A nanomedicine to treat ocular surface inflammation: performance on an experimental dry eye murine model.

MUC5AC is a glycoprotein with gel-forming properties, whose altered expression has been implicated in the pathogenesis of dry eye disease. The aim of our study was to achieve an efficient in vivo transfection of MUC5AC, restore its normal levels in an inflamed ocular surface and determine whether restored MUC5AC levels improve ocular surface inflammation. Cationized gelatin-based nanoparticles (NPs) loaded with a plasmid coding a modified MUC5AC protein (pMUC5AC) were instilled in healthy and experimental dry eye (EDE) mice. MUC5AC expression, clinical signs, corneal fluorescein staining and tear production were evaluated. Ocular specimens were processed for histopathologic evaluation, including goblet cell count and CD4 immunostaining. Neither ocular discomfort nor irritation was observed in vivo after NP treatment. Expression of modified MUC5AC was significantly higher in ocular surface tissue of pMUC5AC-NP-treated animals than that of controls. In healthy mice, pMUC5AC-NPs had no effect on fluorescein staining or tear production. In EDE mice, both parameters significantly improved after pMUC5AC-NP treatment. Anterior eye segment of treated mice showed normal architecture and morphology with lack of remarkable inflammatory changes, and a decrease in CD4+ T-cell infiltration. Thus, pMUC5AC-NPs were well tolerated and able to induce the expression of modified MUC5A in ocular surface tissue, leading to reduction of the inflammation and, consequently improving the associated clinical parameters, such as tear production and fluorescein staining. These results identify a potential application of pMUC5AC-NPs as a new therapeutic modality for the treatment of dry eye disease.

Topographical distribution and characterization of epithelial cells and intraepithelial lymphocytes in the human ocular mucosa.

The conjunctiva plays a key role in the protection of the ocular surface by initiating and regulating immune responses. In this study, we analyze the relative proportion of intraepithelial lymphocytes (IELs), apoptotic cells, and proliferative state in three different topographical regions of the normal human conjunctiva. Superior tarsal, superior bulbar, and inferior tarsal-bulbar-fornical conjunctival cells were collected by brush cytology from 63 healthy donors. Flow cytometry analysis showed higher levels of CD3⁺ and CD8⁺ IELs in both upper tarsal and bulbar conjunctiva than in the inferior tarsal-bulbar-fornix, where the CD19⁺ B cells were increased. For all zones two different cell populations (by cell size and complexity) were present in the apoptosis assay. The more complex cells were reduced within the inferior tarsal-bulbar-fornix when compared with the superior bulbar and tarsal areas. Less complex cells were more predominant in the inferior conjunctiva and were mainly alive. The mean proliferation index of the conjunctival epithelium was significantly lower in the superior bulbar conjunctiva than in superior tarsal and inferior fornical conjunctivas. These findings suggest that each topographical zone from normal human conjunctiva has a unique profile of immunophenotype, viability, and proliferative state that could be related to a differentiated regional functionality.

Dry eye disease in chronic graft-versus-host disease: results from a Spanish retrospective cohort study.

We describe the incidence, clinical features, and final outcome of dry eye disease (DED) associated with chronic graft-versus-host disease (GVHD) over a 5-year period. We reviewed 109 clinical charts from patients undergoing hematopoietic stem cell transplantation (HSCT) between January 2000 and December 2005, abstracting data from the 57 patients who developed chronic GVHD and survived at least 180 days after allogenic HSCT. DED occurred in 22 (40%) patients at an average of 16.5 months after HSCT. Photophobia, irritation, and foreign body sensation were the most frequent symptoms, while punctate keratitis was the most common sign. DED tended to improve over time, with fewer than 5% of patients requiring more than two topical medications at the end of follow-up. Our study represented a formal evaluation of DED incidence among Spanish chronic GVHD patients. It is of utmost importance to assure patients will undergo a comprehensive ophthalmologic evaluation throughout their follow-up.

[Lack of concordance between dry eye syndrome questionnaires and diagnostic tests]. / Falta de concordancia entre los cuestionarios y las pruebas diagnósticas en el síndrome de ojo seco.

PURPOSE: To report the prevalence of dry eye syndrome (DES) in a subset of patients > 50 years old in Valladolid, Spain, calculate internal validity of two DES screening questionnaires, and correlate the results with DES diagnostic tests. METHODS: Patients > 50 years-old were randomly selected from the medical network census in Valladolid; they answered the modified McMonnies questionnaire (Q1) and the Ocular Surface Index questionnaire (Q2) and then underwent an ophthalmic evaluation which included: tear break-up time (TBUT), fluorescein and Rose Bengal staining, and Schirmer with anesthesia were performed. Descriptive analysis, test concordance analysis, and Q1 and Q2 reliability using ROC curves were performed. RESULTS: A total of 270 subjects (58.2% female; 41.8% male) with an average age of 64.5 years (95% CI: 63.3 - 65.7) were enrolled. The prevalence of DES based on the Schirmer-Tear break-up time tests combination was 24.2%. The mean Q1 score was 1.2, and 1.4 for Q2. Grittiness and discomfort in dry environments (28.9%) were the most common symptoms recorded for Q1. For Q2 "grittiness" (99.2%) and "burning sensation" (98.9%)were the most common symptoms. With the Schirmer-TBUT combination, DES prevalence in Valladolid was 24.2%, 45.8% with TBUT, and 45.6% with Schirmer test. Values for the areas under the ROC curve for Q1, Q2, and most objective tests were < 0.5, except for Q2 and TBUT (0.503). Cronbach alpha was > 0.7 for all Q1 and Q2 items. Statistically significant concordance was reported for Schirmer - TBUT (κ=0.14) and TBUT- fluorescein staining (κ=0.09); (P<0.05). CONCLUSIONS: Q1 and Q2 were unreliable for DES diagnosis and showed poor internal consistency. Fluorescein and Rose Bengal staining were the most concordant tests for DES diagnosis. Finally, there was no correlation between either screening questionnaire and objective DES diagnostic tests.

Falta de concordancia entre los cuestionarios y las pruebas diagnósticas en el síndrome de ojo seco / Lack of concordance between dry eye syndrome questionnaires and diagnostic tests

PropósitoReportar la prevalencia del síndrome de ojo seco (SOS) y calcular la validez interna de dos cuestionarios para diagnosticar el síndrome de ojo seco y correlacionarlo con los resultados de varias pruebas diagnósticas.Material y métodoSe seleccionaron pacientes mayores de 50 años del sistema de salud de Castilla y León, en Valladolid. Contestaron los cuestionarios modificados de McMonnies (Q1) y Ocular Surface Index questionnaire (OSDI, Q2) y se les realizó: tiempo de ruptura lagrimal (TBUT), tinciones con fluoresceína y rosa de bengala y Schirmer con anestesia. Se registraron los resultados y se realizó análisis descriptivo, de concordancia y fiabilidad de Q1 y Q2 mediante áreas bajo la curva ROC.ResultadosUn total de 270 sujetos (58,2% mujeres; 41,8% hombres) con edad promedio de 64,5 años (IC 95%: 63,3 - 65,7) se enrolaron. El promedio de Q1 fue de 1,2 y la “sensación de arenillas” y las “molestias en ambientes secos” los síntomas más frecuentes. Para Q2 99,2% fueron “sensación de arenillas” y 98,9% “sensación de ardor”. Con la combinación Schirmer-TBUT, la prevalencia del SOS en Valladolid fue del 24,2%, 45,8% con TBUT y 45,6% con Schirmer. Utilizando Schirmer-TBUT, la prevalencia del SOS fue de 27,6% para mujeres (p<0,15) y 19,6% para hombres. Los valores del área bajo la curva ROC para Q1, Q2 y la mayoría de las pruebas diagnósticas fueron<0,5, excepto para Q2 y TBUT (0,503). Para Schirmer-TBUT, el valor de Q1 fue de 0,49 y 0,45 para Q2. Los valores de Cronbach alfa fue > 0,7 para todos los ítems de Q1 y Q2. Se obtuvieron concordancias estadísticamente significativas (p<0,05) con la combinación Schirmer – TBUT (κ=0,14) y TBUT- tinción fluoresceína (κ=0,09)(AU)

ConclusionesQ1 y Q2 no son fiables para el diagnóstico del SOS y tienen pobre consistencia interna. Las tinciones con fluoresceína y rosa de bengala resultaron ser las más concordantes para el diagnóstico del SOS. No hay correlación entre los cuestionarios y las pruebas diagnósticas objetivas del SOS(AU)

PurposeTo report the prevalence of dry eye syndrome (DES) in a subset of patients > 50 years old in Valladolid, Spain, calculate internal validity of two DES screening questionnaires, and correlate the results with DES diagnostic tests.MethodsPatients > 50 years-old were randomly selected from the medical network census in Valladolid; they answered the modified McMonnies questionnaire (Q1) and the Ocular Surface Index questionnaire (Q2) and then underwent an ophthalmic evaluation which included: tear break-up time (TBUT), fluorescein and Rose Bengal staining, and Schirmer with anesthesia were performed. Descriptive analysis, test concordance analysis, and Q1 and Q2 reliability using ROC curves were performed.ResultsA total of 270 subjects (58.2% female; 41.8% male) with an average age of 64.5 years (95% CI: 63.3 - 65.7) were enrolled. The prevalence of DES based on the Schirmer-Tear break-up time tests combination was 24.2%. The mean Q1 score was 1.2, and 1.4 for Q2. Grittiness and discomfort in dry environments (28.9%) were the most common symptoms recorded for Q1. For Q2 “grittiness” (99.2%) and “burning sensation” (98.9%)were the most common symptoms. With the Schirmer-TBUT combination, DES prevalence in Valladolid was 24.2%, 45.8% with TBUT, and 45.6% with Schirmer test. Values for the areas under the ROC curve for Q1, Q2, and most objective tests were < 0.5, except for Q2 and TBUT (0.503). Cronbach alpha was > 0.7 for all Q1 and Q2 items. Statistically significant concordance was reported for Schirmer – TBUT (κ=0.14) and TBUT- fluorescein staining (κ=0.09); (P<0.05).ConclusionsQ1 and Q2 were unreliable for DES diagnosis and showed poor internal consistency. Fluorescein and Rose Bengal staining were the most concordant tests for DES diagnosis. Finally, there was no correlation between either screening questionnaire and objective DES diagnostic tests(AU)

Uso prudente de antimicrobianos y propuestas de mejora en veterinaria / Prudent use of antimicrobial agents and proposals for improvement in veterinary medicine

Las alertas que se produjeron en las dos últimas décadas del siglo XX en relación con las resistencias a antimicrobianos en ganadería, han conducido al desarrollo de diferentes medidas que han mejorado sustancialmente el uso de los antimicrobianos en veterinaria. Estas medidas tienen que ver fundamentalmente con la aplicación de la nueva ley del medicamento y del paquete de higiene. Aunque el nivel de concienciación de los organismos reguladores es muy satisfactorio, y los sistemas de vigilancia implantados y la promoción del uso prudente comienzan a dar sus frutos, es necesario seguir insistiendo en ellas, al mismo tiempo que se promueven acciones alternativas al empleo de antimicrobianos, especialmente las dirigidas a la prevención de las enfermedades bacterianas, tanto de forma específica, mediante vacunación y mejora de la bioseguridad (utilización de insecticidas, desinfectantes y repelentes), como de forma general mejorando las instalaciones ganaderas y el bienestar animal. Todas estas alternativas conducirían a una menor necesidad de utilización de antimicrobianos en animales. Estas actuaciones deben complementarse con las acciones formativas y de concienciación de todos los agentes implicados (veterinarios, ganaderos, industria farmacéutica, distribuidores de medicamentos y grandes distribuidores de alimentos), así como con el establecimiento de cauces de cooperación más eficaces entre la medicina humana y la veterinaria

The alarms that have sounded in the last two decades of the 20th century in the feld of antimicrobial resistance in livestock farming have led to the development of various measures that have substantially improved the use of antimicrobial agents in veterinary medicine. These measures mainly involve the implementation of new pharmaceutical legislation and the hygiene package provisions. Even though awareness of the regulations is high, surveillance systems have been implemented and the promotion of appropriate use has begun to show results, it is necessary to maintain these measures while promoting alternatives to the use of antimicrobials. In particular, alternatives are needed for those agents used for prevention of bacterial diseases, either specifically, through vaccination and improvement of biosafety (use of insecticides, disinfectants and insect repellents), or generally by improving farm facilities and animal welfare. All of these alternatives would reduce the need to use antimicrobial agents in animals. These actions should be complemented by training for all those players involved (veterinarians, farmers, the pharmaceutical industry, veterinary medicines suppliers and food retailers), and by establishing more effective cooperation between human and veterinary medicine

Autoimmunity at the ocular surface: pathogenesis and regulation.

A healthy ocular surface environment is essential to preserve visual function, and as such the eye has evolved a complex network of mechanisms to maintain homeostasis. Fundamental to the health of the ocular surface is the immune system, designed to respond rapidly to environmental and microbial insults, whereas maintaining tolerance to self-antigens and commensal microbes. To this end, activation of the innate and adaptive immune response is tightly regulated to limit bystander tissue damage. However, aberrant activation of the immune system can result in autoimmunity to self-antigens localized to the ocular surface and associated tissues. Environmental, microbial and endogenous stress, antigen localization, and genetic factors provide the triggers underlying the immunological events that shape the outcome of the diverse spectrum of autoimmune-based ocular surface disorders.

Expression of OCTN2 and OCTN3 in the apical membrane of rat renal cortex and medulla.

Immunological assays and transport measurements in apical membrane vesicles revealed that the apical membrane of rat kidney cortex and medulla presents OCTN2 and OCTN3 proteins and transports L-[(3)H]-carnitine in a Na(+)-dependent and -independent manner. OCTN2 mediates the Na(+)/L-carnitine transport activity measured in medulla because (i) the transport showed the same characteristics as the cortical Na(+)/L-carnitine transporter and (ii) the medulla expressed OCTN2 mRNA and protein. The Na(+)-independent L-carnitine transport activity appears to be mediated by both OCTN2 and OCTN3 since: (i) Na(+)-independent L-carnitine uptake was inhibited by both, anti-OCTN2 and anti-OCTN3 antibodies, (ii) kinetics studies revealed the involvement of a high- and a low-affinity transport systems, and (iii) Western and immunohistochemistry studies revealed that OCTN3 protein is located at the apical membrane of the kidney epithelia. The Na(+)-independent L-carnitine uptake exhibited trans-stimulation by intravesicular L-carnitine or betaine. This trans-stimulation was inhibited by anti-OCTN3 antibody, but not by anti-OCTN2 antibody, indicating that OCTN3 can function as an L-carnitine/organic compound exchanger. This is the first report showing a functional apical OCTN2 in the renal medulla and a functional apical OCTN3 in both renal cortex and medulla.

[Prudent use of antimicrobial agents and proposals for improvement in veterinary medicine]. / Uso prudente de antimicrobianos y propuestas de mejora en veterinaria.

The alarms that have sounded in the last two decades of the 20(th) century in the feld of antimicrobial resistance in livestock farming have led to the development of various measures that have substantially improved the use of antimicrobial agents in veterinary medicine. These measures mainly involve the implementation of new pharmaceutical legislation and the hygiene package provisions. Even though awareness of the regulations is high, surveillance systems have been implemented and the promotion of appropriate use has begun to show results, it is necessary to maintain these measures while promoting alternatives to the use of antimicrobials. In particular, alternatives are needed for those agents used for prevention of bacterial diseases, either specifically, through vaccination and improvement of biosafety (use of insecticides, disinfectants and insect repellents), or generally by improving farm facilities and animal welfare. All of these alternatives would reduce the need to use antimicrobial agents in animals. These actions should be complemented by training for all those players involved (veterinarians, farmers, the pharmaceutical industry, veterinary medicines suppliers and food retailers), and by establishing more effective cooperation between human and veterinary medicine.

Ontogeny regulates creatine metabolism in rat small and large intestine.

The ontogeny of intestinal CRT, AGAT and GAMT was investigated in foetuses, newborn, suckling, weaning and adult rats. In the colon, CRT mediates creatine transport because it was Na(+)- and Cl(-) dependent and inhibited by creatine and GPA. In addition, Northern assays showed two CRT transcripts (2.7-kb and 4.2-kb) and the in situ hybridisation revealed that CRT mRNA is restricted to the colon epithelial cells. The immunohistochemistry revealed that CRT protein was at the apical membrane of colon epithelia. Maturation decreased colonic CRT activity to undetectable levels and increased CRT mRNA abundance. Western assays revealed 57-, 65-, 80- and 116-kDa polypeptides at the intestinal apical membrane. The abundance of the 65-, 80- and 116-kDa polypeptides decreased with age, and that of 57-kDa was only observed in adult rats. The small and large intestine express AGAT and GAMT mRNAs. Maturation decreased AGAT mRNA abundance without affecting that of GAMT. For comparison, renal AGAT mRNA levels were measured and they were increased with age. The study reports for the first time that: i) the apical membrane of rat colon have an active CRT, ii) development down-regulates CRT activity via post-transcriptional mechanism(s), iii) the intestine might synthesize creatine and iv) intestinal and renal creatine synthesis is ontogenically regulated at the level of AGAT gene expression.

[Limbal transplantation: multicenter retrospective case series analysis]. / Estudio multicéntrico retrospectivo sobre trasplante de limbo.

PURPOSE: To report the results of limbal transplantation (LT) in patients with limbal stem cell deficiency (LSCD) in the context of ocular surface diseases. MATERIALS AND METHODS: A multicenter (5 centers) retrospective case series analysis of patients who underwent LT between 1996 and 2004 was performed. Data were collected by the same researcher using a customized database. Success was defined by the absence of a persistent corneal epithelial defect, on-going inflammation or recurrence of a pterygium. RESULTS: Data from 72 LT performed in 61 patients (65 eyes) with a mean follow-up of 20.8 months (SD 23.5; range, 3-115) were analyzed. There were 33 males and 28 females with a mean age of 55.8 years (SD: 15.6; range, 20-89). Fifty-eight (80.6%) LT were autografts (40 pterygia, 12 alkali burns, 3 iatrogenic cases, 2 viral infections, 1 neoplasia case) and 14 (19.4%) were allografts from cadaveric donors (7 immune-based disorders, 6 alkali burns, 1 iatrogenic case); all patients receiving allografts also received systemic immunosuppression. Of the total number of LT, 48 (66.7%) were successful. This proportion increased to 81.0% (47/58) when autografts were used. However, only 7.1% (1/14) of all allografts were successful. The success rate was higher (80.0%) when performed for a pterygium and lower when done for immune-based inflammation (14.3%). CONCLUSION: Autograft tissue for LT is always preferable to allografts to surgically treat LSCD, as clinical success is significantly higher, and systemic immunosuppression is avoided. As expected, immune-based disorders are the most difficult cases to treat. LT has been shown to be an excellent option for recurrent pterygium, although prospective studies need to be performed to further corroborate these results.

Estudio multicéntrico retrospectivo sobre trasplante de limbo / Limbal transplantation: multicenter retrospective case series analysis

Objetivo: Revisar el resultado de los trasplantes de limbo (TL) realizado en pacientes con Síndrome de insuficiencia límbica (SIL) en el contexto de varias enfermedades de la superficie ocular. Materiales y métodos: Se realizó un estudio retrospectivo y multicéntrico (cinco centros) de los TL realizados entre 1996 y 2004. Los datos fueron recogidos por el mismo investigador, en una base de datos especialmente diseñada para el estudio. Se consideró como «éxito» del TL a la ausencia de: defectos epiteliales, inflamación y recurrencia del pterigión cuando éste fue la causa del TL. Resultados: Se analizaron un total de 72 TL realizados en 61 pacientes (65 ojos) con tiempo de seguimiento de 20,8 meses (DS 23,5; rango, 3-115).Hubo 33 hombres y 28 mujeres, con una media de55,8 años (DS: 15,6; rango, 20-89). Se realizaron58 (80.6%) TL autólogos (40 pterigión, 12 causticaciones, tres iatrogénicas, dos infecciones virales, una neoplasia) y 14 (19,4%) TL alogénicos de donante cadáver (siete inflamaciones inmunes, seis causticaciones, uno iatrogénico). Todos los pacientes a los que se les realizó TL alogénicos recibieron inmunosupresión sistémica. Al final del seguimiento,48 (66,7%) TL se consideraron un éxito. Este porcentaje fue del 81,0% (47/58) en los TL autólogos y del 7,1% (1/14) en los TL alogénicos. El porcentaje de éxito también dependió de la etiología, siendo alto en los casos de pterigión (80,0%) y menor en las patologías inmunológicas (14,3%).Conclusiones: El TL autólogo es preferible al alogénico, pues la tasa de fracasos disminuye notablemente y, además, se evita el uso de inmunosupresión oral mantenida. Además, el pronóstico es siempre peor en los casos de patología inflamatoria inmune. El TL autólogo parece ser una buena elección para el tratamiento del pterigión recurrente, aunque se necesitan estudios prospectivos que corroboren estos resultados

Purpose: To report the results of limbal transplantation (LT) in patients with limbal stem cell deficiency (LSCD) in the context of ocular surface diseases. Materials and methods: A multicenter (5 centers) retrospective case series analysis of patients who underwent LT between 1996 and 2004 was performed. Data were collected by the same researcher using a customized database. Success was defined by the absence of a persistent corneal epithelial defect, on-going inflammation or recurrence of a pterygium. Results: Data from 72 LT performed in 61 patients(65 eyes) with a mean follow-up of 20.8 months(SD 23.5; range, 3-115) were analyzed. There were33 males and 28 females with a mean age of 55.8years (SD: 15.6; range, 20-89). Fifty-eight (80.6%)LT were autografts (40 pterygia, 12 alkali burns, 3iatrogenic cases, 2 viral infections, 1 neoplasiacase) and 14 (19.4%) were allografts from cadaveric donors (7 immune-based disorders, 6 alkali burns, 1 iatrogenic case); all patients receiving allografts also received systemic immunosuppression. Of the total number of LT, 48 (66.7%) were successful. This proportion increased to 81.0% (47/58)when autografts were used. However, only 7.1%(1/14) of all allografts were successful. The success rate was higher (80.0%) when performed for apterygium and lower when done for immune-based inflammation (14.3%).Conclusion: Autograft tissue for LT is always preferable to allografts to surgically treat LSCD, as clinical success is significantly higher, and systemic immunosuppression is avoided. As expected, immune-based disorders are the most difficult casesto treat. LT has been shown to be an excellent option for recurrent pterygium, although prospective studies need to be performed to further corroborate these results

Ontogeny up-regulates renal Na(+)/Cl(-)/creatine transporter in rat.

Creatine plays a role in energy storage and transport/shuttle of high-energy phosphate in heart, brain, retina, testis and skeletal muscle. These tissues take creatine from the plasma via a 2Na(+)/1Cl(-)/1creatine cotransporter (CRT). We have previously demonstrated that renal apical membrane presents a 2Na(+)/1Cl(-)/1creatine cotransport activity. The goal of this study was to determine whether this transporter is ontogenically regulated. Na(+)/Cl(-)/creatine transport activity was evaluated by measuring [(14)C]-creatine uptake into renal brush-border membrane vesicles. CRT mRNA expression was measured by Northern and real-time PCR assays. E20 foetuses, newborn, suckling, weaning and adult (2- and 8-month-old) Wistar rats were used. The results revealed that neither the vesicular volume, the binding of creatine to the brush-border membrane vesicles, nor the purity of the brush-border membrane vesicle preparations was affected by maturation. Fetal and neonatal kidneys contained a creatine transporter that was qualitatively indistinguishable from that in the adult: it was concentrative, Na(+)- and Cl(-)-dependent, electrogenic and inhibited by guanidinopropionic acid. Maturation increased this transport activity by increasing the maximal rate of transport (V(max)) without significantly changing the apparent K(m). Northern analysis revealed two transcripts for CRT of 2.7 kb and 4.2 kb in all the ages tested. Northern and real-time PCR assays showed that, as seen with NaCl-dependent creatine transport activity, maturation increased CRT mRNA expression. This study reports for the first time that: (i) an apical renal Na(+)/Cl(-)/creatine cotransporter is already active in rat foetuses and (ii) development regulates Na(+)/Cl(-)/creatine cotransport activity by increasing the density and/or turnover of the transporters.

Nonspecific provocation of target organs in allergic diseases: EAACI-GA(2)LEN consensus report.

It is widely accepted that nonspecific tissue reactivity is a distinct pathophysiological hallmark of allergic diseases, influenced by genetic and environmental factors different from those involved in causing sensitization and allergen response of target organs. This consensus document aims at reviewing procedures currently used for nonspecific provocation of the bronchi, nose and eye and for measuring their responsiveness to nonspecific stimuli.

OCTN3: A Na+-independent L-carnitine transporter in enterocytes basolateral membrane.

L-carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+-free conditions chicken enterocytes take up L-carnitine until the cell to medium L-carnitine ratio is 1. This uptake was inhibited by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L-3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+-independent, (ii) trans-stimulated by intravesicular L-carnitine, and (iii) cis-inhibited by TEA and cold L-carnitine. L-3H-carnitine efflux from L-3H-carnitine preloaded enterocytes was also Na+-independent, and trans-stimulated by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L-carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl-L-carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L-carnitine transport activity measured in the three experimental conditions.

Enfermedad de Behçet ocular: Estudio retrospectivo / Ocular Behçet disease. retrospective study

Objetivo: Estudiar las características clínicas, pronóstico visual y respuesta terapéutica a fármacos inmunosupresores en pacientes con Enfermedad de Behçet Ocular (EB). Pacientes y método: Estudio retrospectivo desde Mayo de 1996 a Mayo de 2003, donde fueron analizadas las historias clínicas de 17 pacientes (33 ojos) con enfermedad de Behçet. Resultados: La relación entre hombre/mujer fue de 12/5. Las manifestaciones extraoculares más frecuentes fueron las úlceras orales (100%) y lesiones cutáneas (88%). A nivel ocular: uveítis anterior en un 59%, uveítis posterior en 76% y panuveítis en un 88%. La agudeza visual mejoró en 11 (33,3%) ojos, se mantuvo estable en 18 (54,5%) ojos y cuatro (12,1%) ojos perdieron dos líneas o más de visión. El tratamiento más utilizado fue con colchicina y ciclosporina (59%). Siete (41,1%) pacientes tuvieron que recibir tres fármacos simultáneamente. Conclusiones: La EB puede producir pérdida irreversible de la visión, sin embargo el pronóstico visual es bueno (85%) de mediar tratamiento inmnosupresor(AU)

Objective: To study clinical features, visual prognosis and response to immunosuppressive drugs in patients with ocular Behçet Disease (BD). Patients and methods: clinical data of 17 patients (33 eyes) were retrospectively reviewed from May 1996 to May 2003. Results: The male/female ratio was 12/5. The commonest manifestations were oral ulceration (100%) and skin lesions (88%). Anterior uveitis was present in 59%, posterior uveitis was in 76% and panuveitis was in 88.1%. Visual acuity improved in 11 (33.3%) eyes, remind stable in 18 (54.5%) eyes and four (12.1%) eyes lost two or more lines. Main drug therapy consisted of colchicine and cyclosporine (59%). Seven patients had received three drugs simultaneously. Conclusions: BD can lead to an irreversible visual loss; however, the visual prognosis is good (85%) if immunosuppressor treatment is implemented(AU)

[Ocular Behçet disease. Retrospective study]. / Enfermedad de Behçet ocular. Estudio retrospectivo.

OBJECTIVE: To study clinical features, visual prognosis and response to immunosuppressive drugs in patients with ocular Behçet Disease (BD). PATIENTS AND METHODS: clinical data of 17 patients (33 eyes) were retrospectively reviewed from May 1996 to May 2003. RESULTS: The male/female ratio was 12/5. The commonest manifestations were oral ulceration (100%) and skin lesions (88%). Anterior uveitis was present in 59%, posterior uveitis was in 76% and panuveitis was in 88.1%. Visual acuity improved in 11 (33.3%) eyes, remind stable in 18 (54.5%) eyes and four (12.1%) eyes lost two or more lines. Main drug therapy consisted of colchicine and cyclosporine (59%). Seven patients had received three drugs simultaneously. CONCLUSIONS: BD can lead to an irreversible visual loss; however, the visual prognosis is good (85%) if immunosuppressor treatment is implemented.

Levels of mucin gene expression in normal human conjunctival epithelium in vivo.

PURPOSE: Conjunctival impression cytology (CIC) samples were used to determine the mean and normal range of mRNA levels of human MUC1, MUC2, MUC4, MUC5AC, and MUC7 mucin genes. METHODS: Real time PCR was performed to determine normal mRNA levels in CIC samples of 24 male and 19 female healthy donors. Correlation coefficients between gene expression levels were obtained. RESULTS: All five mucin genes were expressed in the CIC samples. MUC1 and MUC4 were present at the highest level and MUC2 was at the lowest. There were no gender differences. Significant positive correlations existed between MUC2 and MUC4 and between MUC2 and MUC7 levels. CONCLUSIONS: Normal levels and ranges of mRNAs for MUC1, MUC2, MUC4, MUC5AC and MUC7 conjunctival mucin genes have been established for the first time. These data may serve as the normal threshold values for future comparisons in different experimental and pathological conditions involving the ocular surface.

[Normal human conjunctival epithelium expresses MUC13, MUC15, MUC16 and MUC17 mucin genes]. / Expresión de los genes de mucinas MUC13, MUC15, MUC16 y MUC17 en el epitelio conjuntival normal humano in vivo.

PURPOSE: The ocular surface epithelia express at least five mucin genes of the total of 17 human mucin genes that have been identified so far. This study was designed to determine the expression profile of mucin genes in conjunctival impression cytology (CIC) samples from healthy subjects. METHODS: Two polyethersulfone filters were applied to the superior conjunctiva of both eyes from eight healthy donors. Polymerase chain reaction (PCR) was performed using isolated and retrotranscripted total RNA obtained from the CIC samples to study the expression of all known human mucin genes. Following amplification, PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide to confirm that only a single band was obtained when amplifying all cDNAs with the convenient primers. RESULTS: Transcripts of the previously reported conjunctival mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC7 were detected in all samples. In addition, transcripts of MUC13, MUC15, MUC16 and MUC17 mucin genes also were detected. Amplified products by conventional PCR showed the expected amplicon size. Transcripts of MUC3A, MUC3B, MUC5B, MUC6, MUC8, MUC11, and MUC12 mucin genes were not detected. CONCLUSION: The expression of four additional mucin mRNA (MUC13, MUC15, MUC16, and MUC17) has been proved in human conjunctival epithelium from healthy donors for the first time. The function of these genes remains to be further elucidated.