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The outer membrane of Gram-negative bacteria contains some major proteins, called porins, with particular chemical and physical characteristics which reflect some specialized functions peculiar to the outer membrane. In recent years the biological properties of Salmonella typhimurium SH5014 porins have been the focus of our experimental studies. The aim of the present research was to make a comparative investigation of the biological activities of porins extracted from other microorganisms classified in the Enterobacteriaceae, together with a taxonomically different one, Eikenella corrodens frequently isolated from gum pockets of patients with periodontal disease. Porins from E. coli K12, Proteus mirabilis and Eikenella corrodens were extracted, purified and separated from LPS by means of phenol extraction. The purity of preparation was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis in slabs. We studied the interactions of these proteins with some cellular and humoral systems within the host. Mouse peritoneal macrophages in the presence of these proteins show morphological and functional modifications, depending upon the type of porins and the concentrations used. The ability of porins to interact with the complement system was investigated in vitro. A pool of sera from 20 blood donors served as a source of human complement. The results obtained with porins of various sources were shown comparatively.

Porins, a family of hydrophobic proteins located in the outer membrane of cell-wall of Gram-negative bacteria, were shown to stimulate the synthesis and release of platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine mediator of inflammation and endotoxic shock produced by polymorphonuclear neutrophils. PAF synthesis was independent either from contamination by LPS or generation of TNF. Experiments with labeled precursors demonstrated that PAF was synthesized via the remodeling pathway that involves acetylation of 1-O-alkyl-sn-glyceryl-3-phosphorylcholine generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins, indeed, induced a sustained PLA2-dependent mobilization of [14C]arachidonic acid that was inhibited by p-bromodiphenacylbromide. p-Bromodiphenacylbromide, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase. The addition of 2-lyso-PAF restored PAF synthesis. The activity of acetyl CoA:2-lyso-PAF acetyltransferase was transiently increased in porin-stimulated PMN and the [3H]acetyl group was incorporated in the synthetized PAF after cell preincubation with [3H]acetyl CoA. The activation of PAF synthesis by porins as well as its release were dependent on extracellular Ca2+. Porins by forming trans-membrane channels determined a sustained influx of 45Ca2+ into the cytosol. As shown by inhibitors of Ca(2+)-calmodulin complexes, calmodulin mediated the Ca(2+)-dependent activation of enzymes involved in PAF synthesis.

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 g/ml for 24 h. Flow cytometry revealed that OMF caused significant G2 phase arrest, which was associated with marked decrease in cyclin B1/p34cdc2 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. © 2009 Blackwell Publishing Ltd.

Background FKBP51 immunophilin is abundantly expressed by immune cells. Co-inhibitory immune receptor signalling generates the splicing isoform FKBP51s. Tregs stained by FKBP51s are increased in melanoma patients and their counts are associated with anti-CTLA-4 response. An expansion of FKBP51s+PD-L1+ monocytes was measured in a group of non-responding patients to anti-CTLA-4. The aim of this work was to confirm the predictive value of response of FKBP51s+Tregs in a cohort of patients undergoing anti-PD1 treatment and shed light on a monocyte subset co-expressing PD-L1/FKBP51s. Methods Co-cultures of organoids and autologous lymphocytes were used to confirm that tumour T-cell interaction can induce FKBP51s. PBMC immunophenotype and flow cytometry served to assess and monitor FKBP51s+Treg and FKBP51s+PD-L1+ monocytes in 22 advanced melanoma patients treated with anti-PD1. Silencing and overexpression of FKBP51s in human macrophages served to address the protein role in the tolerant macrophages’ behaviour. Results FKBP51s+Tregs count was increased in responders and had a prognostic value. Non-responders showed an early increase in FKBP51s+ PD-L1+ monocytes during anti-PD1 treatment. Manipulation of FKBP51s modulated the macrophage–phenotype, with forced protein expression promoting aspects associated with tolerance. Conclusions FKBP51s may guide in the selection and monitoring of melanoma patient candidates to immune-checkpoint-targeted therapy. Manipulation of FKBP51s may overcome resistance.

Recent advances in tumor immunology, fostered by dramatic outcomes with cancer immunotherapy, have opened new scenarios in cancer metastasis. The cancer stemness/mesenchymal phenotype and an excess of immune suppressive signals are emerging as Intertwined aspects of human tumors. This review examines recent studies that explored the mechanistic links between cancer cell stemness and immunoevasion, and the evidence points to these key events in cancer metastasis as two sides of the same coin. This review also covers the mechanisms involved in tumor expression of programmed cell death ligand 1 (PD-L1), a major factor exploited by human neoplasias to suppress immune control. We highlight the convergence of mesenchymal traits and PD-L1 expression and examine the functions of this immune inhibitory molecule, which confers cancer cell resistance and aggressiveness.

Despite Glioblastoma (GBM) frequently expressing programmed cell death ligand-1 (PD-L1), treatment with anti-programmed cell death-1 (PD1) has not yielded brilliant results. Intratumor variability of PD-L1 can impact determination accuracy. A previous study on mouse embryonic fibroblasts (MEFs) reported a role for cyclin-D in control of PD-L1 expression. Because tumor-cell growth within a cancer is highly heterogeneous, we looked at whether PD-L1 and its cochaperone FKBP51s were influenced by cell proliferation, using U251 and SF767 GBM-cell-lines. PD-L1 was measured by Western blot, flow cytometry, confocal-microscopy, quantitative PCR (qPCR), CCND1 by qPCR, FKBP51s by Western blot and confocal-microscopy. Chromatin-Immunoprecipitation assay (xChIp) served to assess the DNA-binding of FKBP51 isoforms. In the course of cell culture, PD-L1 appeared to increase concomitantly to cyclin-D on G1/S transition, to decrease during exponential cell growth progressively. We calculated a correlation between CCND1 and PD-L1 gene expression levels. In the temporal window of PD-L1 and CCND1 peak, FKBP51s localized in ER. When cyclin-D declined, FKBP51s went nuclear. XChIp showed that FKBP51s binds CCND1 gene in a closed-chromatin configuration. Our finding suggests that the dynamism of PD-L1 expression in GBM follows cyclin-D fluctuation and raises the hypothesis that FKBP51s might participate in the events that govern cyclin-D oscillation.

Introduction Polycystic ovary syndrome is characterized by anovulation (amenorrhea, oligomenorrhea, irregular menstrual cycles) combined with symptoms of androgen excess (hirsutism, acne, alopecia). The clear definition and diagnosis in adolescents could be challenging considering that most of symptoms occur as part of the expected physiological hormonal imbalance of puberty. Therefore, different diagnostic criteria have been elaborated. Polycystic ovary syndrome could be associated to obesity, diabetes mellitus, and metabolic syndrome. In adolescents with polycystic ovary syndrome, adiposity is associated with higher androgen concentrations and greater menstrual irregularity. Polycystic ovary syndrome in youth is considered a risk factor for type 2 diabetes mellitus in adulthood. On the other hand, increased prevalence of polycystic ovary syndrome has been shown in type 1 diabetes mellitus. Evidence The treatment of polycystic ovary syndrome in adolescents is controversial considering that adequate trials are lacking. First line treatment comprises lifestyle modification (preferably multicomponent including diet, exercise and behavioural strategies) that should be recommended overall in the patients with polycystic ovary syndrome and overweight, central obesity and insulin resistance. Beyond non-pharmacological therapy, pharmacological agents include combined hormonal contraceptives, metformin and antiandrogens, used separately or in combination. The aim of therapy is to bring back ovulation, to normalize menses, to reduce hirsutism and acne, to reduce weight. Other important goal is the treatment of hyperlipidaemia and of hyperglycaemia. Conclusions This narrative review aims to review the most pertinent literature about polycystic ovary syndrome in adolescents with obesity or diabetes. We overviewed the diagnostic criteria, the pathophysiology and the possible treatment approaches.

Thrombocytopenia after TAVI is common and clinically detrimental. Retrospectively, we observed Portico recipients had a more profound platelet drop than Evolut recipients. We thus investigated periprocedural platelet damage and/orpro-inflammatory state in 64 TAVI recipients at baseline and after implantation. Platelet damage was assessed by annexin V staining and monocyte-phagocytic phenotype was assessed according to CD14/CD36 expression. Serum cytokines were measured in 20 patients. The formaldehyde-based storage solution altered platelets. When, before being loaded onto the delivery system, Portico underwent one additional flushing to those recommended, the receiving patients showed thrombocytopenia, platelet damage, and CD36-monocyte count were mitigated. A general increase in IL-6 was recorded in overall TAVI recipients, but a high serum level of IL-8, a potent thrombocytopenia inducer, was measured in Portico recipients only, including those with extra-rinsed valve. Our study suggests a platelet-injury effect by storage-solution and generates the hypothesis of a role for the biomaterial in stimulating innate-immunity. Larger prospective studies are needed. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12265-021-10117-9.

Magnetic resonance imaging (MRI) is the gold standard for glioblastoma (GBM) patient evaluation. Additional non-invasive diagnostic modalities are needed. GBM is heavily infiltrated with tumor-associated macrophages (TAMs) that can be found in peripheral blood. FKBP51s supports alternative-macrophage polarization. Herein, we assessed FKBP51s expression in circulating monocytes from 14 GBM patients. The M2 monocyte phenotype was investigated by qPCR and flow cytometry using antibodies against PD-L1, CD163, FKBP51s, and CD14. MRI assessed morphologic features of the tumors that were aligned to flow cytometry data. PD-L1 expression on circulating monocytes correlated with MRI tumor necrosis score. A wider expansion in circulating CD163/monocytes was measured. These monocytes resulted in a dramatic decrease in patients with an MRI diagnosis of complete but not partial surgical removal of the tumor. Importantly, in patients with residual tumor, most of the peripheral monocytes that in the preoperative stage were CD163/FKBP51s− had turned into CD163/FKBP51s+. After Stupp therapy, CD163/FKBP51s+ monocytes were almost absent in a case of pseudoprogression, while two patients with stable or true disease progression showed sustained levels in such circulating monocytes. Our work provides preliminary but meaningful and novel results that deserve to be confirmed in a larger patient cohort, in support of potential usefulness in GBM monitoring of CD163/FKBP51s/CD14 immunophenotype in adjunct to MRI.

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.

In this study we evaluated the in vitro activities of cefditoren--a broad-spectrum oral cephalosporin--and other comparator agents against 2,396 fresh isolates from community-acquired respiratory tract infections, collected from 6 clinical Italian microbiology laboratories. On penicillin-susceptible pneumococci and Streptococcus pyogenes, cefditoren demonstrated to be the most active antibiotic (MIC(90)values of 0.03 and 0.06 mg/L respectively), showing only a slight decrease in potency on penicillin-intermediate and resistant pneumococci (MIC(90)value 0.5 mg/L, 1.0 mg/L respectively). All the other comparators displayed MIC(90 )values of 4 - 8 mg/L for penicillins and of 4 to64 mg/L for the oral cephalosporins. Cefditoren and levofloxacin were the most active against MSSA (MIC(90)0.5 mg/mL). Cefditoren displayed a uniformly potent inhibitory activity (MIC(90)of 0.03 mg/L) against all strains of Haemophilus influenzae, regardless of their ampicillin resistance (mediated or not by beta-lactamase production), while against Moraxella catarrhalis MIC(90)values were higher against beta-lactamase-positive (0.25 mg/L). Cefditoren was active also against Klebsiella pneumoniae and Escherichia coli : in this case its activity was comparable with that of levofloxacin. In conclusion, cefditoren, due to its potent activity, is a new effective therapeutic option for the treatment of respiratory tract infections.

Epithelia in the human airways, from the nasal aperture to the alveoli, are covered in a protective film of fluid containing a number of antimicrobial proteins. Defensins are single-chain, strongly cationic peptides and are one of the most extensively studied classes of antimicrobial peptides. Moxifloxacin (MXF) is a fluoroquinolone that acts against both Gram positive and Gram negative bacteria. In this study, we evaluated the effects of HBD2, MXF and the association MXF/HBD2 on some cytokines and on the ICAM-1 expression in LPS-stimulated A549 cells. Our results suggest that by lowering the epithelial cell-derived IL-1beta, IL-6, IL-8 and ICAM-1 expression, the MXF/HBD2 association interferes with the multifunctional cytokine network evolving during inflammatory processes of the respiratory tract; this anti-inflammatory potential could be of great value in the treatment of inflammatory disorders.

At least two populations of microorganisms are found in skin microbiota: a resident flora and a transient flora. Colonization and invasion by pathogenous microorganisms is counteracted both by the host defenses and by the resident flora. Most skin infections are therefore self-limiting in healthy subjects and are defined as primary infections. Secondary infections develop on preexisting skin lesions and are usually polymicrobial and caused by microorganisms that in themselves have little pathogenic power. When immune defenses are low, secondary infections arise readily and develop rapidly. This article describes the main bacterial and viral skin diseases.

Quinine sulfate (QS), the first antimalarial agent, was derived from the cinchona tree. Interestingly, antimalarials were recently found effective as additional therapy for AIDS and other viral diseases. The antiviral action of some drugs is associated with the induction of heat shock protein synthesis, in particular of the 70 kDa heat shock protein (Hsp70). In the current study, we examined the effects of QS on HSV-1 replication in HaCat cells. We also estimated the effects of this drug on the Hsp70, ICP27 and VP16 production and on the activation of NF-kB in HaCat cells that are HSV-1 infected to elucidate the possible mechanisms of the antiviral action of QS.

Constitutional 11q deletion is a chromosome imbalance possibly found in MCA/MR patients analyzed for chromosomal anomalies. Its role in determining the phenotype depends on extension and position of deleted region. Loss of heterozygosity of 11q (region 11q23) is also associated with neuroblastoma, the most frequent extra cranial cancer in children. It represents one of the most frequent cytogenetic abnormalities observed in the tumor of patients with high-risk disease even if germline deletion of 11q in neuroblastoma is rare. Hereby, we describe a 18 months old girl presenting with trigonocephaly and dysmorphic facial features, including hypotelorism, broad depressed nasal bridge, micrognathia, synophrys, epicanthal folds, and with a stage 4 neuroblastoma without MYCN amplification, carrying a germline 11q deletion (11q14.1-q22.3), outside from Jacobsen syndrome and from neuroblastoma 11q critical regions. The role of 11q deletion in determining the clinical phenotype and its association with neuroblastoma development in the patient are discussed.

Malassezia pachydermatis is a normal inhabitant of canine and feline skin that can spread to other pets. The outer layer or epidermis is made up primarily of keratinocytes, which are capable of releasing various factors and expressing receptors that are significantly involved in the immune regulation. Little is known about the mechanism by which M. pachydermatis overcomes the natural barrier of the skin. The aim of this study was to evaluate the direct in vitro interaction between human keratinocytes and a clinical strain of live M. pachydermatis isolated as a pure culture from an otitic cat. Human keratinocytes (HaCat) were infected with M. pachydermatis to analyse the modulation of the innate immune response. Gene expression was analysed by real-time PCR. We demonstrated that M. pachydermatis invaded HaCat cells and modulated the expression of TLR2 after 24. h infection, while HBD-2, IL-1β TNF-α, IL-6 and IL-8 were modulated both at 24 and 48. h. Thus, our results demonstrated that M. pachydermatis is able to stimulate the innate immune response in infected human keratinocytes indicating a possible role of this yeast as a human opportunistic pathogen.

The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure-activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1. H-indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however, the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15, apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.

Malignant pleural mesothelioma is a fatal malignancy linked to asbestos exposure. The main challenge for mesothelioma treatment is to go beyond the drug resistance, in particular against cisplatin (CDDP), one of the most used chemotherapeutic drug. 3-O-methylfunicone (OMF) is a metabolite produced by the fungus Penicillium pinophilum; its antiproliferative properties have been previously studied in vitro. Particularly, OMF is able to inhibit mesothelioma cell motility. To improve the effects of CDDP by-passing the resistance of mesothelioma cells to this drug, in the present study we investigated the combined treatment of OMF with CDDP respectively in an established mesothelioma cell line (NCI) and primary mesothelioma cells (Mest). As compared to the effect of single treatments, the combination of OMF and CDDP resulted in a stronger inhibition of NCI and Mest cell proliferation. OMF combination with CDDP was also able to affect the migratory ability of NCI and Mest cells by down-regulating αv and β5 expression and reducing metalloproteinase 2 (MMP-2) production. In addition, this association was effective in modulating VEGF gene expression. This finding highlights the possibility to use OMF and CDDP together to regulate angiogenesis and tumour progression in mesothelioma.

Keratinocytes stimulated by microbial organisms secrete not only a variety of cytokines, chemokines and growth factors, but also antimicrobial peptides such as beta-defensins (HBDs) such as HBD-2 and HBD-3. AV119, a patented blend of avocado sugar, triggers the up-regulation of HBD-2 in skin epithelia upon contact with AV119, but the signalling mechanisms involved are not completely understood. The purpose of this study was to determine if AV119 was able to induce also the expression of HBD-3 in human keratinocytes. In addition, the receptor and intracellular pathways involved in the AV119 up-regulation of HBD-2 and HBD-3 were investigated. Our results demonstrated that AV119 induces a significantly increase of the expression of HBD-3. In addition, the HBD-2 and HBD-3 AV119-induced gene expression and release are TLR-2 dependent. Finally, we demonstrated that AV119 induced ERK/MAPK phosphorylation in human keratinocytes, thus providing evidence that HBD-2 and HBD-3 secretion is through the same transductional pathway. The ability of AV119 to induce also HBD-3 may amplify its therapeutic potential against a broader spectrum of bacterial and yeast strains responsible for human skin disorders.

Keratinocytes play an active role in innate immune responses by secreting a variety of cytokines and chemokines. The release of critical proinflammatory cytokines, which are necessary to activate the immune response, is induced by the stimulation of Toll-like receptors (TLRs) by molecules present on pathogenic micro-organisms such as lipopolysaccharide (LPS). AV119, a patented blend of avocado sugars, induced the aggregation of Malassezia furfur, a dimorphic, lipid-dependent yeast that is part of the normal human cutaneous commensal flora and inhibited its penetration into the keratinocytes. In the present study, the anti-inflammatory effects of AV119 were investigated in LPS-induced inflammation of human keratinocytes. In particular, we analysed the modulation of the LPS-induced expression of proinflammatory cytokines and heat shock protein 70 (HSP70) by AV119 and the involvement of TLR-4. Our data show that AV119 is able to modulate significantly the proinflammatory response in human keratinocytes, blocking the NF-kB activation in human keratinocytes.

Porins are a family of hydrophobic proteins located in the outer membrane of the cell wall in Gram-negative bacteria. The effect of porins on the biosynthesis of platelet-activating factor (PAF) by cultured human umbilical-cord-vein-derived endothelial cells (HUVEC) was investigated. The results demonstrate that porins were able to induce a dose-dependent synthesis of PAF in HUVEC. PAF, synthesized after stimulation with porins, was mainly cell associated and the synthesis peaked at 15 min, decreasing rapidly thereafter. Experiments with radiolabeled precursors demonstrated that PAF, a 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, was synthesized via the remodeling pathway involving the acetylation of 1-O-alkyl-2-lyso-sn-glyceryl-3-phosphorylcholine (2-lysoPAF) generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity. The activation of phospholipase A2 in HUVEC stimulated by porins was detected by observing the mobilization of [14C]arachidonic acid. In addition, the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphorylcholine 2-O-acetyltransferase was transiently increased in porin-stimulated HUVEC and, after incubation with [3H]CoASAc or [3H]acetate, the [3H]acetyl group was incorporated into newly synthesized PAF. Porins, by forming transmembrane channels, induced a sustained influx of extracellular 45Ca2+ into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did not occur in the absence of extracellular Ca2+.

Human keratinocytes synthesize and secrete non-neuronal acetylcholine, which acts as a local cell signaling molecule, regulating functions like proliferation, cell adhesion, motility, desmosomal cell contact, and glandular activity. The keratinocyte acetylcholine axis is composed of the enzymes mediating acetylcholine synthesis (acetyltransferase) and degradation (acetylcholinesterase), and two classes of acetylcholine receptors. In this study we investigated the effect of captopril, an ACE-inhibitor, on acetylcholinesterase and acetylcholine secretion in human keratinocytes. We analyzed the level of acetylcholinesterase in HaCat and NHEK cells by RT-PCR and Western blotting analysis. In addition, the effect of captopril on AChE activity was evaluated. We found that captopril induces a strong AChE up-regulation leading to ACh degradation and reduced secretion. Our results suggest that acantholysis induced by ACE-inhibitors might be linked to altered level of Ach.

The aim of this study was to evaluate the in vitro antibiotic susceptibility of respiratory pathogens recently isolated in Italy to commonly used antibiotics including cefditoren. Six clinical microbiological laboratories collected, between January and September 2009, a total of 2,510 respiratory pathogens from subjects with community-acquired respiratory tract infections (CARTI). Ceftditoren, out of all the beta-lactams studied, had the lowest MIC(90 )against 965 strains of Streptococcus pneumoniae examined, followed by cefotaxime and ceftriaxone (2% resistance in penicillin-resistant S. pneumoniae (PRSP)). Against 470 Haemophilus influenzae , independently of their production of beta-lactamases or ampicillin resistance, cefditoren was the oral cephalosporin with the best in vitro activity, comparable to that of the injectable cephalosporins and levofloxacin. Higher MIC(90)s were found for the macrolides (4 - 16 mg/l) and cefaclor (4 - 32 mg/l). As was foreseeable, Streptococcus pyogenes (225 strains) was uniformly sensitive to all the beta-lactam antibiotics, but the elevated MIC(90 )values reduced (75%) susceptibility of this pathogen to macrolides. Beta-lactamase-negative Moraxella catarrhalis (100 strains) had reduced susceptibility only to the macrolides, while the 250 beta-lactamase-producing strains also had reduced susceptibility to cefuroxime. Levofloxacin showed the lowest MIC(50)/MIC(90 )values in the producing strains, whereas cefditoren, cefotaxime and ceftriaxone in the non-producers. As regards the enterobacteriaceae, cefditoren and levofloxacin had the lowest MIC(90)s against Klebsiella pneumoniae. Cefditoren and the third-generation injectable cephalosporins had the lowest MIC(90)s against Escherichia coli (100% susceptibility) while levofloxacin was less active (86% susceptibility).In conclusion, cefditoren's wide spectrum and high intrinsic activity, as well as its capacity to overcome most of the resistance that has become consolidated in some classes of antibiotics widely used as empiric therapy for CARTI, allows us to suggest that cefditoren might be included in the european guidelines as one of the first-choice antibiotics in the treatment of CARTI.

Methicillin-resistant staphylococci (MRS) were isolated from nasal swabs of 56 of 159 (35.2%; 95% confidence interval [CI]: 27.9–43.2%) healthy horses. Two nasal swabs were collected from each horse; 43 of 159 (27%; 95% CI: 20.5–34.8%) of the cohort were colonized by MRS strains in 1 nostril, while in the remaining 13 of 159 (8.2%; 95% CI: 4.6–13.9%), different or identical MRS strains were isolated in both nostrils. Of the 29 humans in close contact with the horses tested, 4 (13.8%; 95% CI: 4.5–32.6%) were found to be carriers of MRS. All isolates were coagulase negative with the exception of 2 coagulase-positive MRS strains, Staphylococcus aureus and Staphylococcus pseudintermedius, both isolated from horses. To assay the methicillin resistance, a susceptibility test to oxacillin with standardized disk diffusion method, a PBP-2a latex agglutination test, and a methicillin resistance gene ( mecA) polymerase chain reaction assay were performed. Pulsed-field gel electrophoresis patterns of isolates from horses and humans in close contact with the horses revealed similarity. The results suggest evidence of transmission between animals, from animals to humans, and vice versa.

Osteopontin (OPN) is a phosphorylated acidic glycoprotein produced by cells of the immune system, epithelial tissue, smooth muscle cells, osteoblasts, and tumor cells. OPN interacts with integrins and CD44 to enhance Th1 and inhibit Th2 cytokine expression. The involvement of this molecule in the onset of psoriasis has not previously been studied. Here, we demonstrate that OPN is expressed in peripheral blood mononuclear cells and in skin biopsies of psoriatic patients. The study was conducted on 30 patients affected with plaque psoriasis, and on 11 healthy donors. Two blood samples and two skin samples from patients affected with atopic dermatitis were used as control for Th2 typical inflammatory skin disease. The analysis of IL-1beta, IFN-gamma, TauNuF-alpha, IL-8, and ICAM-1 showed the characteristic Th1 pattern in all the psoriatic blood and skin samples analyzed. This study offers an opportunity for understanding inflammation in psoriasis and supports the hypothesis that OPN could represent a potential target for therapeutic intervention in psoriatic patients.

Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is released upon microbial invasion and contributes to mucosal and epithelial defense modulating both innate and adaptive immunity. We found that hBD-2 stimulates chemotaxis of human endothelial cells with an extent similar to that exerted by the vascular endothelial growth factor (VEGF). The hBD-2-dependent chemotaxis is dose-dependent, maximal effect being reached at 500 ng/ml concentration. In the absence of any growth factor, hBD-2 favors wound healing of endothelial cells, causing an about 2-fold increase in the speed of wound closure with respect to the control. Furthermore, hBD-2 promotes endothelial cell proliferation, although at a minor extent as compared to VEGF. When plated on matrigel enriched with angiogenic factors, endothelial cells form a three-dimensional network of tubes that gives rise to capillary-like structures. Similarly to VEGF, hBD-2 promotes capillary-like tube formation of human endothelial cells. Pro-angiogenic effect promoted by hBD-2 is dose-dependent, peaks at a 500 ng/ml hBD-2 concentration and is prevented by blocking anti-alphavbeta3 monoclonal antibody. However, hBD-2-induced pro-angiogenic activity is not due to endogenously produced VEGF because it is not prevented by neutralizing anti-VEGF antibodies. Overall, our findings suggest that hBD-2 could link inflammation and the host defense through its pro-angiogenic activity.

Objectives:Helicobacter pylori infection is a major health problem worldwide, and effective eradication of the infection is mandatory. The efficacy of recommended eradication regimens is approximately 70%. To avoid treatment failure and the consequent development of secondary resistance(s), it is important to choose the most appropriate first-line treatment regimen. This choice should also be made based on the knowledge of the antimicrobial resistance peculiar to a given geographical area. We evaluated the prevalence of antimicrobial-resistant H pylori strains isolated from naive patients and from patients with previous unsuccessful treatments.Methods:This study examined 109 H pylori-infected subjects (Group 1) who had never received an eradication treatment and 104 H pylori-infected subjects (Group 2) who had failed one or more eradication treatments. Resistance to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MET) and levofloxacin (LEV) was determined using the epsilometer test. The significance of differences was evaluated by the χ2 test.Results:The prevalence of antimicrobial resistance was 0% versus 3.1% to AMO, 0% versus 2% to TET, 27% versus 41.3% to MET (pH pylori strains resistant to multiple antimicrobials.Conclusions:This study confirms the high prevalence of H pylori strains resistant to CLA and MET, and indicates that unsuccessful treatments significantly increase resistance. Choosing eradication regimens other than standard triple therapy as a first-line therapy should be advisable in areas with high primary antimicrobial resistance prevalence.

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. © 2007 Wiley-Liss, Inc.

Recent evidence assigns integrins and metalloproteinases (MMPs) an important role in regulating tumor cell progression. Here, we demonstrate that 3-O-methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, affects cell proliferation and motility of breast cancer MCF-7 cells, downregulating avb5 integrin, and inhibiting MMP-9 secretion. This effect was absent when the non-tumoral MCF-10 cell line was used. Inhibition of cell motility was also associated to modifications in cell shape and in the distribution of tubulin fibers of OMF-treated MCF- 7 cells. In addition, a possible effect on survivin and hTERT was also investigated. We found that OMF strongly inhibits survivin and hTERT gene expression. The results of this study indicate that OMF-induced inhibition of cell motility may be mediated through the modulation of avb5 integrin and MMP-9secretion. In addition, the inhibition of typical markers of tumor progression such as hTERT and survivin in MCF-7 and their inactivity towards MCF10 provide strong evidence for a potential use of OMF in anticancer therapy Recent evidence assigns integrins and metalloproteinases (MMPs) an important role in regulating tumor cell progression. Here, we demonstrate that 3-O-methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, affects cell proliferation and motility of breast cancer MCF-7 cells, downregulating αvβ5 integrin, and inhibiting MMP-9 secretion. This effect was absent when the non-tumoral MCF-10 cell line was used. Inhibition of cell motility was also associated to modifications in cell shape and in the distribution of tubulin fibers of OMF-treated MCF-7 cells. In addition, a possible effect on survivin and hTERT was also investigated. We found that OMF strongly inhibits survivin and hTERT gene expression. The results of this study indicate that OMF-induced inhibition of cell motility may be mediated through the modulation of αvβ5 integrin and MMP-9 secretion. In addition, the inhibition of typical markers of tumor progression such as hTERT and survivin in MCF-7 and their inactivity towards MCF10 provide strong evidence for a potential use of OMF in anticancer therapy. © 2007 Wiley-Liss, Inc.

Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the beta-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human beta-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity.

Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

Study objectives: Levofloxacin is a fluoroquinolone antimicrobial agent for which pharmacodynamic relationships between the maximum serum antibiotic concentration (Cmax)/minimum inhibitory concentration (MIC) ratio and/or the area under the serum concentration-time curve during a 24-h dosing period (AUC0–24)/MIC ratio and clinical and/or microbiological outcomes have been developed. In this study we examined the relationship between the in vitro bacterial susceptibility to levofloxacin, the achieved levofloxacin serum and sputum concentrations, and the in vivo bacterial eradication in patients with acute exacerbations of chronic bronchitis. Patients and interventions: Thirty patients received levofloxacin, 500 mg/d po for 7 days. Samples of venous blood and sputum for the determination of levofloxacin concentrations were collected on day 1 immediately prior to dosing, and then at 1, 4, 8, 12, and 24 h. Results: The mean peak concentration in serum (6.5 mg/L) was found 1 h after administration, and at 4 h after administration in sputum (5.1 mg/L). Levofloxacin was always detectable 24 h after administration from both samples. Successful treatment occurred in 90% (27 of 30 patients) when assessed both clinically and bacteriologically. Treatment was successful in eight patients when the AUC0–24/MIC ratio was > 40 for serum, and in nine patients when it was > 30 for sputum. Treatment was also successful in seven patients when the Cmax/MIC ratio was > 5.01 for serum, and in nine patients when the Cmax/MIC ratio was > 4.01 for sputum. Treatment was successful in 90% (27 of 30 patients) when the AUC0–24/MIC ratio was > 125 for serum and > 100 for sputum, and when Cmax/MIC was > 10.01 for serum and > 8.01 for sputum following the first dose. Conclusions: The pharmacodynamics values that we have obtained in sputum with levofloxacin may be used as predictors of therapy outcomes. (CHEST 2005; 128:2093–2098)

Previous studies showed that long-wave ultraviolet (UVA) radiation induces severe skin damage through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. Recent results from our laboratory indicate a dramatic increase of both lipid peroxidation products (TBARS) and abnormal L-isoaspartyl residues, marker of protein damage, in UVA-irradiated human melanoma cells. In this study, the effects of hydroxytyrosol (DOPET), the major antioxidant compound present in olive oil, on UVA-induced cell damages, have been investigated, using a human melanoma cell line (M14) as a model system. In UVA-irradiated M14 cells, a protective effect of DOPET in preventing the uprise of typical markers of oxidative stress, such as TBARS and 2'7'-dichlorofluorescein (DCF) fluorescence intensity, was observed. In addition, DOPET prevents the increase of altered L-isoAsp residues induced by UVA irradiation. These protective effects are dose dependent, reaching the maximum at 400 microM DOPET. At higher concentrations, DOPET causes an arrest of M14 cell proliferation and acts as a proapoptotic stimulus by activating caspase-3 activity. In the investigated model system, DOPET is quantitatively converted into its methylated derivative, endowed with a radical scavenging ability comparable to that of its parent compound. These findings are in line with the hypothesis that the oxidative stress plays a major role in mediating the UVA-induced protein damage. Results suggest that DOPET may exerts differential effects on melanoma cells according to the dose employed and this must always be taken into account when olive oil-derived large consumer products, including cosmetics and functional foods, are employed.

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‐G(1) 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.

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.

The purpose of this study was to determine the in vitro susceptibility to cetyltrimethylammonium naproxenate for various aerobic and anaerobic micro-organisms responsible for oral and pharyngeal diseases by assessing the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) or minimum fungicidal concentrations (MFCs) and by determining kill-times. The MICs of cetyltrimethylammonium naproxenate for 46 tested strains (25 reference strains and 21 clinical isolates) ranged from 8 to 500 micrograms/ml. The MIC was found to be 31.25 micrograms/ml for 36% of the reference strains. Even lower MIC values (15.63 micrograms/ml) were observed for some anaerobic strains, for Haemophilus influenzae and for Candida tropicalis. MIC and MBC values corresponded for the majority of strains tested while the MFC for C. tropicalis and C. albicans was much higher. Only 9.5% of the clinical isolates gave a MIC value of 31.25 micrograms/ml. Enterococcus faecalis, Streptococcus pyogenes and Staphylococcus aureus showed MIC at 62.5 micrograms/ml. The MIC and MBC values among the isolates were comparable, while the MFC value for the yeasts was greater. A concentration of 125 micrograms/ml of cetyltrimethylammonium naproxenate inhibited the growth of all bacteria, except Enterobacteriaceae and Pseudomonaceae, and yeasts. Cetyltrimethylammonium naproxenate shows very rapid kill-time for S. sanguis (0"), and rapid (15") for S. pyogenes, S. dysgalactiae and S. mutans and for Moraxella catarrhalis, while a longer kill-time was necessary for the other microbes tested.

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.

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 µg/ml/106 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)-β1 (about 10 pg/ml), and tumour necrosis factor (TNF)-α (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-β1, and TNF-α. When monocytes were co-stimulated with LPS and amikacin, the PDGF-AB and TGF-β1 values almost overlapped with those from the stimulation of cells with LPS alone, while TNF-α 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-α. 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.

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.

Studies were carried out on some biological activities of Helicobacter pylori porins in vitro. We extracted and purified a porin with an apparent molecular mass of 30 kDa. Human polymorphonuclear leukocytes preincubated with H. pylori porins showed a decrease of chemotaxis, of adherence to nylon wool, and of chemiluminescence. Used as chemotaxins in place of zymosan-activated serum or as chemotaxinogens in place of zymosan, the porins induced polymorphonuclear leukocyte migration. Human monocytes and lymphocytes cultivated in the presence of H. pylori porins released cytokines. Release of the various cytokines studied was obtained with differentiated kinetics and at various porin concentrations. Starting only 3 h after culture, tumor necrosis factor alpha is released quickly, reaching a peak at 18 h, at a porin concentration of 1 microgram/ml/10(6) cells. Interleukin-6 (IL-6) appears later, with a peak at 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells. Lymphocytes stimulated by H. pylori porins release gamma interferon after 18 h of culture at higher concentrations of porins (20 micrograms/ml/10(6) cells). Granulocyte macrophage colony-stimulating factor is released from 6 to 48 h at a concentration of 1 microgram/ml/10(6) cells, while both IL-3 and IL-4 are released after 18 h of culture at different porin concentrations (0.1 and 1 microgram/ml/10(6) cells, respectively). Our results lead us to think that during H. pylori infection, surface components, porins in particular, are able to induce a series of chain reactions ranging from the inflammatory to the immunological responses.

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 μg/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-1α, 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.

The major toxic components in the outer membranes of gram-negative bacteria are lipopolysaccharides (LPS) (endotoxins) and porins. The latter are hydrophobic proteins (mass, about 35,000 Da) that account for more than 50% (18, 35) of the total membrane proteins and that are named porins for their ability to form transmembrane channels for the passive diffusion of small molecules across the cell membrane (26, 27). Purified porins possess immunomodulatory and procoagulant activities and are considered pathogenicity determinants. Depending on the dose, LPS and porins are either frankly toxic to a number of target cells or significantly alter normal cell functions (10, 45). By acting on human polymorphonuclear leukocytes (PMNs), subtoxic concentrations of porins, for example, inhibit phagocytosis and intracellular killing of Salmonella typhimurium (45), decrease oxidative burst and cell hydrophobicity, and cause significant morphological changes in the target cells (46). Subcutaneous injection of porins in rat hind paws causes local inflammation without complement activation (12, 13), a process that is known to be triggered in vitro by these proteins. Finally, it has recently been demonstrated that nontoxic concentrations of porins stimulate the synthesis and release of platelet-activating factor from different types of human cells (PMNs and mesangial and endothelial cells) (6, 41, 42) and promote proinflammatory and immunomodulatory cytokine release from immunocompetent cells or other cellular sources (11). Porins from different microorganisms have similar effects (8, 23, 43, 44). The cell damage from severe infections by gram-negative bacteria comes mainly from exotoxins and enterotoxins, even though the occurrence of LPS and porins in the bacterial microenvironment contributes significantly. Exposure of target cells to high concentrations of these substances leads to a rapid lytic death of the cells (1, 5, 10, 34, 45). In contrast, lower concentrations of LPS produce a less dramatic type of death: programmed cell death (PCD), or apoptosis (49). Classic morphological and biochemical signs of apoptosis in the toxin-injured cells are cell shrinkage, extensive blebbing of the cell surface, disappearance of microvilli, chromatin condensation, internucleosomal DNA fragmentation, presence of large intracytoplasmic vacuoles, increase of intracellular calcium ion and free radical levels, enhancement of endonuclease and protease activities, cell cycle arrest, and modified transcriptional activity of different apoptosis-linked genes (p53, bcl-2, c-myc, etc.) (2, 7). An increase in the expression of type II transglutaminase (tissue transglutaminase [tTGase]) has also been reported to be associated with apoptosis in several cell types, and an important role for tTGase in this process has been proposed (15). The ability of bacterial endotoxin to kill cells by necrosis and/or apoptosis has been found to play a key role in the pathogenesis of many alterations occurring in subjects with infections by gram-negative bacteria, including disseminated intravascular coagulation, septic shock, degenerative processes in different tissues, and reduced fertility or sterility (4, 10, 24, 48). The tissue lesions produced by Pseudomonas aeruginosa are of particular interest. P. aeruginosa is an opportunistic pathogen that commonly invades immunocompromised patients. The main targets of the LPS and porin released by this gram-negative rod during its lysis or active growth are the endothelial and epithelial cells present in many anatomical regions, including the skin, eyes, genitourinary tract, and heart valves. In particular, it has been reported that low levels of natural porin or LPS in patients with mild chronic infections severely damage the human spermatozoa in vitro and lead to reduced fertility or sterility (29). On the basis of these data and considerations, and taking into account the scarce information available on the cytotoxic effects of very low concentrations of LPS or porin on specific target cells, we were prompted to investigate the possible involvement of these substances in the induction of apoptosis in a cell line (SVC1) derived from the rat seminal vesicle secretory epithelium (36, 37, 49).

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. 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.

CHARGE association is characterized by ocular Coloboma, Heart malformations, choanal Atresia, Retardation of growth and development, Genital abnormalities and inner and external Ear abnormalities. Growth failure is a frequent find mainly associated with feeding difficulties or systemic diseases. To date, GH deficiency has been reported in only few patients with CHARGE association however long-term effects of GH treatment, up to final height, have never been reported. We describe a patient with CHARGE association and GH deficiency treated with GH from the age of 3 years and 10 months up to adult height.