Mercè Martí

Thyroglobulin Peptides Associate In Vivo to HLA-DR in Autoimmune Thyroid Glands

Endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express HLA class II (HLA-II) molecules that are presumably involved in the maintenance and regulation of the in situ autoimmune response. HLA-II molecules thus expressed by thyroid cells have the “compact” conformation and are therefore expected to stably bind autologous peptides. Using a new approach to study in situ T cell responses without the characterization of self-reactive T cells and their specificity, we have identified natural HLA-DR-associated peptides in autoimmune organs that will allow finding peptide-specific T cells in situ. This study reports a first analysis of HLA-DR natural ligands from ex vivo Graves’ disease-affected thyroid tissue. Using mass spectrometry, we identified 162 autologous peptides from HLA-DR-expressing cells, including thyroid follicular cells, with some corresponding to predominant molecules of the thyroid colloid. Most interestingly, eight of the peptides were derived from a major autoantigen, thyroglobulin. In vitro binding identified HLA-DR3 as the allele to which one of these peptides likely associates in vivo. Computer modeling and bioinformatics analysis suggested other HLA-DR alleles for binding of other thyroglobulin peptides. Our data demonstrate that although the HLA-DR-associated peptide pool in autoimmune tissue mostly belongs to abundant ubiquitous proteins, peptides from autoantigens are also associated to HLA-DR in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response.

TCR Bias of In Vivo Expanded T Cells in Pancreatic Islets and Spleen at the Onset in Human Type 1 Diabetes

Autoreactive T cells, responsible for the destruction of pancreatic β cells in type 1 diabetes, are known to have a skewed TCR repertoire in the NOD mouse. To define the autoreactive T cell repertoire in human diabetes, we searched for intraislet monoclonal expansions from a recent onset in human pancreas to then trace them down to the patient’s peripheral blood and spleen. Islet infiltration was diverse, but five monoclonal TCR β-chain variable expansions were detected for Vβ1, Vβ7, Vβ11, Vβ17, and Vβ22 families. To identify any sequence bias in the TCRs from intrapancreatic T cells, we analyzed 139 different CDR3 sequences. We observed amino acid preferences in the NDN region that suggested a skewed TCR repertoire within infiltrating T cells. The monoclonal expanded TCR sequences contained amino acid combinations that fit the observed bias. Using these CDR3 sequences as a marker, we traced some of these expansions in the spleen. There, we identified a Vβ22 monoclonal expansion with identical CDR3 sequence to that found in the islets within a polyclonal TCR β-chain variable repertoire. The same Vβ22 TCR was detected in the patient’s PBMCs, making a cross talk between the pancreas and spleen that was reflected in peripheral blood evident. No other pancreatic monoclonal expansions were found in peripheral blood or the spleen, suggesting that the Vβ22 clone may have expanded or accumulated in situ by an autoantigen present in both the spleen and pancreas. Thus, the patient’s spleen might be contributing to disease perpetuation by expanding or retaining some autoreactive T cells.

Biomarker detection of global infectious diseases based on magnetic particles.

Infectious diseases affect the daily lives of millions of people all around the world, and are responsible for hundreds of thousands of deaths, mostly in the developing world. Although most of these major infectious diseases are treatable, the early identification of individuals requiring treatment remains a major issue. The incidence of these diseases would be reduced if rapid diagnostic tests were widely available at the community and primary care level in low-resource settings. Strong research efforts are thus being focused on replacing standard clinical diagnostic methods, such as the invasive detection techniques (biopsy or endoscopy) or expensive diagnostic and monitoring methods, by affordable and sensitive tests based on novel biomarkers. The development of new methods that are needed includes solid-phase separation techniques. In this context, the integration of magnetic particles within bioassays and biosensing devices is very promising since they greatly improve the performance of a biological reaction. The diagnosis of clinical samples with magnetic particles can be easily achieved without pre-enrichment, purification or pretreatment steps often required for standard methods, simplifying the analytical procedures. The biomarkers can be specifically isolated and preconcentrated from complex biological matrixes by magnetic actuation, increasing specificity and the sensitivity of the assay. This review addresses these promising features of the magnetic particles for the detection of biomarkers in emerging technologies related with infectious diseases affecting global health, such as malaria, influenza, dengue, tuberculosis or HIV.

Induction of intercellular adhesion molecule-1 but not of lymphocyte function-associated antigen-3 in thyroid follicular cells

We investigated the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) by thyrocytes and their regulation by cytokines. Immunofluorescence studies on cryostat sections and on freshly dispersed cell preparations showed that ICAM-1 and LFA-3 are barely detectable in non-autoimmune thyrocytes. However, thyrocytes acquired ICAM-1 expression in culture. IFN-gamma, IL-1 beta and TNF-alpha produced a clear enhancement of ICAM-1 expression. When tested in combination, IL-1 beta and TNF-alpha were additive to the IFN-gamma effect. LFA-3 expression was not modulated by these cytokines. In the HT93 thyroid cell line generated by transfection with SV40, ICAM-1 and LFA-3 were both constitutively expressed at high levels. Cytokines modulated ICAM-1 expression similarly, but to a greater extent than in normal thyrocytes. LFA-3 remained unmodified. These results support the notion that normal thyrocytes are immunologically silent cells. The capability of cytokines to induce ICAM-1 together with HLA class I and class II-expression on thyrocytes suggests that under their influence, these cells may express all the surface molecules required for antigen presentation and/or for being recognized as target cells in the context of thyroid autoimmune disease.

Electrochemical magneto-actuated biosensor for CD4 count in AIDS diagnosis and monitoring.

The counting of CD4(+) T lymphocytes is a clinical parameter used for AIDS diagnosis and follow-up. As this disease is particularly prevalent in developing countries, simple and affordable CD4 cell counting methods are urgently needed in resource-limited settings. This paper describes an electrochemical magneto-actuated biosensor for CD4 count in whole blood. The CD4(+) T lymphocytes were isolated, preconcentrated and labeled from 100 μL of whole blood by immunomagnetic separation with magnetic particles modified with antiCD3 antibodies. The captured cells were labeled with a biotinylated antiCD4 antibody, followed by the reaction with the electrochemical reporter streptavidin-peroxidase conjugate. The limit of detection for the CD4 counting magneto-actuated biosensor in whole blood was as low as 44 cells μL(-1) while the logistic range was found to be from 89 to 912 cells μL(-1), which spans the whole medical interest range for CD4 counts in AIDS patients. The electrochemical detection together with the immunomagnetic separation confers high sensitivity, resulting in a rapid, inexpensive, robust, user-friendly method for CD4 counting. This approach is a promising alternative for the costly standard flow cytometry and suitable as diagnostic tool at decentralized practitioner sites in low resource settings, especially in less developed countries.

IL-13 pathway alterations impair iNKT cell-mediated regulation of T effector cells in Type 1 Diabetes

Many studies have shown that human natural killer T (NKT) cells can promote immunity to pathogens, but their regulatory function is still being investigated. Invariant NKT (iNKT) cells have been shown to be effective in preventing type 1 diabetes in the NOD mouse model. Activation of plasmacytoid dendritic cells, modulation of B-cell responses, and immune deviation were proposed to be responsible for the suppressive effect of iNKT cells. We studied the regulatory capacity of human iNKT cells from control subjects and patients with type 1 diabetes (T1D) at disease clinical onset. We demonstrate that control iNKT cells suppress the proliferation of effector T cells (Teffs) through a cell contact–independent mechanism. Of note, suppression depended on the secretion of interleukin-13 (IL-13) by iNKT cells because an antibody blocking this cytokine resulted from the abrogation of Teff suppression; however, T1D-derived iNKT cells showed impaired regulation that could be attributed to the decrease in IL-13 secretion. Thus, alteration of the IL-13 pathway at disease onset may lead to the progression of the autoimmune response in T1D. Advances in the study of iNKT cells and the selection of agonists potentiating IL-13 secretion should permit new therapeutic strategies to prevent the development of T1D.

CD4 quantification based on magneto ELISA for AIDS diagnosis in low resource settings

The Acquired Immune Deficiency Syndrome (AIDS) affects the life of millions of people around the world. Although rapid and low cost screening tests are widely available for the diagnosis of HIV infection, the count of CD4+ T lymphocytes remains a drawback in the areas mostly affected by the HIV, being this control imperative for assessing the deterioration of the immunological system and the progression towards AIDS, when the counting of cells falls down 200cellsμL(-1). This paper describes a high-throughput, simple and rapid method for CD4+ T lymphocytes quantification, directly in whole blood, based on a magneto ELISA. The CD4 cells are separated and preconcentrated from whole blood in magnetic particles, and labeled with an enzyme for the optical readout performed with a standard microplate reader. The magneto ELISA is able to reach the whole CD4 counting range of medical interest, being the limit of detection as low as 50 CD4+ cells per μL of whole blood, without any pretreatment. This method is a highly suitable alternative diagnostic tool for the expensive flow cytometry at the community and primary care level, providing a sensitive method but by using instrumentation widely available in low-resource settings laboratories and requiring low-maintenance, as is the case of a microplate reader operated by filters.

Controlled degradability of PCL-ZnO nanofibrous scaffolds for bone tissue engineering and their antibacterial activity

Up to date, tissue regeneration of large bone defects is a clinical challenge under exhaustive study. Nowadays, the most common clinical solutions concerning bone regeneration involve systems based on human or bovine tissues, which suffer from drawbacks like antigenicity, complex processing, low osteoinductivity, rapid resorption and minimal acceleration of tissue regeneration. This work thus addresses the development of nanofibrous synthetic scaffolds of polycaprolactone (PCL) - a long-term degradation polyester - compounded with hydroxyapatite (HA) and variable concentrations of ZnO as alternative solutions for accelerated bone tissue regeneration in applications requiring mid- and long-term resorption. In vitro cell response of human fetal osteoblasts as well as antibacterial activity against Staphylococcus aureus of PCL:HA:ZnO and PCL:ZnO scaffolds were here evaluated. Furthermore, the effect of ZnO nanostructures at different concentrations on in vitro degradation of PCL electrospun scaffolds was analyzed. The results proved that higher concentrations ZnO may induce early mineralization, as indicated by high alkaline phosphatase activity levels, cell proliferation assays and positive Alizarin-Red-S-stained calcium deposits. Moreover, all PCL:ZnO scaffolds particularly showed antibacterial activity against S. aureus which may be attributed to release of Zn2+ ions. Additionally, results here obtained showed a variable PCL degradation rate as a function of ZnO concentration. Therefore, this work suggests that our PCL:ZnO scaffolds may be promising and competitive short-, mid- and long-term resorption systems against current clinical solutions for bone tissue regeneration.

Interferon gamma transcript detection on T cells based on magnetic actuation and multiplex double-tagging electrochemical genosensing

Interferon-γ is a proinflammatory cytokine, and its production is related with effective host defense against intracellular pathogens. Therefore, the level of interferon-γ is considered a good biomarker for intracellular infections. It is also useful for the assessment, treatment progression and follow-up of non-communicable diseases, including cancer and autoimmune disorders, among others. This work addresses the development of a novel interferon-γ release assay (IGRA) to evaluate the expression of interferon-γ transcripts as biomarker produced by isolated T cells, as a main advantage. The method sequentially combined three different types of magnetic separation, including the immunomagnetic separation of the T cells performed on antiCD3 modified magnetic particles, the retrotranscription and multiplex double-tagging PCR on polydT-modified magnetic particles and, finally, the electrochemical genosensing on streptavidin magnetic particles as a support. This approach is able to quantify the levels of cellular interferon-γ produced by as low as 150 T cells with outstanding analytical features. The detection of interferon-γ transcripts is performed from only 100 μL of whole blood which can be potentially obtained by fingerprick, demonstrating a further clear advantage to be considered as a promising strategy for the quantification of this important biomarker in several clinical applications.