Mario E. Cancino-Diaz

TLRs and NODs mRNA expression pattern in healthy mouse eye.

Aims: To look for TLR and NOD mRNA expression in the healthy eye and in other immune privileged and non-immune privileged mouse organs. Methods: Semiquantitative RT-PCR was performed to look for TLR1–9 and NOD1 and NOD2 mRNA expressions in the whole eye, in the anterior (AP) and posterior (PP) portions of the eye, in corneal fibroblasts (CF) and in ovary, brain, testis, heart, lung, and spleen. Results: All the TLR mRNAs were expressed in the whole eye of Balb/c mice. NIH and C57BL/6 did not express TLR9 and TLR8, respectively. NIH expressed higher levels of TLR1, 2, 3, and 6 than the other strains. C57BL/6 expressed the lowest levels of all TLRs. TLR9, 5, and 4 were the less expressed in all strains. All TLRs were expressed in Balb/c PP and TLR1 was not expressed in AP. In NIH and Balb/c CF the majority of TLRs were overexpressed with LPS. In testis, expression of most TLRs was absent. Non-immune privileged organs expressed most of the TLRs. All the organs expressed NOD1 and NOD2. In PP NOD2 was not expressed. Conclusion: TLRs and NODs are expressed in the eye, and could have an important role in the innate immunity.

Gatifloxacin, Moxifloxacin, and Balofloxacin Resistance due to Mutations in the gyrA and parC Genes of Staphylococcus epidermidis Strains Isolated from Patients with Endophthalmitis, Corneal Ulcers and Conjunctivitis

Aims:Staphylococcus epidermidis is considered a commensal bacterium; however, it is frequently isolated from ocular infections showing a multidrug resistance. Ciprofloxacin-resistant strains have been isolated from ocular infections; however, resistance to quinolone, such as gatifloxacin and moxifloxacin, is not often studied, consequently the resistance mechanism is unknown. Our aim was to address the quinolone resistance and to explore the resistance mechanism in S. epidermidis strains isolated from ocular infections. Methods:S. epidermidis strains were isolated from patients with conjunctivitis (n = 23), endophthalmitis (n = 14) and corneal ulcers (n = 7). Minimum inhibition concentrations were determined by broth and agar dilution methods for moxifloxacin, gatifloxacin, balofloxacin, rufloxacin and pazufloxacin. Mutations were identified by sequencing the gyrA and parC genes, and their expression was determined by reverse transcriptase polymerase chain reaction. Results: We found that 13.6% (6/44) of the strains were quinolone resistant. In endophthalmitis, 21.4% were gatifloxacin, moxifloxacin and balofloxacin resistant. In corneal ulcers, 14.2, 14.2 and 28.5% were gatifloxacin, moxifloxacin and balofloxacin resistant, respectively, and in conjunctivitis only 4.3% were gatifloxacin resistant. The 6 strains with quinolone resistance showed mutations at Ser84Phe for the gyrA gene, and Ser80Phe for the parC gene. Gatifloxacin did not change the expression levels of gyrA and parC genes. Conclusion:S. epidermidis strains isolated from three ocular pathologies were gatifloxacin and moxifloxacin resistant due to mutations on the gyrA and parC genes.

LL-37 regulates the overexpression of vascular endothelial growth factor (VEGF) and c-IAP-2 in human keratinocytes

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.

Expression of CRAMP via PGN‐TLR‐2 and of α‐defensin‐3 via CpG‐ODN‐TLR‐9 in corneal fibroblasts

Aims: To search for the induction of the expression of antimicrobial peptides in corneal fibroblasts treated with bacterial components. Methods: RT-PCR was performed to search for mRNAs expression of antimicrobial peptides and toll-like receptors (TLRs) in murine primary cultures of corneal fibroblast (PCCF) treated with lipopolysaccharide (LPS) from Escherichia coli, peptidoglycan from Staphylococcus aureus, and cytosine-phosphorous-guanine oligonucleotide (CpG-ODN). Cellular activation was blocked with anti-TRL antibodies. Results: LPS did not induce expression of antimicrobial peptide in corneal fibroblasts. Cathelin related antimicrobial peptide (CRAMP) and α-defensin 3 were overexpressed in a time and dose dependent manner in corneal fibroblasts treated with peptidoglycan and with CpG-ODN, respectively. CRAMP expression was blocked when PCCF were treated with anti-TLR-2 antibodies. α-Defensin 3 was not expressed in NIH murine corneal fibroblasts (which do not express the TLR-9 molecule) treated with CpG-ODN. Conclusion: Results suggest that corneal fibroblasts, which are the second cellular barrier of the cornea, can play an important part in the innate immunity of the eye via TLR stimulation.

D-Amino acids inhibit biofilm formation in Staphylococcus epidermidis strains from ocular infections

Biofilm formation on medical and surgical devices is a major virulence determinant for Staphylococcus epidermidis. The bacterium S. epidermidis is able to produce biofilms on biotic and abiotic surfaces and is the cause of ocular infection (OI). Recent studies have shown that d-amino acids inhibit and disrupt biofilm formation in the prototype strains Bacillus subtilis NCBI3610 and Staphylococcus aureus SCO1. The effect of d-amino acids on S. epidermidis biofilm formation has yet to be tested for clinical or commensal isolates. S. epidermidis strains isolated from healthy skin (n = 3), conjunctiva (n = 9) and OI (n = 19) were treated with d-Leu, d-Tyr, d-Pro, d-Phe, d-Met or d-Ala and tested for biofilm formation. The presence of d-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 way d-Met inhibited most of the strains (26/31), followed by d-Phe (21/31). Additionally, the use of d-Met inhibited biofilm formation on a contact lens. The use of l-isomers caused no defect in biofilm formation in all strains tested. In contrast, when biofilms were already formed d-Met, d-Phe and d-Pro were able to disrupt it. In summary, here we demonstrated the inhibitory effect of d-amino acids on biofilm formation in S. epidermidis. Moreover, we showed, for the first time, that S. epidermidis clinical strains have a different sensitivity to these compounds during biofilm formation.

The NALP3/Cryopyrin-Inflammasome Complex is Expressed in LPS-Induced Ocular Inflammation

In the inflammosome complex, NALP3 or NALP1 binds to ASC and activates caspase-1 which induces IL-1β. In murine LPS-induced ocular inflammation, the production of IL-1β is increased. We suggest that NALP3- or NALP1-inflammasome complex can be participating in the LPS-induced ocular inflammation. In this work, eye, brain, testis, heart, spleen, and lung were obtained from C3H/HeN mice treated with LPS for 3 to 48 hours, and the expression of NALP1b, NALP3, ASC, caspase-1, IL-1β, and IL-18 was determined. Infiltrated leukocytes producing IL-1β in the anterior chamber were found at 12-hour posttreatment. A high upregulated expression of NALP3, ASC, caspase-1, IL-1β, and IL-18 was found at the same time when infiltrated leukocytes were observed. NALP1b was not detected in the eye of treated mice. NALP3 was also overexpressed in heart and lung. These results suggest that NALP3-, but not NALP1-inflammosome complex, is participating in the murine LPS-induced ocular inflammation.

Staphylococcus epidermidis with the icaA " /icaD " /IS256 " genotype and protein or protein/extracellular-DNA biofilm is frequent in ocular infections

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.

Cross Talk between Proliferative, Angiogenic, and Cellular Mechanisms Orchestred by HIF-1α 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-1α in the cross talk between the cytokines and cells of the immunological system involved in the pathogenesis of psoriasis is discussed.

Keratinocytes treated with peptidoglycan from Staphylococcus aureus produce vascular endothelial growth factor, and its expression is amplified by the subsequent production of interleukin-13

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.

Peptidoglycan from Staphylococcus aureus has an anti‐apoptotic effect in HaCaT keratinocytes mediated by the production of the cellular inhibitor of apoptosis protein‐2

Colonization of epithelium by microorganisms leads to inflammatory responses. In some cases an anti‐apoptotic response involving the cellular inhibitor of apoptosis protein‐2 (cIAP‐2) also occurs. Although strong expression of cIAP‐2 has been observed in lesional skin from psoriatic patients and in HaCaT keratinocytes treated with peptidoglycan (PGN) from Staphylococcus aureus, anti‐apoptotic responses induced in the skin by cIAP‐2 have seldom been studied. In this study, the effect of PGN on TNF‐α‐induced apoptotic HaCaT keratinocytes was assessed. Morphological analysis, quantification of cells with DNA fragmentation and active caspase‐3 detection was performed to assess apoptotic cell death. Greater LL‐37 and cIAP‐2 production was found in keratinocytes stimulated with PGN than in non‐treated cells (P < 0.05). In comparison with cells treated with TNF‐α only, a significant reduction in apoptotic cell death was observed when HaCaT were pretreated with PGN before inducing apoptosis with TNF‐α (P < 0.05). In addition, an inhibitor of cIAP‐2 activity (LCL161) stopped the PGN effect. These findings show that PGN from S. aureus has an anti‐apoptotic effect in keratinocytes mediated by cIAP‐2 production, suggesting that this anti‐apoptotic activity could favor proliferation of keratinocytes in psoriasis.