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.

3-O-Methylfunicone, a metabolite produced by Penicillium pinophilum, modulates ERK1/2 activity, affecting cell motility of human mesothelioma cells.

OBJECTIVES: 3-O-methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, affects cell proliferation and motility in a variety of human solid tumours. The aim of this study was to demonstrate whether OMF has the ability to arrest cell division and motility, in a human mesothelioma cell line. Malignant mesothelioma is an aggressive cancer that does not respond to standard therapies the cells of which are considered to be highly resistant to apoptosis. MATERIAL AND METHODS: Cell motility and invasion were measured using a modified Boyden chamber. Gene expression was examined by RT-PCR, while ERK1/2 was investigated by Western blot analysis. All experiments were also performed on primary cultures of mesothelial cells. RESULTS: The present study shows that OMF inhibited motility of the NCI mesothelioma cell line by modulating ERK signalling activity, and affected alphaVbeta5 integrin and MMP-2 expression, inducing marked downregulation at both mRNA and protein levels. Substantial downregulation of VEGF gene expression was also demonstrated. These effects were not observed in normal mesothelial cell cultures. CONCLUSION: OMF may have potential as a naturally derived anti-tumour drug for treatment of mesothelioma.

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.

3-O-methylfunicone, a metabolite of Penicillium pinophilum, inhibits proliferation of human melanoma cells by causing G(2) + M arrest and inducing apoptosis.

OBJECTIVES: Melanoma cells take advantage of impaired ability to undergo programmed cell death in response to different external stimuli and chemotherapeutic drugs; this makes prevention of tumour progression very difficult. The aim of this study was to demonstrate whether 3-O-methylfunicone (OMF), a metabolite of Penicillium pinophilum, has the ability to arrest cell population growth and to induce apoptosis in A375P (parental) and A375M (metastasis derivatived) melanoma cell lines. MATERIALS AND METHODS: Cell proliferation and apoptosis were analysed by flow cytometry, DNA fragmentation, caspase-3 and caspase-9 activation, and PARP-1 cleavage. RESULTS: We demonstrated that OMF affected cell proliferation in a time- and dose-dependent manner, reaching the best effect at concentration of 80 microg/ml for 24 h. Flow cytometry revealed that OMF caused significant G(2) phase arrest, which was associated with marked decrease in cyclin B1/p34(cdc2) complex and p21 induction. OMF also induced marked decrease of survivin expression. Reduced levels of apoptosis were evident after silencing p21 expression in both cell lines. Finally, the effect exercised by OMF on hTERT and TEP-1 gene expression confirmed the ability of this molecule to interfere with replicative ability of cells. CONCLUSIONS: The results reported here seem to suggest that OMF as a promising molecule to include in strategies for treatment of melanoma.

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.

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.

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.

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.

Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction.

Pemphigus is an autoimmune disease where both endogenous (genetic) and exogenous (environmental) factors play a part. Viral infections, in particular herpesvirus infections, have been identified as a possible triggering factor for pemphigus. In this study, using the polymerase chain reaction, we studied peripheral blood mononuclear cells (PBMC) and skin biopsies from patients with pemphigus, and in some of these were able to demonstrate the presence of DNA sequences of herpes simplex virus 1/2 (50% in PBMC and 71% in skin biopsies), Epstein-Barr virus (15% in PBMC and 5% in skin biopsies) and human herpesvirus 6 (20% in PBMC only). However, the inability to detect herpesvirus DNA consistently in these cases suggests that viral infection may only be an occasional factor triggering the outbreak or exacerbation of the disease. The possible role of interferons and interleukins in the pathogenesis of virus-induced pemphigus is discussed.

Porin from Pseudomonas aeruginosa induces apoptosis in an epithelial cell line derived from rat seminal vesicles.

Micromolar concentrations of porin, purified from the outer membranes of Pseudomonas aeruginosa, induced in vitro the classic morphological and biochemical signs of apoptosis in an epithelial cell line (SVC1) derived from the rat seminal vesicle secretory epithelium. The programmed cell death (PCD) was p53 independent and associated with significant decrease of bcl-2 expression, a marked increase of c-myc transcriptional activity, and an absence of the mRNA coding for tissue transglutaminase. The Ca(2+) influx, caused by the porin treatment of SVC1 cells, appears to play an important role in the triggering of apoptosis in our biological model. The possibility that the porin property of inducing PCD plays a role in the infertility of individuals chronically infected by gram-negative bacteria is discussed.

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.

Effects of a new topic amikacin formulation on chemotaxis and release of profibrotic factors by human monocytes.

Aminoglycosides, widely used because of their large-spectrum antibiotic effects, should not interfere with the healing process of an ulcer or an infected wound. We evaluated the effects of amikacin or the excipients present in the topic formulation BG 90, powder 2. 5% (Boniscontro e Gazzone S.r.l., Rome, Italy), on human monocyte chemotaxis and the release of profibrotic factors by resting or lipopolysaccharide (LPS)-activated monocytes. The chemotactic response of monocytes to zymosan-activated serum is not modified in vitro by pre-incubation of the cells with amikacin (2 and 10 microg/ml/10(6) cells) or excipients. Unstimulated monocytes did not secrete appreciable amounts of cytokines. Vice versa, amikacin-stimulated cells released platelet-derived growth factor AB (PDGF-AB) (about 340 pg/ml), transforming growth factor (TGF)-beta1 (about 10 pg/ml), and tumour necrosis factor (TNF)-alpha (over 1,100 pg/ml); among excipients, ZnO and vitamin E induced PDGF-AB release (about 320 and, respectively, 200 pg/ml), while stimulation of monocyte monolayers by the other excipients did not lead to appreciable cytokine release. As expected, LPS-activated human monocytes produced PDGF-AB, TGF-beta1, and TNF-alpha. When monocytes were co-stimulated with LPS and amikacin, the PDGF-AB and TGF-beta1 values almost overlapped with those from the stimulation of cells with LPS alone, while TNF-alpha production was slowly reduced. The results show a stimulating effect of aminoglycoside on the production of profibrotic factors and, therefore, on the healing process of wounds in addition to a modulating effect on the production of pro-inflammatory cytokines like TNF-alpha. Moreover, ZnO and tocopherol (free-radical scavengers), used as excipients in the topic formulation, induce the release of growth factors with profibrotic activity (PDGF-AB). Further research is warranted to explore the effects of this formulation in vivo, verifying whether the association of the antibiotic with scavengers has a double advantage in topical amikacin: on the one hand, it could limit the damage from free radicals, and on the other it could favour tissue healing.

Porins of Pseudomonas aeruginosa induce release of tumor necrosis factor alpha and interleukin-6 by human leukocytes.

The aim of this study was to examine the ability of Pseudomonas aeruginosa components to induce release of cytokines from human leukocytes. Human whole-blood cultures were incubated with several concentrations of purified P. aeruginosa products, including porins, exomucopolysaccharide, lipopolysaccharide, and toxin A. Supernatants were assayed for tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) activities. All of the P. aeruginosa components except toxin A were able to stimulate the release of both cytokines. On a weight basis, porins were as effective as lipopolysaccharide and significantly more effective than exomucopolysaccharide in inducing IL-6 release (P < 0.05). Moreover, porins were more potent than either exomucopolysaccharide or lipopolysaccharide in inducing TNF-alpha release (P < 0.05). Further experiments using isolated leukocytes suggested that monocytes were the cell population predominantly responsible for the production of both cytokines. These data indicate that P. aeruginosa porins are able to induce significant cytokine production. These components may be responsible for the chronically overactive inflammatory response associated with persistent lung infection in cystic fibrosis patients.

Bacterial porins stimulate bone resorption.

Porins are abundant outer membrane proteins of gram-negative bacteria involved in transport of low-molecular-mass molecules. During the past decade, porins from a number of bacteria have also been shown to have proinflammatory activities including inducing the synthesis of proinflammatory mediators (cytokines, platelet-activating factor, and nitric oxide) in cultured cells and inducing inflammation in vivo. With this range of actions, it was possible that porins could also interact with bone cells to cause aberrant bone remodeling and that this could contribute to the bone destruction seen in gram-negative bone infections. By using purified preparations of Salmonella typhimurium and Pseudomonas aeruginosa porins, in the presence of polymyxin B, it was possible to induce concentration-dependent loss of calcium from cultured murine calvaria at porin concentrations in the range of 1 to 10 nM. The mechanism of action of the porins was determined by the inclusion of inhibitors of cyclooxygenase or inflammatory cytokines in the culture media. The bone-resorbing activity of both porins was not inhibited by the cyclooxygenase inhibitor indomethacin or by neutralizing the activity of tumor necrosis factor. Indeed, relatively high concentrations of these agents produced an unexpected increase in the bone resorption induced by the porins. In contrast, porin-induced bone resorption could be inhibited by relatively high concentrations of the natural inhibitor of interleukin-1 (IL-1 receptor antagonist). It appears that these porins stimulate bone resorption by a mechanism distinct from that of lipopolysaccharide, and the possibility therefore exists that porins play a role in bone destruction in gram-negative bacterial infections of bone.

Rat seminal vesicle protein SV-IV and its transglutaminase-synthesized polyaminated derivative SPD2-SV-IV induce cytokine release from human resting lymphocytes and monocytes in vitro.

Micromolar amounts of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, induce in vitro a marked release of a variety of cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin 6, and granulocyte-monocyte colony-stimulating factor) from human resting peripheral blood mononuclear cells as well as from isolated resting lymphocytes and monocytes. This effect was found to be significantly higher when the spermidine adduct of SV-IV (Spd2-SV-IV), synthesized in vitro by the enzyme transglutaminase, was used instead of the native protein. Furthermore, the pretreatment of monocytes with transglutaminase caused an increase of the inducing effect of both native and modified SV-IV on the release of interleukin 6 from these cells. The inducing effect of these proteins on the cytokine release was markedly inhibited by actinomycin D and cycloheximide.

Survival to lipopolysaccharide, cytokine release and phagocyte functions in mice treated with different total parenteral nutrition regimens.

Effects on host defenses of Total Parenteral Nutrition (TPN) with long- (LCT) and medium-chain triglycerides (MCT) were studied. Survival to lipopolysaccharide (LPS) challenge, blood clearance of Escherichia coli, in vivo and in vitro production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were investigated. In BALB/c mice, LCTs produced a 25% reduction in mortality, compared with controls. TPN performed with a LCT plus MCT mixture reduced mortality by 50%. Spasms appeared after 18 h and 12 h respectively in mice treated with LCT-MCT mixture or LCTs alone, respect to controls (8 h). The LCT-MCT mixture produced a 67% blood clearance of E. coli after 1 h, while the treatment with LCTs alone had no significant effects compared to controls (about 40%). The LCT-MCT mixture induced a 50% increase in chemiluminescence respect to controls. A 33% increase was observed in rats treated with LCTs alone. TNF-alpha serum levels after challenge with LPS were not modified by any of the triglycerides or their combinations. IL-6 increased by 43% with LCT-MCT mixture and by 39% with LCTs alone versus controls. After a 3 h in vitro treatment with LCTs, human monocytes were stimulated to release TNF-alpha at levels higher than those stimulated with the LCT-MCT mixture, and respect to controls. In contrast after 3 h the stimulation with LCT-MCT mixture induced a higher IL-6 release than controls and cells stimulated with LCTs alone, or with LPS.

Pulmonary penetration of dirithromycin in patients suffering from acute exacerbation of chronic bronchitis.

The aim of this study was to evaluate the concentrations of dirithromycin, a new macrolide antibiotic, in bronchial secretions (BS), bronchial mucosa (BM), epithelial lining fluid (ELF) and serum in 25 patients with acute exacerbation of chronic bronchitis after a 5-day, once-daily, dirithromycin regimen. All patients received dirithromycin, 500 mg (two 250 mg tablets) given orally once daily at 08.00 fasted, for 5 consecutive days. They were divided into five groups (n = 5 in each group) according to sampling time (24, 48, 72, 96 and 120 h, after the last dose). Mean serum concentrations remained low throughout the study (0.44 microgram/ml at 24 h, 0.31 microgram/ml at 48 h, 0.33 microgram/ml at 72 h, 0.12 microgram/ml at 96 h and 0.11 microgram/ml at 120 h, respectively), although they were higher than the MICs for Moraxella catarrhalis for up to 72 h and than that for Streptococcus pneumoniae for up to 120 h after the last dose. By contrast, in all other samples, mean concentrations were higher than the MICs for many relevant respiratory pathogens for at least 3 days, and higher than that for S. pneumonia and M. catarrhalis for up to 120 h (mean concentrations measured 2.67, 2.15, 1.74, 0.27 and 0.17 micrograms/ml, respectively, in BS; 2.59, 2.59, 1.96, 0.41 and 0.27 micrograms/g, respectively, in BM; 2.21, 2.25, 1.57, 0.22 and 0.15 micrograms/ml, respectively, in ELF). These findings demonstrate that dirithromycin is concentrated in each of these potential sites of infection for up to 3 days after a 5-day course of therapy. Therefore, short-term therapy with dirithromycin may be useful for many respiratory infections.

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.