Prospective, Randomized, Double-Blind Parallel Group Nutritional Study to Evaluate the Effects of Routine Intake of Fresh vs. Pasteurized Yogurt on the Immune System in Healthy Adults.

The immune system is affected by the dietary products humans intake. Immune system regulation by nutrition has uses in the clinical context, but it can also benefit healthy populations by delaying or preventing the emergence of immune-mediated chronic illnesses. In this study, the purpose was to describe and compare the modulator effects on the immune system of the routine ingestion of fresh vs. pasteurized yogurt. A unicentral, prospective, randomized, double-blind, parallel group 8-week nutritional study was carried out comparing the ingestion of 125 g of the products in healthy adults three times a day. A complete battery of in vitro tests on the activity of the immune system, processes and phenomena was performed. Exclusive immune-modulatory effects of fresh yogurt with respect to base line were found in terms of increased systemic IgM (primary immune responses), increased synthesis of IFN-gamma upon stimulation (Th1) and increased peripheral T cells (mainly "naive" CD4s). In the three interventions, we observed an increased phagocytic activity and burst test in granulocytes, together with increased secretion of IL-6, IL-1 ß and IL-8 (pro-inflammatory) and increased CD16 expression (FcR favoring phagocytosis) in granulocytes. Overall, it is concluded that regardless of bacteria being alive or thermally inactivated, yogurt has common effects on the innate system, but the presence of live bacteria is necessary to achieve a potentiating effect on the specific immune response.

Conjunctival Intraepithelial Lymphocytes, Lacrimal Cytokines and Ocular Commensal Microbiota: Analysis of the Three Main Players in Allergic Conjunctivitis.

Conjunctival intraepithelial lymphocytes, tear soluble molecules and commensal microbiota have important roles in the ocular mucosal immune response in healthy and diseased subjects. For the purpose of this study, the cellular and microbial populations of the conjunctiva and the lacrimal soluble molecules were analyzed to find the main biomarkers in allergic conjunctivitis. A total of 35 healthy subjects, 28 subjects with seasonal allergic conjunctivitis and 32 subjects with perennial allergic conjunctivitis were recruited to obtain peripheral blood, conjunctival brush cytology, tear fluid and microbiota samples. Flow cytometry for lymphocytes, multiplex bead assays for cytokines and high-throughput DNA sequencing for microbiome analysis were used. For perennial allergic conjunctivitis, an increased proportion of Th2 and NKT lymphocytes was found, while CD3+TCRγδ+ lymphocytes and double negative MAIT cells were decreased. In contrast, seasonal allergic conjunctivitis was distinguished by an increase in Th17 and Th22 cell proportions, while the Th1 cell proportion decreased. Among tear fluid, the vast majority of pro-inflammatory cytokines (especially Th2 and Th17 cytokines) in perennial allergies and MMP-9 together with IgA in seasonal allergies were increased. In contrast, TGF-ß2 was decreased in both forms of conjunctivitis. Finally, fungal (Malassezia species) and bacterial (Kocuria and Propionobacterium acnes species) colonization were observed in the perennial allergic conjunctivitis group. These results provide the basis for the development of a disease profile for perennial allergic conjunctivitis and open the door to new therapeutic and diagnostic strategies.

Characterisation of Zamorano-Leonese Donkey Milk as an Alternative Sustainably Produced Protein Food.

The Zamorano-Leonese donkey is the local breed of the Castilla y León region of Spain and is a protected endangered species. The best way to preserve it is to explore viable alternatives such as milk production. Unlike other donkey breeds, this one has not been previously characterised. The aim of this work is the complete nutritional characterisation of its milk for human consumption, either directly or as an ingredient, to meet the new consumer expectations of sustainability and health concerns. This breed did not differ from others in terms of amino acid and protein profile. Its low concentration of ß-lactoglobulin may be correlated to a low allergenicity. The presence of lactozyme and lactoferrin, which are potent antimicrobials, stand out among the proteins. This milk presented a higher content of unsaturated fatty acids, being oleic fatty acid the main one. Zamorano-Leonese donkey milk did have a higher content of vitamin C, riboflavin, folic acid and vitamin E than the other donkey breeds. It also had a high concentration of vitamin D despite its low-fat content. However, its mineral concentration was lower than other donkey breeds in line with its lower ash content. In terms of micronutrients, it had a high amount of zinc and selenium. Based on these results we can conclude that donkey milk is a food and/or ingredient with beneficial effects on cardiovascular health and the proper functioning of the immune system, as well as being a good source of protein. Therefore, donkey milk from this local species from Spain is a food and/or ingredient with beneficial nutritional properties and sustainable from an environmental point of view.

Occupational hypersensitivity pneumonitis caused by fossil-containing rocks.

AIMS: We present a hypersensitivity immune response to inhalation of antigens from fossil soils frequently used in tile manufacture. We found that the soil polished by a worker affected by pneumonitis was a paleosol containing bivalves from the cretaceous period called Hippurites. METHODS: We made a diagnostic study for pneumonitis (analysis, microbiology, radiology, high-resolution CT, bronchoalveolar lavage, pulmonary biopsy. A biochemical study of the polishing materials used (magnesium hexafluorosilicate crystallizer), steel spoilage, washing liquid and Bilbao red limestone) after scraping of the same. Allergy study included skin tests with extracts from fossil soils, determination of IgG and IgE to mollusks, IgE-immunodetection with soil extracts with the patient's serum and non-atopic controls. Histology was made using scanning electron microscopy of the lung biopsy and the fossil soil to determine the presence of remains of mollusks, fungi, pollen or other fossil elements. RESULTS: SDS-PAGE IgE Immunoblotting assay detecting IgE binding in soil extract between 66 and 35 kDa. Likewise, IgE-Immunblotting assay with extracts from bivalve mollusks (razor shell, mussel and scallop) and gastropod (sea snail), detecting IgE binding between 100 kDa - 30 kDa, as well as in some bands with molecular mass between 20 and 14 kDa, proving sensitization to mollusks. CONCLUSIONS: Bivalve proteins preserved in fossil soils may produce an immune hypersensitivity response. This may impact on the precautions exposed workers, in this case fossil soil cutters and polishers, should take.

Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis.

Retinal pigment epithelial (RPE) cells are currently in the "spotlight" of cell therapy approaches to some retinal diseases. The analysis of the expressed proteins of RPE primary cells is an essential step for many of these approaches. But the emission of autofluorescence by RPE cells produces higher background noise interference thereby creating an impediment to this analysis. Trypan Blue (TB), a routinely used counterstain, has the capacity to quench this autofluorescence, if it is used in optimized concentration. The results from the method developed in our study indicate that incubation of the cultured RPE cells with 20 µg/ml of TB after immunolabelling (post-treatment) as well as incubation of the retinal tissue specimens with same concentration before paraffin embedding, sectioning and immunolabelling (pre-treatment) can be applied to effectively quench the autofluorescence of RPE cells. Thus it can facilitate the evaluation of expressed cellular proteins in experimental as well as in pathological conditions, fulfilling the current requirement for developing a method which can serve to eliminate the autofluorescence of the cells, not only in cell cultures but also in tissues samples. This method should significantly increase the quality and value of RPE cell protein analysis, as well as other cell protein analysis performed by Flow cytometry (FC) and Immunohistochemistry (IHC) techniques.

Epithelial component and intraepithelial lymphocytes of conjunctiva-associated lymphoid tissue in healthy children.

Conjunctiva-associated lymphoid tissue (CALT) plays a key role in protecting the eye surface by initiating and regulating immune responses. The aim of this study was to investigate in healthy children the proportion of intraepithelial lymphocytes (IELs), the degree of viability and/or apoptosis and cell proliferation in three different topographic areas of the conjunctiva. Superior tarsal, superior bulbar, and inferior tarsal-bulbarfornix conjunctival cells were collected by brush cytology (BC) from 24 healthy paediatric subjects (13 boys and 11 girls, mean age 6±2 years) who were to undergo strabismus correction surgery under general anaesthesia. Subsequently, these cells were analysed phenotypically and functionally by flow cytometry (FC). Flow cytometry analysis showed that not all the cells obtained by BC were of the epithelial lineage, but that there was a population of CD45+ cells (IELs) regularly present in the conjunctiva of healthy children. These IELs were mostly T-lymphocytes (CD3+) and B-lymphocytes (CD19+), with higher levels of T-lymphocytes (CD3+) in the upper areas than in the inferior tarsal-bulbar-fornix, where the highest levels of B-lymphocytes (CD19+) were found. In the apoptosis assay, two groups of cell populations were differentiated by cell size and complexity (cytoplasmic granularity), with more complex cells predominating in the upper areas of the conjunctiva and less complex cells being more abundant in the inferior tarsal-bulbar-fornix. Finally, the proliferative capacity of the conjunctival epithelium was significantly higher in the upper tarsal zone than in the rest of the zones analysed. These results suggest that the epithelial component and the IELs of CALT are also regularly present in the conjunctiva of the healthy child, varying in phenotype, viability and cell proliferation according to the different conjunctival regions analysed, which could lead us to believe that each conjunctival zone plays a different, specific role in the regulation of the immune response at the ocular level.

Characterization and short-term culture of cells recovered from human conjunctival epithelium by minimally invasive means.

PURPOSE: To characterize conjunctival cells obtained by brush cytology (BC) and establish short-term cultures. METHODS: Human tarsal and bulbar conjunctival cells were obtained by BC and transported in 3 different media: serum-free medium (DK-SFM) with low [Ca(2+)], 10% fetal bovine serum (FBS) supplemented medium (FBSm10), and 20% FBS-supplemented medium (FBSm20). Recovered cells were counted and initial viability assessed. Flow cytometry established epithelial or immune lineage, viability, apoptosis, and cell cycle stage. To establish short-term cultures, tarsal conjunctival cells were seeded onto Permanox or denuded amniotic membrane (dAM) and cultured in the 3 media. Living adherent cells were assessed on Days 1, 2, and 5 by fluorescence microscopy. RESULTS: Initial cell recovery was significantly lower with DK-SFM than in the other two culture media. Flow cytometry showed that 3.8+/-0.4% of recovered tarsal cells were CD45+ leukocytes and 67.9+/-1.6% were CK7+ secretory epithelial cells. S-phase cells composed 3.5+/-0.3% of the recovered tarsal cells and 2.1+/-0.2% of the bulbar cells (p=0.0006). The percentage of viable, apoptotic, and dead cells was similar for tarsal and bulbar cells. Two different cell populations were observed in both locations. About 24% consisted of smaller, less complex cells with high viability, and the remainder was composed of larger, more complex cells with poor viability. Significantly more living cells were supported by FBSm10 on the dAM substratum (p=0.011) than by the other media on either dAM or Permanox. CONCLUSIONS: Conjunctival BC recovers proliferating cells that can be maintained on dAM in FBSm10 for up to 5 days.

Component-resolved diagnosis in allergic disease: Utility and limitations.

Component resolved diagnosis (CRD) is a microarray-based diagnostic solution capable of simultaneously analysing specific IgE antibodies against 112 allergenic components, providing sensitivity patterns for multi-sensitised or complex patients. The CRD is indicated for these patients, especially those with concomitant respiratory and food allergies. This study reivews the method, its utility, limitations, and our experience in allergic diseases with difficult etiologic diagnosis (eosinophilic esophagitis, occupational asthma and drug allergy).

Effects of competitive learning tools on medical students: A case study.

OBJECTIVE: Competitive learning techniques are being successfully used in courses of different disciplines. However, there is still a significant gap in analyzing their effects in medical students competing individually. The authors conducted this study to assess the effectiveness of the use of a competitive learning tool on the academic achievement and satisfaction of medical students. METHODS: The authors collected data from a Human Immunology course in medical students (n = 285) and conducted a nonrandomized (quasi-experimental) control group pretest-posttest design. They used the Mann-Whitney U-test to measure the strength of the association between two variables and to compare the two student groups. RESULTS: The improvement and academic outcomes of the experimental group students were significantly higher than those of the control group students. The students using the competitive learning tool had better academic performance, and they were satisfied with this type of learning. The study, however, had some limitations. The authors did not make a random assignment to the control and experimental groups and the groups were not completely homogenous. CONCLUSION: The use of competitive learning techniques motivates medical students, improves their academic outcomes and may foster the cooperation among students and provide a pleasant classroom environment. The authors are planning further studies with a more complete evaluation of cognitive learning styles or incorporating chronometry as well as team-competition.

In Vitro Model for Predicting the Protective Effect of Ultraviolet-Blocking Contact Lens in Human Corneal Epithelial Cells.

PURPOSE: To develop an in vitro method to determine the protective effect of UV-blocking contact lenses (CLs) in human corneal epithelial (HCE) cells exposed to UV-B radiation. MATERIALS AND METHODS: SV-40-transformed HCE cells were covered with non-UV-blocking CL, UV-blocking CL or not covered, and exposed to UV-B radiation. As control, HCE cells were covered with both types of CLs or not covered, but not exposed to UV-B radiation. Cell viability at 24, 48 and 72 h, after UV-B exposure and removing CLs, was determined by alamarBlue(®) assay. Percentage of live, dead and apoptotic cells was also assessed by flow cytometry after 24 h of UV-B exposure. Intracellular reactive oxygen species (ROS) production after 1 h of exposure was assessed using the dye H(2)DCF-DA. RESULTS: Cell viability significantly decreased, apoptotic cells and intracellular ROS production significantly increased when UVB-exposed cells were covered with non-UV-blocking CL or not covered compared to non-irradiated cells. When cells were covered with UV-blocking CL, cell viability significantly increased and apoptotic cells and intracellular ROS production did not increase compared to exposed cells. CONCLUSIONS: UV-B radiation induces cell death by apoptosis, increases ROS production and decreases viable cells. UV-blocking CL is able to avoid these effects increasing cell viability and protecting HCE cells from apoptosis and ROS production induced by UV-B radiation. This in vitro model is an alternative to in vivo methods to determine the protective effect of UV-blocking ophthalmic biomaterials because it is a quicker, cheaper and reliable model that avoids the use of animals.

Mutations of CD40 ligand in two patients with hyper-IgM syndrome.

Two patients with the X-linked form of the hyper-IgM syndrome have been studied. Both patients present: 1. Mutations in the CD40L gene (a nonsense point mutation that introduces a termination codon at the extracellular domain of the protein, and a deletion that eliminates exon 4 as consequence of an abnormal splicing). 2. Lack of CD40L expression on the lymphocyte surface after stimulation with ionomycin and PMA. 3. Altered lymphocytic proliferation in response to anti-CD3. 4. Hyper IgM, low IgG and IgA levels and neutropenia. One of the patients shows, in addition, low Natural Killer cell numbers and severe herpetic infections, which distinguishes this case from the common hyper-IgM syndrome phenotype. Finally, a hyper-IgM stable phenotype has been immortalized by Herpes virus Saimiri for the first time.

Fatal disseminated Scedosporium prolificans infection initiated by ophthalmic involvement in a patient with acute myeloblastic leukemia.

Scedosporium prolificans is an opportunistic saprophytic fungus that rapidly disseminates and is intrinsically resistant to currently available antifungal drugs. We report a fatal case of disseminated S. prolificans infection that started with orbital and ocular involvement in a patient with secondary acute myeloblastic leukemia.

Flow cytometry assessment of the purity of human retinal pigment epithelial primary cell cultures.

Culturing of human retinal pigment epithelial cells (hRPE) is the initial step in cell therapy of some retinal diseases. To transfer these cells into clinical use, it is necessary to guarantee that they are well differentiated and contamination free. Fluorescence microscopy is the easiest method to do this, but it is associated with operator subjectivity, and the results are highly variable. The aim of this study was to demonstrate the practicality of implementing flow cytometry (FC) analysis to determine the purity of human RPE primary cell cultures. An ARPE19 cell line, human skin fibroblasts, hRPE, and human corneal epithelial cells were analysed by FC to determine the percentage of the hRPE population expressing RPE65 and epithelial and fibroblast proteins. The cell viability and DNA content also were determined. FC analysis showed that the hRPE cells were healthy, stable, and expressed RPE65 protein in the study working conditions. The density of RPE65 protein expression decreased during passages 2 to 10, which was confirmed using a Western blot technique. However, the hRPE cells did not express the 112-kDa epithelial and fibroblast proteins in the current working conditions. These findings suggested that FC facilitates a detailed analysis of human RPE primary cell cultures, a necessary step in developing new cell therapies for retinal diseases.

In vitro simulation of corneal epithelium microenvironment induces a corneal epithelial-like cell phenotype from human adipose tissue mesenchymal stem cells.

PURPOSE: Transplantation of autologous corneal stem cells in not possible in cases of bilateral limbal stem cell deficiency (LSCD). To restore the ocular surface in these patients, an autologous extraocular source of stem cells is desirable to avoid dependence on deceased donor tissue and host immunosuppression of allogenic transplants. While bone marrow-derived mesenchymal stem cells (MSCs) can acquire certain characteristics of corneal epithelial cells, subcutaneous adipose tissue (AT) is more readily available and accessible. The aim of this study was to determine if extraocular human AT-derived MSCs (hAT-MSCs) can acquire in vitro some features of corneal epithelial-like cells. METHODS: hAT-MSCs were isolated from human lipoaspirates and expanded up to 3-4 passages. We studied the immunophenotype of MSCs and demonstrated its multipotent capacity to differentiate toward osteoblasts, adipocytes and chondrocytes. To test the capacity of differentiation of hAT-MSCs toward corneal epithelial-like cells, hAT-MSCs were cultured on substrata of plastic or collagen IV. We used basal culture medium (BM), BM conditioned with human corneal epithelial cells (HCEcBM) and BM conditioned with limbal fibroblasts (LFcBM). RESULTS: The hAT-MSCs incubated for 15 days with HCEcBM acquired more polygonal and complex morphology as evaluated by phase-contrast microscopy and flow cytometry. Additionally, the expression of transforming growth factor-ß receptor CD105 and corneal epithelial marker CK12 got increased as evaluated by flow cytometry, real-time reverse-transcription polymerase chain reaction, western blot and immunostaining. These changes were absent in hAT-MSCs incubated with unconditioned BM or with LFcBM. CONCLUSIONS: Corneal epithelial-like cells can be induced from extraocular hAT-MSCs by subjecting them to an in vitro microenvironment containing conditioning signals derived from differentiated human corneal epithelial cells. Our results suggest that hAT-MSCs could provide a novel source of stem cells that hold the potential to restore sight lost in patients suffering from bilateral ocular surface failure due to LSCD.

Differential cell proliferation, apoptosis, and immune response in healthy and evaporative-type dry eye conjunctival epithelia.

PURPOSE: To assess cell viability and cell cycle kinetics of the conjunctival epithelium and to identify intraepithelial lymphocyte (IEL) subsets in patients with evaporative-type dry eye disease (ev-DED) caused by meibomian gland dysfunction and in healthy subjects. The effect of topical treatment and correlations between clinical symptoms and signs and epithelial and immune variables were also determined. METHODS: Inferior fornix and tarsal conjunctival cells were collected by brush cytology (BC) from patients with mild to moderate ev-DED (n = 23) before and after 2 months of treatment that included lid hygiene, artificial tears, and a 3-week course of topical unpreserved steroids. Healthy subjects (n = 17) served as controls. Two symptom questionnaires were self-answered, and multiple DED-related clinical tests were performed. Epithelial or immune lineage, IEL subtypes, cell viability, apoptosis, and cell cycle stage of the BC-recovered cells were determined by flow cytometry. RESULTS: Conjunctival cell viability was dramatically decreased in ev-DED patients compared with controls. For both groups, two different cell populations, differentiated by cell size and complexity, were present in the apoptosis assay. After 2 months of treatment, 87% of subjects subjectively improved, CD8 cells increased, and CD4 cells and the CD4/CD8 ratio significantly decreased. The pretreatment and posttreatment proliferative capacity of the conjunctival epithelium was significantly lower in ev-DED patients than in healthy controls. CONCLUSIONS: The viability and proliferative capacity of ev-DED patient conjunctival cells were reduced, suggesting a potential role for these parameters as disease biomarkers.

Dry eye disease as an inflammatory disorder.

PURPOSE: Dry eye disease (DED) is a prevalent inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface disease, impaired quality of vision, and a wide range of complications, eventually causing a reduction in quality of life. It still is a frustrating disease because of the present scarcity of therapies that can reverse, or at least stop, its progression. METHODS: A comprehensive literature survey of English-written scientific publications on the role of inflammation in DED. RESULTS: New investigations have demonstrated that a chronic inflammatory response plays a key role in the pathogenesis of human DED. Additionally, correlations between inflammatory molecules and clinical data suggest that inflammation can be responsible for some of the clinical symptoms and signs. CONCLUSIONS: Research efforts to clarify its pathophysiology are leading to a better understanding of DED, demonstrating that inflammation, in addition to many other factors, plays a relevant role.