Emilio De Simone

Potential Role of Fibroblast-Like Synoviocytes in Joint Damage Induced by Brucella abortus Infection through Production and Induction of Matrix Metalloproteinases

ABSTRACT Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators.

Development of ELISAs for the measurement of IgM and IgG subclasses in sera from llamas (Lama glama) and assessment of the humoral immune response against different antigens

Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.

Efecto del alendronato sobre el perfil de citoquinas y metaloproteinasas 2 y 9 en un modelo múrido de artritis experimental

Resumen Introduccion La artritis es una de las artropatias mas frecuentes, se caracteriza por el dano que produce en el cartilago articular y la resorcion osea subcondral. El diagnostico temprano es de crucial importancia para instaurar una terapia preventiva, ya que, en ocasiones, la enfermedad es diagnosticada al presentarse lesiones oseas de dificil resolucion. Objetivos Caracterizar, en un modelo murido de artritis experimental producida por adyuvante, el perfil de distintos biomarcadores articulares, interleuquinas (IL-4, IL-6 y TNF-α) y metaloproteasas (MMP-2 y MMP-9), que permitan seguir la evolucion de la enfermedad y analizar sus diferencias, al aplicar el tratamiento con alendronato en forma preventiva o curativa. Materiales y metodos El alendronato, en forma preventiva, se aplico el dia 0 y de manera curativa a los dos meses posadyuvante. Se realizo un puntaje de los sintomas clinicos; al sacrificio, se determinaron los marcadores articulares y se realizaron los estudios histopatologicos y radiograficos. Resultados Lo mas destacable fue que el grupo que recibio el alendronato, de manera preventiva, alcanzo un puntaje clinico normal de manera mas temprana que el grupo control con adyuvante. Asimismo, los animales tratados con alendronato presentaron valores significativamente mas bajos de metaloproteasas. Conclusiones Nuestros resultados sugieren que, aparentemente, el alendronato disminuye la actividad de proteasas vinculadas a la fisiopatologia de la enfermedad articular, lo cual podria resultar sumamente beneficioso para la terapeutica a instaurar.

Structural analysis of effector functions related motifs, complement activation and hemagglutinating activities in Lama glama heavy chain antibodies

Heavy chain antibodies (HCAbs), devoid of the light chains and the CH(1) domain, are present in the serum of camelids. IgG(2) and IgG(3) are HCAbs; whereas IgG(1) has the conventional structure. In order to study the immunological properties of llama HCAbs, from which to date little is known, llamas (Lama glama) HCAbs cDNA were cloned, sequenced and compared with other mammalian Igs. The sequence analysis showed that llama HCAbs cDNA organization is similar to other mammalian Igs and the presence of conserved binding motifs to Protein A, Protein G, FcγRI, FcγRIII and C1q in HCAbs were observed. In a previous work, different IgG isotypes purified by Protein A and Protein G chromatography, were assayed for their ability to fix complement. Both IgG(1) and the total serum were able to fix complement, whereas IgG(2) and IgG(3) fixed complement even in the absence of antigen (anti-complementary activity). Therefore, in this work we performed the complement activating activity of the different IgG isotypes purified under physiological conditions using Sephadex G-150 and their ability to induce hemagglutination. Llamas were immunized with sheep red blood cells (RBC) stroma and the different isotypes were purified from sera. Whole serum and IgG(1) could activate complement; however, HCAbs (IgG(2)+IgG(3)) could not, despite the presence of the C1q binding motif in their primary sequence. Unlike IgG(1), the fraction corresponding to IgG(2)+IgG(3) did not display hemagglutinating activity. Our findings suggest that HCAbs cannot crosslink efficiently with different antigens and that the C1q binding site might be hindered by the proximity of the variable domains.

Determination of reference values for cytokines and matrix metalloproteinases in the synovial fluid of horses of different ages

Osteoarthritis (OA) is the most common joint disease in sport horses. Inflammatory and anti-inflammatory cytokines and metalloproteinases (MMPs) play a crucial role in the degradation of the cartilage extracellular matrix. The study of inflammatory molecular biomarkers could be useful in the diagnosis and choice of therapeutic strategies. However, little is known about the normal values of inflammatory biomarkers at different ages. Moreover, early diagnosis of OA is crucial to ensure early treatment in order to prevent chronic damage. The aim of this study was to establish a reference range for values of pro-inflammatory cytokines (IL-1β, IL-4, IL-6 and TNF-α) and MMPs (MMP-2 and MMP-9) in the synovial fluid of horses of different ages. The old horses group presented higher values of IL-4 and TNF-α than the rest of the groups studied (P<0.001 vs. all other groups). IL-1β levels were higher in foals than in all the other groups (P<0.001). No statistically significant differences were found between the groups as regards the mean IL-6 values. Synovial MMP-2 levels were statistically lower in the old horses group (P<0.001) and higher in the foals group (P<0.05). Higher MMP-9 levels were found in the foals group (55.45 ± 58.1 percentage of proteolysis) (P<0.001 vs. all other groups). This study demonstrates the existence of variations in the levels of pro-inflammatory markers in the synovial fluid of horses at different ages.