**2. Features of the immune status of patients with multiple sclerosis**

The development of ADs is characterized by spontaneous generation of primary Abs to proteins, nucleic acids and their complexes, polysaccharides, nucleotides, etc. [8, 16–21, 28– 32]. The origin of natural Abzs is complex. On the one hand, they are similar to artificial Abzs may be directed against analogues of transitional states of catalytic reagents or even against substrates of enzymes acting as haptens. Some antigens may change conformation when they associate with other proteins, and their structure in such complexes could mimic that of a transitional state of the antigenic reaction. On the other hand, later in ADs anti-idiotypic Abs can be induced by a primary antigen and may show some of the characteristics of catalytic activity [8, 33, 34].

MS is known as a chronic demyelinating disease of the central nervous system. Its etiology to date is unclear, and the most widely accepted theory of MS pathogenesis assigns the main role in the destruction of myelin to the inflammation related to autoimmune (AI) reactions [32]. Activated CD4+ myelin-reactive T cells are generally considered as major mediators of MS. Several recent findings imply an important role of B cells and auto-Abs against myelin autoantigens in the pathogenesis of MS [32, 33, 35, 36]. Different studies suggest that a crucial role in MS immunopathogenesis can belong to auto-Abs against myelin autoantigens exercis‐ ing Ab-mediated demyelination [36]. Auto-antibodies against oligodendrocyte progenitor cell surface protein could block remyelination by eliminating or incapacitating these cells [37]. An important dual role of auto-Abs is suggested: They may be harmful in lesion formation but also potentially beneficial in the repair [35]. It is appropriate to mention here that the main targets of both above-mentioned auto-Abs are glycoproteins: myelin oligodendrocyte glyco‐ protein that is expressed preferentially on the surface of the myelin sheath [38] and progenitor cell-specific surface glycoprotein AN2 [37], respectively. Elevated level of oligoclonal IgGs in the cerebrospinal fluid (CSF) and B cell accumulation in the CSF and in lesions of MS patients provide evidence for antibody involvement in demyelination [39].

After cloning, the IgG repertoire directly from active plaques and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease for understanding MS pathogenesis new keys have been proposed [40]. It was shown that in the MS patients' high-affinity anti-DNA Abs are the major components of intrathecal IgG response. Furthermore, DNA-specific monoclonal Abs obtained from patients with MS and DNA-specific Ab derived from a SLE patient bound efficiently to the oligoden‐ drocytes and surface of neuronal cells. Cell-surface recognition of these Abs was DNAdependent. The results obtained indicate that anti-DNA Abs may promote important neuropathological mechanisms in chronic inflammatory disorders, such as MS and SLE [40]. It should be mentioned that SLE and MS demonstrated some similarities in the development of the same medical, biochemical, and immunological indexes including anti-DNA antibodies. Relative levels of auto-Abs to native (nat) and denatured (den) DNA in the blood of 49 MS patients and healthy donors were compared [41, 42]. The levels of anti-nat-DNA and anti-den-DNA Abs in 18% and 53% of the patients, respectively, were significantly higher than in controls. The titters of Abs to den-DNA in MS patients are usually higher than to nat-DNA. The correlation coefficients between titters of Abs to nat-DNA and den-DNA for a complete group (0.88) and its subgroups with remission (0.81), primary progressing (0.88), and secon‐ dary chronic-progressive (0.89) state of the disease were estimated [41, 42]

Interestingly, the titters of Abs against human myelin basic protein (MBP) in SLE patients 2.2 fold higher than in healthy individuals, but 2.1-fold lower than in patients with MS [43, 44]. In the case of 49 patients with MS, a possible correlation between titters of Abs to DNA [41, 42] and to MBP [44] and 13 different standard clinical parameters including Poser criteria (indexes for evaluation of damage to functional systems: pyramidal functions; cerebellar functions; functions of brain stem; sensitive functions; functions of intestines and urinary bladder; visual functions; cerebral (psychical) functions and sum of these characteristics) [45] was carried out [21, 44]. High percent of MS patients showed significantly higher anti-MBP and anti-DNA Abs levels as compared to healthy subjects. For the whole group of MS patients, the absolute values of positive CCs between titters of anti-DNA or anti-MBP Abs and clinical Poser indexes were very low (between 0.01 and 0.19), absent (~0), or were negative (−0.02 to −0.07) and statistically insignificant. Several CCs become higher increasing values up to 0.1–0.55 and −0.04 to −0.47 after the cohort was divided into subgroups of patients with primary progressing, secondary progressing, and remitting course of the disease [44].

The groups of secondary progressing, primary progressing, and remitting course of MS patients were not "homogenous" in relation to the characteristics of the patients, and their further subdivision using cluster and factorial analysis showed high statistically significant correlation coefficients [44]. For example, a direct dependence between titters of anti-MBP and symptoms of damage of the pyramidal tract for one sub-subgroup of the remitting course subgroup was observed (CC = 0.92). In some cases, correlations of the opposite sign were found for the same pairs of analyzed parameters for the three subgroups with different MS courses and their sub-subgroups obtained by cluster analysis.

Interestingly, in the case of different MS subgroups, the level of anti-DNA Abs correlates with various clinical parameters, more often with the disturbance in the function of cerebrum, bladder, and intestines, and, to a less extent, cerebellum [41, 42]. In contrast to anti-DNA Abs, a positive correlation of anti-MBP titters with disturbed brain stem function was revealed in a relatively large fraction of patients (~37%), while a negative correlation was shown with pyramid function and with the rate of disease progression [42, 44]. The correlation between Autoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 103

Abs to DNA and to MBP in MS patients was also very weak. On overall, the correlation coefficients between the same two analyzed parameters can be either positive or negative in the case of whole group, different subgroups, and their sub-subgroups [41, 42, 44]. The absence of a definite dependence between titters of anti-DNA and anti-MBP Abs and these parameters with the standard clinical indices may be due to the several reasons. Taken together, during analysis of biochemical, immunological and clinical indices must taken into account the current phase of the disease. It should be mentioned; quite different characteristics of pathologic processes can be obtained in individual patients, as the disease progresses against the back‐ ground of the continually changing immunoregulation including exhaustion of different compensatory and adaptive mechanisms and systemic metabolic changes. This causes the clinical course in individual MS patients hardly predictable [46, 47]. Since each patient can be characterized by an individual combination of genetic, environmental, chronic, inflammatory, autoimmune, demyelinating, neurodegenerative, and other factors [41, 42, 44], it is not surprising that we could not find statistically significant correlation of titters of antibodies to DNA and MBP with the standard clinical parameters in the whole group of 49 MS patients.

### **3. Catalytic antibodies of MS patients**

intrathecal IgG response. Furthermore, DNA-specific monoclonal Abs obtained from patients with MS and DNA-specific Ab derived from a SLE patient bound efficiently to the oligoden‐ drocytes and surface of neuronal cells. Cell-surface recognition of these Abs was DNAdependent. The results obtained indicate that anti-DNA Abs may promote important neuropathological mechanisms in chronic inflammatory disorders, such as MS and SLE [40]. It should be mentioned that SLE and MS demonstrated some similarities in the development of the same medical, biochemical, and immunological indexes including anti-DNA antibodies. Relative levels of auto-Abs to native (nat) and denatured (den) DNA in the blood of 49 MS patients and healthy donors were compared [41, 42]. The levels of anti-nat-DNA and anti-den-DNA Abs in 18% and 53% of the patients, respectively, were significantly higher than in controls. The titters of Abs to den-DNA in MS patients are usually higher than to nat-DNA. The correlation coefficients between titters of Abs to nat-DNA and den-DNA for a complete group (0.88) and its subgroups with remission (0.81), primary progressing (0.88), and secon‐

Interestingly, the titters of Abs against human myelin basic protein (MBP) in SLE patients 2.2 fold higher than in healthy individuals, but 2.1-fold lower than in patients with MS [43, 44]. In the case of 49 patients with MS, a possible correlation between titters of Abs to DNA [41, 42] and to MBP [44] and 13 different standard clinical parameters including Poser criteria (indexes for evaluation of damage to functional systems: pyramidal functions; cerebellar functions; functions of brain stem; sensitive functions; functions of intestines and urinary bladder; visual functions; cerebral (psychical) functions and sum of these characteristics) [45] was carried out [21, 44]. High percent of MS patients showed significantly higher anti-MBP and anti-DNA Abs levels as compared to healthy subjects. For the whole group of MS patients, the absolute values of positive CCs between titters of anti-DNA or anti-MBP Abs and clinical Poser indexes were very low (between 0.01 and 0.19), absent (~0), or were negative (−0.02 to −0.07) and statistically insignificant. Several CCs become higher increasing values up to 0.1–0.55 and −0.04 to −0.47 after the cohort was divided into subgroups of patients with primary progressing, secondary

The groups of secondary progressing, primary progressing, and remitting course of MS patients were not "homogenous" in relation to the characteristics of the patients, and their further subdivision using cluster and factorial analysis showed high statistically significant correlation coefficients [44]. For example, a direct dependence between titters of anti-MBP and symptoms of damage of the pyramidal tract for one sub-subgroup of the remitting course subgroup was observed (CC = 0.92). In some cases, correlations of the opposite sign were found for the same pairs of analyzed parameters for the three subgroups with different MS courses

Interestingly, in the case of different MS subgroups, the level of anti-DNA Abs correlates with various clinical parameters, more often with the disturbance in the function of cerebrum, bladder, and intestines, and, to a less extent, cerebellum [41, 42]. In contrast to anti-DNA Abs, a positive correlation of anti-MBP titters with disturbed brain stem function was revealed in a relatively large fraction of patients (~37%), while a negative correlation was shown with pyramid function and with the rate of disease progression [42, 44]. The correlation between

dary chronic-progressive (0.89) state of the disease were estimated [41, 42]

progressing, and remitting course of the disease [44].

102 Trending Topics in Multiple Sclerosis

and their sub-subgroups obtained by cluster analysis.

Natural Abzs from the sera of patients with various diseases are usually polyclonal in origin and may be products of different immuno-competent cells ([13–24] and references cited here). Natural abzyme purification is one of the most complicated aspects of their study; it was discussed in detail in reviews [13, 19]. In study of Abzs with different activities from the sera and CSF of MS patients and healthy donors, electrophoretically and immunologically homo‐ geneous Ab fraction (IgG+IgM + IgA) was first purified by affinity chromatography of the serum or CSF proteins on Protein A-Sepharose under conditions that remove nonspecifically bound proteins. Then IgMs were separated from IgAs and IgGs by FPLC gel filtration [48– 56]. 150 kDa IgG, 170 kDa IgA, and ~900 kDa IgM did not contain any contaminating proteins detectable by silver staining under non-reducing and reducing conditions (e.g., **Figure 1**) [48– 56].

The application of rigid criteria allowed the authors of the first article concerning natural Abzs [9] to conclude that vasoactive intestinal peptide-hydrolyzing activity is an intrinsic property of Abs from the sera of patients with asthma. Later several additional rigid criteria were proposed (for review see [13, 19]). We applied a set of strict criteria worked out previously [9, 13–21] for the analysis of DNase [52, 54–56], MBP-hydrolyzing [48–51, 53] and amylase [57, 58] activities as intrinsic properties of IgGs, IgAs, and IgMs from the sera and IgGs from the CSF of MS patients [52, 53]. They may be summarized as follows: (a) the IgGs, IgAs, and IgMs were electrophoretically homogeneous (e.g., **Figure 1**); (b) FPLC gel filtration of these Abs using conditions dissociating strong noncovalent complexes in an acidic buffer did not eliminate analyzed activities, and the peaks of the activities and intact Abs exactly coincided (**Figure 2A**); (c) immobilized polyclonal mouse IgGs against the light chains of human antibodies completely absorbed the activities; peaks of these activities coincided with the peak of IgGs ( IgA or IgM) eluted with an acidic buffer (e.g., **Figure 2B**). (d) F(ab) and F(ab)2 fragments obtained using digestion by proteases of corresponding catalytic IgGs showed comparable levels of the activities comparing with intact Abs. The fulfillment of these criteria was observed for MS Abzs with all activities mentioned above.

**Figure 1.** SDS-PAGE analysis of IgGmix (7 μg) corresponding to equimolar mixtures of 13 IgG preparations of CSFs and 13 IgGs from sera of MS patients, and 10 healthy donors in 3–16% gradient gel before (A) and after treatment with DTT (B) followed by silver staining [52, 54–56]. The arrows (lane C) indicate the positions of molecular mass markers. In situ gel assay of DNase activity of IgGmix (15 μg) corresponding to CSFs and to sera of MS patients, and healthy donors in a gel containing DNA before (C) and after treatment with DTT (D). DNase activity was revealed by ethidium bromide staining as a dark band on the fluorescent background. A part of the gel corresponding to Panels C and D was stained with Coumassie R250 to show the position of intact IgGs and separated light and heavy chains (the arrows of Panels C and D).

To exclude possible artifacts due to the hypothetical traces of contaminating enzymes, IgGs from sera and CSF were subjected to SDS-PAGE in a gel co-polymerized with calf thymus DNA, and their DNase activity was detected by incubating the gel in the standard reaction buffer (**Figure 1C** and **D**). Ethidium bromide staining of the gels after the electrophoresis and refolding of IgGs revealed sharp dark bands against a fluorescent background of DNA. In addition, after incubation of IgGs with DTT only light chains of MS csf-IgGmix and serum-IgGmix demonstrated DNase activity (**Figure 1D**). Since SDS dissociates all protein complexes, the revealing of the DNase activity in the gel zones of only to intact IgGs (Panel C) and separated light chains (Panel D) together with the absence of any other activity or protein bands (**Figure 1**), guaranty direct evidence that IgGs from sera and CSFs of MS patients cleavage DNA and they do not contain canonical DNases [52, 54–56].

MS IgGs and IgAs were separated by SDS-PAGE, respectively, under non-reducing and reducing conditions, and their MBP-hydrolyzing and amylase activities were detected after the extraction of Abs from excised gel slices (for example, **Figure 2C**) [44, 48–51, 53, 57, 58]. Autoimmune Processes in Multiple Sclerosis: Production of Harmful Catalytic Antibodies Associated with Significant Changes in the Hematopoietic Stem Cell Differentiation and Proliferation http://dx.doi.org/10.5772/63824 105

The detection of MBP-hydrolyzing and amylase activities in the gel region corresponding only to intact IgGs, together with the absence of any other bands of the activity or protein, provided direct evidence that IgG possesses MBP-hydrolyzing and amylase activities [44, 48–51, 57, 58]. Similar results were obtained for MS IgAs- and IgMs-hydrolyzing MBP [49]. In addition, it was shown that the fractions of IgGs (IgAs or IgMs) having affinity to MBP-Sepharose hydrolyze effectively only human and MBPs, but not other control proteins [48–50].

fragments obtained using digestion by proteases of corresponding catalytic IgGs showed comparable levels of the activities comparing with intact Abs. The fulfillment of these criteria

**Figure 1.** SDS-PAGE analysis of IgGmix (7 μg) corresponding to equimolar mixtures of 13 IgG preparations of CSFs and 13 IgGs from sera of MS patients, and 10 healthy donors in 3–16% gradient gel before (A) and after treatment with DTT (B) followed by silver staining [52, 54–56]. The arrows (lane C) indicate the positions of molecular mass markers. In situ gel assay of DNase activity of IgGmix (15 μg) corresponding to CSFs and to sera of MS patients, and healthy donors in a gel containing DNA before (C) and after treatment with DTT (D). DNase activity was revealed by ethidium bromide staining as a dark band on the fluorescent background. A part of the gel corresponding to Panels C and D was stained with Coumassie R250 to show the position of intact IgGs and separated light and heavy chains (the arrows of

To exclude possible artifacts due to the hypothetical traces of contaminating enzymes, IgGs from sera and CSF were subjected to SDS-PAGE in a gel co-polymerized with calf thymus DNA, and their DNase activity was detected by incubating the gel in the standard reaction buffer (**Figure 1C** and **D**). Ethidium bromide staining of the gels after the electrophoresis and refolding of IgGs revealed sharp dark bands against a fluorescent background of DNA. In addition, after incubation of IgGs with DTT only light chains of MS csf-IgGmix and serum-IgGmix demonstrated DNase activity (**Figure 1D**). Since SDS dissociates all protein complexes, the revealing of the DNase activity in the gel zones of only to intact IgGs (Panel C) and separated light chains (Panel D) together with the absence of any other activity or protein bands (**Figure 1**), guaranty direct evidence that IgGs from sera and CSFs of MS patients cleavage

MS IgGs and IgAs were separated by SDS-PAGE, respectively, under non-reducing and reducing conditions, and their MBP-hydrolyzing and amylase activities were detected after the extraction of Abs from excised gel slices (for example, **Figure 2C**) [44, 48–51, 53, 57, 58].

DNA and they do not contain canonical DNases [52, 54–56].

was observed for MS Abzs with all activities mentioned above.

104 Trending Topics in Multiple Sclerosis

Panels C and D).

**Figure 2.** Analysis of strict criteria fulfilment to show that the enzymatic activities are intrinsic properties of IgGs from cerebrospinal fluid of MS patients. FPLC gel filtration of mixture of equal amounts of electrophoretically homogeneous IgGs from 15 preparations of CSF (csf-IgGmix) on a Superdex 200 column in an acidic buffer (pH 2.6) destroying im‐ munocomplexes after Abs incubation in the same buffer (A) and csf-IgGmix affinity chromatography on Sepharose bearing mouse IgGs against human IgGs (B): (—), absorbance at 280 nm (A280); relative activity (RA) of IgGs in the hydrolysis of DNA (○), BMP (■), and maltoheptaose (∆). In-gel assay of MBP-hydrolyzing (■) and amylase (∆) activi‐ ties of csf-IgGmix (15 μg) (C). The relative MBP-hydrolyzing and amylase activities (RA, %) were revealed using the extracts of 2–3-mm fragments of one longitudinal slice of the gel. The RA of IgGs corresponding to complete hydroly‐ sis of the substrates was taken for 100%. The second control longitudinal slice of the same gel was stained with Coo‐ massie Blue (Panel C, lane 1); lane C shows positions of protein markers. The average error in the initial rate determination from three experiments did not exceed 7–10%.

It is known that catalytic centers of nuclease and proteolytic Abzs are usually located on the light chain, while the heavy chain is more often responsible for specific antigen recognition and increased antigen affinity for Abs [13–21]. It was shown that catalytic centers of DNase, protease, and amylase Abzs from MS patients are located on the light chains of these Abs [48– 53, 54, 56–58]. Intact proteins usually interact with both light and heavy chains of Abzs, thus ensuring the specificity of the target protein recognition and its cleavage. Overall, it is obvious that nuclease, amylase, and MBP-hydrolyzing activities are intrinsic properties of Abzs from sera and CSF of MS patients and do not due to the admixture of any possible canonical enzymes.
