Quality of life in adult patients using dialyzable leukocyte extract for allergic rhinitis.

Allergic rhinitis (AR) has considerable impact on the general health of individuals. Therefore, treatment trials should include an evaluation of quality of life. We aimed to determine changes in the quality of life of moderate/severe AR patients treated with standard treatment in addition to dialyzable leukocyte extract (DLE), a peptide-based immunomodulator. In a prospective, non-controlled trial, DLE was added to the standard treatment regimen for patients with moderate/severe AR. DLE was administered orally at 2 mg per day for 5 days, followed by 4 mg per week for 5 weeks, and then 2 mg per week for 5 weeks. The primary endpoints were overall improved Standardized Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) scores, domain scores, and individual item scores of 0.5 points or higher. Statistical significance was defined as P < .05. Thirty patients (50% female) aged 14 to 60 years old (33.4 ± 11.9) were enrolled in this study. The mean overall basal quality of life score was 3.41 ± 1.22. After 11 weeks, the mean RQLQ score was 1.74 ± 1.09 ( P < .0001; 95% confidence interval [CI], 1.05-2.33), and all the domain scores improved (daily activities P < .001, 95% CI 0.91-2.15, sleep P < .001, 95% CI 0.9-2.26, non-hay fever symptoms P = .001, 95% CI 0.51-1.82, practical problems P < .001, 95% CI 1.55-2.85, nasal symptoms P < .001, 95% CI 1.36-2.67, ocular symptoms P < .001, 95% CI 1.05-2.17, emotional P < .001, 95% CI 1.23-2.55). Each of the 28 individual item scores on the RQLQ showed clinical (minimal important difference [MID] ≥ 0.5) and statistical ( P < .05) improvements. DLE might be a beneficial adjuvant treatment for AR. Our results provide preliminary data for future research. Clinical trials registration ID: NCT02506998.

[Immunological evaluation during treatment of a case of borderline lepromatous leprosy]. / Evaluación inmunológica durante el tratamiento de un caso de lepra dimorfa lepromatosa.

BACKGROUND: Leprosy is a chronic granulomatous infection that affects skin and peripheral nerves. Its prevalence has declined, but is still observed mainly in poor rural areas. CASE REPORT: A male city dweller with photophobia and chronic dermatosis in the face: nodular and erythematous lesions, pustules, keratitis and entropion, partial eyebrows loss, and edema on eyelids, chin, and nose bridge. The rest of the body had no lesion or lymphadenopathy. Biopsy revealed Langhans giant cell proliferation in the superficial dermis without epidermal atrophy. BAAR staining for detection were positive, no Virschow cells were observed, and Fite-Franco staining (leprosy-specific) was negative. Cutaneous tuberculosis was diagnosed. Rifampicin/isoniazid/pyrazinamide and dialysate leukocyte extract were prescribed. A month later, the swelling had decreased significantly. Polymerase chain reaction (PCR) test was positive for Mycobacterium leprae. Flow cytometry showed CD4 count normalization. Long-term treatment with rifampicin, clofazimine, and dapsone was established. CONCLUSIONS: The host's immune response determines the clinical features of the disease: if response is bad there will be vacuolated macrophages filled with bacilli (lepromatous leprosy). Clinical and histopathological findings help typing.

Antecedentes: La lepra es una infección granulomatosa crónica que afecta piel y nervios periféricos. Aunque su prevalencia ha disminuido, se sigue observando principalmente en el medio rural pobre. Caso clínico: Hombre residente de una ciudad, con fotofobia y dermatosis crónica en la cara: lesiones nodulares y eritematosas, pústulas, queratitis y entropión, pérdida parcial de las cejas y edema de párpados, barbilla y puente nasal. El resto del cuerpo sin lesiones ni adenomegalias. La biopsia reveló proliferación de células gigantes de Langhans en la dermis superficial, sin atrofia epidérmica. Las tinciones para búsqueda de BAAR fueron positivas. No se observaron células de Virschow y la tinción de Fite-Franco (específica de lepra) fue negativa. Se diagnosticó tuberculosis cutánea. Se prescribió rifampicina-isoniazida-pirazinamida y extracto dializado de leucocitos. Un mes después, la inflamación había disminuido de forma importante. La reacción en cadena de la polimerasa fue positiva para Mycobacterium leprae. Con la citometría de flujo de seguimiento se observó normalización de la cuenta de CD4. Se estableció tratamiento a largo plazo con rifampicina, clofazimina y dapsona. Conclusiones: La respuesta inmune del huésped determina las características clínicas de la enfermedad: si la respuesta es mala habrá macrófagos vacuolados llenos de bacilos (lepromatosa). Los hallazgos clínicos e histopatológicos ayudan a la tipificación.

Tissue and cellular characterisation of nucleolin in a murine model of corneal angiogenesis.

PURPOSE: Corneal neovascularisation (CNV), with consequent loss of transparency, is due to an imbalance of proangiogenic factors. Cell-surface nucleolin (NCL) has been associated with neo-angiogenesis. There are studies identifying NCL translocation from nucleus to the cell surface, which is essential for endothelial cell proliferation. To find the possible role of NCL in the generation of corneal neovessels, the aim of this study is to characterise the NCL presence and cell-localisation in non-injured corneas, as well as to describe the changes in NCL cell and tissue localisation in CNV, and to analyse the effect of bevacizumab on NCL cellular and tissular distribution. METHODS: Suture-induced CNV was performed in mice. The corneal tissues were obtained and the histological and co-immunofluorescence assays were performed using different proteins, such as CD31, cadherin and isolectin B4. To determine the possible role of VEGF in NCL presence and localisation in our CNV model, bevacizumab was concomitantly used. RESULTS: Nucleolin was principally observed in the nucleus of the basal epithelial cells of normal corneas. Interestingly, angiogenesis-induced changes were observed in the localisation of NCL, not only in tissue but also at the cellular level where NCL was extranuclear in epithelial cells, stromal cells and neovessels. In contrast, these changes were reverted when bevacizumab was used. Besides, NCL was able to stain only aberrant corneal neovessels in comparison with retinal vessels. CONCLUSIONS: NCL mobilisation outside the nucleus during angiogenesis could have a possible role as a proangiogenic molecule in the corneal tissue.

Pemphigus: An Ophthalmological Review.

OBJECTIVE: To describe the epidemiology, etiology, pathogenesis, clinical characteristics, and management of pemphigus, with an emphasis on ocular involvement. METHODS: Literature review. RESULTS: Pemphigus is an autoimmune epithelial blistering disease of the skin and mucous membranes. The typical pathological finding is acantholysis of the epidermis that leads to blister formation. Immunofluorescence techniques show autoantibody deposition on the epidermal intercellular substance. Although a genetic background is necessary, environmental factors are crucial for the onset and perpetuation of the disease. Exposure to some drugs, toxic agents, and foods and associations with other autoimmune diseases and lymphoproliferative conditions should be assessed. Generally, the skin is the most commonly affected tissue. Ocular involvement might be present and exhibit a clinical course that is independent of skin compromise. Visual function may be affected depending on the severity of the presentation. In untreated cases, mortality is high because of bacterial sepsis and hydroelectrolyte imbalance. A multidisciplinary approach should be used involving a dermatologist, ophthalmologist, and immunologist. Immunosuppressive agents are the mainstay of treatment; corticosteroids typically with azathioprine or mycophenolate mofetil are the drugs of choice. Surgical treatment of trichiasis and malposition of the eyelids and tectonic procedures for corneal perforation are sometimes required in very severe and recalcitrant cases. CONCLUSIONS: Pemphigus is a potential life- and sight-threatening disease. Understanding the disease facilitates the adequate assessment of the modifiable factors and the prompt initiation of immunotherapy. Ocular involvement can develop in patients with pemphigus. Adequate ophthalmological care is needed, in particular, prevention of infections, scarring, and corneal perforation.

Ophthalmic indications of amniotic membrane transplantation in Mexico: an eight years Amniotic Membrane Bank experience.

Amniotic membrane, the inner layer of the placenta, has biological properties (e.g. promotes epithelization, reduces fibrosis, secretes antimicrobial products and inhibits immune responses) which make it a useful option for several ophthalmologic procedures, especially those involving the ocular surface. Its use in eye surgery has been reported by other authors. To our knowledge, there is a lack of descriptive studies on surgical indications using amniotic membrane in Mexican population. Here we describe the eight years Amniotic Membrane Bank experience in Mexico, including a detailed protocol of the donors selection, tissue harvesting, preparation, storage and distribution of amniotic membrane since its establishment in 2007. Moreover, we describe the Ophthalmological indications of amniotic membrane transplantation of the total of 1686 amniotic membranes fragments used during eight years. The five most common indications for amniotic membrane transplantation were pterygium (46 %), corneal ulcers (12.6 %), conjunctival surface repair (11.1 %), neoplasms (7.4 %), and persistent epithelial defects (7.3 %). In addition, we compared the indications of amniotic membrane use in two different types of Institutions: general hospitals and ophthalmologic reference hospitals. We found interesting differences between the indications and use rates between these institutions, although pterygium was the most frequent pathology that amniotic membrane fragments were used in both institutions, there was up to a five-fold increase in the use of amniotic membrane for correction of persistent epithelial defects in reference hospitals which could be explained due to the more complex and severe ophthalmological pathologies admitted in reference hospitals. In conclusion, Amniotic Membrane is used in a numerous ocular pathologies and especially on pterygium in our Mexican population.

Triple subconjunctival bevacizumab injection for early corneal recurrent pterygium: one-year follow-up.

PURPOSE: The aim of the study was to evaluate the effect of 3 subconjunctival bevacizumab injections in patients with an early corneal pterygium recurrence. METHODS: This study was a nonrandomized single center trial. Patients with an early corneal pterygium recurrence were selected. All patients received 3 subconjunctival bevacizumab (2.5 mg/0.1 mL) injections (basal, 2 and 4 weeks) in the recurrence area of the pterygium. The corneal and corneal-conjunctival neovascularization areas and the corneal opacification area of each pterygium were determined using digital slit lamp pictures. RESULTS: Thirty-eight patients were enrolled into the study; all patients were injected within 3 months of the diagnosed pterygium recurrence. Interestingly, the bevacizumab injections had a significant effect (P<0.05) on the reduction of corneal, corneal-conjunctival area of neovascularization determined as pixels and on the corneal opacification area determined as mm(2) when comparing the basal values, to the values obtained after 15 days, 1 month, 3 months, 6 months, and 12 months after injections. CONCLUSIONS: The vascularized area in all recurrent pterygia and the corneal opacification area with this triple regimen of subconjunctival bevacizumab injections were reduced, which remained until the end of the study. These results suggest that bevacizumab subconjunctival injections could be useful to treat recurrent pterygium.

Stem cells isolated from the human stromal limbus possess immunosuppressant properties.

PURPOSE: Mesenchymal stromal stem cells (MSC) are non-hemopoietic cells with the capacity to self-renewal and to differentiate into various cell lineages of mesenchymal origin. More recently, the immune regulatory potential of MSC has been focused on. Furthermore, mesenchymal stem cells obtained from diverse tissues possess immunomodulatory properties and inhibit proinflammatory immune reactions. The aim of this study was to determine the immunosuppressive characteristics of mesenchymal stem cells isolated from human limbal (L-MSC) tissue. METHODS: L-MSC were enzymatically obtained from cadaveric sclero-corneal rims and expanded in vitro. The cells were characterized by flow cytometry using specific antibodies to mesenchymal stem cells markers. Clonogenic and tissue transdifferentiation in vitro assays were performed. The effect of L-MSC soluble factors on T cell proliferation was determined by flow cytometry. Cytokines such as transforming growth factor-b1 (TGF-ß1) and Interleukin-10 (IL-10) on supernatants from L-MSC were identified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Herein, we described that L-MSC cells in vitro-expanded were positive for the expression of vimentin, CD29, CD34, CD39, CD73 and CD105 mesenchymal stem cells markers; meanwhile, this cell population was negative to CD45 and HLA-DR hemopoietic markers as well as to cytokeratin expression. Clonogenic assays showed that these cells were able to form colonies. In addition, this L-MSC population had the ability to transdifferentiate into neurons and chondrocytes and to form tubular networks on matrigel in the presence of vascular endothelial growth factor (VEGF). These results indicated that these cells were stem cells. Additionally, soluble factors secreted by L-MSC were capable of mediating the suppression of T-cell receptor (TCR)-engagement lymphocyte proliferation. In an attempt to identify the possible immunosuppressive factors secreted by L-MSC, TGFß1 and IL-10 cytokines were determined in the L-MSC supernatants by ELISA; interestingly, TGFß1 was constitutively secreted by this cell population; in contrast, IL-10 was not detectable. Moreover, TGFßRII neutralizing antibodies were able to revert the TCR-engagement lymphocyte proliferation inhibition mediated by L-MSC. Thus, TGFß1 secreted by L-MSC was able to suppress T cell proliferation. CONCLUSIONS: Taken together these results, explain in part the immunosuppressive features of this cell population obtained from the human limbus. All these characteristics make this cell population an excellent source to be used in the regenerative medicine.

Amniotic membrane is an immunosuppressor of peripheral blood mononuclear cells.

UNLABELLED: Amniotic membrane (AM) is the inner layer of the placenta, which is in contact with the fetus; it has been used for transplantation in ocular surface diseases. It has been reported that amniotic membrane promotes epithelialization, inhibits angiogenesis and diminishes ocular inflammation. A persistent epithelial defect is the delay in epithelial wound healing caused by infiltrating inflammatory cells into the cornea and amniotic membrane transplantation has been successfully used in its treatment, however the mechanism of action in inhibiting inflammation it is not well understood. This study was aimed at determining whether denuded amniotic membrane (dAM) induces anti-inflammatory effects on peripheral blood mononuclear cells. METHODS: Human peripheral blood mononuclear cells (PBMC) were cultured on dAM. Proliferation and apoptosis assays were performed on PBMC; and synthesis and secretion of pro-inflammatory cytokines by these cells was analyzed. RESULTS: dAM induced apoptosis and inhibited cell proliferation of PBMC; and abolished the synthesis and the secretion of pro-inflammatory cytokines even when they were LPS stimulated. In contrast, when PBMC were cultured on hydrophilic membrane cell culture inserts, apoptosis was not significantly induced, cell proliferation was conserved, and synthesis and secretion of pro-inflammatory cytokines were not affected. CONCLUSIONS: Taken together, these results could explain the anti-inflammatory in vivo effects observed when the amniotic membrane is used as a transplant.

Study of the expression of CD30 in pterygia compared to healthy conjunctivas.

PURPOSE: The pterygium is characterized by a fibrovascular neoformation from the bulbar conjunctiva into the cornea. The recent discovery that abnormal markers associated with tumor diseases are identified in the pterygium strengthens the theory that the pterygium is a tumor-like disease rather than a degenerative disease. The CD30 molecule has been identified in neoplastic and normal epithelial proliferating cells. The aim of this study was to determine the expression of the CD30 molecule in the pterygium. METHODS: Immunohistochemical staining using an antibody to CD30 and to the Ki-67 nuclear antigen was performed on 25 pterygial specimens and 10 healthy conjunctivas. RESULTS: Strong immunostaining against CD30 was observed in all pterygium specimens, in contrast to the healthy conjunctivas that showed weak immono-positivity in only three cases. The staining was diffused, predominantly to the basal epithelium. The Ki-67 antigen was observed in the nucleus of the basal epithelium of the pterygial specimens, and no staining was observed in the healthy conjunctiva. When serial sections were stained with CD30 and Ki-67, the cells that expressed CD30 also expressed Ki-67. CONCLUSIONS: The present study identified for the first time the existence of CD30 molecules in the basal epithelium of a pterygium. The fact that the positivity to Ki-67 in the basal epithelium of the pterygium correlated with the CD30 reactivity suggested that this protein could be associated with the uncontrolled cell proliferation of the epithelium in this pathology. The CD30 molecule could therefore be a suitable target to be inhibited using chimerical antibodies in pterygium diseases.

La interleuquina 6 está involucrada en la inmunopatogénesis de la queratoconjuntivitis por adenovirus / Interleukin 6 is a cytokine involved in theimmunopathogenesis of adenoviral keratoconjunctivitis

Los receptores tipo toll (RTT) desempeñan un papel importante en la respuesta inmune innata en las infecciones oculares, particularmente las producidas por adenovirus (Ad). Los objetivos de este estudio fueron determinar si la infección adenoviral induce citocinas proinflamatorias en células epiteliales corneales (CEC) humanas y evaluar la contribución de los RTT al microambiente pro inflamatorio por las células epiteliales limbales (CEL) humanas. Métodos: Las CEC fueron aisladas de córneas humanas sanas y cultivadas en medio epitelial hormonal suplementado hasta su confluencia. Las CEL fueron obtenidas a partir de rodetes limbales humanos y cultivadas en medio KSFM hasta su confluencia. Posteriormente, las CEC fueron infectadas con Ad5 y cultivadas en diferentes tiempos (24 h, 48 h, 72 h). Las CEL fueron estimuladas con agonistas de RTT por 24 h. Los sobrenadantes de cultivo fueron recuperados y analizados por ELISA para determinar las concentraciones de interleucina (IL) 1b, IL-6, factor de necorsis tumoral alfa (FNT-α) e interferón alfa (IFN-α). Resultados: La infección de CEC por Ad5 indujo la producción de IL-6 desde las 24 h. No se detectó IL-1b, FNT-α o IFN-α en los sobrenadantes de cultivo de las células infectadas en ningún tiempo de cultivo. La estimulación por agonistas de RTT en CEL indujo la producción de IL-6 a través de RTT8> RTT4 = RTT2. Conclusiones: La infección por Ad5 indujo la producción de IL-6 por CEC. El RTT8 podría estar implicado con la producción de esta citocina en CEL.

Toll-like receptors (TLRs) play an important role in theinnate immune response to ocular infections particularlyby adenovirus (Ad). The objectives of this study were todetermine whether adenoviral infection inducesproinflammatory cytokines by human corneal epithelialcells (CEC) and to evaluate TLR contribution to the proinflammatorymicroenvironment by human limbalepithelial cells (LEC). Methods: CEC were isolated fromhuman healthy corneas and grown in supplementedhormonal epithelial medium until confluence. LEC wereobtained from human limbal rims, and cultured in KSFMmedium until confluence. Then CEC were infected withAd5 and cultured at different times (24 h, 48 h, 72 h).LEC were stimulated with TLRs-agonist for 24 h. Culturessupernatants were recovered and analyzed by ELISA todetermine IL-1b, IL-6, TNF-α and IFN-α concentrations.Results: Ad5 infection of CEC induced the production ofIL-6 since 24 h. Nor IL-1b, TNF-α, or IFN-α were detectedin the culture supernatants of infected cells in any timeof culture. TLRs-agonist stimulation of LEC induced IL-6production through TLR8 > TLR4 = TLR2. Conclusions:Ad5 infection induced IL-6 production by CEC. TLR8 couldbe implicated in the production of this cytokine in LEC.

[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.

Pars planitis is associated with an increased frequency of effector-memory CD57+ T cells.

AIM: To evaluate the frequency, phenotype and the potential function of CD57+ T cell subsets in patients with pars planitis. METHODS: CD4+CD57+ and CD8+CD57+ T cells were quantitated in peripheral blood from 15 patients with pars planitis and 15 healthy controls. To evaluate the phenotype and potential function of CD57+ T cell subsets CCR7, CD27, CD28, CD45RA, CD45RO, intracellular IFN-gamma, IL-4, perforin and granzyme-A expression were assessed by flow cytometry. RESULTS: CD57+ T cells subsets were increased in patients with pars planitis (p = 0.002). The majority of CD4+CD57+ T cells were CCR7-CD27-CD28-CD45RO+, while the most CD8+CD57+ T cells were CCR7-CD27-CD28-CD45RA+. The number of cells positive for intracellular IFN-gamma and IL-4 was higher in the CD57+ T cell populations. A greater number of CD8+CD57+ T cells than CD8+CD57- T cells were positive to perforin (p = 0.006) and granzyme-A (p = 0.01). CONCLUSIONS: CD57+ T cells had a phenotype associated with peripheral memory (CCR7-CD27-CD28-). Cytokine production by CD57+ T cells suggests that these cells may play a role in helper cell regulation. High expression of intracellular proteins involved in cytotoxicity suggests that CD8+CD57+ T cells may play an effector role. Taken together, this study proposes that CD57+ T cells function as memory-effector T cell subsets during pars planitis pathogenesis.

[Uveitis immunopathology: currents knowledge, clinical correlation and research perspectives in the immue-ocular area]. / Inmunopatologia de la uveítis: conocimientos actuales, correlación clinica y perspectivas de investigación en el área inmuno-ocular.

The eye has multiple mechanisms of immune regulation implicated in the maintenance of ocular immune privilege. However, sometimes diseases or disorders appear and can cause clinical manifestations of intraocular inflammation; usually those diseases are collectively named "uveitis". Despite the uveitis is the main cause of eye morbidity and lost of visual function, the vast majority of the immune mechanisms involved in its generation remains unknown. In this article, the authors reviews the process of immune regulation inside the eye, the immunological mechanisms of damage implicated in the generation of uveitis, as well as the clinical aspects associated with the immune pathogenesis; in the last part of this paper we present the most recent trends in ocular research related to immunological damage in uveitis.