytes on Herpes simplex virus type 1 (HSV-1) infection. GH and PRL increased cell susceptibility to infection which was related to a slight TNF-alpha expression and release. Insulin had no significant effect. Cells activated with lipopolysaccharide (LPS) and then treated with PRL showed a lower susceptibility to HSV infection related to a significant increase in TNF-alpha expression and release. On the contrary, GH and insulin increased the susceptibility to infection of activated cells but did not modify TNF-alpha expression with respect to cells treated only with hormones.

Overexpression of a hydrogen peroxide-resistant periplasmic Cu,Zn superoxide dismutase protects Escherichia coli from macrophage killing.

We have studied the effect of H2O2 on the activity of the Escherichia coli Cu,ZnSOD showing that, unlike the bovine enzyme, this bacterial Cu,ZnSOD is highly resistant to inactivation by hydrogen peroxide. In view of the key role played by oxygen radicals in bacterial killing by phagocytes, we have tested the ability of E. coli strains expressing different amounts of Cu,ZnSOD in the periplasmic space to survive the phagocytic attack of activated macrophages. Overexpression of the enzyme effectively protected the bacterial cell from macrophage killing. The results obtained support the hypothesis that in pathogenic bacteria periplasmic Cu,ZnSOD may reduce the oxyradical damages induced by the respiratory burst and therefore be important in virulence.

Identification of Listeria monocytogenes by colony hybridization test using the virulence-associated hly and inlA genes as probes.

We developed a method of identification of Listeria monocytogenes based on colony hybridization with nonradioactively labeled DNA probes, represented by the hly and inlA virulence-associated genes. The procedure described in this paper results simple, rapid, specific and reproducible. Since it can be performed in a short time, the above technique can be applied to detect L. monocytogenes from different source and constitutes a noteworthy and alternative tool to identify this gram-positive pathogenic bacterium.

Ultrastructural and biochemical studies on Candida albicans after prolonged incubation in sea water.

Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.

Growth hormone modulates IL-alpha and IFN-gamma release by murine splenocytes activated by LPS or porins of Salmonella typhimurium.

The effect of growth hormone (GH) on the release of IL-1alpha and IFN-gamma from murine splenocytes was investigated. Their release from splenocytes activated by Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) 0.5 microg/ml was increased by c. 65% in the presence of GH 100 pg/ml. With splenocytes activated by S. Typhimurium porins 5 microg/ml, GH increased the production of both IL-1alpha and IFN-gamma by c. 56%. Polymyxin treatment abolished the cytokine-releasing activity of LPS but had no effect on the activity of the porin preparation.

Interleukin-1 and interleukin-6 gene expression in human monocytes stimulated with Salmonella typhimurium porins.

The aim of this study was to verify whether Salmonella typhimurium porins can affect the expression of interleukin-1 (IL-1) and interleukin-6 (IL-6) genes. Human monocytes were treated with porins, and total RNAs were analysed by Northern blotting to evaluate the expression of IL-1 alpha, IL-1 beta and IL-6 in both treated and untreated cell cultures. Porins induced a significant increase in IL-1 and IL-6 transcripts. This increase was related to the dose of porins, and it peaked 5 hr after treatment. The same results were obtained when polymyxin B was added to the porin preparation to eliminate eventual traces of lipopolysaccharide (LPS) associated with porins. The porins-mediated increase in interleukin transcripts did not require de novo protein synthesis, and it was because of the enhanced half-life of IL-1 and IL-6 mRNAs, rather an increased rate of gene transcription. These data suggest that porins may affect inflammatory and immunological responses by enhancing the expression of cytokine genes.

Invasion of cultured human cells by Streptococcus pyogenes.

The invasive capacity of streptococcal strains belonging to groups A and B was evaluated by infecting human epithelial and endothelial cells and monitoring the number of viable intracellular bacteria at different times postinfection. All strains tested entered eukaryotic cells (HeLa, HEp2 and HUVE), with Streptococcus pyogenes exhibiting a higher invasion efficiency than group B streptococci (GBS). No intracellular multiplication was observed, and GBS remained viable 24 h postinfection, whereas S. pyogenes were gradually killed. We found that cytochalasin D almost completely inhibited internalization of all bacterial strains, whereas colchicine had no effect, indicating that host microfilaments play a major role in bacterial internalization. Moreover, the use of the lysosomotropic agent ammonium chloride enabled us to demonstrate that a pH increase in the intracellular vesicles did not affect streptococcal entry. These results were documented by electron microscopic observations which revealed the different steps in the invasion pathway, including a fusion event between phagosomes containing S. pyogenes and lysosomes.

Effect of growth hormone, prolactin and insulin on the release of IL-1 alpha, IFN-gamma and IL-4 by staphylococcal enterotoxin A-stimulated splenocytes.

The regulation by peptide hormones (Growth Hormone, Prolactin, Insulin) of cytokine secretion by splenocytes stimulated with Staphylococcal Enterotoxin A was studied. Growth hormone increases the release of IFN-gamma from splenocytes stimulated with Enterotoxin A by 50% but considerably decreases IL-1 alpha release by 93%. Prolactin decreases the release of IL-1 alpha by 80%, but has no significant effects on IFN-gamma release. Insulin causes a 50% decrease in IFN-gamma and 95% decrease in IL-1 alpha. IL-4 release was not changed. The results are discussed in terms of the possibility of an interesting function for these endocrine peptides which expands their range of biologic activities within the immune system.