David Calzada

Association of Immunological Cell Profiles with Specific Clinical Phenotypes of Scleroderma Disease

This study aimed to search the correlation among immunological profiles and clinical phenotypes of scleroderma in well-characterized groups of scleroderma patients, comparing forty-nine scleroderma patients stratified according to specific clinical phenotypes with forty-nine healthy controls. Five immunological cell subpopulations (B, CD4+ and CD8+ T-cells, NK, and monocytes) and their respective stages of apoptosis and activation were analyzed by flow cytometry, in samples of peripheral blood mononuclear cells (PBMCs). Analyses of results were stratified according to disease stage, time since the diagnosis, and visceral damage (pulmonary fibrosis, pulmonary hypertension, and cardiac affliction) and by time of treatment with corticosteroids. An increase in the percentages of monocytes and a decrease in the B cells were mainly related to the disease progression. A general apoptosis decrease was found in all phenotypes studied, except in localized scleroderma. An increase of B and NK cells activation was found in patients diagnosed more than 10 years ago. Specific cell populations like monocytes, NK, and B cells were associated with the type of affected organ. This study shows how, in a heterogeneous disease, proper patients stratification according to clinical phenotypes allows finding specific cellular profiles. Our data may lead to improvements in the knowledge of prognosis factors and to aid in the analysis of future specific therapies.

Characterization of immune cell phenotypes in adults with autism spectrum disorders

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impairments in verbal and non-verbal communication, impaired social interactions and repetitive behaviors. There is evidence of a link between ASD symptoms and immune dysfunction, but few studies have been performed in adult patients to confirm this. In this work, we used flow cytometry to study immunological differences in peripheral blood mononuclear cells from 59 adult patients and 26 healthy control subjects to identify possible immune cell profiles related with this group of disorders. We analyzed six immune cell subpopulations (ie, B-cells, CD4+ and CD8+ T-cells, NK, NKT cells, and monocytes) and their corresponding stages of apoptosis and activation. The most noteworthy results showed that, compared to healthy controls, patients had increased percentages of CD8+ T-cells and B-cells, and a decrease in the percentage of NKT cells. Regarding CD25 expression, we found overall CD25+ overexpression, primarily in NK and NKT cells. Apoptosis percentage showed an increasing trend only in monocytes of patients. These data support a link between ASD and immune dysfunction, suggesting that specific cellular phenotypes and/or activation status of immune cells may be relevant in adult ASD.

Data set on a study of gene expression in peripheral samples to identify biomarkers of severity of allergic and nonallergic asthma

This article contains information related to the research article entitled “Biomarkers associated with disease severity in allergic and nonallergic asthma” (S. Baos, D. Calzada, L. Cremades, J. Sastre, J. Quiralte, F. Florido, C. Lahoz, B. Cárdaba, In press). Specifically, the clinical criteria stablished for selecting the study population (n=104 subjects) are described. Moreover, this article describes the criteria for selecting the 94 genes to be analyzed in PBMCs (peripheral blood mononuclear cells), it is provided a description of these genes and a Table with the genes most differentially expressed by clinical phenotypes and, finally it is detailed the experimental methodology followed for studying the protein expression of MSR1 (macrophage scavenger receptor 1), one of the genes evaluated in the research.

Therapeutic targets for olive pollen allergy defined by gene markers modulated by Ole e 1-derived peptides.

Two regions of Ole e 1, the major olive-pollen allergen, have been characterized as T-cell epitopes, one as immunodominant region (aa91-130) and the other, as mainly recognized by non-allergic subjects (aa10-31). This report tries to characterize the specific relevance of these epitopes in the allergic response to olive pollen by analyzing the secreted cytokines and the gene expression profiles induced after specific stimulation of peripheral blood mononuclear cells (PBMCs). PBMCs from olive pollen-allergic and non-allergic control subjects were stimulated with olive-pollen extract and Ole e 1 dodecapeptides containing relevant T-cell epitopes. Levels of cytokines were measured in cellular supernatants and gene expression was determined by microarrays, on the RNAs extracted from PBMCs. One hundred eighty-nine differential genes (fold change >2 or <-2, P<0.05) were validated by qRT-PCR in a large population. It was not possible to define a pattern of response according the overall cytokine results but interesting differences were observed, mainly in the regulatory cytokines. Principal component (PCA) gene-expression analysis defined clusters that correlated with the experimental conditions in the group of allergic subjects. Gene expression and functional analyses revealed differential genes and pathways among the experimental conditions. A set of 51 genes (many essential to T-cell tolerance and homeostasis) correlated with the response to aa10-31 of Ole e 1. In conclusion, two peptides derived from Ole e 1 could regulate the immune response in allergic patients, by gene-expression modification of several regulation-related genes. These results open new research ways to the regulation of allergy by Oleaceae family members.

Immunological Mechanisms in Allergic Diseases and Allergen Tolerance: The Role of Treg Cells

The immune system regulates itself to establish an appropriate immune response to potentially harmful pathogens while tolerating harmless environmental antigens and self-antigens. A central role in this balance is played by regulatory T cells (Tregs) through various ways of actions. By means of molecule secretion and cell-cell contact mechanisms, Tregs may have the capacity to modulate effector T cells and suppress the action of proinflammatory cytokines across a broad range of cell types. As a result, abnormal regulatory T cell function has been pointed as a main cause in the development of allergic diseases, a major public health problem in industrialized countries, with a high socioeconomic impact. This prevalence and impact have created an international interest in improving the allergy diagnosis and therapy. Additionally, research has sought to gain a better understanding of the molecular mechanisms underlining this kind of disease, in order to a better management. At this respect, the role of Treg cells is one of the most promising areas of research, mainly because of their potential use as new immunotherapeutical approaches. Therefore, the aim of this review is to update the existing knowledge of the role of Tregs in this pathology deepening in their implication in allergen-specific therapy (AIT).

New Treatments for Allergy: Advances in Peptide Immunotherapy

BACKGROUND Nowadays, allergen-specific immunotherapy (AIT) is the only treatment able to modulate the course of allergic diseases. Although it has been applied for the last 100 years, treatment with whole allergen extracts is not without its drawbacks: AIT can cause local and systemic adverse events and may produce new IgE sensitization against other allergens present in the extract. Furthermore, the lengthy treatment duration (3-5 years), frequent administration, and high cost of treatment are other disadvantages. For these reasons, there is a need for safer and more effective AIT strategies. One promising approach is the use of synthetic peptides representing the B- or T-cell epitopes of allergens. OBJECTIVE This review summarizes the main advances in peptide immunotherapy, from preclinical models to early clinical trials, focusing on house dust mite, bee venom, cat allergy, and Oleaceae pollinosis. RESULTS Following an extensive review of the relevant literature, we summarize how peptide therapies may change the course of allergic diseases and promote allergen tolerance, thereby ameliorating the main disadvantages of AIT. Although the molecular mechanisms involved are not yet fully defined, they seem to depend on structure, length, peptide sequence, and route of administration. This novel immunotherapy has been demonstrated to modulate the immune system, promoting regulatory T-cell induction and Th2 inhibition. This tolerance-inducing potential has led this therapy to be termed SPIRE (synthetic peptide immuno-regulatory epitopes). CONCLUSION Experimental models and clinical trials have demonstrated the usefulness of SPIRE treatment to cure these diseases, opening a new era in allergen therapeutics.

Nonallergic Asthma and Its Severity: Biomarkers for Its Discrimination in Peripheral Samples

Asthma is a complex and heterogeneous respiratory disorder characterized by chronic airway inflammation. It has generally been associated with allergic mechanisms related to type 2 airway inflammation. Nevertheless, between 10 and 33% of asthmatic individuals have nonallergic asthma (NA). Several targeted treatments are in clinical development for patients with Th2 immune response, but few biomarkers are been defined for low or non-Th2-mediated inflammation asthma. We have recently defined by gene expression a set of genes as potential biomarkers of NA, mainly associated with disease severity: IL10, MSR1, PHLDA1, SERPINB2, CHI3L1, IL8, and PI3. Here, we analyzed their protein expression and specificity using sera and isolated peripheral blood mononuclear cells (PBMCs). First, protein quantification was carried out using ELISA (in sera) or Western blot (proteins extracted from PBMCs by Trizol procedure), depending on the biomarker in 30 healthy controls (C) subjects and 30 NA patients. A receiver operating characteristic curve analysis was performed by using the R program to study the specificity and sensitivity of the candidate biomarkers at a gene- and protein expression level. Four kinds of comparisons were performed: total NA group vs C group, severe NA patients vs C, moderate–mild NA patients vs C, and severe NA patients vs moderate–mild NA patients. We found that all the single genes showed good sensitivity vs specificity for some phenotypic discrimination, with CHI3L1 and PI3 exhibiting the best results for C vs NA: CHI3L1 area under the curve (AUC) (CI 95%): 0.95 (0.84–1.00) and PI3 AUC: 0.99 (0.98–1.00); C vs severe NA: PI3 AUC: 1 (0.99–1.00); and C vs moderate–mild NA: CHI3L1 AUC: 1 (0.99–1.00) and PI3 AUC: 0.99 (0.96–1.00). However, the results for discriminating asthma disease and severity with protein expression were better when two or three biomarkers were combined. In conclusion, individual genes and combinations of proteins have been evaluated as reliable biomarkers for classifying NA subjects and their severity. These new panels could be good diagnostic tests.

Polymorphisms of Tumor Necrosis Factor-α, Transforming Growth Factor-β, and Interleukin-10 in Asthma Associated with Olive Pollen Sensitization

Sensitization to specific olive pollen-allergens (Ole e 2 and 10) has been correlated with a clinical pattern of asthma. This study analyzes the association between several polymorphims of TNFA (G-308A, C-857T, and C-1031T), IL10 (C-571A and A-1117G), and TGFB (C-509-T) and these sensitizations. These polymorphisms were genotyped by allelic discrimination, in olive pollen-allergic patients (phenotyped for specific Ole e 2 and 10 sensitizations) and healthy controls. Levels of serum-soluble cytokines were correlated with specific genotypes and clinical phenotypes. The results showed that heterozygous TGFB C-509T genotype, besides having the lowest sera TGF- levels, was significantly increased in olive pollen-allergic patients compared with controls. According specific sensitizations, CC genotype of IL10 C-571A could be a protective factor for Ole e 2 sensitization and mainly for asthmatic Ole e 2 sensitized patients compared with asthmatic non-Ole e 2 sensitized patients (OR: 0.26, P = 0.008). In contrast, heterozygous CA genotype was increased in Ole e 2 asthmatic subjects compared to asthmatic non-Ole e 2 sensitized patients. Lastly, heterozygous TNFA G-308A genotype was associated with Ole e 10 sensitization (OR: 2.5, P = 0.04). In conclusion, these results suggest a role of TGF-β1 in olive-pollen sensitization and TNF-α and IL-10 genotypes in the asthma induced by specific olive-pollen allergens.

New Challenges for Old Diseases: The Impact of -Omics Technologies in the Understanding of Allergic Diseases

Allergic diseases are an adverse reaction of the immune system against otherwise innocuous substances and are characterized by their high complexity. Patients can be asymptomatic or their involvement could be as severe as asthma. The complex nature of the phenotypes involved seems to point to genetic and environmental factors implication. Familiar aggregation or genetic implication in the development of these diseases is well reported, and experts seem to agree that atopic diseases affect homozygotic twins more than dizigotic twins (Ownby, 1990, Duffy et al., 1990). Allergic diseases are characterized by a Th2 inflammatory response involving several possible modulator factors (genetics and environmental factors), subject-related or antigenrelated modulators such as adjuvants, solubility in the microenvironment of mucosa, size of the sensitization agent, mucosa permeability, viral infections, and the greater or lesser ability of effectors cells to liberate mediators. Other factors include atmospheric pollution, exposure to tobacco, lifestyle-related diet and hygiene habits and maternal effects. The interaction between these factors produces the clinical picture of allergic disease. Great advances have been performed in the understanding, diagnosis and treatment of these diseases but the search of specific protective or risk biomarkers is an unsolved field. Since completion of the human genome project a rapid progress in genetics and bioinformatics have enabled the development of multiple tools as well as a large public databases, which include genetic and genomic data linked to clinical health data. The scientific revolution represented by the description of the human genome was largely facilitated by the use of DNA microarray technology, which made it possible to build a catalog of all genes within a given organism. The human genome project found that humans have an average of between 20,000 and 25,000 protein-coding genes (IHGSC, 2004). In addition, genetic variability between individuals is approximately 1% (Venter et al., 2001), suggesting that interactions between genes, proteins and the environment contribute to differences in human phenotype, maintenance of health and susceptibility to disease. On the other hand, the emergence of gene expression microarray technology in the mid-1990s has enabled genome-wide measurement of gene expression in a single experiment. This is turn