Immunobiotic and Paraprobiotic Potential Effect of Lactobacillus casei in a Systemic Toxoplasmosis Murine Model.

One of the main characteristics of probiotics is their ability to stimulate and modulate the immune response regardless of their viability. Lactobacillus casei (Lc) can stimulate local and systemic immunity, in addition to the activation of macrophages at sites distant from the intestine. Activated macrophages limit the replication of intracellular protozoa, such as Toxoplasma gondii, through the production of nitric oxide. The present study aimed to evaluate the protection generated by treatment with viable and non-viable Lc in the murine systemic toxoplasmosis model. CD1 male mice were treated with viable Lc (immunobiotic) and non-viable Lc (paraprobiotic), infected with tachyzoites of Toxoplasma gondii RH strain. The reduction of the parasitic load, activation of peritoneal macrophages, inflammatory cytokines, and cell populations was evaluated at 7 days post-infection, in addition to the survival. The immunobiotic and paraprobiotic reduced the parasitic load, but only the immunobiotic increased the activation of peritoneal macrophages, and the production of interferon-gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin-6 (IL-6) while the paraprobiotic increased the production of monocyte chemoattractant protein-1 (MCP-1) and T CD4+CD44+ lymphocytes. Viable and non-viable Lc increases survival but does not prevent the death of animals. The results provide evidence about the remote immunological stimulation of viable and non-viable Lc in an in vivo parasitic model.

Steam roller maneuver in pneumatic retinopexy. Does it work?

OBJECTIVE: To identify the efficacy and safety of the steam-roller maneuver, in patients treated with pneumatic retinopexy. METHOD: Experimental, prospective, comparative, longitudinal study in patients with retinal detachment, treated with pneumatic retinopexy. Patients were assigned to one of two groups: without steam roller maneuver (group 1) or with it (group 2). The proportions of single-intervention anatomical success, visual improvement, anatomical success with reintervention, and adverse events were compared between groups (chi squared); preoperative and postoperative visual acuity in logMAR was compared within groups (Wilcoxon's t). RESULTS: 40 eyes were evaluated (mean age 55.9 ± 13.3 years); 15 were assigned to group 1, 25 to group 2. The proportions of single-intervention anatomical success, visual improvement, anatomical success with reintervention, and adverse events did not differ between groups (p > 0.05). At the end of follow up, visual acuity improved in both groups; however, it only improved in group 2, in eyes with single intervention anatomical success (mean log MAR before surgery 1.72 ± 1.64; after surgery 0.61 ± 0.61; p = 0.008). CONCLUSIONS: The steam roller maneuver is efficient for improving visual acuity in patients with pneumatic retinopexy, who achieve single intervention anatomical success; furthermore, the maneuver does not impair prognosis in eyes that require reintervention.

OBJETIVO: Determinar la eficacia y la seguridad de la maniobra steam roller en pacientes tratados con retinopexia neumática. MÉTODO: estudio experimental, prospectivo, comparativo y longitudinal en pacientes con desprendimiento de retina primario, tratados mediante retinopexia neumática. Los sujetos se asignaron a uno de dos grupos: sin maniobra de steam roller (grupo 1) o con ella (grupo 2). Se compararon entre grupos las proporciones de éxito anatómico con una sola intervención, mejoría visual, éxito anatómico con reintervención y eventos adversos (prueba de ji al cuadrado). Se comparó en cada grupo la agudeza visual preoperatoria con la posoperatoria (prueba t de Wilcoxon). RESULTADOS: 40 ojos (edad 55.9 ± 13.3 años); 15 se asignaron al grupo 1 y 25 al grupo 2. Las proporciones de éxito anatómico con una sola intervención, mejoría visual, éxito anatómico con reintervención y eventos adversos no difirieron entre grupos (p > 0.05). La agudeza visual mejoró en ambos grupos, pero en los ojos que presentaron éxito con una sola intervención solo mejoró en el grupo 2 (promedio logMAR preoperatorio 1.72 ± 1.64; postoperatorio 0.61 ± 0.61; p = 0.008). CONCLUSIONES: La maniobra steam roller es eficaz para mejorar la agudeza visual en pacientes con retinopexia neumática, que alcanzan éxito anatómico con una sola intervención, y no deteriora el pronóstico en quienes requieren reintervención.

Dexamethasone implant in silicone oil: in vitro behavior.

BACKGROUND: To determine the effect of the silicone on the dexamethasone intravitreal implant. METHODS: Basic, experimental, prospective and transversal study performed at the hospital "Nuestra Señora de la Luz" in Mexico City. One dexamethasone implant was placed in a test tube with 4 mL of each tamponade medium: 1000cS, 5000cS and heavy silicone oil; basic saline solution was used as the control medium. Photographs were taken weekly for 12 months. 200 µL samples were taken from each medium at 24 h, 1, 2 weeks and monthly for 12 months. ELISA test was performed to quantify dexamethasone release in every sample. An inflammatory stimulus was created and later exposed it to every sample in order to test their anti-inflammatory capacity by cytokine analysis using cytometric bead array. Statistically significant results were obtained with p < 0.05. RESULTS: Photographic follow-up showed disintegration of the implant in control medium. Implants in silicone oil suffered no changes during follow-up. Dexamethasone levels in control medium showed stability from month 2 to 12. Silicone oil mediums showed irregular dexamethasone release during the 1 year period. Dexamethasone in control medium had inhibitory effects on TNF-α starting at 24 h (p < 0.001) and remained stable. Dexamethasone in 1000cS silicone oil showed inhibitory effects from month 2 (p < 0.001) until month 6 (p < 0.001). Implants in denser silicone oils showed no inhibitory effects in any of the samples. CONCLUSIONS: Denser mediums altered the implant pharmacokinetics and showed no anti-inflammatory effects even when concentrations were quantified at levels similar to control medium in vitro.

Defensin production by human limbo-corneal fibroblasts infected with mycobacteria.

Epithelial cells of the cornea and the conjunctiva constitutively produce antimicrobial peptides; however, the production of defensins by other cell types located around the eye has not been investigated. We analyzed the production of beta-defensins (hBD) and cathelicidin LL-37 during the infection of primary limbo-corneal fibroblasts with M. tuberculosis (MTB), M. abscessus (MAB), and M. smegmatis (MSM). The intracellular survival of each mycobacterium, the production of cytokines and the changes on the distribution of the actin filaments during the infection were also analyzed. Fibroblasts produce basal levels of hBD1 and LL-37 and under PMA stimulation they produce hBD2, hBD3 and overexpress hBD1 and LL-37. MAB induced the highest levels of hBD1 and LL-37 and intermediate levels of IL-6; however, MAB was not eliminated. In addition, MAB induced the greatest change to the distribution of the actin filaments. MTB also produced changes in the structure of the cytoskeleton and induced low levels of hBD1 and IL-6, and intermediate levels of LL-37. The balance of these molecules induced by MTB appeared to contribute to the non-replicative state observed in the limbo-corneal cells. MSM induced the lowest levels of hBD1 and LL-37 but the highest levels of IL-6; MSM was eliminated. The results suggest that mycobacterial infections regulate the production of antimicrobial peptides and cytokines, which in conjunction can contribute to the control of the bacilli.

[Down regulation of IL-8 and IL-6 in human limbal epithelial cells cultured with human dialyzable leukocyte extracts]. / Células madre epiteliales limbales de IL-8 e IL-6 reguladas hacia la baja en cultivos con dializados de extractos leucocitarios humanos.

INTRODUCTION: Human Limbal Epithelial Cells (hLEC) are stem cells that give rise to corneal epithelium. After corneal damage, hLEC produce large amounts of IL-8 and IL-6, inducing inflammation in cornea and conjunctiva. Despite inflammation is necessary to repair the ocular surface since this process may be potentially harmful and could lead to corneal opacity. Ophthalmic infectious diseases have been treated with human dialyzable leukocyte extracts (hDLE). Clinical observations in hDLE-treated patients, have suggested an apparent control of ocular inflammatory injuries, without changes in the re-epithelialization process. OBJECTIVE: To determine the inflammatory cytokine profile in supernatants (SN) of hLEC cultured with hDLE. METHODS: hLEC were obtained from cadaver donors. hDLE were added to the hLEC cultures, and SN were collected at different times (1h, 3h, 6h, and 24h). IL-1?, IL 6, IL-8, IL-12p70 and TNF-? were measured in SN with cytometric bead arrays. RESULTS: The majority of isolated cells were CK19+/vimentin+/p63+, indicating that cultured-cells were limbal epithelial stem cells. Limbal cells responded to hDLE by diminishing the secretion of IL-8 and IL-6. Secretion of IL-8 and IL-6 was down-regulated significantly at 24h of culture with hDLE. Interestingly, hDLE did not induce secretion of IL-1 ?, TNF-?, and IL-12p70 in hLEC at any evaluated times. CONCLUSIONS: hDLE down-regulates secretion of IL-8 and IL-6 without induction of IL-1 ?, TNF-a, and IL-12p70 in hLEC. Our results provide a basis to understand some clinical effects, related to control ocular inflammation, that have been observed in patients treated with hDLE.

[In vitro phenotypic characterization of human limbal epithelial cells]. / Caracterización fenotípica de células epiteliales limbales humanas expandidas in vitro.

INTRODUCTION: Human limbal epithelial cells (huLEC) have been used for clinical purposes in ocular surface diseases to promote rapid re-epithelisation and restore corneal epithelium integrity. However, in Mexico this technique has not been fully developed. This study was conducted to characterize the huLEC phenotype expanded in vitro using a cell culture technique. MATERIAL AND METHODS: Cells were obtained from limbal tissue, cultured in KSFM medium and analyzed for the expression of vimentin, K, K19, p63, K12, by flow cytometry and immuno-fluorescence. RESULTS: The phenotype of cultured cells was vimentin+K+K19+ p63+K12-. CONCLUSIONS: Our results suggest that under these culture conditions huLEC maintained their stem cell phenotype. This culture technique could be used for clinical purposes in Mexico.

Caracterización fenotípica de células epiteliales limbales humanas expandidas in vitro / In vitro phenotypic characterization of human limbal epithelial cells

Introducción: Las células epiteliales limbales humanas (CELhu) han sido utilizadas en la clínica para promover la rápida reepitelización en enfermedades de la superficie ocular con la finalidad de restaurar la integridad del epitelio corneal. Sin embargo, esta técnica aún no ha sido desarrollada en nuestro país. El objetivo de este estudio fue desarrollar una técnica de cultivo de CELhu in vitro y caracterizar su fenotipo. Material y métodos: Las células fueron obtenidas de tejido limbal, cultivadas en medio KSFM y analizadas para la expresión de vimentina, K, K19, p63 y K12 por citometría de flujo e inmunofluorescencia. Resultados: El fenotipo de las células cultivadas fue vimentina+K+K19+p63+K12-. Conclusiones: Nuestros resultados sugieren que bajo estas condiciones de cultivo, las CELhu mantuvieron el fenotipo de célula pluripotencial. Esta técnica de cultivo podría ser utilizada con propósitos clínicos en nuestro país.

INTRODUCTION: Human limbal epithelial cells (huLEC) have been used for clinical purposes in ocular surface diseases to promote rapid re-epithelisation and restore corneal epithelium integrity. However, in Mexico this technique has not been fully developed. This study was conducted to characterize the huLEC phenotype expanded in vitro using a cell culture technique. MATERIAL AND METHODS: Cells were obtained from limbal tissue, cultured in KSFM medium and analyzed for the expression of vimentin, K, K19, p63, K12, by flow cytometry and immuno-fluorescence. RESULTS: The phenotype of cultured cells was vimentin+K+K19+ p63+K12-. CONCLUSIONS: Our results suggest that under these culture conditions huLEC maintained their stem cell phenotype. This culture technique could be used for clinical purposes in Mexico.