Tärkeä tavoite MS-taudin hoidon kehittämisessä on tunnistaa taudin immunologiset alatyypit, mikä edistää yksilöllisen hoidon edellytyksiä ja tarkkuuslääketieteen käyttöönottoa. Lisäksi löydettiin useita ennustavia ehdokasbiomarkkereita, joiden yli-ilmentyminen näyttää liittyvän MS-taudin kehittymisriskiin.
Overview of multiple sclerosis
- Epidemiology of multiple sclerosis
- Clinical subtypes
- Diagnosis
- Disease-modifying therapies of multiple sclerosis
Initially, the Schumacker criteria were based on clinical evidence of two recurrences separated in space (lesions at the different locations in the CNS) and time (at least three months apart) (Schumacker et al., 1965). The clinical efficacy of GA has been explained by a shift from pro-inflammatory responses to anti-inflammatory responses (Farina et al., 2005; Johnson, 2010).
Pathogenesis and neuropathology
Neuropathology of multiple sclerosis
A normal sclera can be considered highly abnormal in patients with MS, especially in patients in the progressive phase of the disease (Allen and McKeown, 1979; Kutzelnigg et al., 2005). The histopathological features of these cortical lesions are significantly different from white matter lesions (Calabrese et al., 2010).
Immunopathogenesis of multiple sclerosis
- Activation of immune cells
- Transmigration of immune cells to the central nervous system
- Mechanisms of neural tissue damage
- Apoptosis
The BBB and BCB epithelial cells limit the access of immune cells to the CNS (Holman et al., 2011). Figure adapted from Krammer et al., 2007, reproduced with permission of the publisher.
Cytokines and chemokines
- Cytokines
- Chemokines
Elevated levels of IFN- have been detected in the CNS at the peak of EAE disease, and treatment of patients with IFN- was detrimental to MS patients (Panitch et al., 1987). In the CNS, TRAIL has been shown to mediate apoptosis of human neurons and oligodendrocytes (Nitsch et al., 2000; Matysiak et al., 2002).
Biomarkers in multiple sclerosis
- Biomarkers of inflammation
- Biomarkers of blood-brain barrier function
- Biomarkers of degeneration
- Biomarkers of regeneration
- Magnetic resonance imaging as a surrogate marker for multiple
Most MS lesions are typically located in the periventicular white matter of the brain as well as superficial areas of the spinal cord, which share a close anatomical relationship with CSF space (Tumani et al., 2009). Under physiological conditions, blood-derived proteins enter the CSF compartment by passive diffusion across the blood-CSF barrier ( Tumani et al., 2009 ). Intrathecal immunoglobulin synthesis and the presence of OCB in MS patients were the most important and earliest evidence suggesting a role for B cells and antibodies in the pathology of MS (Awad et al., 2010).
In another study, serum anti-NF antibodies were significantly elevated in PPMS (Ehling et al., 2004). Furthermore, CSF levels of Tau tended to be highest in the early stage of the disease (Martinez-Yelamos et al., 2004; Brettschneider et al., 2005). In MS patients, reduced levels of brain-derived neurotrophic factor (BDNF) have been reported in blood and CSF (Azoulay et al., 2005; Frota et al., 2009).
The levels of BDNF, neural cell adhesion molecule (NCAM) and ciliary neurotrophic factor (CNTF) in the CSF of MS patients are associated with disease activity (Weinstock-Guttman et al., 2007; Frota et al., 2009). The changes observed by MRI in MS patients reflect the underlying pathology: inflammation and axonal degeneration (Katz et al., 1993; Bruck et al., 1997).
Subjects and ethical considerations (Studies I-IV)
The study was approved by the Tampere University Hospital Ethics Committee and all subjects gave written informed consent. Abbreviations: RRMS relapsing-remitting multiple sclerosis; SPMS secondary progressive multiple sclerosis; Primary progressive multiple sclerosis PPMS.
Molecular biology methods
- Blood sampling and RNA isolation from peripheral blood
- Gene expression analysis by Lightcycler (Study I)
- QRT-PCR using low density array (Study III)
- Flow cytometry analysis (Study I)
- Enzyme-linked immunosorbent assay (Studies I, II and IV)
- Luminex (Studies I, II and IV)
Primers and probes for LightCycler PCR were designed and manufactured by TIB MolBiol (Berlin, Germany) (Table 7). Reference gene PCR was performed at the same time and PCR conditions as the target gene with a LightCycler h-G6PDH housekeeping gene kit (Roche Diagnostic GmbH, Mannheim, Germany). The reference gene was selected by a LightCycler h-Housekeeping Gene Selection Set, which included five alternative reference genes.
The human apoptosis array was used to determine the expression of 93 apoptotic molecules and three housekeeping genes by PBMC. To normalize the results, the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used and two healthy control samples were used as calibrators in the data analysis. A five-parameter regression formula was used to calculate sample concentration from the Human sepsis/apoptosis LINCOplex Kit and the High-Sensitivity Human Cytokine LINCOplex Kit, and a four-parameter regression formula was used for the Human Cytokine LINCOplex Kit.
The same batch of monoclonal antibodies for the Bio-Plex Cytokine Assay System was used between experiments; the manufacturer reports that between-test and intra-test values are lower than 15%. The percent recovery of the standards ranged from 90% to 110%, which was used as the limit of detection for each protein.
Magnetic resonance imaging (Studies II and IV)
Cluster analysis (Study III)
Results were visualized with the Treeview program (Michael Eisen, http://rana.lbl.gov/Eisensoftvare.htm).
Statistical analyses (Studies I-IV)
Candidate immunological biomarkers (Studies I- III)
Characterization of immune profiles in different subtypes of
Abbreviations: CDMS clinically definite MS (RRMS, SPMS, and PPMS); RRMS relapsing remitting MS; SPMS secondary progressive MS; PPMS primary progressive MS; CIS clinically isolated syndrome; HC healthy controls; Fas L Fas ligand; MIF macrophage migration inhibitory factor; TRAIL tumor necrosis factor-related apoptosis-inducing ligand; CXCL C-X-C motif chemokine ligand; CCL C-C motif chemokine ligand; IL interleukin; TNF tumor necrosis factor. Gene expression profiles of apoptosis-related genes in early MS patients were also examined (Study III). In RRMS, six genes, including the Bcl-2 family (BAD, BBC3, BCL2L14), the death receptor pathway (TNFRSF25), and the NF-B family (IKBKE, NFKBID), were upregulated compared to controls (Table 9).
We then compared the expression of all genes in RRMS, CIS and controls using cluster analysis (Figure 8). Comparison between CIS and controls showed up-regulation of several genes in two regions in the dendrogram in CIS (Figure 8B). Hierarchical clustering showing relative gene expression comparing RRMS (A) and CIS (B) patients with healthy controls.
Gene expression patterns were used to group patterns and genes by two-way hierarchical clustering. Genes that showed a 1.5- or 0.67-fold change in expression level compared to control were considered up-regulated (red) and down-regulated (green), respectively.
Immunological biomarkers in relation to the clinical and
- Candidate biomarkers of disease activity
- Candidate biomarkers of disease progression
- Conversion-related markers
The median levels of sTRAIL (A), sFas(B), sFasL (C) and MIF (D) in sera from all MS patients (n=91), untreated MS patients in remission (n=32), IFN-treated MS patients during remission (n=27), untreated MS patients during relapse (n=22), IFN-treated MS patients during relapse (n=10) and healthy controls (n=26). Therefore, to analyze the relationship between measured biomarkers and disease progression over the year, CDMS patients were divided into two groups: patients with disability progression (EDSS score increased more than 0.5, n=20) and those with stable EDSS score ( EDSS score unchanged). , n=44). In the worsening patients, the levels of sFas and MIF were higher than in the stable group (Figure 10 B and C).
To further assess the relationship between immunological responses and MRI changes consistent with neurodegeneration, patients with RRMS and SPMS were divided into two groups: those with increased T1 volumes over the year (advanced, T1 1.0 cm3, n=19) and the stable ones. (stable, T1< 1.0, n=27). Upregulated sFas levels were found in patients with increased T1 lesion volumes compared to the stable group (Figure 10 D). -C) Increased levels of sFas and MIF were found in patients with progressive disability in the CDMS group.
CDMS patients were divided into two groups: patients with disability progression (EDSS score increased more than 0.5, n=20) and those without disability progression (EDSS score unchanged, n=44) over one year follow-up. D) Upregulated levels of sFas were found in those RRMS and SPMS patient whose T1 lesion volumes increased compared to stable group of patients. RRMS and SPMS patients were divided into the two groups: those with increased T1 volumes from baseline to one-year follow-up (progressor, n=19) and those with unchanged T1 volumes (stable, n=27). The length of the box represents the interquartile range within which 50% of the values were located.
Reconstitution of immune activity after pregnancy (Study IV)
Primary progressive multiple sclerosis
Current DMTs have little or no beneficial effect in the progressive stage of the disease. It has therefore been suggested that neurodegenerative events in chronically progressive patients develop independently of inflammation. The involvement of the Fas-FasL system in the pathogenesis of MS has been suggested by several authors (Zipp, 2000; Pender, 2007), but the exact role of sFas and sFasL in the pathogenesis of MS is still unknown.
Fas-FasL is one of the T cell death receptor-ligand systems that are crucial in the apoptotic signaling pathway. In particular, defects in the death receptor Fas have been reported in T lymphocytes from patients with multiple sclerosis, particularly in the chronically progressive subtype. TNF- is a well-known trigger of oligodendrocyte apoptosis (Watzlawik et al., 2010), while CCL2 is involved in the recruitment of monocytes and T cells from the blood to the brain parenchyma (Mahad and Ransohoff, 2003).
Overall, our findings of increased levels of sFas, TNF and CCL2 in sera from PPMS patients, together with previous evidence, are consistent with the involvement of these molecules in the pathogenesis of this subtype. This study also supports the hypothesis that the inflammation is also present in the PPMS subtype.
Relapsing-remitting multiple sclerosis and clinically isolated
These defects are thought to promote the survival of autoreactive T cells (Comi et al., 2000; Sharief, 2000a). Soluble forms of Fas have been shown to inhibit apoptotic events in T cells (Aktas et al., 2006). Our observation of increased sFas in PPMS is consistent with previous data, favoring the concept of dysregulated Fas-mediated apoptosis in MS (Ciusani et al., 1998; Boylan et al., 2001).
In addition, TNF- and CCL2 have been identified in active MS lesions (Selmaj et al., 1991) and increased levels of TNF- in serum and CSF together with intrathecal synthesis of CCL2 have been reported in PPMS (Sharief and Henges, 1991 ; Hohnoki et al., 1998). TNFRSF25 can either activate NF-B and MAP-kinase signaling or alternatively trigger caspase activation and programmed cell death (Chinnaiyan et al., 1996; Wen et al., 2003). NF-B-related genes are essential in inducing proinflammatory events in immune cells (Krammer et al., 2007).
In summary, aberrant gene expression profiles were seen in RRMS as well as CIS patients, suggesting that apoptotic events are an important part of MS pathogenesis. Upregulation of these pro- and anti-apoptosis molecules indicates the regulatory effect of the immune system to maintain peripheral homeostasis and thereby prevent pathogenetic events.
Immunological biomarkers in relation to the multiple sclerosis
Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS) initiated by recruitment of activated T cells and macrophages to the brain (McFarland and Martin, 2007).
