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Over the years, the increasing acquisition of antibiotic resistance genes has led to the emergence of highly resistant bacterial strains and the loss of standard antibiotics’ efficacy, including β-lactam/β-lactamase inhibitor combinations and the last line carbapenems. Klebsiella pneumoniae is considered one of the major exponents of a group of multidrug-resistant ESKAPE pathogens responsible for serious healthcare-associated infections. In this study, we proved the antimicrobial activity of two analogues of Temporin L against twenty carbapenemase-producing K. pneumoniae clinical isolates. According to the antibiotic susceptibility assay, all the K. pneumoniae strains were resistant to at least one other class of antibiotics, in addition to beta-lactams. Peptides 1B and C showed activity on all test strains, but the lipidated analogue C expressed the greater antimicrobial properties, with MIC values ranging from 6.25 to 25 µM. Furthermore, the peptide C showed bactericidal activity at MIC values. The results clearly highlight the great potential of antimicrobial peptides both as a new treatment option for difficult-to-treat infections and as a new strategy of drug-resistance control.

Cotula cinerea, belonging to the tribe Anthemideae, is a plant widespread in the Southern hemisphere. It is frequently used in folk medicine in North African countries for several of its medical properties, shown by its extracts and essential oils. The dichloromethane extract obtained from its aerial parts demonstrated antibiotic activity against Enterococcus faecalis and was fractionated by bioguided purification procedures affording five main sesquiterpene lactones. They were identified by spectroscopic methods (NMR and ESIMS data) as guaiantrienolides, i.e., 6-acetoxy-1β-,6-acetoxy-1α-, and 6-acetoxy-10-β-hydroxyguaiantrienolide (1–3), and germacrenolides, i.e., haagenolide and 1,10-epoxyhaagenolide (4 and 5). The absolute configuration was assigned by applying the advanced Mosher’s method to haagenolide and by X-ray diffraction analysis to 1,10-epoxyhaagenolide. The specific antibiotic and antibiofilm activities were tested toward the clinical isolates of Enterococcus faecalis. The results showed that compounds 3–5 have antibacterial activity against all the strains of E. faecalis, while compound 2 exhibited activity only toward some strains. Compound 1 did not show this activity but had only antibiofilm properties. Thus, these metabolites have potential as new antibiotics and antibiofilm against drug resistant opportunistic pathogens.

The rapid development of antimicrobial resistance is pushing the search in the discovering of novel antimicrobial molecules to prevent and treat bacterial infections. Self-assembling antimicrobial peptides, as the lipidated peptides, are a novel and promising class of molecules capable of meeting this need. Based on previous work on Temporin L analogs, several new molecules lipidated at the N- or and the C-terminus were synthesised. Our goal is to improve membrane interactions through finely tuning self-assembly to reduce oligomerisation in aqueous solution and enhance self-assembly in bacterial membranes while reducing toxicity against human cells. The results here reported show that the length of the aliphatic moiety is a key factor to control target cell specificity and the oligomeric state of peptides either in aqueous solution or in a membrane-mimicking environment. The results of this study pave the way for the design of novel molecules with enhanced activities.

Green and alternative strategies for the preparation of silver nanoparticles (AgNP) are the focus of considerable attention, given their wide range of applications. A clean, eco-friendly, low-cost, scalable and straightforward protocol for the production of AgNP is reported, using hydrolyzed spent coffee grounds (HSCG) both as a reducing agent and a stabilizing matrix. The sample was easily recovered in 50–60% yield by centrifugation and lyophilization. Size and morphological analyses combined with X-ray diffraction analysis indicated ca. 6% w/w incorporation of AgNP of about 20 nm size into the HSCG matrix. AgNP-HSCG exhibited efficient antioxidant properties in several chemical assays and antimicrobial activities (MIC< 0.1 mg/mL) against Gram-negative bacteria such as E. coli and P. aeruginosa. Antibacterial devices made of chitosan films containing AgNP-HSCG were also prepared that showed significant inhibition zones against the same bacterial strains, even at the lowest loading (0.3% w/w) of AgNP, and remarkable antioxidant properties in aqueous media. An economic evaluation of the whole process was carried out, demonstrating that manufacturing of both HSCG and chitosan-based antibacterial devices is a convenient and easy-to-perform process, with the potential of full industrial scalability. Overall, the adopted circular approach, designed to reuse a cheap and abundant agri-food waste while avoiding the use of toxic or noxious chemicals, makes HSCG a practical and easily accessible multifunctional material for the clean and sustainable synthesis of AgNP with adequate size and stability for incorporation in efficient antioxidants and antimicrobial devices.

Purpose Human leukocyte antigen (HLA)-G is a non-classic major histocompatibility complex HLA class I molecule. HLA-G may have tolerogenic properties which are linked to epigenetic-sensitive pathways. There is a correlation of sHLA-G levels and graft acceptance in transplantation studies. There are previous data on correlation of sHLA-G with graft rejection as well as with viral infections such as hepatitis C virus (HCV) in kidney transplanted patients. Here, we report the sHLA-G expression in patients on the waiting list for kidney transplantation, with and without anti-HCV compared to a control group. Methods Serum of 67 patients on the waiting list for kidney transplantation (n = 43 with anti-HCV and n = 24 without anti-HCV) was analyzed. Among these patients, n = 39 were on the waiting list for the first transplantation, while n = 28 were patients who returned in the list. The control group included n = 23 blood donors with anti-HCV (n = 13) and without anti-HCV (n = 10). Results The expression of sHLA-G was significantly lower in the control group (39.6 ± 34.1 U/ml) compared to both - patients on the waiting list for the first transplantation (62.5 ± 42.4 U/ml, p=0.031) and patients who returned in the list (76.7 ± 53.9 U/ml, p=0.006). No significant differences were observed in all anti-HCV positive groups. A positive linear correlation between sHLA-G and TNF-α, and patient age was observed. Conclusions Serum sHLA-G values were significantly increased in both - patients on the waiting list for the first transplantation and patients who returned in the list, as compared to control group. Our findings confirm the key tolerogenic role of sHLA-G levels as epigenetic-related marker for measuring the state of kidney allograft acceptance.

The identification of human leukocyte antigen (HLA) antibodies in the sera of candidates awaiting organ transplantation has evolved over time. This has been possible because of the introduction of more sensitive techniques and to the increasing focus on the structural aspects of the HLA epitopes. The use of the HLAMatchmaker algorithm in the analysis of positive sera and the verification of HLA ABC epitopes in the HLA Epitope Registry website provide new stimuli on the interpretation of antibody reactivity. The epitopes defined by eplet pairs often involve a nonself-eplet and a self-eplet (nonself-self paradigm), suggesting that the antibody response to an HLA mismatch must have an auto-reactive component. Here, we report an application of the nonself-self paradigm that provides a basis for better knowledge and interpretation of HLA-antibody reactivity in Luminex assays with single alleles.

Serological assays are still considered the most useful tests in the diagnosis of syphilis. Since no single serological assay is able to provide a satisfactory result, in our laboratory we have evaluated the usefulness of a commercially-available immunoblot to diagnose syphilis infection among blood donors. From October 2012 to June 2013, 4572 blood donors were screened for syphilis with an automated chemiluminescent microparticle immunoassay (CMIA). To confirm the presence of treponemal antibodies, CMIA-reactive sera were tested by standard Treponema pallidum haemagglutination assay (TPHA). In addition, an alternative confirmatory test – the immunoblot INNO-LIA assay was introduced in our laboratory. Since two additional positives among CMIA-reactive-TPHA-negative samples were found, we concluded that the INNO-LIA immunoblot allowed a better detection of syphilis compared to TPHA. A confirmatory strategy based on the use of two treponemal assays could meet the screening requirements for blood donors as well as in our centre.

The search of HLA antibodies is currently more accessible by solid-phase techniques (Luminex) in the immunized patients leading to an expansion of the antibody patterns. The aim of this study was to investigate low median fluorescence intensity value in unexpected reactivity patterns. Here, we performed HLAMatchmaker analyses to evaluate the potential functional epitopes that can elicit HLA-specific alloantibody responses in a pregnancy-sensitized woman with an epitope defined by the 82LR. Surprisingly, in according to the registry of HLA epitopes, we found that 82LR epitope covered all allelic specificities of our unexpected antibody patterns, shared between Bw4-positive HLA-B antigen and HLA-A23, -A24, -A25 and -A32. This finding is consistent with the verification of HLA ABC epitope recorded in the website-based HLA Epitope Registry and addresses the importance of determining HLA antibody epitope-specificities on Luminex technique-dependent antibody reactivity. 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

Background Patients with end-stage liver disease (ESLD) have a compromised nutritional status because of the liver crucial role in regulating metabolic homeostasis and energy balance. Data sources A systematic review of literature based on extensive relevant articles published from 2001 to 2017 in English in PubMed database was performed by searching keywords such as liver disease, non-alcoholic liver disease, alcoholic liver disease, malnutrition, epigenetics, gut microbiota, and probiotics. Results Liver transplantation would be one eligible therapy for ESLD patients, even if, the clinical outcome is negatively influenced by malnutrition and/or infections. The malnutrition is a condition of nutrient imbalance with a high incidence in ESLD patients. An accurate evaluation of nutritional status could be fundamental for reducing complications and prolonging the survival of ESLD patients including those undergoing liver transplantation. In addition, the interaction among nutrients, diet and genes via epigenetics has emerged as a potential target to reduce the morbidity and mortality in ESLD patients. The malnutrition induces changes in gut microbiota causing dysbiosis with a probable translocation of bacteria and/or pathogen-derived factors from the intestine to the liver. Gut microbiota contribute to the progression of chronic liver diseases as well as hepatocellular carcinoma. The administration of probiotics modulating gut microbiota could improve all chronic liver diseases. Conclusions This review provides an update on malnutrition status linked to epigenetics and the potential benefit of some probiotics on the management of ESLD patients. In support of this view and to reveal the constant and growing interest in this field, some clinical trials are reported.

Intravenous immunoglobulin (IVIG) products are known to have beneficial immunomodulatory effects on several inflammatory and autoimmune disorders. These effects could be attributed to a different inhibitory action on complement factors, but other mechanisms could be implicated, e.g., immunocomplexes development and/or anti-idiotypic antibodies. Positive results on the reduction of anti-Human Leukocyte Antigens (HLA) antibodies in highly sensitized patients have also been found. The present study focuses on the effect of IVIG on the reduction of Panel Reactive Antibody level and crossmatch positivity in sensitized patients awaiting kidney transplantation.The study was performed adapting an in vitro assay on sensitized patients' sera in waiting list for kidney transplantation. Sera of twelve highly sensitized patients were evaluated for the cytotoxicity inhibition after 10% IVIG treatment.A reduction of anti- HLA antibody levels was observed in 75% (9/12) of treated patients in vitro, while 25% (3/12) resulted unresponsiveness. Particularly, our data showed a significantly higher Panel Reactive Antibody reduction for T lymphocytes (p0.010) than B lymphocytes (p0.032).In this study, we have used an in vitro assay to investigate susceptibility to desensitization with IVIG treatment of sensitized patient sera. These findings reveal that the variable effect of IVIG on reducing Panel Reactive Antibody in our immunized patients could be attributed to a different inhibitory action on complement, likely due to the type and the titre of anti-HLA antibodies.

Human leukocyte antigen (HLA) antibodies represent a significant risk factor for transplant failure. It is very important to characterize anti-HLA antibodies as epitopes rather than antigens so that this knowledge can be applied clinically. The aim of the study was to investigate the extra reactivity patterns in sensitized multipare. Here, we have used the HLAMatchmaker program, a theoretical algorithm, to explain these unexpected antibody reactivity patterns in multipare awaiting for heart transplant. The patient was sensitized during pregnancy by alleles HLA-A(*)24:02, HLA-DRB1(*)07:01, HLA-DRB4(*)01:01, DQB1(*)02:02 and DQA1(*)02:01 mismatches with development of respective antibodies. However, the patient' sera were shown an unexpected reactivity not directed toward HLA mismatches of daughters: A(∗)23:01, A(*)24:03 and B(*)15:12 for class I and DRB4(*)01:03, DRB1(*)09:02, DRB1(*)09:01, DQB1(*)03:01, DQB1(*)03:03, DQB1(*)03:02, DQB1(*)04:02, DQB1(*)04:01 and DQB1(*)02:01 for class II. By HLAMatchmaker analysis we found that these antibodies reacted with eplet shared by antigens in single allele Luminex panels. These eplets were: 62EE, 66GKH, 70KAH, 71HS, 127K, 113YH, 144KR, 150AAH, 151AHV, 163TG and 167DG for class I and 4Q, 74RRAE, 71RRA, 98KN, 120N, and 135G, 25FT, 34HE, 41ER, 47EK2, 48LF for class II. Thus, HLAMatchmaker software together with to solid phase techniques could open new horizons for a more precise characterization of the HLA-antibodies.

Chlamydia pneumoniae, an intracellular bacterium, is associated with respiratory diseases, reinfectivity and chronic diseases such as cardiovascular disease, hypertension and stroke. The risk of infection is higher and infections are a serious clinical problem in patients with type 1 (insulin-dependent) diabetes mellitus (T1DM). Although diabetes mellitus and hyperglycaemia are considered possible risk factors for various types of aetiological agents, the epidemiological evidence concerning C. pneumoniae infection is scanty. The aim of the present study was to evaluate the impact of glycosylated haemoglobin (HbA1c) levels, an indicator of a hyperglycaemic state, on C. pneumoniae infection and disease chronicity; in addition we compared the duration of diabetes with the occurrence of C. pneumoniae infection. C. pneumoniae blood real time PCR and serology (IgG, IgA and IgM) assays by an ELISA method were performed. C. pneumoniae DNA was detected in 46.5 % [95 % confidence interval (CI) = 35.1-57.9 %] of the patients with T1DM; this prevalence is higher (P0.05) than in non-diabetic paediatric controls, 10.5 % (95 % CI = 3.6-17.4 %). IgG/IgA C. pneumoniae antibody positivity was significantly (P≤0.05) more common in patients in poor metabolic control (HbA1c9 %) versus patients in good metabolic control (HbA1c7 %), suggesting that the metabolic control of the disease is compromised in the patients with T1DM. In conclusion, adolescents with T1DM were more likely to show signs of infection with C. pneumoniae compared with healthy adolescents and the results suggest an increased risk of progressing from an acute C. pneumoniae infection to a chronic form.

Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process. Porphyromonas gingivalis, a Gram-negative anaerobic black-pigmented rod, which produces several virulence factors that stimulate human periodontal ligament cells (HPLCs) to produce various inflammatory mediators, has been implicated as a crucial etiologic agent in the initiation and progression of periodontitis. Since natural polyphenols such as resveratrol have growth-inhibitory effects on some bacterial pathogens and have shown chemo-preventive, anti-inflammatory and antioxidant activity, in the present study we used an HPLC model stimulated with lipopolysaccharide (LPS) of P. gingivalis to simulate the in vivo conditions such as those found in diseased periodontal sites. To determine whether resveratrol interferes with P. gingivalis LPS-activity and reduces the production of pro-inflammatory molecules, we investigated its effect on the cytokines IL-1b, IL-6, IL-8, IL-12 and TNF-a and NO production of HPLCs. The results showed that resveratrol treatment decreased in a dose- and time-dependent manner the NO expression induced by P. gingivalis LPS, correlated to an increased viability of infected HPLCs, and decreased the production of pro-inflammatory cytokines in HPLCs stimulated by P. gingivalis LPS. These results suggest that the ability of resveratrol to determine immunomodulatory effects could provide possible therapeutic applications for the treatment of periodontitis.

Chlamydia pneumoniae is an obligate intracellular Gram-negative bacterium that causes recurrent pharyngitis, pneumonia and chronic inflammation induced by cycles of persistence and productive infection that might also explain the association with chronic diseases. The aim of this study was to determine whether C. pneumoniae can invade and survive within human osteoblasts and whether this infection elicits the secretion of proinflammatory cytokines. Our results demonstrated that C. pneumoniae was able to infect the SaOS-2 osteoblastic cell line and to replicate in the osteoblasts in a time-dependent manner and was associated to an increase in the cell number and cell viability. In addition, infection of the SaOS-2 cell line with C. pneumoniae at MOI of 4 is correlated to a proinflammatory response. Infected osteoblasts produced increased levels of cytokines IL-6, IL-8, IL-17, and IL-23. The production of cytokines increased with subsequent interaction between osteoblasts and monocytes and the maximum levels of cytokines released were detected 72 h after infection with C. pneumoniae. Thus, controlling the release of chemokines, e.g., IL-23, may be a therapeutic strategy for preventing inflammatory bone disease and counteract inflammation and bone destruction.

Monocytes are pivotal effector cells of the innate immune system that are vital for recognizing and eliminating invasive microbial pathogens. When microbial products bind to pathogen-recognition receptors, monocytes are activated and release a broad array of cytokines and defensins that orchestrate the host innate and adaptive immune responses. The aim of the present study is to investigate whether Toll-like receptor-4 (TLR4) mediates human beta-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells. We showed that TLR4 is expressed in U937 cells and monocytes infected with viable microorganisms in a time-dependent fashion, while heat-inactivated microorganisms induced a lesser expression, albeit still significant, of TLR4 compared with viable organisms; flow cytometric analysis, in particular, revealed a higher level of TLR4 expression at 48 and 72 h postinfection. In addition, U937 cells and monocytes responded to C. pneumoniae in a TLR4-dependent manner with induction of mRNA and protein of the antimicrobial peptide HBD-2. The treatment of cells with TLR4-neutralizing antibody resulted in a decrease in C. pneumoniae-induced HBD-2 production. This study reveals that TLRs not only recognize ligands but also the types of effector molecules induced, namely, antimicrobial peptides. An understanding of the importance of the TLR-mediated antimicrobial mechanisms may provide new avenues for the development of therapeutic regimens aimed at activating the body's own defenses by stimulating TLR-dependent pathways.

Chlamydia pneumoniae is an obligate intracellular Gram-negative bacterium with a unique biphasic developmental cycle that can cause persistent infections. In humans, Chlamydia causes airway infection and has been implicated in chronic inflammatory diseases, such as asthma and atherosclerosis. In addition, recent studies demonstrated that patients with severe periodontitis can harbor C. pneumoniae, which can increase the risk for a host inflammatory response with weighty clinical sequelae. Previous studies have established that periodontal pathogenic bacteria (i.e. Gram-negative bacteria) can induce the synthesis and release of cytokines and other inflammatory mediators in human gingival fibroblasts. HGF are resident cells of the periodontium that respond to receptor stimulation by producing a variety of substances including cytokines and growth factors. Our results demonstrate that after 48 hr of incubation with viable C. pneumoniae HGF showed a proliferative response, as seen by both colorimetric MTT assay and direct cell count (30% and 35%, respectively). In addition, HGF incubated with viable or UV light-inactivated C. pneumoniae organisms showed an increase in the levels of IL-6 and IL-10, but not IL-4; on the contrary, HGF infected with heat-killed bacteria did not show a significant production of any of the cytokines considered. In conclusion, the present study suggests that C. pneumoniae may modulate the expression of IL-6 and IL-10 by human gingival fibroblasts. Further studies are warranted to clarify the molecular mechanisms of C. pneumoniae in the regulation of cytokine expression by host cells and to elaborate the relevant clinical implications.

Tri- and tetranuclear clusters of formula C[RuCo3(CO)12] (C = NEt4, NEt4-3; C = bmim, bmim-3; C = H, H-3), [Co4(CO)12] (4), [Co4(CO)10(μ-dppm)] (dppm = Ph2PCH2PPh2) (5), [Co4(CO)8(μ-dppa)2] (dppa = Ph2PNHPPh2) (6), [HFeCo3(CO)11(PPh3)] (7), and [(PNP)PtCo2(CO)7] (PNP = dppa, 8-H; PNP = C6H13SCH2CH2N(PPh2)2, 8-S; PNP = PhN(PPh2)2, 8-Ph) were used as precatalysts in the intramolecular Pauson−Khand reaction of diethyl (allylpropargyl)malonate (1) under 8 bar of CO at 70 °C, which afforded the cyclopentenone 2. The best results with 2% precatalyst loading were obtained with NEt4-3 and bmim-3, which gave isolated yields of 93 and 94%, respectively. With a 10% loading, H-3 afforded a yield of 98%. Whereas the diphosphane-substituted Co4 clusters 5 and 6 were inactive, the PtCo2 clusters 8 (at 5% loading) afforded yields ranging from 85 to 91%. The enyne-substituted clusters 9 with a butterfly RuCo3 metal core were isolated from the reaction of NEt4-3 with 1 and were characterized by ESI-MS and spectroscopic met...

A combined ESI-MS/NMR mechanistic study on the palladium-catalysed cross-coupling reaction between aryldiazonium salts and aryltrifluoroborates with bis(μ-acetato)bis(4,4′-difluoroazobenzene-C2,N)dipalladium(II) (4) as the precatalyst is reported. The reaction follows a Pd0/PdII cycle after reduction of 4 to a molecular Pd0 species (I), which according to the combined ESI-MS and 19F NMR studies, bears an arylated azobenzene ligand. Oxidative addition by the diazonium salt generates the arylpalladium(II) intermediate (II), which was also detected in solution. The catalytic cycle is completed with a transmetallation between II and theorganoborate, which is followed by fast reductive elimination of the cross-coupling product to restore the molecular Pd0 species I. A concurrent activation path was also observed which consists of the formation of (4,4′-difluoroazobenzene-C2,N)dipalladium(II) tetrafluoroborate (7) by the reaction of 4 with the diazonium salt and subsequent reduction by the aryltrifluoroborate to give I. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Chlamydia pneumoniae , a gram-negative bacterium, is an important human intracellular pathogen; studies of C. pneumoniae pathogenesis have shown that the organism can infect many cell types associated with both respiratory and vascular sites, including arterial smooth muscle cells, macrophages and vascular endothelium. Recently, the recognition of atherosclerosis as an inflammatory disease in its genesis, progression and ultimate clinical manifestations has created an interesting area of vascular research. We assessed the hypothesis that growth factors from THP-1 macrophages infected with C. pneumoniae are involved in the regulation of cell proliferation in HUVEC. The induction of these factors were dependent on time of infection, as medium harvested 48 h after infection had a greater activity than media harvested at 12 or 24 h after infection. Heat-killed C. pneumoniae produced similar results to those of live bacteria. In addition, conditioned medium filtered sterile from infected macrophages induced the proliferation of HUVEC, thus demonstrating its angiogenic potential. Moreover, pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded almost comparable results, suggesting that bacterium cell-attachment is sufficient for VEGF, IL-1β and IL-8 induction. Further studies are necessary to elucidate the biological role of chlamydial involvement in the complex and mutifactored processes of angiogenesis and possibly contribute to the development of therapeutic strategies.

Chlamydophila pneumoniae is an important human intracellular pathogen; however, the pathogenesis of C. pneumoniae infection is poorly understood and the immune control mechanism versus host cells is not completely known. The role of the nitric oxide (NO) synthase pathway in inhibiting the ability of C. pneumoniae to infect macrophage J774 cells and the ability of NO to damage isolated C. pneumoniae were investigated. Exposure of infected cultures to recombinant murine gamma interferon (MurIFN-γ) resulted in increased production of NO and reduced viability. Addition of 2-(N,N-diethylamino)-diazenolase-2-oxide before infection of J774 cells or during chlamydial cultivation released NO, both resulting in a reduction in the viability of C. pneumoniae in a dose-dependent way. These results indicate that immune control of chlamydial growth in murine macrophage cells may trigger a mechanism that includes NO release with effects on the multiplication of the microorganism, thus suggesting that NO may play a role in preventing the systemic spread of Chlamydia.Key words: Chlamydophila pneumoniae, J774 cells, NO.

The system comprising a palladium complex in a 1-butyl-3-methylimidazolium tetrafluoroborate/methanol mixture efficiently catalyses the cross-coupling reaction between p-tolyldiazonium tetrafluoroborate and potassium phenyltrifluoroborate at room temperature. The presence of methanol (or water) in the reaction mixture is necessary in order to achieve quantitative conversions, due to its scavenging behaviour towards the BF3 formed during the reaction. Yields higher than 90% were obtained using Pd2(dba)3 or the azapalladacycle 10 as the palladium source. With the latter complex a turnover frequency of about 6000 h−1 was attained in the coupling of aryldiazonium tetrafluoroborates with potassium vinyltrifluoroborate. Recycling of the catalytic solution could be performed provided that a slight excess of diazonium salt was used in the first run. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Chlamydia trachomatis and Chlamydophila pneumoniae are human pathogens; the former being the etiologic agent for trachoma as well as a prevalent sexually transmitted bacterium, while C. pneumoniae is a respiratory pathogen responsible for community-acquired pneumonia. Patients with reactive arthritis show evidence of present or past Chlamydial infection. Chlamydia spp., has been strongly implicated as a triggering factor for reactive arthritis. We describe the simultaneous occurrence of C. pneumoniae and C. trachomatis infections in a subject with reactive arthritis. We suggest treatment for a patient with Chlamydia-associated arthritis to define a means by which persistent organisms can be induced to return to the active developmental cycle, thereby making them more accessible to antibiotic activity.

Automated chemiluminescent immunoassays (CLIAs) are useful for the detection of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus 1/2 antigen/antibodies (HIV 1/2 Ag/Ab) in blood donor screening. Eight hundred and forty serum samples were tested for hepatitis B surface antigen (HBsAg), HCV antibodies (anti-HCV), and HIV1/2 Ag/Ab in parallel using 2 different CLIAs (Abbott Architect i2000SR and Roche Cobas e411). The concordance between the 2 systems was high (Cohen's kappa 0.97 for HBsAg, 0.77 for anti-HCV, 0.92 for HIV1/2 Ag/Ab) and the specificity and the positive predictive value were comparable. Among the 12 discrepant results, 11 were false-positive and 1 (reactive by Architect) was true-positive for anti-HCV. Positivity for HBV DNA, HCV RNA, and HIV RNA was recorded in 90.9%, 38.9%, and 100% of true-positive samples, respectively. This study represents the first stringent comparison between Architect i2000SR and Cobas e411 in blood donors. We observed a good correlation and high agreement among HBV, HCV, and HIV with the 2 automated systems.

Matrix metalloproteinases (MMP) are a family of host-derived enzymes involved in the turnover of extracellular matrix (ECM) molecules, and, in particular, it is demonstrated that the 92 KDa gelatinase MMP-9 is often expressed in atherosclerotic plaques by macrophages and smooth muscle cells. Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerosis lesions. In this study, we analyzed the TLR2/TLR4 expression in HUVEC infected with C. pneumoniae and correlated it to the production of VEGF and MMP-9. The results obtained showed an increased VEGF and MMP-9 production correlated with a time-dependent increase in cellular proliferation in HUVEC infected with C. pneumoniae at a multiplicity of infection (MOI) of 2 IFU/cell. HUVEC preincubated with VEGF antibody did not release MMP-9, as detected by zymography assessment and ELISA assay. In addition, we demonstrated that TLR2/TLR4 are expressed in HUVEC infected with viable microorganisms (25#x0025; and 17#x0025;, respectively), while UV-inactivated microorganisms induced a lesser expression (20#x0025; and 11#x0025;, respectively) compared to control cells and HUVEC exposed to heat-killed bacteria showed a percentage of TLR-expressing cells similar to the control cells. In addition, the cells preincubated for 60 min with TLR2/TLR4 neutralizing antibodies showed a decrease in C. pneumonia-induced VEGF and MMP-9 production.

Flavonoids are phenolic compounds widely distributed in almost every plant and act as pharmacologically active constituents in many herbal medicines. They have multiple biological, pharmacological, and medicinal properties including anti-inflammatory and cytoprotective effects. In the present study, the experiments were performed to evaluate the effect of resveratrol and quercetin on proliferation, viability, nitric oxide (NO) production, and apoptosis in Salmonella enterica serovar Typhimurium-infected U937 cells and monocytes (MN). The results showed in a time- and dose-dependent manner that both resveratrol and quercetin reduced S. enterica serovar Typhimurium-induced NO production. In addition, the vegetable extracts resveratrol and quercetin inhibited cell viability and proliferation in S. enterica serovar Typhimurium-infected cells. S. enterica serovar Typhimurium-induced apoptosis was also blocked by resveratrol and quercetin. The results obtained indicate that flavonoids modulate the host response during salmonellosis by protecting the host cells from the toxic effects of bacterial infection and also by decreasing programmed cell death. Hence, these polyphenols can be considered potential candidates against S. enterica serovar Typhimurium-related gastric pathogenic processes, and further attention should be given to their application as a treatment for infectious diseases.

Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process affecting tooth-supporting tissues and resulting in periodontal pocket formation, alveolar bone resorption and, eventually, tooth loss. The continuous challenge of host immune and resident cells by periodontopathogens and their virulence factors, such as lipopolysaccharide (LPS), results in enhanced and uncontrolled secretion of cytokines. The latter directly or indirectly participate in tissue destruction and bone resorption. Metronidazole (MTZ) is a widely used antimicrobial agent. The immunomodulatory effects of antibiotics might influence the degree of the local response to infection on the human periodontal ligament cell (HPLC). HPLCs play a role in the immune response of the oral cavity. In addition, HPLC can produce cytokines that increase the inflammatory response and that supply for normal communication. MTZ has also been proposed in the field of periodontal therapy either with a systemic administration or with local biodegradable sustained-release agents. The local administration of MTZ in the form of gel significantly reduces the systemic side effects. The aim of the present study, was to simulate the in vivo conditions occurring in diseased periodontal sites, and to evaluate the effects of MTZ on the viability of isolated HPLCs. The ability of MTZ to modulate the release of interleukin (IL)-1 beta, IL-6, IL-8, IL-12 and tumor necrosis factor alpha (TNF-alpha) in HPLC, treated or not with LPS of Porphyromonas gingivalis was also evaluated. The results obtained showed that MTZ had no cytotoxic effect on HPLC and was able to inhibit the production of pro-inflammatory cytokines analyzed. The ability of MTZ to determine immunomodulatory effects could provide possible therapeutic applications in the field of periodontal research. (C) 2010 Elsevier B.V. All rights reserved.

Human beta-defensin 2 is an antimicrobial peptide that is produced by several epithelial cells after stimulation with micro-organisms and inflammatory mediators. Gram-negative bacteria, which are typically detected in periodontal pockets in periodontitis, elicit a stronger antibacterial peptide response of human beta-defensin 2 by epithelial cells. In this study, we investigated whether Chlamydia pneumoniae is able both to enter and grow in human gingival fibroblasts (HGF), to modify the production of cytokines, and is involved in regulation of beta-defensin 2 expression. Gingival fibroblasts discarded from periodontal procedures on healthy young individuals were infected with viable C. pneumoniae or with heat- or ultraviolet-inactivated organisms at a multiplicity of infection of 4 inclusion-forming units per cell. Our results demonstrate that after 48 h of incubation with viable C. pneumoniae, gingival fibroblasts showed a proliferative response as seen by both colorimetric assay and direct cell count (40% and 45%, respectively). Moreover, cells incubated with viable or ultraviolet light-inactivated C. pneumoniae organisms showed an increase in the levels of interleukin-6, interleukin-10 and human beta-defensin 2 in a time-dependent fashion, while the cells infected with heat-killed bacteria did not show a significant production either of the cytokines or beta-defensin 2 at any time. In conclusion, we demonstrate the correlation between multiplication of C. pneumoniae in human gingival fibroblasts and release of interleukin-6, interleukin-10 and up-regulation of beta-defensin 2, suggesting that gingival fibroblasts may be a periodontium niche for obligate intracellular C. pneumoniae and may play a role in innate gingival immune system and inflammatory response mechanisms of periodontitis.

Chlamydia pneumoniae is an intracellular pathogen and an important cause of respiratory tract infections in humans and more recently it has been associated with chronic diseases such as atherosclerosis. Numerous studies have been performed to show the "infectious" hypothesis of atherosclerosis by direct detection of the organisms within atheromatous plaques by seroepidemiological estimation and by animal, immunological and antibiotic interventional studies. In this work we investigated the relation between chronic chlamydial infection, inflammatory markers, Interleukin 7 (IL-7) production and coronary heart disease. We studied 60 patients with coronary heart diseases (CHD), 45 of whom were men and 15 women, with a mean age of 65+/-5 years, and a control group of 20 healthy subjects, 15 men and 5 women, with a mean age of 60+/-7 years. Detailed histories including symptoms, risk factors and demographic data were obtained from patients and healthy subjects by administering a standardized questionnaire. Our results demonstrate that the enzyme-linked immunoassay (ELISA) test appears to have a greater sensitivity than the microimmunofluorescence (MIF) technique. 80% of patients had positive IgG to C. pneumoniae and 58% positive IgA to C. pneumoniae with ELISA, while the MIF test showed 68% and 55% positive IgG and IgA to C. pneumoniae, respectively. The control subjects showed 55% positive IgG and 10% IgA to C. pneumoniae by ELISA and 35% positive IgG and 5% IgA to C. pneumoniae by MIF. The combination of positive IgG and IgA to C. pneumoniae was present more frequently than in the control group. Serum levels of IL-7 measured by ELISA were also significantly higher in patients compared to healthy subjects. In conclusion, our study shows that C. pneumoniae IgG and IgA seropositivity, inflammatory markers such as IL-7, fibrinogen, C-reactive protein were significantly correlated with CHD.

Ionic liquids (ILs) are suitable media for the Co 2 (CO) 8 -catalysed intramolecular and intermolecular Pauson–Khand (PK) annelation, provided that the reaction is carried out under a CO pressure of 10 bar. Two diethyl allyl propargyl malonates were quantitatively converted into the relevant cyclopentenones, whereas heteroatom tethered enynes gave lower yields in their cyclocarbonylation products. A moderate yield in the corresponding Pauson–Khand product was obtained reacting phenylacetylene with norbornene.

Pseudomonas aeruginosa is a Gram-negative, aerobic bacillus causing infections of the respiratory and other organ systems in susceptible hosts. Although it does not cause pulmonary infections in immunocompetent individuals, P. aeruginosa causes chronic lung infection in individuals with cystic fibrosis and nosocomial pneumonia resulting in significant morbidity and mortality. Exogenous administration of an important P. aeruginosa virulence factor, lipase, present in P. aeruginosa culture supernatant, induces potent mononuclear cell activation leading to the production of numerous proinflammatory cytokines. In particular, P. aeruginosa culture supernatant stimulated increased proliferation of THP-1 cells and monocytes (MN). The addition of culture supernatant to THP-1 cells and MN also induced Interleukin (IL)-23 and vascular endothelial growth factor (VEGF) release in a time-dependent manner. To investigate whether any compounds present in the supernatant lipase contributed to releasing IL-23 and VEGF, the culture supernatant from P. aeruginosa containing lipase was treated with hexadecylsulfonylfluoride (AMSF). The AMSF-treated culture supernatant (CS) did not show any induction on the IL-23 and VEGF release compared to the cells treated with CS without AMSF. We also showed that Toll-like receptors (TLR)2/TLR4 are expressed in THP-1 cells and MN treated with P. aeruginosa CS in a time-dependent fashion. Flow cytometry analysis revealed a higher TLR4 and a lower TLR2 expression at 48 and 72 h of treatment. The treatment of cells with TLR4 neutralizing antibody, and to a lesser extent with TLR2 neutralizing antibody, resulted in a decrease in P. aeruginosa CS-induced IL-23 and VEGF production.