Cinthia Farina

Mechanisms of action of glatiramer acetate in multiple sclerosis

Glatiramer acetate (GA, Copaxone [Teva Pharmaceuticals, Kansas City, MO], formerly known as copolymer-1) and interferon- (IFN)-β are both used for the immunomodulatory treatment of multiple sclerosis, but they act in different ways. Four major mechanisms of GA have been identified: 1) competition with myelin-basic protein (MBP) for binding to major histocompatibility complex (MHC) molecules; 2) competition of GA/MHC with MBP/MHC for binding to the T-cell receptor; 3) partial activation and tolerance induction of MBP-specific T cells (action as an altered peptide ligand); and 4) induction of GA-reactive T-helper 2- (TH2)-like regulatory cells. Of these four mechanisms, 1 and 2 presumably occur only in vitro and are therefore irrelevant for the in vivo effects of GA. In contrast, mechanisms 3 and 4 could occur in vivo and both could contribute to the clinical effects of GA.

Preferential expression and function of Toll-like receptor 3 in human astrocytes

In contrast to other tissues, the central nervous system (CNS) is essentially devoid of MHC expression and shielded from antibodies by the blood-brain barrier. Therefore, a rapid local innate immune response by resident brain cells is required to effectively fight infectious agents. This study analyzed the expression and function of Toll-like receptors (TLRs) in cultured human astrocytes. Quantitative PCR for TLRs 1 to 10 showed a basal expression of TLR3 that could be enhanced by IFN-gamma, IL-1beta, and IFN-beta. The other TLRs were barely detectable and not inducible by the same cytokines. IFN-gamma-activated astrocytes responded to TLR3 ligand poly (I:C) engagement with IL-6 production, while ligands of other TLRs, like LPS, lipoteichoic acid, peptidoglycan, flagellin, and CpG, had no effect. Poly (I:C) also triggered astrocyte production of TNF-alpha and the chemokines CCL2/MCP-1, CCL5/RANTES, CCL20/MIP-3alpha, and CXCL10/IP-10. The adapter molecules MyD88 (full length and short isoform), TIRAP/Mal, and TICAM-1/TRIF, which are required for TLR signaling, were all expressed by astrocytes. Thus, resting and activated human astrocytes express preferentially TLR3 and, upon TLR3 engagement, produce IL-6 and chemokines active on T cells, B cells, monocytes, and dendritic cells. These data indicate that astrocytes function as sentinels for viral infections in the CNS.

Glatiramer acetate in multiple sclerosis: update on potential mechanisms of action

Glatiramer acetate is a synthetic random copolymer approved for the immunomodulatory therapy of relapsing-type multiple sclerosis (MS). Previous work has focused on the effects of this drug on T cells, especially the glatiramer-acetate-induced shift of the cytokine profile towards those characteristic of T-helper-2 (Th2) cells. Glatiramer acetate was thought to bring about this Th2 shift by acting like an altered peptide ligand but more recent work has shown that the drug notably affects the properties of antigen-presenting cells, such as monocytes and dendritic cells. These new observations might offer an explanation for the previously observed Th2 shift. In this review, we focus on these new findings. We address several controversial issues, including the possible neurotrophic effects of glatiramer acetate, the potential role of neutralising antibodies to the drug, and attempts to develop biomarkers of the treatment response. Finally, we will think about how a better understanding of glatiramer acetate might help the development of new immunomodulatory agents for MS.

CCL19 is constitutively expressed in the CNS, up-regulated in neuroinflammation, active and also inactive multiple sclerosis lesions

CCL19 and CCL21 bind to CCR7, which is crucial for both inducing an immune response and establishing immunological tolerance. We report that in the normal human brain CCL19, but not CCL21, is transcribed, and detectable as a protein in tissue lysates and in cerebrospinal fluid. In both active and inactive multiple sclerosis (MS) lesions CCL19 transcripts were elevated. In cerebrospinal fluid from MS and OIND patients CCL19 protein was increased. In relapsing-remitting and secondary progressive MS patients CCL19 correlated with intrathecal IgG production. This study suggests that CCL19 plays a role in both the physiological immunosurveillance of the healthy CNS and the pathological maintenance of immune cells in the CNS of MS patients.

Fingolimod May Support Neuroprotection via Blockade of Astrocyte Nitric Oxide

Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFκB pathway and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration. Here we investigated the role of the crosstalk between sphingosine‐1‐phosphate (S1P) and cytokine signaling cascades in astrocyte activation and inflammation‐mediated neurodegeneration, and addressed the effects of fingolimod on astrocyte–neuron interaction and NO synthesis in vivo.

Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine.

Metastatic melanoma patients treated with an autologous DNP-modified tumor cell vaccine develop inflammatory responses in metastatic tumors characterized by infiltration of CD8+ T cells. To further define this immune response, we analyzed T cell receptor beta-chain variable (TCRBV) region repertoire in biopsy specimens and peripheral blood lymphocytes of six patients. After administration of DNP vaccine, a restricted set of TCRBV gene families was found to be expanded compared with prevaccine metastases. In several postvaccine lesions of one patient, obtained over a 2-yr period, TCRBV14+ T cells were clonally expanded and identical T cell clonotypes could be detected. Two major recurring clones were biased toward the use of TCRBJ1S5. Furthermore, T cell lines derived from two such infiltrated skin lesions and, enriched in TCRBV14+ T cells, displayed HLA-class I-restricted lysis of the autologous melanoma cells. Clonal expansion of T cells was demonstrated in the T cell-infiltrated, postvaccine metastasis of a second patient as well. These results indicate that vaccination with autologous, DNP-modified melanoma cells can expand selected clones of T cells at the tumor site and that such clones are potentially destructive to the tumor.

iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF

The possibility of generating neural stem/precursor cells (NPCs) from induced pluripotent stem cells (iPSCs) has opened a new avenue of research that might nurture bench-to-bedside translation of cell transplantation protocols in central nervous system myelin disorders. Here we show that mouse iPSC-derived NPCs (miPSC-NPCs)-when intrathecally transplanted after disease onset-ameliorate clinical and pathological features of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Transplanted miPSC-NPCs exert the neuroprotective effect not through cell replacement, but through the secretion of leukaemia inhibitory factor that promotes survival, differentiation and the remyelination capacity of both endogenous oligodendrocyte precursors and mature oligodendrocytes. The early preservation of tissue integrity limits blood-brain barrier damage and central nervous system infiltration of blood-borne encephalitogenic leukocytes, ultimately responsible for demyelination and axonal damage. While proposing a novel mechanism of action, our results further expand the therapeutic potential of NPCs derived from iPSCs in myelin disorders.

Mouse DREAM/calsenilin/KChIP3: gene structure, coding potential, and expression.

Ca2+-binding proteins containing EF-hands are important constituents of intracellular signaling pathways. Recently, three new members of the Neuronal Calcium Sensor subgroup have been cloned in humans. Calsenilin interacts with presenilins, DREAM is a calcium-regulated transcriptional repressor and KChIP3 binds and modulates A-type potassium channels. Here we describe the mouse full-length cDNA and the genomic locus, demonstrating that the three proteins are encoded by the same unique gene. Various mechanisms contribute to the coding potential of this locus. These include alternate translation starts in the first exon and alternative splicing yielding transcripts lacking the EF-hand domains. In situ hybridization, RT-PCR, and Northern blotting reveal nervous system-restricted expression largely coinciding with the distribution of the Kv4.2 alpha-subunit of potassium channels. The presence of transcripts in early embryonic stages suggests roles for the protein also during development.

Astrocytes: Key Regulators of Neuroinflammation.

Astrocytes are crucial regulators of innate and adaptive immune responses in the injured central nervous system. Depending on timing and context, astrocyte activity may exacerbate inflammatory reactions and tissue damage, or promote immunosuppression and tissue repair. Recent literature has unveiled key factors and intracellular signaling pathways that govern astrocyte behavior during neuroinflammation. Here we have re-visited in vivo studies on astrocyte signaling in neuroinflammatory models focusing on evidences obtained from the analysis of transgenic mice where distinct genes involved in ligand binding, transcriptional regulation and cell communication have been manipulated in astrocytes. The integration of in vivo observations with in vitro data clarifies precise signaling steps, highlights the crosstalk among pathways and identifies shared effector mechanisms in neuroinflammation.

Treatment with glatiramer acetate induces specific IgG4 antibodies in multiple sclerosis patients

We analysed the humoral immune response to glatiramer acetate (GA, Copaxone) in 20 multiple sclerosis patients treated with GA, 20 patients not treated with GA and 20 normal control subjects. Using an ELISA for detection of total GA-reactive immunoglobulins (all isotypes), all treated patients but also 3/20 untreated and 8/20 healthy subjects scored positive at 1:20 plasma dilutions. At higher dilutions, 5/20 treated patients and two healthy donors had relatively high levels of anti-GA antibodies. Isotype and IgG subclass analysis revealed that the two antibody-positive normal subjects had IgM and small titers of IgG1 or IgG2 antibodies. In contrast, 18 of 20 GA-treated patients, had low but significant titers of GA-reactive IgG4 antibodies. This finding is consistent with the previously described GA-mediated induction of T-helper 2 (TH2)-like regulatory T cells.