Regulatory T cells control strain specific resistance to Experimental Autoimmune Prostatitis

This deposit is composed by a publication in which the IGC' authors have had the role of collaboration (it's a collaboration publication). This type of deposit in ARCA is in restrictedAccess (it can't be in open access to the public), and could only be accessed by two ways: either by requesting a legal copy to the author (the email contact present in this deposit) or by visiting the following link: https://www.nature.com/articles/srep33097Susceptibility to autoimmune diseases results from the encounter of a complex and long evolved genetic context with a no less complex and changing environment. Major actors in maintaining health are regulatory T cells (Treg) that primarily dampen a large subset of autoreactive lymphocytes escaping thymic negative selection. Here, we directly asked whether Treg participate in defining susceptibility and resistance to Experimental Autoimmune Prostatitis (EAP). We analyzed three common laboratory strains of mice presenting with different susceptibility to autoimmune prostatitis upon immunization with prostate proteins. The NOD, the C57BL/6 and the BALB/c mice that can be classified along a disease score ranging from severe, mild and to undetectable, respectively. Upon mild and transient depletion of Treg at the induction phase of EAP, each model showed an increment along this score, most remarkably with the BALB/c mice switching from a resistant to a susceptible phenotype. We further show that disease associates with the upregulation of CXCR3 expression on effector T cells, a process requiring IFNγ. Together with recent advances on environmental factors affecting Treg, these findings provide a likely cellular and molecular explanation to the recent rise in autoimmune diseases incidence.Argentinean Agency for Promotion of Science and Technology; CONICET; National University of Córdoba grants; Cooperation Program in Science & Technology between Argentina and Portugal FCT–MINCYT

CD8 T cells are dispensable for experimental autoimmune prostatitis induction and chronic pelvic pain development

IntroductionChronic Pelvic Pain Syndrome or Chronic Prostatitis (CPPS/CP) is the most prevalent urologic affliction among young adult men. It is a challenging condition to treat, which significantly decreases patient quality of life, mostly because of its still uncertain aetiology. In that regard, an autoimmune origin is a prominent supported theory. Indeed, studies in patients and in rodent models of Experimental Autoimmune Prostatitis (EAP) have provided compelling evidence suggesting a key role of CD4 Th1 cells in disease pathogenesis. However, the implication of other prominent effectors of the immune system, such as CD8 T cells, has yet to be studied.MethodsWe herein analyzed the induction of prostatitis and the development of chronic pelvic pain in EAP using CD8 T cell-deficient animals.ResultsWe found similarly elevated PA-specific immune responses, with high frequencies of specific IFNg+CD4+ and IL17+CD4+ T cells in prostate draining lymph nodes from PA-immunized either CD8 KO or wild type animals with respect to controls. Moreover, these peripheral immune responses were paralleled by the development of significant chronic pelvic pain, and accompanied by prostate histological lesions, characterized by hemorrhage, epithelial cell desquamation, marked periglandular leukocyte infiltration, and increased collagen deposition in both, PA-immunized CD8 KO and wild type animals. As expected, control animals did not develop prostate histological lesions.DiscussionOur results indicate that CD8 T cells do not play a major role in EAP pathogenesis and chronic pelvic pain development. Moreover, our results corroborate the previous notion that a CD4 Th1 associated immune response drives the induction of prostate tissue inflammation and the development of chronic pelvic pain

IL-10 Producing B Cells Dampen Protective T Cell Response and Allow Chlamydia muridarum Infection of the Male Genital Tract

A significant proportion of individuals develop chronic, persistent and recurrent genital tract infections with Chlamydia trachomatis, which has been attributed to the numerous strategies that the bacterium uses to subvert host immune responses. Animal chlamydia models have demonstrated that protective immune response is mediated by CD4+ Th1 cytokine responses. Herein, we demonstrate that early after infecting the male genital tract, C. muridarum triggers the production of IL-10 by splenic and lymph node cells. In addition, C. muridarum triggers IL-6 and TNFα secretion. Data obtained from in vitro and in vivo experiments revealed B cells as the major IL-10 contributors. Indeed, purified B cells produced high amounts of IL-10 and also exhibited enhanced expression of inhibitory molecules such as CD39, PD-L1 and PD1 after C. muridarum stimulation. In vitro experiments performed with sorted cell subsets revealed that Marginal Zone B cells were the main IL-10 producers. In vitro and in vivo studies using TLR-deficient mice indicated that TLR4 signaling pathway was essential for IL-10 production. In addition, in vivo treatments to neutralize IL-10 or deplete B cells indicated that IL-10 and B cells played a significant role in delaying bacterial clearance ability. Moreover, the latter was confirmed by adoptive cell transfer experiments in which the absence of IL-10-producing B cells conferred the host a greater capability to induce Th1 responses and clear the infection. Interestingly, NOD mice, which were the least efficient in clearing the infection, presented much more Marginal Zone B counts and also enhanced TLR4 expression on Marginal Zone B cells when compared to B6 and BALB/c mice. Besides, treatment with antibodies that selectively deplete Marginal Zone B cells rendered mice more capable of inducing enhanced IFNγ responses and clearing the infection. Our findings suggest that B cells play a detrimental role in C. muridarum infection and that activation by innate receptors like TLR4 and IL-10 production by these cells could be used by Chlamydia spp. as a strategy to modulate the immune response establishing chronic infections in susceptible hosts

Detección óptica ultrasensible de antígenos de relevancia en Ciencias de los Alimentos utilizando nanopartículas de plata mediante inmunoensayos

RESUMEN            La combinación de las propiedades ópticas de las nanopartículas de plata (Ag NPs) con algunos elementos utilizados en el método estándar ELISA, uno de los inmunoensayos más ampliamente difundidos para la detección de antígenos, ha permitido en nuestros laboratorios el desarrollo de un nuevo método óptico, libre de enzimas, de bajo costo, rápido y más sensible que el ELISA, denominado IDILA (del inglés, Intensity Depletion Immuno-Linked Assay). El mismo se puede realizar directamente en dispersión coloidal, sin la necesidad de inmovilizar el anticuerpo específico de captura en una placa o la utilización de anticuerpos secundarios y primarios sobre un sustrato. En este trabajo se demuestra la capacidad del IDILA para cuantificar antígenos de relevancia en Ciencias de los Alimentos a muy bajas concentraciones. En particular, utilizando Ag NPs de 58 nm de diámetro, describiremos cómo la técnica IDILA se puede emplear para la detección ultrasensible de gliadina, proteína a la que son sensibles las personas con enfermedad celíaca. Los resultados de los experimentos realizados se comparan con ELISA sándwich, la técnica estándar homologada por el codex alimentarius, demostrando que el IDILA es casi 1000 veces más sensible que el ELISA, teniendo también límites de detección más bajos.            Utilizando las condiciones apropiadas, el IDILA demuestra que es capaz de detectar concentraciones femtomolares del antígeno, además de ser robusto, confiable, de bajo costo, rápido (alrededor de 2 horas) y de fácil implementación utilizando el equipo estándar y los reactivos biomoleculares utilizados para el ELISA. PALABRAS CLAVESNanopartículas Plasmónicas – Bioconjugación – Sensor – Inmunoensayo – IDILA – ELISA –Gliadina - Enfermedad Celíaca - Tecnología de los Alimentos    ABSTRACTThe combination of the optical properties of silver nanoparticles (Ag NPs) with some elements commonly used in ELISA , immunoassay widely used for antigen detection, has given rise to an enzyme-free, low cost, fast and more sensitive optical method developed by our group denoted as Intensity Depletion Immuno-linked Assay (IDILA). IDILA can be performed directly in colloidal dispersion without any immobilization of the capture antibody, antigen, or secondary and primary antibodies on a substrate. The capabilities of the method for quantifying antigens at ultralow concentrations in colloidal dispersions as well as its robust performance are demonstrated for specific antigens of importance in food chemistry. In this work, using conveniently functionalized 58 nm diameter Ag NPs, we describe how the application of the IDILA methodology can be used for the ultrasensitive detection of gliadin, a protein to which people bearing celiac disease are sensitive. Results of the experiments performed were compared with ELISA, the standard technique approved by the codex alimentarius, demonstrating that the IDILA assay is ~1000 times more sensitive than ELISA, and also with a lower detection limit.            Using the appropriate conditions, the IDILA assay has shown to be able to detect femtomolar concentrations of the antigen, besides of being robust, reliable, cheap, fast (around 2 hours) and of easy implementation using the standard equipment and biomolecular reagents used for ELISA.                            KEYWORDSPlasmonic Nanoparticles – Bioconjugation – Sensing – Immunoassay – IDILA – ELISA - Gliadin - Celia Disease - Food Technology

Enzyme-Free Immunoassay Using Silver Nanoparticles for Detection of Gliadin at Ultralow Concentrations

Determination of biomarkers in clinical or food samples is of crucial importance for monitoring, prevention, and care of public health. The standard procedure used for this purpose is the enzyme-linked immunosorbent assay (ELISA), which makes use of the specific antibody-antigen biorecognition and the catalytic effect of the enzymes. One of the main shortcomings of this technique is the use of enzymes that often present low chemical and thermal stabilities compared to other chemicals. Other drawbacks include the nonspecific binding process that could lead to false-positive results, the use of relatively large amounts of the sample, and the number of time-consuming steps involved. Recently, an enzyme-free and ultrasensitive analytical method for antigen detection denoted as intensity depletion immunolinked assay (IDILA) has been proposed by our laboratory. The assay is based on the inhibition to form Ag nanosphere dimers linked by a specific antibody in the presence of the corresponding antigen. In this work, we go a step further demonstrating how the performance of this method could be improved by using silver nanoparticles (Ag NPs) of different diameters (58 and 78 nm). The experiments are performed for detecting gliadin, an antigen of utmost importance in celiac disease, and the results are compared with ELISA, the standard technique homologated by the Food Codex Alimentarius. It is found that the IDILA assay could be around 1000 or 10 000 times more sensitive than ELISA, also having lower limits of detection, depending on the conditions explored (fraction of dimers and Ag NP diameter). Using the appropriate conditions, the IDILA assay is shown to be able to detect femtomolar concentrations of the antigen, besides being robust, reliable, cheap, rapid (around 2 h), and of easy implementation using the standard equipment and biomolecular reagents used for the ELISA assay.Fil: Mercadal, Pablo Agustin. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Fraire, Juan Carlos. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Motrich, Ruben Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Coronado, Eduardo A.. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentin

Fra-1 and c-Fos expression in malignant breast tumor biopsies and non-pathological samples.

<p>(<b>A</b>). Fra-1 (first row), c-Fos (second row) and PCNA content was determined by WB in total homogenate prepared from 5 normal (N1 to N5) and 8 tumor samples (T1 to T8) randomly selected from the collection of breast tissue samples. Note the abundant Fra-1, c-Fos and PCNA expression in all tumor samples contrasting with the very low or undetectable expression levels in the non-pathological ones. Tubulin was used as a loading control (lower panel). (<b>B</b>) TH, MF and SF obtained as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053211#pone-0053211-g002" target="_blank">Figure 2</a> from tumors #5 (left panel) and #6 (right panel) were examined for Fra-1 and c-Fos content. In addition, Fra-1 and c-Fos content was examined in MF and SF obtained after stripping MF with 1M KCl prior to centrifugation. Results shown are from one representative experiment out of three performed with essentially the same results.</p

Human malignant breast carcinomas show abundant Fra-1 and c-Fos expression co-localizing with the ER marker calnexin.

<p>Expression of Fra-1 (<b>A</b>), c-Fos (<b>B</b>), the ER marker calnexin, and the nuclear marker of proliferating cells PCNA was examined by triple labeling in malignant human breast tumor specimens (n = 210) and non-pathological samples (n = 37). Six representative samples of invasive ductal carcinomas from a tissue array immunostained for Fra-1 (A, green) or c-Fos (B, green), calnexin (red) and PCNA (blue) and three non-pathological samples (bottom row) are shown for each onco-protein examined. Note that all actively proliferating cells (PCNA positive cells) show abundant Fra-1 and c-Fos expression which in all cases is observed co-localizing with the ER marker as evidenced by the yellow color of the merged, triple-labeled images.</p

Both Fra-1 and c-Fos are capable of sustaining cell proliferation.

<p>(<b>A</b>). MDA-MB231 cells non-transfected (first column), control-transfected (second column) or transfected to block the expression of c-Fos (third column), or of Fra-1 (fourth column), or of both proteins (last column) were examined for cell proliferation. Proliferation was determined as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053211#s2" target="_blank">Materials and Methods</a> and expressed as arbitrary fluorescent units of DNA. Results are the mean ± SD of 3 experiments performed in sextuplicate; 10,000 cells were plated for each proliferation assay. *: p<0.001. (<b>B</b>) WB determination of the expression of Fra-1 (top row), c-Fos (second row), PCNA (third row) and Tubulin used as a loading control (bottom row) of the samples used in (A) to determine cell proliferation.</p

Nuclear AP-1-Fra-1/c-Fos and cytoplasmic Fra-1/c-Fos are required at early and late stages of cell proliferation, respectively.

<p>(<b>A</b>) MDA-MB231 cells were cultured+or – NLSP that blocks AP-1 nuclear import, in the presence or the absence of anti-Fra-1 or anti-c-Fos antibodies added at the indicated times (0 h or 16 h) after FBS addition and examined for proliferation. Controls were performed that received FITC-IgG antibody (last column). Proliferation was determined as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053211#s2" target="_blank">Materials and Methods</a> and expressed as arbitrary fluorescent units of DNA. Results are the mean ± SD of 3 experiments performed in quintuplicate; 10,000 cells were plated for each proliferation assay. Note that nuclear import of Fra-1/c-Fos-AP-1 is required only during the first 16 h after FBS addition whereas cytoplasmic Fra-1/c-Fos are required at all time points (compare column 5 with columns 6,7 or 8). (<b>B</b>) TH form MDA-MB231 cells cultured as in (A) were assayed for phospholipid synthesis capacity. Results are the mean cpm incorporated into phospholipids/mg protein ± SD of 3 experiments performed in triplicate. *: p<0.001.</p

Schematic representations of c-Fos and Fra-1 and comparison of their Basic Domains (BD).

<p>c-Fos is a 380 amino acid (aa) protein whereas Fra-1 contains 271 aa. The BD of c-Fos spans from aa 139 to 159 whereas that of Fra-1 spans from aa 107 to aa 127. Comparison of both BDś shows that of the 12 basic aa that each contain (schematized in bold letters), these are highly homologous showing only two conservative substitutions of the basic aa that are underlined in the scheme. c-Fos contains a C-terminal trans-activating domain (TAD) that is not present in Fra-1.</p