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Mesangial cells (MC) maintain the architecture and cellular communication and indirectly join in the glomerular filtration rate for the correct functioning of the glomerulus. Consequently, these cells are activated constantly in response to changes in the intraglomerular environment due to a metabolic imbalance or infection. IL-36, a member of the IL-1 family, is a cytokine that initiates and maintains inflammation in different tissues in acute and chronic pathologies, including the skin, lungs, and intestines. In the kidney, IL-36 has been described in the development of tubulointerstitial lesions, the production of an inflammatory environment, and is associated with metabolic and mesangioproliferative disorders. The participation of IL-36 in functional dysregulation and the consequent generation of the inflammatory environment by MCs in the presence of microbial stimulation is not yet elucidated. In this work, the MES SV40 cell cultures were stimulated with classical pathogen-associated molecular patterns (PAMPs), mimicking an infection by negative and positive bacteria as well as a viral infection. Lipopolysaccharide (LPS), peptidoglycan (PGN) microbial wall components, and a viral mimic poly I:C were used, and the mRNA and protein expression of the IL-36 members were assessed. We observed a differential and dose-dependent IL-36 mRNA and protein expression under LPS, PGN, and poly I:C stimulation. IL-36β was only found when the cells were treated with LPS, while IL-36α and IL-36γ were favored by PGN and poly I:C stimulation. We suggest that the microbial components participate in the activation of MCs, leading them to the production of IL-36, in which a specific member may participate in the origin and maintenance of inflammation in the glomerular environment that is associated with infections.

Marine obligate actinobacteria produce a wide variety of secondary metabolites with biological activity, notably those with antibiotic activity urgently needed against multi-drug-resistant bacteria. Seventy-five marine actinobacteria were isolated from a marine sediment sample collected in Punta Arena de La Ventana, Baja California Sur, Mexico. The 16S rRNA gene identification, Multi Locus Sequence Analysis, and the marine salt requirement for growth assigned seventy-one isolates as members of the genus Salinispora, grouped apart but related to the main Salinispora arenicola species clade. The ability of salinisporae to inhibit bacterial growth of Staphylococcus epidermidis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacer baumannii, Pseudomonas aeruginosa, and Enterobacter spp. was evaluated by cross-streaking plate and supernatant inhibition tests. Ten supernatants inhibited the growth of eight strains of S. epidermidis from patients suffering from ocular infections, two out of the eight showed growth inhibition on ten S. epidermidis strains from prosthetic joint infections. Also, it inhibited the growth of the remaining six multi-drug-resistant bacteria tested. These results showed that some Salinispora strains could produce antibacterial compounds to combat bacteria of clinical importance and prove that studying different geographical sites uncovers untapped microorganisms with metabolic potential.

Staphylococcus epidermidis is a coagulase-negative bacterium capable of causing recurrent relapses in prosthetic joint infection (PJI). The aim of this study was to determine if Staphylococcus epidermidis isolates from patients with recurrent relapses of prosthetic joint infection (PJI) changed genotypically (pulsed-field gel electrophoresis (PFGE) pattern analysis and genes involved in biofilm formation) and phenotypically (antimicrobial resistance, biofilm formation) during the different episodes. Four patients with PJI recurrent relapses were evaluated clinically and microbiologically. Genotypic and phenotypic characteristics of 31 S. epidermidis isolates were determined. In all cases, PJI was treated with antimicrobial therapy and resection of the prosthesis without reimplantation. Months later, all patients had a relapse episode and treated with rifampin plus vancomycin and surgical debridement. Changes in the antibiotics resistance profile in isolates from patients 1 and 2 were observed in the two episodes. Patient 1 had four clones A, B, C, and D that were distributed differentially in the two episodes. Similarly, patients 2 and 3 had two clones and subclones (E-E1 and F-F1, respectively), and patient 4 had only the clone G in both episodes. The clone F formed small-colony variants (SCVs). High level of biofilm formation was found in all clones, except for clones D and G. Clones/subclones showed a genotypic variation in icaA, sdrF, bap, sesI, and embp genes. The principal coordinate analysis showed that all clones/subclones were different. These results showed that the initial infective clone of S. epidermidis from PJI, changed genotypically and phenotypically after a second relapse as a response to the treatment.

Currently, the treatment of infections by Staphylococcus epidermidis (S. epidermidis) represents a challenge because some strains have multidrug-resistance to antimicrobial products (antibiotic and biocides) and can produce biofilms. These biofilms protect bacterial cells from both antimicrobials and the host immune response. Therefore, it is crucial to encourage research on the development of new treatments. One method is immunotherapy, targeting components of S. epidermidis, such as S. epidermidis surface (Ses) proteins. Ses is expressed constitutively in most strains, and they participate in biofilm formation. This review is an update on Ses, regarding their structure, biological function, their relationship with S. epidermidis biofilm formation, and its possible role as therapeutic targets to develop immunotherapeutic treatments to prevent infections by S. epidermidis.

Staphylococcus epidermidis can cause prosthetic joint infections. Strategies to differentiate between healthy skin and prosthetic joint infections isolates are relatively ineffective, which makes necessary to search for new differential biomarkers. Staphylococcus epidermidis has eleven surface proteins, denoted as Ses proteins. In this work, ses genes are used as biomarkers to differentiate between prosthetic joint infections and healthy skin isolates.All prosthetic joint infections (n = 51) and healthy skin (n = 51) isolates were genotyped by pulsed-field gel electrophoresis. icaA, embp, sesA-I, and sdrF genes were determined by PCR. The phenotypic data included biofilm production and antibiotic resistance.10 pulsed-field gel electrophoresis profiles were identified: four profiles were exclusive of prosthetic joint infections isolates, three profiles presented a higher proportion in prosthetic joint infections isolates and three profiles presented a higher proportion in healthy skin isolates. sesA, sesB, sesC, sesD, sesE, sesG, and sesH genes were more prevalent in healthy skin isolates than in prosthetic joint infections isolates (p .05). Prosthetic joint infections isolates were more resistant to oxacillin (78%), ciprofloxacin (60%), levofloxacin (60%), and moxifloxacin (57%). The principal coordinate analysis and a discriminant analysis found that prosthetic joint infections isolates had as discriminant biomarker the biofilm formation, the icaA gene, oxacillin, ciprofloxacin, levofloxacin, moxifloxacin, and gentamicin resistance. In contrast, the healthy skin isolates had as discriminant biomarkers the embp, sesA, sesB, sesC, sesD, sesE, sesG, and sesH genes.These data suggest that ses genes can be considered biomarkers to differentiate between S. epidermidis commensal and prosthetic joint infections clinical.

Staphylococci have quorum-sensing (QS) systems that enable cell-to-cell communication, as well as the regulation of numerous colonization and virulence factors. The accessory gene regulator (Agr) operon is one of the Staphylococcus genus QS systems. Three groups (I, II, and III) are present in Staphylococcus epidermidis Agr operon. To date, it is unknown whether Agr groups can interact symbiotically during biofilm development. This study analyzed a symbiotic association among Agr groups during biofilm formation in clinical and commensal isolates. Different combinations among Agr group isolates was used to study biofilm formation in vitro and in vivo (using a mouse catheter-infection model). The analysis of Agr groups were also performed from samples of human skin (head, armpits, and nostrils). Different predominant coexistence was found within biofilms, suggesting symbiosis type. In vitro, Agr I had a competition with Agr II and Agr III. Agr II had a competition with Agr III, and Agr II was an antagonist to Agr I and III when the three strains were combined. In vivo, Agr II had a competition to Agr I, but Agr I and II were antagonists to Agr III. The associations found in vitro and in vivo were also found in different sites of the skin. Besides, other associations were observed: Agr III antagonized Agr I and II, and Agr III competed with Agr I and Agr II. These results suggest that, in S. epidermidis, a symbiotic association of competition and antagonism occurs among different Agr groups during biofilm formation.

Epidemiological studies comparing clinical and commensal Staphylococcus epidermidis isolates suggest that biofilm formation is a discriminant biomarker. A study showed that four non‐biofilm‐forming clinical S. epidermidis isolates could form an induced biofilm by trypsin treatment, suggesting that S. epidermidis can form biofilms in a protease‐independent way and in a trypsin‐induced way. In this study, the trypsin capacity to induce biofilm formation was evaluated in non‐biofilm‐forming S. epidermidis isolates (n = 133) in order to support this mechanism and to establish the importance of total biofilms (meaning the sum of protease‐independent biofilm and trypsin‐induced biofilm). Staphylococcus epidermidis isolates from ocular infections (OI; n = 24), prosthetic joint infections (PJI; n = 64), and healthy skin (HS‐1; n = 100) were screened for protease‐independent biofilm formation according to Christensen's method. The result was that there are significant differences (p, Epidemiological studies comparing clinical and commensal Staphylococcus epidermidis isolates suggest that biofilm formation is a discriminant biomarker. In this study, the capacity of trypsin to induce biofilm formation was evaluated in non‐biofilm‐forming S. epidermidis isolates. Trypsin is capable to induce biofilm production in non‐biofilm‐forming commensal S. epidermidis isolates with the icaA −/aap+ genotype

High-density lipoproteins (HDL) comprise a heterogeneous family of lipoprotein particles divided into subclasses that are determined by density, size and surface charge as well as protein composition. Epidemiological studies have suggested an inverse correlation between High-density lipoprotein-cholesterol (HDL-C) levels and the risk of cardiovascular diseases and atherosclerosis. HDLs promote reverse cholesterol transport (RCT) and have several atheroprotective functions such as anti-inflammation, anti-thrombosis, and anti-oxidation. HDLs are considered to be atheroprotective because they are associated in serum with paraoxonases (PONs) which protect HDL from oxidation. Polyphenol consumption reduces the risk of chronic diseases in humans. Polyphenols increase the binding of HDL to PON1, increasing the catalytic activity of PON1. This review summarizes the evidence currently available regarding pharmacological and alternative treatments aimed at improving the functionality of HDL-C. Information on the effectiveness of the treatments has contributed to the understanding of the molecular mechanisms that regulate plasma levels of HDL-C, thereby promoting the development of more effective treatment of cardiovascular diseases. For that purpose, Scopus and Medline databases were searched to identify the publications investigating the impact of current therapies focused on high-density lipoproteins.

Previous studies have shown that biofilm-forming bacteria are deficient in tricarboxylic acid (TCA) cycle metabolites, suggesting a relationship between these cellular processes. In this work, we compared the proteomes of planktonic vs biofilm cells from a clinical strain of Staphylococcus epidermidis using LC-MS/MS. A total of 168 proteins were identified from both growth conditions. The biofilm cells showed enrichment of proteins participating in glycolysis for the formation of pyruvate; however, the absence of TCA cycle proteins and the presence of lactate dehydrogenase, formate acetyltransferase, and acetoin reductase suggested that pyruvate was catabolized to their respective products: lactate, formate and acetoin. On the other hand, planktonic cells showed proteins participating in glycolysis and the TCA cycle, the pentose phosphate pathway , gluconeogenesis , ATP generation and the oxidative stress response. Functional networks with higher interconnection were predicted for planktonic proteins. We propose that in S. epidermidis , the relative absence of TCA cycle proteins is associated with the formation of biofilms and that lactate, formate and acetoin are the end products of partial glucose metabolism .

Recent advances in the field of flow cytometry (FCM) have highlighted the importance of incorporating it as a basic analysis tool in laboratories. FCM not only allows the identification of cell subpopulations by detecting the expression of molecules in the cell membrane or cytoplasm, but it can also quantify and identify soluble molecules. The proper functioning of the FCM requires six fundamental systems, from those related to the transport of events to the systems dedicated to the analysis of information. In this review, we have identified the main considerations that every FCM user must know for an optimal antibody panel design, the quality systems that must govern the FCM protocols to guarantee reproducible results in research or clinical laboratories. Finally, we have introduced the current evidence that highlights the relevance of FCM in the investigation and clinical diagnosis of respiratory diseases, establishing important advances in the basic and clinical study of diseases as old as Tuberculosis along with the recent proposals for the monitoring and classification of patients infected with the new SARS-CoV2 virus.

Pregnancy is a complex process where several physiological pathways interact. The down-regulated inflammatory response and the abundance of anti-inflammatory molecules during gestation may explain the acceptance of the fetus and the lack of immune response against it, even though it is a foreign tissue for the mother. NF-κB is a key regulator of the transcription of inflammatory genes, such as IL-8, IL-1β, TNF-α, or IL-6. Increased NF-κB activity that leads to the production of proinflammatory cytokines may induce obstetric disorders, such as preterm birth or abortion. Low activity of this transcription factor is associated with the beneficial anti-inflammatory environment during fetus development until delivery. Galectin-1 (Gal-1) is a lectin-type glycan-binding protein that is able to down-regulate inflammation. It has been shown that Gal-1 is abundantly expressed at the feto-maternal interface in humans, where it promotes maternal immune tolerance to the fetal semi-allograft. Gal-1 tolerance-promoting mechanisms have been established for adaptive immune cells, such as T cells and dendritic cells. However, the role of this lectin has not been established in non-immune cells at the feto-maternal interface. Here, we determined that Gal-1 is able to block the stimulating effect of LPS on IL-6 in human decidua cells. Our results show that Gal-1 acts by inhibiting the stimulation of the LPS-induced IκBζ expression, an NF-κB regulator involved in IL-6 gene transcription.

Outer membrane vesicles (OMVs) are released from the outer membrane of Gram-negative bacteria. Moreover, Gram-positive bacteria also produce membrane-derived vesicles. As OMVs transport several bacterial components, especially from the cell envelope, their interaction with the host cell, with other bacteria or as immunogens, have been studied intensely. Several functions have been ascribed to OMVs, especially those related to the transport of virulence factors, antigenic protein composition, and development as acellular vaccines. In this work, we review some of the recent findings about OMVs produced by specific pathogenic bacterial species.

Biofilm formation on medical and surgical devices is a major virulence determinant forStaphylococcus epidermidis. The bacteriumS.epidermidisis able to produce biofilms on biotic and abiotic surfaces and is the cause of ocular infection (OI). Recent studies have shown thatd-amino acids inhibit and disrupt biofilm formation in the prototype strainsBacillus subtilisNCBI3610 andStaphylococcus aureusSCO1. The effect ofd-amino acids onS.epidermidisbiofilm formation has yet to be tested for clinical or commensal isolates.S.epidermidisstrains isolated from healthy skin (n = 3), conjunctiva (n = 9) and OI (n = 19) were treated withd-Leu,d-Tyr,d-Pro,d-Phe,d-Met ord-Ala and tested for biofilm formation. The presence ofd-amino acids during biofilm formation resulted in a variety of patterns. Some strains were sensitive to all amino acids tested, while others were sensitive to one or more, and one strain was resistant to all of them when added individually; in this wayd-Met inhibited most of the strains (26/31), followed byd-Phe (21/31). Additionally, the use ofd-Met inhibited biofilm formation on a contact lens. The use ofl-isomers caused no defect in biofilm formation in all strains tested. In contrast, when biofilms were already formedd-Met,d-Phe andd-Pro were able to disrupt it. In summary, here we demonstrated the inhibitory effect ofd-amino acids on biofilm formation inS.epidermidis. Moreover, we showed, for the first time, thatS.epidermidisclinical strains have a different sensitivity to these compounds during biofilm formation.

Purpose. Staphylococcus epidermidis ATCC12228 lipoteichoic acid (LTA) inhibits TNFα production from keratinocytes that are activated with poly I:C. However, this effect has not been proven in clinical or commensal isolates. Methodology. The

Staphylococci produce a large number of extracellular proteases, some of which are considered as potential virulence factors. Staphylococcus epidermidis is a causative agent of nosocomial infections in medical devices by the formation of biofilms. It has been proposed that proteases contribute to the different stages of biofilm formation. S. epidermidis secretes a small number of extracellular proteases, such as serine protease Esp, cysteine protease EcpA, and metalloprotease SepA that have a relatively low substrate specificity. Recent findings indicate a significant contribution of extracellular proteases in biofilm formation through the proteolytic inactivation of adhesion molecules. The objective of this work is to provide an overview of the current knowledge of S. epidermidis' extracellular proteases during pathogenicity, especially in the different stages of biofilm formation.

In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 μg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR

Keratitis caused by Staphylococcus aureus often leads to Vascular Endothelial Growth Factor (VEGF)-dependent neovascularization, but contribution of peptidoglycan (PGN), muramyl dipeptide (MDP) and lipoteichoic acid (LTA) from S. aureus to VEGF-dependent neovascularization has not been well-studied. This work was focused on the analysis of S. aureus cell wall components in the production of VEGF family members (VEGF-A, VEGF-B, VEGF-C and VEGF-D) in ocular limbal fibroblasts. Primary culture of human limbal fibroblasts (PCHLFs) were stimulated with PGN, MDP, and LTA, and VEGF family; toll-like receptor 2 (TLR2), nucleotide-binding oligomerization domain 1 (NOD1), and NOD2 expression were determined by RT-PCR. Anti-TLR2 antibody, epidermal growth factor receptor (EGFR) signaling inhibitors (AG1478 and PD98059), and NFκB activation were used to analyze VEGF-A by ELISA. TLR2 and NOD1 expression were analyzed by flow cytometry. The stimulation of PCHLFs with PGN and MDP increased the levels of VEGF-A expression (mRNA and protein) in a time-dependent and dose-dependent manner. VEGF-B, VEGF-C and VEGF-D were expressed constitutively, and no further induction was observed in stimulated PCHLFs. LTA did not increase the expression levels of the VEGF family. TLR2 mRNA and protein were increased only when PCHLFs were stimulated with PGN. Treatment with an anti-TLR2 antibody blocked the interaction of PGN with the receptor, inhibiting VEGF-A over-expression; the presence of anti-TLR2 antibodies did not affect the over-production of VEGF-A after MDP treatment. PCHLFs stimulated with PGN and MDP, but not with LTA, activated NFκB. MDP stimulated the production of NOD1 and NOD2 mRNAs in a time-dependent and dose-dependent manner, and NOD2 protein was only increased by MDP. Treatment of PCHLFs with AG1478 and PD98059 inhibitors prior to stimulation with MDP resulted in the inhibition of VEGF-A over-production, compared with PCHLFs stimulated with MDP alone. Taken together, these results suggest that limbal fibroblasts produce VEGF-A through PGN-TLR2-NFκB and MDP-NOD2-EGFR.

The in vitro and keratinocyte (HaCAT cells) culture expression of four putative genes coding for secreted aspartyl proteases of Candida dubliniensis-CdSAP1, CdSAP2, CdSAP3, and CdSAP4 (CdSAP1-4) - is reported for the first time. In addition, CdSAP7, 8, 9, and 10, orthologous genes of Candida albicans, were recognized in C. dubliniensis genome. There are no orthologs of C. albicans SAP5 and 6 in C. dubliniensis. The expression of CdSAP1 and 2 was independent of the morphological stage of C. dubliniensis; they are expressed at both pH 4 and pH 7, and were induced with albumin as nitrogen source. CdSAP3 expression was regulated by the pH, and was related to the infection process of keratinocytes. Expression of CdSAP4 predominated during the mycelial phase and the initial stage of keratinocyte infection. During infection of the HaCaT cell line, only genes CdSAP3-4 were expressed, and keratinocytes were affected in their number and shape by the infection with C. dubliniensis; however, this effect decreased in the presence of pepstatin A (aspartyl protease inhibitor). Pepstatin A was not able to inhibit keratinocyte damage. Based on the aforementioned, we suggest that the Saps from C. dubliniensis could be considered a virulence factor just as those from C. albicans, and participants in the nitrogen metabolism of the yeast for nutrient acquisition.

Background A feature of psoriasis is the rapid proliferation of keratinocytes, during which apoptosis is blocked and angiogenesis starts. It is known that tumor hypoxic cells produce histone deacetylase-1 (HDAC-1), which up-regulates hypoxia-inducible factor-1alpha (HIF-1alpha) and down-regulates von Hippel-Lindau (VHL) protein by up-regulating vascular endothelial growth factor (VEGF) expression. It has been reported recently that the porcine peptide PR39 (homologous to human LL-37) has angiogenic and antiapoptotic activity. Thus, LL-37, induced by insulin-like growth factor-1 (IGF-1), could help in the production of VEGF. PR39 also induces the expression of inhibitor of apoptosis protein-2 (IAP-2), which blocks apoptosis. The purpose of this work was to analyze whether these genes and their proteins are expressed in psoriatic biopsies. Methods Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) messenger RNA (mRNA) expression and immunohistochemical staining, we studied VHL, IAP-2, and related genes in skin biopsies from psoriatic patients and healthy subjects. Results An over-expression of the mRNA for HDAC-1, HIF-1alpha, LL-37, and IGF-1 in psoriatic skin, in comparison with skin from healthy subjects, was found. The antiangiogenic VHL mRNA and protein were under-expressed in psoriatic skin and highly expressed in healthy skin. The antiapoptotic IAP-2 was over-expressed in dermal endothelial cells from psoriatic skin. The pro-apoptotic Bax, Fas, and FasL mRNAs were expressed. Conclusions These findings suggest that there could be an association of HDAC-1, HIF-1alpha, LL-37, VHL, and IAP-2 with angiogenic and apoptotic mechanisms in psoriasis.

Psoriasis is a chronic inflammatory skin disease where the altered regulation in angiogenesis, inflammation, and proliferation of keratinocytes are the possible causes of the disease, and the transcription factor “hypoxia-inducible factor 1-alpha” (HIF-1α) is involved in the homeostasis of these three biological phenomena. In this review, the role of HIF-1αin the cross talk between the cytokines and cells of the immunological system involved in the pathogenesis of psoriasis is discussed.

Although several cytokines and their receptors have been involved in the development of psoriasis, the etiology is still unknown. In this study we looked for genes possibly involved in the disease by the reverse transcription-polymerase chain reaction differential display technique in lesional and nonlesional skin biopsies from psoriatic patients. We found the mRNA of the alpha1 chain of the interleukin-13 receptor expressed differentially in psoriatic biopsies. By reverse transcription-polymerase chain reaction, we confirmed an overexpression of the alpha1 chain of the IL-13 receptor and alpha chain of the interleukin-4 receptor mRNA in lesional skin psoriatic biopsies, when compared with skin biopsies from healthy subjects (p0.01). The nonlesional skin obtained from a region close to a lesional zone in psoriatic patients presented also an overexpression of these mRNA in 50% of the samples. Interleukin-13 and interleukin-4 were not detected either as mRNA or as the proteins in any of the biopsies from psoriatic patients or healthy subjects. A monoclonal antibody to the alpha1 chain of the interleukin-13 receptor detected the receptor in the epidermal keratinocytes of psoriatic patients and of healthy subjects; however, the positive antibody reaction was stronger in skin tissue from healthy subjects than in psoriatic lesional skin tissue (p0.01), although the mRNA was overexpressed. As interleukin-13 is a pleiotropic immunoregulatory cytokine with a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes, we suggest, based on our results, that the interleukin-13 receptor possibly plays an important part in the early inflammatory process of psoriasis; however, its function is lost in the psoriatic keratinocytes.

Biofilm formation on medical and surgical devices is the main virulence factor of Staphylococcus epidermidis. A recent study has shown that norspermidine inhibits and disassembles the biofilm in the wild-type Bacillus subtilis NCBI3610 strain. In this study, the effect of norspermidine on S. epidermidis biofilm formation of clinical or commensal strains was tested. Biofilm producing strains of S. epidermidis were isolated from healthy skin (HS; n = 3), healthy conjunctiva (HC; n = 9) and ocular infection (OI; n = 19). All strains were treated with different concentrations of norspermidine, spermidine, putrescine, and cadaverine (1, 10, 25, 50 and 100 μM), and the biofilm formation was tested on microtiter plate. Besides, cell-free supernatants of S. epidermidis growth at 4 h and 40 h were analyzed by gas chromatography coupled to mass spectrometry (GC–MS) to detect norspermidine. Results showed that norspermidine at 25 μM and 100 μM prevented the biofilm formation in 45.16% (14/31) and 16.13% (5/31), respectively; only in one isolate from OI, norspermidine did not have effect. Other polyamines as spermidine, putrescine and cadaverine did not have effect on the biofilm formation of the strains tested. Norspermidine was also capable to disassemble a biofilm already formed. Norspermidine was detected in the 40 h cell-free supernatant of S. epidermidis by GC–MS. Norspermidine inhibited the biofilm development of S. epidermidis on the surface of contact lens. In this work, it was demonstrated that S. epidermidis produces and releases norspermidine causing an inhibitory effect on biofilm formation. Moreover, this is the first time showing that clinical S. epidermidis strains have different sensitivity to norspermidine, which suggest that the composition and structure of the biofilms is varied. We propose that norspermidine could potentially be used in the pre-treating of medical and surgical devices to inhibit the biofilm formation.

In ocular infections (OIs) caused by Staphylococcus epidermidis, biofilms composed mainly of poly-N-acetylglucosamine (PNAG) have been widely studied, but PNAG-independent biofilms have not. Therefore, we searched for a relationship between the ica operon (involved in PNAG-biofilm) and the biochemical composition of biofilms in isolates from OI. Isolates from OI (n = 62), from healthy conjunctiva (HC; n = 45) and from healthy skin (HS; n = 53), were used to detect icaA and icaD genes, and the insertion sequence 256 (IS256) using PCR. The compositions of the biofilms were determined by treatment with NaIO4, proteinase K and DNase I. Multilocus sequence typing (MLST) was performed to characterize the isolates, and the expression of aap and embp genes was determined by real-time qPCR. A strong relationship between the icaA −/icaD −/IS256 − genotype and protein- or protein/extracellular DNA (eDNA)-biofilm composition was found in the isolates from OI (53.6 %), whereas the icaA +/icaD +/IS256 − genotype and carbohydrate-biofilm was most prevalent in isolates from HC (25 %) and HS (25 %). Isolates with an icaA −/icaD −/IS256 − genotype and protein-biofilm phenotype were predominantly of the ST2 lineage, while carbohydrate-biofilm-producing strains were mainly of the ST9 lineage. The protein-biofilm-producing strains had higher expression levels of aap gene than carbohydrate-biofilm-producing strains; while embp gene did not have the same pattern of expression. These results suggest that S. epidermidis strains with icaA− /icaD− /IS256 − genotype and protein- or protein/eDNA-biofilms have a stronger ability to establish in the eye than S. epidermidis strains with icaA+ /icaD+ /IS256 − genotype and PNAG-biofilms.

Purpose: In hypoxic tumoral tissues, vascular endothelial growth factor (VEGF) expression is positively regulated by histone deacetylase 1 (HDAC1) and negatively regulated by the tumour suppressor protein von Hippel-Lindau (VHL) via transforming growth factor-alpha (HIF-1alpha). It has been reported that VEGF, HDAC1 and LL-37, but not VHL, are over-expressed in psoriatic skin. Although HIF-1alpha is constitutively expressed in normal keratinocytes, it is not known if HDAC1 and VHL can regulate VEGF production in these cells. Methods: The participation of HDAC1 and VHL in the regulation of VEGF expression in HDAC-, VHL- and LL-37-transfected HaCaT cells, and in HaCaT cells treated with HDAC1 inhibitors, was studied. Results: The production of VEGF was increased in HDAC1- and LL-37-transfected HaCaT cells and maintained in VHL-transfected cells under hypoxic conditions; meanwhile, VEGF production decreased in HaCaT cells treated with TSA, in cells transfected with HDAC1-siRNA, in cells co-transfected with HIF-1alpha-siRNA and pHDAC-1 and in VHL-transfected HaCaT cells. The levels of cytoplasmic HIF-1alpha were high in pLL37-transfected cells and low in pVHL- and pHDAC1-transfected cells; however, HIF-1alpha was detected in the nucleus of the HDAC1-transfected cells. The expression of VEGF was high in cells co-transfected with pHDAC1- and pLL-37, and the expression decreased when pVHL was present. Conclusions: These data demonstrate that HDAC1, LL-37 and VHL can modulate the production of VEGF via HIF-1alpha in HaCaT cells.

Background Vascular endothelial growth factor (VEGF)-transgenic mice develop psoriasiform plaques that resemble human psoriasis, demonstrating that VEGF is an important factor in the development of psoriasis. In human keratinocytes, VEGF is regulated by human cathelicidin LL37, and the expression of this peptide, as well as interleukin-13 receptor-alpha1 (IL-13Rα1) and VEGF, is increased in psoriatic skin. Peptidoglycan (PGN) stimulates innate immunity, inducing cytokines and antimicrobial peptides (cathelicidins), but PGN can also activate psoriatic T-helper-1 (Th1) lymphocytes. As neither the bacterial component that induces LL37 and VEGF expression nor the function of IL-13 in keratinocytes has been clarified, the role of PGN in the induction of these molecules was evaluated in this work. Methods HaCaT keratinocytes were treated with PGN from Staphylococcus aureus, recombinant VEGF (rVEGF), and recombinant IL-13 (rIL-13). The mRNA expression of LL37, VEGF, and IL-13 was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), and VEGF and IL-13 production was measured by enzyme-linked immunosorbent assay (ELISA). Results Keratinocytes treated with PGN show increased expression of LL37 at 24 h, VEGF at 36 h, and IL-13 at 72 h post-treatment. Anti-Toll-like receptor 2 (anti-TLR2) antibody blocked the production of these molecules. LL37 and VEGF expression in keratinocytes treated with PGN was dose dependent. rVEGF induced IL-13 production and rIL-13 induced VEGF production at 36 h. The anti-Flt-1 antibody blocked IL-13 expression. Conclusions These results suggest that PGN from S. aureus can induce LL37 and VEGF expression in keratinocytes, and that this VEGF production can be amplified by subsequent IL-13 overproduction.

Heat shock protein 60kDa (HSP60) could be involved in glaucoma because its expression in optical nerve, and autoantibodies to HSP60 have been found in glaucomatous patients; however, HSP60 antigenic sites recognized by glaucomatous patients are unknown. Therefore, in this work, the antibody levels in the sera of glaucoma patients, and HSP60 antigenic sites were determined. Analysis by the Hopp and Woods method with GENE 2.2 software identified the possible antigenic sites in the amino acid regions 200-213, 226-237, 278-290, 357-368 and 418-427 of Streptococcus pyogenes HSP60 (HSP60Sp). According to these, polypeptides were generated truncating the hsp60Sp gene by PCR. Antibody levels to HSP60Sp and the generated polypeptides were measured by ELISA in serum of glaucomatous patients and in healthy subjects. Glaucomatous patients showed higher antibody levels to rHSP60Sp (p=0.002) than healthy subjects. We found that the high anti-rHSP60Sp antibody levels are associated with the disease (OR=5.2; IC95% of 1.6-16.7; p

Background The antimicrobial peptide PR39 is a porcine cathelicidin with angiogenic and antiapoptotic activities, as it can regulate the expression of vascular endothelial growth factor (VEGF) and inhibitor apoptosis protein-2 (c-IAP-2) in endothelial cells. The human homolog LL-37 has been found to be highly expressed in human keratinocytes from psoriatic patients, but it is not known whether LL-37 can modulate the expression of VEGF and c-IAP-2 in keratinocytes, as both molecules are involved in the overgrowth of psoriatic skin. Therefore, in this work, we studied the possible role of CAP18/LL-37 in the modulation of VEGF and c-IAP-2 expression in human keratinocytes. Methods The CAP18/LL-37 gene was cloned into a plasmid that contained green fluorescent protein (GFP). This plasmid was called pGFP-CAP18/LL-37. The expression of LL-37, VEGF, and c-IAP-2 was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting in HaCaT cells transfected with pGFP-CAP18/LL-37. Specific DNAzymes were used to break the CAP18/LL-37 mRNA (DNAz-CAP18/LL-37). Results HaCaT cells transfected with pGFP-CAP18/LL-37 showed the upregulation of VEGF and c-IAP-2 mRNAs. Hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA expression did not change during the assays; however, its protein was increased, as well as the VEGF protein. HaCaT cells cotransfected with pGFP-CAP18/LL-37 and DNAz-CAP18/LL-37 showed depleted expression of LL-37, VEGF, and c-IAP-2 mRNAs. Conclusions These results suggest that LL-37 may modulate the expression of VEGF and c-IAP-2 via HIF-1alpha in human keratinocytes.

Corneal neovascularization can be induced by a severe ocular infection, injury or immunological diseases. The vascular endothelial growth factor (VEGF) is the main cytokine involved in this phenomenon, inducing angiogenesis from the vascularized ocular tissues. As the limbal tissue is located between conjunctival and corneal tissues, we suggest that the limbal cells are participating in the production of VEGF induced by bacterial components as LPS. In this work, RT-PCRs and immunoblots were used to investigate the expression of VEGF and other pro-angiogenic genes in primary cultures of human limbal fibroblasts (PCHLF) treated with lipopolysaccharide (LPS) from Escherichia coli. We found that the expression of VEGF was initiated at 6 h and reaches its highest expression at 72 h after stimulation with LPS. Up-regulation of toll-like receptor 4 (TLR4) after 3 h of treatment was also observed. LPS-induced the expression of VEGF in a dose-dependent manner, and the blocking of TLR4 with an anti-TLR4 antibody prevented VEGF expression. We also analyzed the molecules that modulate VEGF expression. LPS did not induce the up-regulation of LL-37 nor the hypoxia induced factor 1 alpha (HIF-1alpha) mRNA expression, however, an up-regulation of interleukin 13 receptor alpha 1 (IL-13Ralpha1) and interleukin 4 receptor alpha (IL-4Ralpha) were observed after 3 and 12 h of stimulation, respectively. The expression of interleukin 13 did not change throughout the treatment. These results suggest that TLR4, IL-13Ralpha1 and IL-4Ralpha induced by LPS in PCHLF could be playing an important role in the corneal neovascularization.

Aims: To determine the levels of IgG class antibodies to recombinant heat shock protein 60 kDa of Yersinia enterocolitica (rHSP60Ye), Klebsiella pneumoniae (rHSP60Kp), Escherichia coli (rHSP60Ec), Shigella flexneri (rHSP60Sf), and Streptococcus pyogenes (rHSP60Sp) in the serum of patients with HLA-B27 associated acute anterior uveitis (HLA-B27 associated AAU), idiopathic acute anterior uveitis (idiopathic AAU), pars planitis, Vogt-Koyanagi-Harada (VKH), and healthy subjects. Methods: The genes that code for HSP60Ye, HSP60Kp, HSP60Ec, HSP60Sf, and HSP60Sp were cloned by PCR from genomic DNA. The rHSPs were purified by affinity using a Ni-NTA resin. The serum levels of IgG class antibodies to rHSP60s were determined by ELISA in patients with uveitis (n = 42) and in healthy subjects (n = 25). Results: The majority of patients with uveitis had higher levels of IgG class antibodies to rHSP60Ye compared with levels of healthy subjects (p = 0.01), although these differences were only observed in the HLA-B27 associated AAU (p = 0.005) and in pars planitis patients (p = 0.001). The levels of IgG antibodies to the rHSP60Kp, rHSP60Sf, rHSP60Ec, and rHSP60Sp were similar in patients with uveitis and in healthy subjects (p>0.05). Conclusion: The results suggest that HSP60Ye could be involved in the aetiology of HLA-B27 associated AAU and pars planitis.

Purpose: HLA class II, p-36 protein, heat shock protein and retinal antigens have been associated with pars planitis (PP), but their participation in the development of the disease are unknown. A search for new molecules related to PP is necessary. This work focused on the identification of peptides recognized by PP patient sera using the phage display method. Methods: Sera of PP patients were used to isolate peptides fused to M13-phage pIII protein. The response of PP and healthy sera to peptides was determined by ELISA. PCR amplification and sequencing of peptide-encoding fragments from clones with high recognition by PP sera were used to characterize displayed peptides. Results: One hundred clones were randomly selected from a phage display library after three panning rounds using serum proteins from a PP patient. The immunologic response level of 100 clones selected were determined with a major number of patients, it was found that one clone was recognized stronger in PP patients sera than in healthy sera (PP vs. healthy; P