Serono Research Grants, 2004

Field of Clinical Research in Endocrinology

Research Project:

Molecular and genetic molecular determinants of metabolic syndrome: chronic activation of immune system.

Main researcher:

Dr. José Manuel Fernández

Project Summary:

Type 2 diabetes mellitus is a highly prevalent disease (it affects 5% of the adult population) with a high morbidity and mortality, mainly cardiovascular, and a high sociosanitary cost. It is phonotypically characterized by hyperglycemia and insulin resistance, frequently associated with other metabolic alterations (high blood pressure, dyslipemia and central obesity), constituting as a whole the so-called metabolic syndrome.

The etiology of metabolic syndrome is polygenic and influenced by environmental factors. In this sense, our team has identified some of the molecular and genetic determinants of the Insulin Resistance Syndrome. Following this line of research, the main aim of the present project is molecular and genetic characterization of new modulating factors of insulin sensitivity in a wide sample of healthy, obese and type 2 diabetic subjects. Specifically, the presence of bacterial DNA residues in the plasma of subjects with no clinical inflammatory pathologies will be studied and characterized in order to analyze the immunostimulating capacity of bacterial DNA, its relationship with cytokine levels and other proinflammatory molecules and their possible implication in the development of some clinical determinants of metabolic syndrome.

The methodology to be used will consist in real time PCR techniques for bacterial DNA residue quantification in the plasma of the subjects under study and sequencing for their specific determination. Likewise, ex vivo cell culture techniques will be used for analyzing cytokine expression in peripheral lymphocytes, both at gene expression level by means of PCR amplification and at plasma level quantification level by means of enzyme-linked immunoabsorbent assay techniques.

Jury:

Field of Assisted Human Reproduction

Research Project:

Leptin receptor function in embryo implantation. Clinical implications in obesity and infertility.

Main researcher:

Dra. Ana Cristina Cervero Sanz

Project Summary:

The leptin system is involved in the regulation of body weight and in the reproductive function acting at endocrine and paracrine levels. The long shape of the leptin (OB-RL) receptor as well as other soluble isoforms have been identified in human endometria, mainly in the epithelial compartment. Furthermore, its expression in human endometria shows cyclic variation with an increase of its expression in the implantation window suggesting its possible implication as an endometrial receptivity marker. The leptin receptor is present along the entire pre-implant development in human embryos, whereas leptin is only expressed in the blastocyte stage. All this suggests that this system could be involved in the mother-embryo dialogue established during human embryo implantation.
Aims: 1) To determine the functional importance of the leptin receptor in embryo implantation. 2) To compare endometrial expression of OB-RL and peripheral concentrations of leptin in slim (BMI<20) and obese (BMI>30) patients with proven fertility and implantation failure of endometrial origin. 3) To sequence the OB-R gene in infertile and/or obese patients with a lack of mRNA expression. We thus hope to find possible mutations at genetic level that explain the absence of said expression.
Methodology: a) Cloning of RNAi sequence in the vector by means of bacterial restriction, ligation and transformation processes. b) Quantitative PCR. c) Western blot and confocal microscopy. d) ELISA assays in order to quantify the amount of blood leptin. e) Endometrial epithelial cell cultures (EEC). f) Temporal transfection of human endometrial epithelial cells. g) In vitro adhesion assays. h) Sequencing.

Jury:

Field of Multiple Sclerosis Clinical Research

Research Project:

Role of IgM intrathecal synthesis in multiple sclerosis physiopathology.

Main researcher:

Dr. José Carlos Álvarez Cermeño

Project Summary:

Multiple sclerosis (MS) is the most frequent demyelinating disease. It has an initial inflammatory phase with relapsing-remittent evolution in which the neurological symptoms are mainly due to demyelinization, being partially or totally reversible. This phase is followed by another progressive phase in a large percentage of patients, in whom a neurodegenerative pathogeny prevails with axonal fragmentation and dysfunction, which is the cause of irreversible neurological deficits. We have described that the intrathecal synthesis of IgM is associated to MS progression and is a prognostic factor for the appearance of new relapses and greater incapacity. It is thus deduced that IgM is associated, either directly or indirectly, with axonal lesion and neurodegeneration.

The aim of this project is to study the role of IgM in MS physiopathology: to analyze possible autoantigens against which it could be directed (proteic, lipidic, etc.) and study the B subpopulations of the LCR that produce it. We also aim to research whether neurological deficit and cerebral atrophy progression correlate with the presence of a certain population of B cells or a certain antigen or group of antigens against which this response could be directed. The possible use of this data as prognostic factors of incapacity will also be studied. Subsequently, the effect of IgM isolated from the patients on murine fetal neuron oligodendrocyte cultures with the aim of finding a model in order to study the lesion mechanisms and its inhibitors

Jury:

Field of Clinical Research in Psoriasis: Clinical Analysis and Immunology

Research Project:

Psoriasis pathogeny: study of the role of CD4/NKG2C+ T cells and their capacity for transferring the disease to xenotransplanted SCID mice with psoriatic biopsies.

Main researcher:

Dr. Salvio Serrano Ortega

Project Summary:

Common psoriasis is an inflammatory disease of the skin characterized by epidermal keratinocyte hyperproliferation accompanied by intense leukocyte infiltrates. Numerous factors are involved in the complex pathogeny of the disease, among which the role of several cytokines and chemokines, some genes that may be related to susceptibility of acquiring the disease and essentially the cells that infiltrate the psoriatic lesion are worth mentioning. Among these, dendritic cells and T lymphocytes seem to be the fundamental elements responsible for the inflammation. It has recently been suggested that psoriasis is a Type 1 inflammatory reaction, i.e. mediated by gamma interferon (IFN) that would occur as a response to the presence mainly of excessive amounts of IL-12 and IL-23. However, the scarce representation in the lesion of the different receptors for antigen (TCR) in T cells strongly suggests the existence of an auto or neoantigen in the psoriatic lesion areas that is recognized exclusively by those TCR and that remains unidentified. Interestingly, the presence has been described of CD4+ T cells in the infiltrates which also express CD94 receptors, known for their capacity to regulate IFN production both positively or negatively in both cytotoxic T cells and collaborator cells. We have recently demonstrated that CD4+ T cells also express CD94 and that the function of these receptors is perpetuation of chronic responses by means of IFN production regulation, the same that seems to occur in psoriasis. Likewise, a CD4+CD94+ T cell line has been described that comes from a psoriatic lesion that has the ability to induce the disease in severely combined immunodeficient mice (xenografted SCID) from 35 clones of CD4+CD94+ T cells obtained from biopsies of psoriatic lesions from newly diagnosed patients not subjected to anti-inflammatory treatment at the time of obtaining the sample.

Jury:

Bolsa Serono de Investigaçâo Biomédica en el Área de Investigación Clinica em Neuroinmunología

Research Project:

Identification of lymphocyte B and T responses against axonal growth inhibitor associated to myelin, Nogo-A, in patients with multiple sclerosis and other conditions of brain white matter.

Main researcher:

Dr. Paulo Pacheco da Fontoura

Project Summary:

Multiple sclerosis (MS) is a demyelinating disease affecting the Central Nervous System and it is the main cause of incapacity from neurological causes in young adults, except for injuries. MS is mainly characterized by the occurrence of periodical episodes of clinical worsening that coincide with the appearance of demyelinating lesions in several CNS locations; even in the absence of clinical symptomatology, however, it is now recognized that several subclinical demyelinating episodes occur during disease progression which due to their being less serious, or due to their location, do not pass the threshold of clinical detection (1). The mechanisms that determine the occurrence of these demyelination episodes are not well known, although several associations with other events that may influence the Immune System are known, as well as some infections. In a second phase of the disease, we observe a progressive increase in neurological deficits even in the absence of demyelination episodes. The relatively recent discovery of the existence of precocious axonal damage in acute demyelinating lesions (2) gave rise to the concept that incapacity in this secondary progressive phase results from the progressive accumulation of axon damage in multiple neuronal circuits, and that therefore MS, after an initial period in which clinical symptoms are derived mainly from the acute interruption of axonal transmission in demyelinating areas, becomes a neurodegenerative disease in its later phases due to the cumulative loss of axons.

Although there is not an absolute consensus regarding MS physiopathology, all current data indicate that the disease results largely from Immune System deregulatory activity. For example, results of neuropathological studies of MS lesions (3) demonstrate that in at least two of the main subtypes of lesions there was evidence of immune system participation, which wants to provide antibodies and complement mediated by T lymphocytes. Likewise, gene expression studies in MS lesions (using gene microarrays) confirm the existence of high levels of expression of several genes that are classically associated to immune response, as well as the participation of new genes the influence of which was unsuspected until now (4, 5). Experimental autoimmune encephalomielitis (EAE), the most frequently used animal model for MS has been for the last 70 years one of the main aids in order to understand the physiopathology of this disease (6). The central role of acquired immune response was discovered based on this model, mediated by T lymphocytes that were autoreactive against protein components of CNS myelin (7), such as myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein and myelin-associated glycoprotein (MBP, MOG, PLP and MAG, respectively). The role of lymphocyte B response against CNS antigens is not well defined, although recent data from the EAE model and from clinical studies have confirmed its relevance in disease progression and in the determination of the rate of outbreaks (8, 9).

Identification of B and T lymphocyte response targets in patients with Multiple Sclerosis thus assumes great physiopathological importance, since it would allow not only to discover some of the mechanisms underlying the origins of the disease, but also eventually to accomplish the development of antigen-specific therapies (10). In peripheral blood of MS patients and in normal controls it is possible to identify T lymphocytes that react against several components of the myelin sheath. Including MBP, MOG and PLP, and in demyelination source areas of patient CNS, the immunodominant peptide of MBP (MBP83-99) is associated to the HLA-DR2 molecule expressed by inflammatory cells (11, 12). It is also possible to extract reactive antibodies against this molecule from the demyelination plaques of patients (13). The characterization of T lymphocyte responses against myelin antigens from peripheral lymphocyte pollutions has allowed identifying immunodominant regions in MBP, MOG and PLP in patients with MS, to study their T helper phenotype, repository restrictions and the ability to produce cytokines (14-22). Based on these studies, although it has not yet been possible to identify antigens that are specific to disease induction, or related to clinical progression, it has been possible to confirm the relationship between inflammatory cytokine (such as gamma interferon) production capacity and clinical progression (23).

The Nogo-A protein is a member of the Reticulon protein family recently identified as being one of the most important inhibitors of axonal regrowth present exclusively in central nervous system (CNS) myelin (24, 25). There are three alternative transcripts of the Nogo gene, among which Nogo-A is the longest of all, having 1163 amino acids. All the isoforms (Nogo-A, B and C) have a common carboxy terminus containing a sequence of 66 amino acids flanked by two hydrophobic regions. This presumably extracellular region called Nogo-66 has been attributed most of this protein’s inhibition capacity for neuronal growth, mediated by interaction with a recently identified receptor present in axons (26, 27). The induction of immune responses against some regions of this protein have been the target of several works, such as the attempt to block its action and thus promote axon regrowth, mostly animal models of CNS injuries (28-31). More recently, we and other groups have studied the possible role of this myelin component in EAE and MS physiopathogenesis. It has already been possible to identify antibodies against the Nogo-A protein in MS patients, although no epitope-mapping study has been yet performed, which would be necessary to identify key residues for the immune response (32). Active immunization against the p472 region of this protein and the adoptive transfer of anti-Nogo immunoglobulins were capable of reducing clinical intensity of demyelinization and axonal loss associated to EAE (33). Our own work demonstrated that using EAE induction protocols intended for breaking the tolerance in disease-sensitive strains, immunization with peptides derived from the extracellular Nogo-66 sequence leads to the occurrence of clinical and histopathological symptoms of demyelinization, as well as the generation of a specific T lymphocyte response against the antigen used in the induction, and of antibodies against the induction antigen but also against several other myelin antigens, constituting an example of epitope diversification (34, 35). Thus, some Nogo-A protein regions seem to have the ability to perform as encephalitogens capable of inducing EAE and join the list of antigenic targets potentially relevant for MS physiopathology. The fact that this protein is one of the most important inhibitors of axonal regrowth also gains particular relevance since its implication in the anti-myelin autoimmune response in MS could be related with progressive axonal loss or the impossibility of recovering from acute demyelinating lesions.

The presence of T lymphocytes that are reactive against myelin antigens in CNS traumatic pathologies is a well-known phenomenon (36, 37), its generation depending probably on the presence in trauma locations of immune system cell components capable of producing antigens locally released to effector cells. The role that these cells could have in traumatic lesion physiopathogeny and evolution is little understood. There are several data from experimental trauma models, however, that seem to indicate that the immune response to trauma consisting of T cells reactive against CNS myelin components could have a neuroprotector role (38, 39). The presence of T lymphocytes reactive against Nogo antigens or antibodies against this protein could, in this context, have a crucial role in the determination of trauma response and in the determination of neurological incapacity.

Jury: