Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments. METHODOLOGY/PRINCIPAL FINDINGS: We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing. CONCLUSIONS/SIGNIFICANCE: Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.

Repeated courses of granulocyte colony-stimulating factor in amyotrophic lateral sclerosis: clinical and biological results from a prospective multicenter study.

Granulocyte colony-stimulating factor (G-CSF) induces a transient mobilization of hematopoietic progenitor cells from bone marrow to peripheral blood. Our aim was to evaluate safety of repeated courses of G-CSF in patients with amyotrophic lateral sclerosis (ALS), assessing disease progression and changes in chemokine and cytokine levels in serum and cerebrospinal fluid (CSF). Twenty-four ALS patients entered an open-label, multicenter trial in which four courses of G-CSF and mannitol were administered at 3-month intervals. Levels of G-CSF were increased after treatment in the serum and CSF. Few and transitory adverse events were observed. No significant reduction of the mean monthly decrease in ALSFRS-R score and forced vital capacity was observed. A significant reduction in CSF levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-17 (IL-17) was observed. G-CSF treatment was safe and feasible in a multicenter series of ALS patients. A decrease in the CSF levels of proinflammatory cytokines MCP-1 and IL-17 was found, indicating a G-CSF-induced central anti-inflammatory response.

Immune system alterations in sporadic amyotrophic lateral sclerosis patients suggest an ongoing neuroinflammatory process.

In this work we show that patients with sporadic amyotrophic lateral sclerosis exhibit immunological alterations in their blood, with respect to healthy controls, such as: i) increased levels of CD4+ cells and decreased levels of CD8+ T lymphocytes, the latter due to the reduced expression of the anti-apoptotic molecule Bcl-2; ii) significantly reduced CD4+CD25+ regulatory T (Treg) cells and monocytes (CD14+) levels in patients at a less severe stage of disease, suggesting their early recruitment towards the CNS area of primary neurodegeneration; iii) reduced expression of HLA-DR and CCR2 expression, as markers of activation, in monocytes. Since resident microglia partially derives from circulating activated monocytes and Treg cells are known to interact with the local microglia, this study strengthens the hypothesis of an involvement of the adaptive immune system associated with a neuroinflammatory process in the pathobiology of ALS.

Nitroproteomics of peripheral blood mononuclear cells from patients and a rat model of ALS.

Increased levels of 3-nitrotyrosine in the central nervous system have been found in patients and mouse models of familial ALS (fALS), suggesting a possible use of nitrated proteins as biomarkers. We analyzed peripheral blood mononuclear cells (PBMCs), easily accessible samples, from sporadic ALS (sALS) patients and a rat model of fALS (a) to establish whether an increased level of nitrated proteins was present in PBMCs, too, and (b) to identify possible candidate biomarkers. With a proteomic approach, we identified for the first time the major overnitrated proteins in PBMCs from patients and rats at different disease stages. In the rats, their increased levels already were measured at a presymptomatic stage. Among them, actin, ATP synthase, and vinculin overlap between sALS patients and the rat model. Interestingly, in a previous study, actin and ATPase have been found overnitrated in the spinal cord of a mouse model of fALS before disease onset, suggesting their possible involvement in motor neuron degeneration. In conclusion, we observed that an increased level of nitrated proteins was not restricted to the spinal cord but also was present in peripheral cells of patients and an animal model, and that nitrated proteins are promising candidate biomarkers for early diagnosis of ALS.

Qualitative difference between the cytotoxic T lymphocyte responses to melanocyte antigens in melanoma and vitiligo.

Vitiligo is a skin disorder characterized by depigmented macules secondary to melanocyte loss. An unusual facet is its relation to melanoma: cytotoxic T lymphocytes directed to melanocyte antigens are found in both conditions and imply a breakdown of tolerance, yet the resulting immune reaction is the opposite. The mechanisms at the basis of these opposite effects are not known. Here, we performed a direct comparison of whole melanocyte-specific T cell populations in the two diseases. We demonstrate that neither precursor frequencies of Melan-A/MART-1-specific T lymphocytes nor their status of activation differ significantly. However, by using a tetramer-based T cell receptor down-regulation assay, we documented a higher affinity of vitiligo T cells. We calculated that the peptide concentration required for 50% of maximal receptor down-regulation differed by 6.5-fold between the two diseases. Moreover, only vitiligo T cells were capable of efficient receptor down-regulation and IFN-gamma production in response to HLA-matched melanoma cells, suggesting that this difference in receptor affinity is physiologically relevant. The differences in receptor affinity and tumor reactivity were confirmed by analyzing Melan-A/MART-1-specific clones established from the two diseases. Our results suggest that the quality, and not the quantity, of the melanocyte-specific cytotoxic responses differs between the two pathologies.

Transfer of efficient anti-melanocyte T cells from vitiligo donors to melanoma patients as a novel immunotherapeutical strategy.

BACKGROUND: Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune-mediated loss of epidermal melanocytes. High frequencies of self-reactive T lymphocytes directed toward melanocyte differentiation antigens are found in vitiligo patients and might be directly responsible for the pathogenesis of the disease. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self antigens shared by normal melanocytes and melanoma cells are found in both conditions, but the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research. PRESENTATION OF THE HYPOTHESIS: Our working hypothesis is that vitiligo patients might represent a unique source of therapeutic cells to be used in allo-transfer for HLA-matched melanoma patients. The adoptive transfer of ex-vivo generated autologous tumor-specific T cells is a therapy that has met with only limited success, essentially because of inability to isolate therapeutically valuable T cells from the majority of tumor patients. Ideally, model systems where strong and efficient responses against the same (tumor) antigens are achieved would represent a better source of therapeutic cells. We believe it is possible to identify one such model in the melanoma-vitiligo dichotomy: T lymphocytes specific for different melanocyte differentiation antigens are found in vitiligo and represent the effective anti-melanocyte reactivity that is often ineffective in melanoma. TESTING THE HYPOTHESIS: Melanocyte-specific T cell clones can be isolated from the peripheral blood of vitiligo patients and tested for their capacity to efficiently expand in vitro without loosing their cytotoxic activity and to migrate to the skin. Cytotoxicity against melanoma patients' non-tumor cells can also be tested. In addition, it would be interesting to attempt an in vivo animal model. If the results obtained from these validation steps will be satisfactory, it might be possible to plan the clinical grade preparation of relevant clones for transfer. IMPLICATIONS OF THE HYPOTHESIS: When translated into a clinical trial, the possibility of in vitro selecting few effective tumor-specific T cell clones for infusion, inherent with this approach, could enhance the therapeutic graft-versus-tumor effect while possibly decreasing the risk of graft-versus-host disease.

Melanocyte-specific, cytotoxic T cell responses in vitiligo: the effective variant of melanoma immunity?

Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune-mediated loss of epidermal melanocytes. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self-antigens shared by normal melanocytes and melanoma cells are found in both conditions and might prove important in melanocyte destruction, yet the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research. In the present article, we will address these issues by reviewing current literature on the subject as well as by posing some speculations.

Molecular and functional bases of self-antigen recognition in long-term persistent melanocyte-specific CD8+ T cells in one vitiligo patient.

Vitiligo patients possess high frequencies of circulating CD8+ T lymphocytes specific for the melanocyte differentiation antigen Melan-A/MART-1. These self-specific T cells exhibit intact functional properties and their T cell receptors are selected for a narrow range of high affinities of antigen recognition, suggesting their important role in the pathogenesis of vitiligo. In order to understand the molecular base for this unexpected, optimal T cell receptor recognition of a self-antigen, a tetramer-guided ex vivo analysis of the T cell receptor repertoire specific for the Melan-A antigen in a patient affected by vitiligo is reported. All T cell receptors sequenced corresponded to different clonotypes, excluding extensive clonal expansions and revealing a large repertoire of circulating Melan-A-specific T lymphocytes. A certain degree of T cell receptor structural conservation was noticed, however, as a single AV segment contributed to the alpha chain rearrangement in 100% of clones and a conserved amino acid sequence was found in the beta chain complementarity determining region 3 of various high affinity cells. We suggest that the conserved alpha chain confers self-antigen recognition, necessary for intrathymic selection and peripheral homeostasis, to many synonymous T cell receptors, whereas the beta chain fine tunes the T cell receptor affinity of the specific cells. In addition, we demonstrate that many high avidity T cell clones from this patient were capable of specifically lysing normal, HLA-matched melanocytes. These autoreactive clones persisted for more than 3 y in the patient's peripheral blood. These data, together with the skin-homing potential of the clones, directly point to the in vivo pathogenic role of melanocyte-specific cytotoxic T lymphocytes in vitiligo.

Dominant TCR-alpha requirements for a self antigen recognition in humans.

TCR-alpha and -beta chains are composed of somatically rearranged V, D, and J germline-encoded gene segments that confer Ag specificity. Recent crystallographic analyses revealed that TCR-alpha has more contacts with peptide than TCR-beta, suggesting the possibility that peptide recognition predominantly relies on TCR-alpha. T cells specific for the self Ag Melan-A/MART-1 possess an exceptionally high precursor frequency in human histocompatibility leukocyte Ag-A2 individuals. This provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at both the pre- and postimmune levels. Molecular and phenotypic analysis of many different Melan-A-specific T cell populations revealed that a structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2, namely the highly preferential use of a particular TCRAV segment, AV2. Examination of CD8 single-positive thymocytes indicated that this preferential use in forming the Melan-A-specific TCR is mainly imposed by intrathymic positive selection. Our data demonstrate a dominant function of TCRAV2 segment in forming the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-alpha.

Human CD8 co-receptor is strictly involved in MHC-peptide tetramer-TCR binding and T cell activation.

Although there has been extensive analysis on the capacity of MHC-peptide tetramers to bind antigen-specific TCR, there have been comparatively few studies regarding the role of the CD4 and CD8 co-receptors in binding and activation by these multimeric molecules. Here, we start from the observation that different antibodies against human CD8 exert opposite effects on MHC-peptide tetramer binding to the TCR: tetramer staining was enhanced by OKT8 antibody, while it was blocked with SK1 antibody. We used these different anti-CD8 antibodies to modulate CD8 function during tetramer staining of Melan-A/MART1-specific CTL clones. We show that CD8 action could be variably modulated during all the phases of interaction, indicating that CD8 participates in both the initial association of the TCR with MHC-peptide tetramers and the stability of this interaction. While the blocking effect of anti-CD8 antibodies was mostly exerted during the initial binding of the TCR with MHC-peptide tetramers, the enhancing effect was exerted by augmenting the duration of this interaction. Blocking anti-CD8 antibodies were also capable of preventing tetramer-mediated T cell activation. The possibility of variably affecting MHC-peptide tetramer binding and T cell activation using anti-CD8 antibodies confirms the critical role exerted by the CD8 co-receptor in this interaction and supports the notion that TCR engagement by MHC-peptide ligands typically involves CD8.