**6. Change of some indexes characterizing development of EAE in C57BL/6 mice**

We tried to compare several different indexes associated with development of ADs in auto‐ immune prone mice MRL-lpr/lpr and experimental autoimmune encephalomyelitis (EAE; C57BL/6) mice, the models of human SLE and MS, respectively. When for acceleration of SLE development in MRL-lpr/lpr DNA is usually used [22–24], the stimulation of EAE in C57BL/ 6mice is usually carried out using mouse myelin oligodendrocyte glycoprotein (MOG35-55), which is an antigen of the myelin sheath [112, 113].

In our study, we have used four experimental groups of C57BL/6 and (CBA × C57BL) F1 or CBA mice:


with MS and other autoimmune diseases in the analysis of their possible role in the patho‐ genesis of these pathologies, since some of them may be very harmful for people health.

**5. Specific reorganization of immune system of SLE prone MRL-lpr/lpr**

According to our data, the catalytic activity of nuclease and protease Abzs in sera of autoim‐ mune patients is usually very easily detectable at the beginning of autoimmune diseases, when concentrations of Abs to DNA or other autoantigens are not yet significantly increased, and correspond to levels in healthy donors [13–21]. It was shown that detection of Abzs with different activities in human serum may be considered a good indicator of the onset or a significant progression of autoimmune reactions associated with several pathologies [13–21]. In this regard, is of great interest to understand what mechanisms underlie the development of autoimmune processes and how they associated with the production of catalytic antibodies. Some observations suggest that autoimmune diseases can be originated from defects in the hematopoietic stem cells (HSCs) [107]. Therefore, one can assume that in the development of different Ads, there may be some common objective laws. Taking this into account, it seems reasonable first to consider what regularities were found by us in the development of SLE in MRL-lpr/lpr mice. First, it was shown that IgGs from the sera of 2-month-old to 7-month-old control non-autoimmune BALB/c and (CBAxC57BL)F1 mice and conditionally healthy 2 month-old to3-month-old autoimmune prone MRL-lpr/lpr mice are catalytically inactive [22–

It is known that MRL-lpr/lpr mice spontaneously developing a lupus-like AI disorder are characterized by visual symptoms of the pathology (pink spots, baldness of head, and parts of the back, general health deterioration, etc.). The sera of these mice contain Abzs with DNase, amylase and ATPase activities [22–24, 108]. Appearance of proteinuria (≥3-mg/ml concentra‐ tion of protein in urine) correlated well with pronounced visual symptoms [22–24]. The highest levels of anti-DNA Abs, catalytic activities of Abzs, visible markers of SLE and proteinuria were observed usually at 5–12 months of age, which agrees with previously reported data for typical period of signs of pathology appearance in MRL-lpr/lpr mice [109]. We have analyzed spontaneously diseased mice with all visible symptoms no older than 7 months. Although the state of "health" in the case of AI-prone mice should be considered very provisional, the mouse SLE pathology is nevertheless spontaneous and autoimmune reactions, leading to deep pathology develop gradually. To distinguish various levels of the pathology development, MRL-lpr/lpr mice demonstrating no typical SLE indices and Abzs activities (similar to healthy control non-autoimmune mice) were conditionally designated (independently of age) as healthy MRL-lpr/lpr mice, whereas the animals demonstrating no visual or biochemical SLE indices but having detectable abzyme activities were conditionally designated as pre-diseased

A specific reorganization of immune system of these mice after spontaneous development of deep SLE-like pathology results in conditions associated with a production of Abs hydrolyzing

**mice**

120 Trending Topics in Multiple Sclerosis

24].

mice.


In MOG-induced EAE mice, first clinical symptoms appear at 5–7 days after immunization, while the maximum stage of the disease is usually manifested at 14–16 days after immunization [112, 113]. A possible change in the relative weight, different immunological and biochemical parameters at 3 months of age (zero time; control) as well as MOG-treated C57BL/6 and CBA mice during consecutive 40 days was analyzed. After 6 days but not at later stages an average decrease in weight of EAE mice was observed in MOG treated compared

to untreated control mice (**Figure 10A**), but the weight of CBA mice increase slower, than untreated mice during 40 days (**Figure 10B**). In other models of autoimmunity including MRLlpr/lpr, the appearance of pronounced visual symptoms usually correlates well with proteinuria (≥3 mg/ml concentration of protein in urine) [22–24]. Control non-autoimmune CBA mice at 7 months demonstrated no proteinuria (<2 mg/ml) [22–24]. At the same time, EAE C57BL/6 mice at three months of age are usually characterized by a significantly higher level of proteinuria (up to 10–12 mg/ml) [114]. We have analyzed the time-dependent change in proteinuria for immunized and untreated C57BL/6 mice (**Figure 10C**) and control CBA mice (**Figure 10D**). In seven mice of the EAE control group, the average proteinuria was 7.2 ± 0.8 mg/ml at time zero (3 months of age) and was nearly the same until day 20, but increased to 12 ± 0.8 mg/ml at 40 days (**Figure 10C**). For control CBA untreated mice, there was no remarkable change in concentration of urine proteins during 40 days (**Figure 10D**). In EAE mice treated with MOG, a steady increase in proteinuria was observed: day 0: 7.2 ± 0.8 mg/ml; day 30: 13.6 ± 2.5 mg/ml; day 40: 21.8 ± 3.8 mg/ml. The increase after 40 days became significantly different from control animals (**Figure 10C**). CBA mice from 0 to 40 days demonstrated increase in urine protein concentration from 1.6 ± 0.08 to 2.5 ± 0.23 mg/ml (**Figure 10D**). High level of EAE mice proteinuria at three months may be due to the fact that these mice demonstrate spontaneous development of autoimmune processes, which is stimulated by their immunization by MOG. Immunization of non-autoimmune CBA mice also leads to remarkable increase in proteinuria, but after 40 days it is lower 3 mg/ml (**Figure 10D**).

**Figure 10.** In time changes in weight of C57B2/6 (A) and CBA (B) mice, as well as relative concentration of urine pro‐ teins of C57B2/6 (C) and CBA (D) mice in the case of MOG-treated and untreated animals (see Panels) [110, 111].

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The sera of healthy humans and mammals usually contain auto-Abs to many various antigens, including DNA [13–21, 59]. For example, the average concentration of anti-DNA Abs for BALB and CBA non-autoimmune mice is approximately 0.03–0.04 A450 units, which is comparable with that for healthy MRL-lpr/lpr mice (0.032 A450 units); then after the spontaneous devel‐ opment of SLE it increases to 0.2 A450 units [24]. Untreated C57BL/6 mice demonstrated an approximately 3.5-fold higher concentration of anti-DNA Abs (0.12 ± 0.04 A450 units) at 3 months of age (day 0) comparing with MRL-lpr/lpr mice. For untreated C57BL/6 mice, the change in anti-DNA Abs during 0–40 days was statistically insignificant (**Figure 11A**). Comparing to healthy control individuals and other neurological diseases of non-AI origin, the sera of MS patients contain anti-DNA Abs at a higher concentration (0.22 A450 units) [19, 21]. Immunization of C57BL/6 mice with MOG during 30 days (0.24 A450 units) led to a nearlinear statistically significant twofold increase in anti-DNA Ab concentration, and then to a further additional increase to 0.42 A450 units, which is higher than that for spontaneous disease (0.12 A450 units) (**Figure 11A**), but lower than that for MRL-lpr/lpr mice immunized with DNA (0.6 A450 units) [24].

to untreated control mice (**Figure 10A**), but the weight of CBA mice increase slower, than untreated mice during 40 days (**Figure 10B**). In other models of autoimmunity including MRLlpr/lpr, the appearance of pronounced visual symptoms usually correlates well with proteinuria (≥3 mg/ml concentration of protein in urine) [22–24]. Control non-autoimmune CBA mice at 7 months demonstrated no proteinuria (<2 mg/ml) [22–24]. At the same time, EAE C57BL/6 mice at three months of age are usually characterized by a significantly higher level of proteinuria (up to 10–12 mg/ml) [114]. We have analyzed the time-dependent change in proteinuria for immunized and untreated C57BL/6 mice (**Figure 10C**) and control CBA mice (**Figure 10D**). In seven mice of the EAE control group, the average proteinuria was 7.2 ± 0.8 mg/ml at time zero (3 months of age) and was nearly the same until day 20, but increased to 12 ± 0.8 mg/ml at 40 days (**Figure 10C**). For control CBA untreated mice, there was no remarkable change in concentration of urine proteins during 40 days (**Figure 10D**). In EAE mice treated with MOG, a steady increase in proteinuria was observed: day 0: 7.2 ± 0.8 mg/ml; day 30: 13.6 ± 2.5 mg/ml; day 40: 21.8 ± 3.8 mg/ml. The increase after 40 days became significantly different from control animals (**Figure 10C**). CBA mice from 0 to 40 days demonstrated increase in urine protein concentration from 1.6 ± 0.08 to 2.5 ± 0.23 mg/ml (**Figure 10D**). High level of EAE mice proteinuria at three months may be due to the fact that these mice demonstrate spontaneous development of autoimmune processes, which is stimulated by their immunization by MOG. Immunization of non-autoimmune CBA mice also leads to remarkable increase in proteinuria, but after 40 days it is lower 3 mg/ml (**Figure 10D**).

122 Trending Topics in Multiple Sclerosis

**Figure 10.** In time changes in weight of C57B2/6 (A) and CBA (B) mice, as well as relative concentration of urine pro‐ teins of C57B2/6 (C) and CBA (D) mice in the case of MOG-treated and untreated animals (see Panels) [110, 111].

**Figure 11.** Relative concentrations of Abs to DNA in the case of C57B2/6 (A) and CBA (B) mice as well as to MOG for C57B2/6 (C) and CBA (D) untreated and treated with MOG (see Panels) [110, 111]. Anti-MOG and anti-MBP antibody concentrations in the sera of C57B2/6 and CBA mice were measured by ELISA (sera was diluted 50-fold). The concen‐ tration of plasma anti-DNA Abs was determined using standard ELISA plates with immobilized double-stranded DNA (sera was diluted 100-fold).

Untreated CBA mice demonstrated at zero time fourfold lower average concentration of anti-DNA Abs (0.03 A450 units) comparing with EAE mice (0.12 A450 units), and their concentra‐ tion was not increase remarkably during 40 days (**Figure 11B**). After CBA mice treatment with MOG, the average concentration of anti-DNA Abs increased ~twofold at 12 days (0.06 A450 units) and then was the same up to 40 days (**Figure 11B**). Finally at 40 days, anti-DNA concentration in treated CBA mice was ~sevenfold lower than for EAE mice immunized with MOG (**Figure 11**). Thus, the immunization of CBA mice with MOG can somehow stimulate their immune response to DNA in these healthy mice, but it is significantly lower than that in the case of autoimmune EAE C57BL/6 mice.

Human healthy donors show a relative average index of anti-MBP Abs – 0.08 ± 0.04 A450, which is ~four- and fivefold lower than that for MS patients (0.32 ± 0.08 A450 units) [44]. The concentration of anti-MOG Abs in the sera of C57BL/6 control (untreated) mice demonstrated a near-linear statistically significant approximately 4.8-fold increase during 40 days (from 0.023 to 0.11 A450 units; **Figure 11C**). After treatment of mice, a significant rise in the titter at 10 days (from 0.023 to 0.083 A450 units) was revealed, followed by a temporary plateau and then an additional increase to 0.18 A450 units. The concentrations of anti-MOG Abs at zero time for C57BL/6 and CBA mice were to some extent comparable (~0.02 A450 units). However, in contrast to EAE mice, CBA mice did not demonstrate remarkable change in anti-MOG Abs over time (**Figure 11D**). The treatment of CBA mice with MOG led to an increase in the average concentration of anti-MOG Abs ~1.4-fold, but this value (0.028 A450 units) is ~6.5- and ~fourfold, respectively lower than those for treated (0.18 A450 units) and untreated (0.11 A450 units) EAE mice (**Figure 11**). Thus, the immunization of CBA mice with MOG stimulates formation of Abs against this antigen, but these healthy mice in contrast to EAE mice do not demonstrate spontaneous development of autoimmune reactions leading to the formation of anti-MOG antibodies. These data indicate that the mice treatment with MOG leads to the formation of Abs against MOG and to DNA. At the same time, the relative concentrations of Abs to different antigens are not always good indicators of the real development of autoim‐ mune diseases, because the relative titters to autoantigens in healthy humans and mammals can be very different [13–21, 59]. The titters of antibodies against autoantigens in sick patients and animals are usually higher than those in healthy individuals only in very late stages of ADs. As we have shown previously, the detection of DNase Abs in human sera may be considered to be a good indicator for the beginning of, or a significant development in, AI reactions associated with several ADs [13–21]. Moreover, like in AI patients, only DNase and ATPase activities of IgGs can be considered as statistically significant indicators of pre-disease conditions of spontaneous SLE in AI prone MRL-lpr/lpr mice [22–24]. Notably, well detectable DNase and ATPase Abs in MRL-lpr/lpr mice can sometimes be revealed 1–2 months earlier than a statistically reliable increase in Abs against DNA, as well as detection of visual and biochemical indexes of mouse SLE [22–24]. We have estimated the relative DNase and proteolytic activities of IgGs from untreated EAE and CBA and MOG-treated mice (**Figure 12**). Surprisingly, detectable levels of IgGs with all activities (in contrast to MRL-lpr/lpr) were revealed the sera of untreated C57BL/6 mice [22–24] even at the beginning of the experiment (at 3 months of age) and then it increased near-linearly. After 40 days, all activities were statistically significantly higher than at day 0: DNase (6.1-fold), MOG-hydrolyzing (1.8-fold), MBP-hydrolyzing (2.8-fold) (**Figure 12**), while there was no statistically significant increase in the concentrations of anti-DNA Abs (**Figure 11**). Furthermore, the detectable increase in abzymes from immunized EAE mice hydrolyzing DNA, MOG, and MBP was observed at a 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 125

time corresponding to the outset and acute phase of EAE (6–8 days), followed by a statistically significant increase in their activities at 14 days, and maximal increases at 22 days after treatment with MOG (**Figure 12**). Interestingly, a significant decrease in the activities was revealed at the transition from the acute (18–22 days) to the severe chronic phase of EAE (40 days) induced by MOG.

MOG, the average concentration of anti-DNA Abs increased ~twofold at 12 days (0.06 A450 units) and then was the same up to 40 days (**Figure 11B**). Finally at 40 days, anti-DNA concentration in treated CBA mice was ~sevenfold lower than for EAE mice immunized with MOG (**Figure 11**). Thus, the immunization of CBA mice with MOG can somehow stimulate their immune response to DNA in these healthy mice, but it is significantly lower than that in

Human healthy donors show a relative average index of anti-MBP Abs – 0.08 ± 0.04 A450, which is ~four- and fivefold lower than that for MS patients (0.32 ± 0.08 A450 units) [44]. The concentration of anti-MOG Abs in the sera of C57BL/6 control (untreated) mice demonstrated a near-linear statistically significant approximately 4.8-fold increase during 40 days (from 0.023 to 0.11 A450 units; **Figure 11C**). After treatment of mice, a significant rise in the titter at 10 days (from 0.023 to 0.083 A450 units) was revealed, followed by a temporary plateau and then an additional increase to 0.18 A450 units. The concentrations of anti-MOG Abs at zero time for C57BL/6 and CBA mice were to some extent comparable (~0.02 A450 units). However, in contrast to EAE mice, CBA mice did not demonstrate remarkable change in anti-MOG Abs over time (**Figure 11D**). The treatment of CBA mice with MOG led to an increase in the average concentration of anti-MOG Abs ~1.4-fold, but this value (0.028 A450 units) is ~6.5- and ~fourfold, respectively lower than those for treated (0.18 A450 units) and untreated (0.11 A450 units) EAE mice (**Figure 11**). Thus, the immunization of CBA mice with MOG stimulates formation of Abs against this antigen, but these healthy mice in contrast to EAE mice do not demonstrate spontaneous development of autoimmune reactions leading to the formation of anti-MOG antibodies. These data indicate that the mice treatment with MOG leads to the formation of Abs against MOG and to DNA. At the same time, the relative concentrations of Abs to different antigens are not always good indicators of the real development of autoim‐ mune diseases, because the relative titters to autoantigens in healthy humans and mammals can be very different [13–21, 59]. The titters of antibodies against autoantigens in sick patients and animals are usually higher than those in healthy individuals only in very late stages of ADs. As we have shown previously, the detection of DNase Abs in human sera may be considered to be a good indicator for the beginning of, or a significant development in, AI reactions associated with several ADs [13–21]. Moreover, like in AI patients, only DNase and ATPase activities of IgGs can be considered as statistically significant indicators of pre-disease conditions of spontaneous SLE in AI prone MRL-lpr/lpr mice [22–24]. Notably, well detectable DNase and ATPase Abs in MRL-lpr/lpr mice can sometimes be revealed 1–2 months earlier than a statistically reliable increase in Abs against DNA, as well as detection of visual and biochemical indexes of mouse SLE [22–24]. We have estimated the relative DNase and proteolytic activities of IgGs from untreated EAE and CBA and MOG-treated mice (**Figure 12**). Surprisingly, detectable levels of IgGs with all activities (in contrast to MRL-lpr/lpr) were revealed the sera of untreated C57BL/6 mice [22–24] even at the beginning of the experiment (at 3 months of age) and then it increased near-linearly. After 40 days, all activities were statistically significantly higher than at day 0: DNase (6.1-fold), MOG-hydrolyzing (1.8-fold), MBP-hydrolyzing (2.8-fold) (**Figure 12**), while there was no statistically significant increase in the concentrations of anti-DNA Abs (**Figure 11**). Furthermore, the detectable increase in abzymes from immunized EAE mice hydrolyzing DNA, MOG, and MBP was observed at a

the case of autoimmune EAE C57BL/6 mice.

124 Trending Topics in Multiple Sclerosis

**Figure 12.** In time changes in the relative average activity of IgGs from sera of untreated and MOG-treated EAE C57B2/6 and CBA mice in the hydrolysis of MBP (A, B), MOG (C, D) and DNA (E, F) [110, 111]. To quantify the MOG-, MBP-, and DNA-hydrolyzing activities of IgGs of different groups, we determined a concentration for each individual IgG preparation (0.03–0.2 mg/ml) and time of incubation (1–24 h) providing hydrolysis (10–40%) of these substrates to their products. Since all measurements (initial rates) were taken within the linear regions of the time courses and Abs concentration curves, the measured RAs for IgGs were normalized to standard conditions and maximal enzymatic ac‐ tivities were taken as 100% of activity analyzed. The average relative activities (RAs) of IgGs corresponding to 7 indi‐ vidual mice for each group are given.

It was shown earlier that Abs from sera of healthy mice and rabbit are completely inactive in the hydrolysis of different substrates including DNA and proteins [13–21]. Immunization of healthy animals with complex DNA and methylated BSA (m-BSA) led to the formation of Abshydrolyzing DNA and RNA [116, 117]. The averages relative activities of Abs from sera of immunized animals are usually 100- to 1000-fold lower those from patients with autoimmune diseases [13–21, 115, 116].

IgGs from sera of CBA untreated mice were completely inactive in the hydrolysis of MBP, MOG, and DNA (**Figure 12**). After treatment of CBA mice, IgGs demonstrated detectable activity in the hydrolysis of these substrates, but their maximal relative activities in the hydrolysis of MBP, MOG, and DNA were approximately 40- to 70-fold lower than those for Abs of immunized EAE mice (**Figure 12**). In addition, IgGs from sera of EAE mice at three months of age were significantly more active than maximal activity of Abs of immunized CBA mice in the hydrolysis of all substrates: MBP (6.4-fold), MOG (6.5-fold), and DNA (2.5-fold) (**Figure 12**). It means that spontaneous autoimmune processes and the appearance of the cells producing different Abzs begin to develop in EAE mice before 3 months of their age.

Interestingly, during 40 days, the average relative DNA-hydrolyzing activity increased in EAE mice by a factor of 6.1 (**Figure 12E**), while statistically significant increase in the titters of anti-DNA Abs, there was not observed (**Figure 11A**). Since it was previously shown that the relative content of Abzs to different antigens in the total pools of Abs is usually ≤ 0.01—3%, this phenomenon is not surprising, [13–21]. Therefore, the production of a small fraction of specific DNase and protease Abzs may have no detectable effect on the total concentration anti-DNA and anti-MOG Abs.

#### **6.1. Proliferation and apoptosis of lymphocytes in different organs of EAE prone mice**

Immunization of healthy and AI-prone mammals usually leads to in an increase in the level of lymphocytes differentiation and proliferation in different organs [19–21]. Different lym‐ phocytes producing Abzs were obtained with a dramatically higher incidence in autoimmune mouse strains than in conventionally used control mouse strains [117, 118]. At the same time, immunization of animals with some antigens usually leads to an increase in apoptosis of some lymphocytes. Apoptotic cells are considered as the primary source of antigens and immuno‐ gens in SLE triggering the recognition, perception, processing, and/or presentation of apoptotic autoantigens by antigen-presenting cells and can cause AI processes [60]. DNA-hydrolyzing DNase Abzs from patients with SLE [68] and MS [16], as well as DNase Bence-Jones proteins from multiple myeloma patients [119] are cytotoxic, cause nuclear DNA fragmentation, and induce cell apoptosis. A significant decrease in apoptosis can be an important factor providing the enhancement in the level of specific lymphocytes producing auto-Abs and Abzs, which are normally eliminated in different mammalian organs. Thus, the total number of lympho‐ cytes of various types in different organs after immunization of animals depends on the ratio of possible levels of cell proliferation and apoptosis. The overall level of cytotoxic (harmful) or beneficial to organisms antibodies may depend on this ratio.

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We have compared possible lymphocyte proliferation (**Figure 13**) and apoptosis (**Figure 14**) in different organs of EAE and CBA mice. Lymphocyte proliferation in different organs at 3 months of age (zero time) for EAE and CBA mice is to some extent comparable (**Figure 13**).

It was shown earlier that Abs from sera of healthy mice and rabbit are completely inactive in the hydrolysis of different substrates including DNA and proteins [13–21]. Immunization of healthy animals with complex DNA and methylated BSA (m-BSA) led to the formation of Abshydrolyzing DNA and RNA [116, 117]. The averages relative activities of Abs from sera of immunized animals are usually 100- to 1000-fold lower those from patients with autoimmune

IgGs from sera of CBA untreated mice were completely inactive in the hydrolysis of MBP, MOG, and DNA (**Figure 12**). After treatment of CBA mice, IgGs demonstrated detectable activity in the hydrolysis of these substrates, but their maximal relative activities in the hydrolysis of MBP, MOG, and DNA were approximately 40- to 70-fold lower than those for Abs of immunized EAE mice (**Figure 12**). In addition, IgGs from sera of EAE mice at three months of age were significantly more active than maximal activity of Abs of immunized CBA mice in the hydrolysis of all substrates: MBP (6.4-fold), MOG (6.5-fold), and DNA (2.5-fold) (**Figure 12**). It means that spontaneous autoimmune processes and the appearance of the cells

producing different Abzs begin to develop in EAE mice before 3 months of their age.

Interestingly, during 40 days, the average relative DNA-hydrolyzing activity increased in EAE mice by a factor of 6.1 (**Figure 12E**), while statistically significant increase in the titters of anti-DNA Abs, there was not observed (**Figure 11A**). Since it was previously shown that the relative content of Abzs to different antigens in the total pools of Abs is usually ≤ 0.01—3%, this phenomenon is not surprising, [13–21]. Therefore, the production of a small fraction of specific DNase and protease Abzs may have no detectable effect on the total concentration anti-DNA

**6.1. Proliferation and apoptosis of lymphocytes in different organs of EAE prone mice**

or beneficial to organisms antibodies may depend on this ratio.

Immunization of healthy and AI-prone mammals usually leads to in an increase in the level of lymphocytes differentiation and proliferation in different organs [19–21]. Different lym‐ phocytes producing Abzs were obtained with a dramatically higher incidence in autoimmune mouse strains than in conventionally used control mouse strains [117, 118]. At the same time, immunization of animals with some antigens usually leads to an increase in apoptosis of some lymphocytes. Apoptotic cells are considered as the primary source of antigens and immuno‐ gens in SLE triggering the recognition, perception, processing, and/or presentation of apoptotic autoantigens by antigen-presenting cells and can cause AI processes [60]. DNA-hydrolyzing DNase Abzs from patients with SLE [68] and MS [16], as well as DNase Bence-Jones proteins from multiple myeloma patients [119] are cytotoxic, cause nuclear DNA fragmentation, and induce cell apoptosis. A significant decrease in apoptosis can be an important factor providing the enhancement in the level of specific lymphocytes producing auto-Abs and Abzs, which are normally eliminated in different mammalian organs. Thus, the total number of lympho‐ cytes of various types in different organs after immunization of animals depends on the ratio of possible levels of cell proliferation and apoptosis. The overall level of cytotoxic (harmful)

diseases [13–21, 115, 116].

126 Trending Topics in Multiple Sclerosis

and anti-MOG Abs.

**Figure 13.** In time changes in lymphocyte proliferation (optical density) in bone marrow, thymus, spleen, and lymph nodes in the case of untreated and MOG-treated EAE and CBA mice; average values corresponding to 7 mice of each group are given [110, 111]. The error in the lymphocyte determination from three independent experiments in the case of every mouse of each group did not exceed 7–10%. Types of mice and their organs are given on panels A–H.

Proliferation of lymphocytes in bone marrow of untreated EAE mice increases constantly over time, and their treatment with MOG slightly decreases the proliferation level (**Figure 13A**). At the same time, untreated CBA mice demonstrate a remarkable decrease in the cell proliferation level over time. However, immunization of these mice with MOG increases their proliferation in the period from 10 to 15 days, but then it is significantly decreased (**Figure 13B**). Maximal observed level of lymphocytes proliferation in bone marrow CBA mice (~26%) is ~threefold lower than that for EAE mice (~77%). The total number of lymphocytes resulting in the brains of EAE mice can be significantly higher than the observed one, since the average level of cell apoptosis in the brains of these mice is about 14.2% and increased up to 22.4% after their treatment with MOG (**Figure 14A**). Interestingly, at three month of age, CBA mice (4.2%) demonstrate ~3.4-fold lower the average level of cell apoptosis comparing with EAE mice, while their immunization with MOG increase it only to 4.6% (**Figure 14B**), 4.9-fold lower than that for EAE mice (**Figure 14A**).

**Figure 14.** In time changes in cell apoptosis (%) in bone marrow, thymus, spleen, and lymph nodes in the case of un‐ treated and MOG-treated EAE and CBA mice; average values corresponding to 7 mice of each group are given [110, 111]. The error in the cell apoptosis determination from three independent experiments in the case of every mouse of each group did not exceed 7–10%. Types of mice and their organs are given on panels A–H.

The level of lymphocyte proliferation in thymus of untreated EAE mice almost does not change in time (**Figures 13C**), while it increases significantly at 20 days and then return to the level observed at zero time at 40 days (**Figure 13D**). The converse situation is observed for levels of the lymphocytes proliferation of EAE and CBA mice after their immunization with MOG (**Figures 13C** and **D**). Interestingly, the level of the thymus cell apoptosis in healthy CBA (~0.9%) at three month of age is about fivefold lower than that for EAE mice (~4.8%); after mice treatment with MOG, it increases in EAE mice up to 22%, but remains relatively low (~4.8%) in CBA animals. Thus, in contrast to EAE animals, the apoptosis does not affect significantly on the level of lymphocytes proliferation in thymus of CBA mice.

in the period from 10 to 15 days, but then it is significantly decreased (**Figure 13B**). Maximal observed level of lymphocytes proliferation in bone marrow CBA mice (~26%) is ~threefold lower than that for EAE mice (~77%). The total number of lymphocytes resulting in the brains of EAE mice can be significantly higher than the observed one, since the average level of cell apoptosis in the brains of these mice is about 14.2% and increased up to 22.4% after their treatment with MOG (**Figure 14A**). Interestingly, at three month of age, CBA mice (4.2%) demonstrate ~3.4-fold lower the average level of cell apoptosis comparing with EAE mice, while their immunization with MOG increase it only to 4.6% (**Figure 14B**), 4.9-fold lower than

**Figure 14.** In time changes in cell apoptosis (%) in bone marrow, thymus, spleen, and lymph nodes in the case of un‐ treated and MOG-treated EAE and CBA mice; average values corresponding to 7 mice of each group are given [110, 111]. The error in the cell apoptosis determination from three independent experiments in the case of every mouse of

each group did not exceed 7–10%. Types of mice and their organs are given on panels A–H.

that for EAE mice (**Figure 14A**).

128 Trending Topics in Multiple Sclerosis

While the level of lymphocytes proliferation in the spleen of untreated EAE mice remarkably increases over time, in CBA mice, it has tendency of only slight decrease (**Figures 13 E** and **F**). At the beginning of autoimmune processes in MOG-immunized EAE mice (7–15 days), there is a strong increase in spleen lymphocytes proliferation for EAE and CBA mice, but the maximum value for autoimmune mice is significantly higher (**Figures 13E** and **F**). Interest‐ ingly, the relative level of spleen cell apoptosis in non-immunized EAE mice at three months is very high (~36%) compared to the bone marrow (14.2%) and especially to the thymus (4.8%), but it remains practically unchanged during the spontaneous development of the EAE pathology (**Figure 14E**). At three months of age, CBA mice are characterized by a very low level of spleen cell apoptosis, 0.5%, and its increases at 14 days to 3%, but it returns to 0.5% at 40 days (**Figure 14F**). The treatment of EAE mice leads first (12 days) to an increase in the proliferation from 36% to 55.6%, and then to significant decrease to 19.5% at 40 days. Approx‐ imately after 20 days of CBA mice treatment with MOG the cell proliferation increase to ~5%, this is ~11-fold lower than that for EAE mice (**Figure 14F**). Thus, immune processes in the spleen of control healthy CBA mice before and after their treatment with MOG vary relatively weak over time. However, a slow increase in the number of lymphocytes in the spleen of EAE mice during spontaneous develop of the disease and its acceleration after treatment with MOG are associated with strong parallel opposite processes in the lymphocytes proliferation and apoptosis (**Figures 13** and **14E** and **F**). These data are consistent with the fact that immunization of MRL-lpr/lpr mice at three months of age with DNA also leads to a very strong activation of the proliferation and apoptosis in their spleen [22–24].

Lymph nodes of EAE and CBA mice demonstrate different profiles of the cell proliferation over time (**Figures 13 G** and **H**). The level of cell proliferation in lymph nodes of EAE mice before and after animals' treatment with MOG is nearly the same; the cell proliferation is constantly decreased from 3 to 40 days. The level of lymphocytes proliferation in the lymph nodes of CBA mice at 6–10 days before and after their treatment is remarkably decreased (**Figure 13H**). Then, untreated and treated CBA mice show remarkable increase in the prolif‐ eration level, but finally control mice at 40 days demonstrate additional increase, while immunized mice—significant decrease in cell proliferation. It should be mentioned that lymph nodes (~36%) similarly to spleen (~36%) of EAE mice are characterized by very high level of cell apoptosis at three months of age, while this level for CBA mice is very low (~1.3%) (**Figure 14H**). Parallel high-level differently directed processes of the cell proliferation and apoptosis in lymph nodes of EAE mice can probably to some extent counterbalance each other. High level of the apoptosis in the lymph nodes of non-treated, but more powerful apoptosis in treated EAE mice can be a reason of a slow decrease in lymphocytes of this organ of autoim‐ mune mice leading to a comparable profiles of the changes over time (**Figures 13** and **14**). The relative changes in the levels of proliferation and apoptosis in lymph nodes of CBA comparing with EAE mice are relatively low (**Figures 13** and **14**).

Overall, the autoimmune EAE and non-autoimmune CBA mice demonstrate very different profiles of differentiation and proliferation of bone marrow stem cells, as well as levels of proliferation and apoptosis of lymphocytes in various organs.

Primary differentiation of lymphocytes begins in the bone morrow of people and animals, and additional differentiation occurs in their various organs. Therefore, possible relative levels of cell differentiation, proliferation, and apoptosis in various organs of mice depends on what type of hematopoietic stem cells was "arrived" to these organs from their bone marrow. As it was mentioned above, autoimmune diseases can originate from defects in hematopoietic stem cells [107]. Therefore, it was very important to understand a possible difference and similarity in differentiation and proliferation of stem cells in the bone marrow of mice, which are prone and unwilling to autoimmune diseases.

#### **6.2. Differentiation of stem cells in the bone marrow of mice**

The specific reorganization of the immune system in a profound SLE-like pathology of MRLlpr/lpr mice during its spontaneous development is associated with changes in the differen‐ tiation profile of bone marrow HSCs, leading to the rise of the level of lymphocyte proliferation in combination with the production of different Abzs [22–24]. Since similar to MRL-lpr/lpr, C57BL/6 mice also reveal features of autoimmune-prone mice, it was reasonable to expect similar changes in the HSCs: erythroid burst-forming unit (early erythroid colonies; BFU-E), erythroid burst-forming unit (late erythroid colonies; CFU-E), granulocytic-macrophagic colony-forming unit (CFU-GM), and granulocytic-erythroid-megacaryocytic-macrophagic colony-forming unit (CFU-GEMM) in these mice before and after their immunization with MOG. But it was also very interesting to compare changes in the HSCs for autoimmune C57BL/ 6 and non-autoimmune CBA mice.

The relative amount of different colonies (BFU-E + CFU-E + CFU-GM + CFU-GEMM) of three monthly autoimmune EAE and normal healthy CBA mice was different. In order to calculate the relative amount of four different types of colonies, we have estimated a relative percent of every type of the colonies with respect to the total number of colonies taken as 100% (**Table 2**). It was shown that the average relative number of BFU-E (57.3 ± 20.1 and 54.5 ± 7.0%), CFU-GM (34.0 ± 3.9 and 28.2 ± 2.0%) colonies, respectively, for C57BL/6 and CBA mice at three months of age was to some extent comparable (**Table 2**). At the same time, the relative average number of CFU-E (3.2 ± 1.7%) for EAE mice was 4.6 lower than that for CBA mice (14.7 ± 10.5%), while in the case of CFU-GEMM, the reverse situation was observed; this average value for EAE mice (5.6 ± 1.7%) was ~2.2-fold higher than that for CBA mice (2.6 ± 0.78%) (**Table 2**). In time changes in the proliferation profiles of the HSCs, during 40 days, it was observed for untreated and MOG-treated autoimmune C57BL/6 and nonautoimmune CBA mice.

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 131


\*

level of the apoptosis in the lymph nodes of non-treated, but more powerful apoptosis in treated EAE mice can be a reason of a slow decrease in lymphocytes of this organ of autoim‐ mune mice leading to a comparable profiles of the changes over time (**Figures 13** and **14**). The relative changes in the levels of proliferation and apoptosis in lymph nodes of CBA comparing

Overall, the autoimmune EAE and non-autoimmune CBA mice demonstrate very different profiles of differentiation and proliferation of bone marrow stem cells, as well as levels of

Primary differentiation of lymphocytes begins in the bone morrow of people and animals, and additional differentiation occurs in their various organs. Therefore, possible relative levels of cell differentiation, proliferation, and apoptosis in various organs of mice depends on what type of hematopoietic stem cells was "arrived" to these organs from their bone marrow. As it was mentioned above, autoimmune diseases can originate from defects in hematopoietic stem cells [107]. Therefore, it was very important to understand a possible difference and similarity in differentiation and proliferation of stem cells in the bone marrow of mice, which are prone

The specific reorganization of the immune system in a profound SLE-like pathology of MRLlpr/lpr mice during its spontaneous development is associated with changes in the differen‐ tiation profile of bone marrow HSCs, leading to the rise of the level of lymphocyte proliferation in combination with the production of different Abzs [22–24]. Since similar to MRL-lpr/lpr, C57BL/6 mice also reveal features of autoimmune-prone mice, it was reasonable to expect similar changes in the HSCs: erythroid burst-forming unit (early erythroid colonies; BFU-E), erythroid burst-forming unit (late erythroid colonies; CFU-E), granulocytic-macrophagic colony-forming unit (CFU-GM), and granulocytic-erythroid-megacaryocytic-macrophagic colony-forming unit (CFU-GEMM) in these mice before and after their immunization with MOG. But it was also very interesting to compare changes in the HSCs for autoimmune C57BL/

The relative amount of different colonies (BFU-E + CFU-E + CFU-GM + CFU-GEMM) of three monthly autoimmune EAE and normal healthy CBA mice was different. In order to calculate the relative amount of four different types of colonies, we have estimated a relative percent of every type of the colonies with respect to the total number of colonies taken as 100% (**Table 2**). It was shown that the average relative number of BFU-E (57.3 ± 20.1 and 54.5 ± 7.0%), CFU-GM (34.0 ± 3.9 and 28.2 ± 2.0%) colonies, respectively, for C57BL/6 and CBA mice at three months of age was to some extent comparable (**Table 2**). At the same time, the relative average number of CFU-E (3.2 ± 1.7%) for EAE mice was 4.6 lower than that for CBA mice (14.7 ± 10.5%), while in the case of CFU-GEMM, the reverse situation was observed; this average value for EAE mice (5.6 ± 1.7%) was ~2.2-fold higher than that for CBA mice (2.6 ± 0.78%) (**Table 2**). In time changes in the proliferation profiles of the HSCs, during 40 days, it was observed for untreated and MOG-treated autoimmune C57BL/6 and non-

with EAE mice are relatively low (**Figures 13** and **14**).

130 Trending Topics in Multiple Sclerosis

and unwilling to autoimmune diseases.

6 and non-autoimmune CBA mice.

autoimmune CBA mice.

proliferation and apoptosis of lymphocytes in various organs.

**6.2. Differentiation of stem cells in the bone marrow of mice**

Total amount of four types of colonies was taken for 100 %; the average meaning± average deviation are given.

\*\* Parameters 1 and 2 from [110] and [111], respectively.

**Table 2.** The average relative content of different colonies of EAE and CBA mice at three month of age (zero time).

It was revealed that there was a constant increase in the relative amount of BFU-E in untreated EAE mice to 28–30 days and then was observe a small decrease (**Figure 15A**). After the treatment of EAE mice with MOG, there was a significant decrease in BFU-E colonies up to 18–20 days and then there was a significant increase (**Figure 15A**). In the case of healthy CBA mice, we revealed quite opposite situation; the relative number of BFU-E in time for untreated mice was decreased, while after treatment with MOG it was significantly increased (**Figure 15B**).

In time changes of CFU-E, before and after treatment of EAE mice was comparable; first there was significant increase from 0 to 10 days and then from 10 to 40 days the number of these colonies slowly, but strongly decreased (**Figure 15C**). CFU-E colonies demonstrated different changes over time in the case of CBA mice. Before and after CBA mice treatment, the relative number of CFU-E cells was remarkably decreased from 0 to approximately 10– 12 days (**Figure 15D**). At the same time, later we observed significant increase in CFU-E for MOG-treated CBA mice, but brightly expressed decrease of the cells in the case of the untreated mice (**Figure 15D**).

Quite opposite types in the differentiation were also observed for CFU-GM forming units in the case of the EAE and CBA mice (**Figures 15 E** and **F**). While the relative number of CFU-GM cells in EAE mice over time was dramatically decreased (0–30 days), in the case of CBA mice, it was remarkably increased (0–40 days). The MOG-treatment of EAE mice led to a slight increase in the number of the CFU-GM colonies, while for CBA mice there was observed a constant decrease the number of these colonies (**Figure 15E** and **F**).

Interestingly, a significant decrease in the CFU-GEMM units in treated EAE mice was observed at 10–40 days, while the level of their decrease for untreated mice was observed only after 20 days (**Figure 15G**). In the case of untreated CBA mice, the relative number of CFU-GEMM units was significantly increased from 0 to 25 days, but then it began to decrease, while treatment of CBA mice with MOG stimulated a constant decrease in the number of these colonies (**Figure 15H**). Thus, the autoimmune C57BL/6 and non-autoimmune CBA mice of three-month age demonstrate comparable percent of BFU-E and CFU-GM colonies, but significant difference in the relative number of CFU-E and CFU-GEMM units (**Figure 15**). In addition, these mice show a completely opposite profile of stem cells differentiation before and after their treatment with MOG (**Figure 15**). However, changes in the differentiation profile in the case of untreated CBA mice are not associated with the appearance of cells producing Abzs; Abs from the sera of these mice are completely catalytically inactive. Immunization of CBA mice with MOG stimulates production of Abs-hydrolyzing MBP, MOG, and DNA, but the activities of these Abzs are very low comparing with those for EAE mice. Specific differ‐ entiation of bone marrow stem cells of EAE mice even before three months of their age leads to the appearance of cells producing abzyme with relatively high activity. During spontaneous development of EAE in C57BL/6 mice, there is a significant increase in such cells and Abzs, while their immunization stimulates a very strong increase in the Abzs activities.

**Figure 15.** In time changes of an average relative content (%) of colony-forming units of bone marrow progenitor colo‐ nies of different type in the case of untreated and MOG-treated mice; average percent of different colonies (BFU-E + CFU-E + CFU-GM + CFU-GEMM) corresponding to 7 mice of each group of autoimmune C57B2/6 and non-autoim‐ mune CBA mice are given; types of progenitor colonies and mice analyzed are shown on panels A–H.

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 133

and after their treatment with MOG (**Figure 15**). However, changes in the differentiation profile in the case of untreated CBA mice are not associated with the appearance of cells producing Abzs; Abs from the sera of these mice are completely catalytically inactive. Immunization of CBA mice with MOG stimulates production of Abs-hydrolyzing MBP, MOG, and DNA, but the activities of these Abzs are very low comparing with those for EAE mice. Specific differ‐ entiation of bone marrow stem cells of EAE mice even before three months of their age leads to the appearance of cells producing abzyme with relatively high activity. During spontaneous development of EAE in C57BL/6 mice, there is a significant increase in such cells and Abzs,

**Figure 15.** In time changes of an average relative content (%) of colony-forming units of bone marrow progenitor colo‐ nies of different type in the case of untreated and MOG-treated mice; average percent of different colonies (BFU-E + CFU-E + CFU-GM + CFU-GEMM) corresponding to 7 mice of each group of autoimmune C57B2/6 and non-autoim‐

mune CBA mice are given; types of progenitor colonies and mice analyzed are shown on panels A–H.

while their immunization stimulates a very strong increase in the Abzs activities.

132 Trending Topics in Multiple Sclerosis

Cuprizone-induced demyelination is a widely used experimental model to study processes of de- and remyelination in the central nervous system [120–123]. We used EAE C57BL/6 mice to assess effects of cuprizone-induced demyelination on different parameters associated with autoimmune inflammation [124]. It was shown that treatment of mice with cuprizone (0–40 days) leads to a significant decrease in the size of the brain corpus callosum when compared with untreated mice. In addition, cuprizone treatment leads to a significant decrease of several indexes, which characterize spontaneous and MOG-induced EAE: increased levels of protei‐ nuria, titters of anti-DNA and anti-MOG antibodies, the generation of Abzshydrolyzing DNA, myelin basic protein (MBP), and MOG. As it mentioned above, spontaneous and MOGinduced EAE is associated with a specific reorganization of the immune system resulting in changes in the profile and level of proliferation of mice HSCs. At day 40 of the experiment, cuprizone decreases the summed proliferation of HSCs (BFU-E, CFU-E, CFU-GM and CFU-GEMM) colony units ~1.2-fold in comparison with untreated mice, but does not significantly change the differentiation profile of BFU-E, CFU-GM, and CFU-GEMM cells. Our data indicate that cuprizone treatment is associated with demyelination, but not with stimulation of autoimmune processes.

It was shown that cuprizone has selective specificity against oligodendrocytes [120, 121]. Acute demyelination following treatment with cuprizone is associated with apoptosis of oligoden‐ drocytes, activation of microglia, and phagocytosis of myelin sheaths [122, 123]. Cuprizone causes demyelination processes all over the brain, with the corpus callosum being the most affected structure. Also distinct foci of demyelination are found in the hippocampus, cerebel‐ lum, putamen, and the ventral part of caudal nuclei.

According to our data, cuprizone-induced demyelination is likely not associated with the development of typical autoimmune processes observed after spontaneous or MOG-induced EAE. It looks more like a specific poisoning of mice with cuprizone, leading to the manifesta‐ tion of some symptoms seen in EAE. The effect of a cuprizone leading to the suppression of cell proliferation and activation of apoptosis [120–123] is consistent with our data on the decrease in the level of lymphocyte proliferation and inhibition of profile differentiation changes of stem cells in the bone marrow. A maximal decrease in the RAs of all Abzs after EAE mice treatment with cuprizone correlates with twofold decrease in the proliferation only CFU-E cells, while its effect on proliferation of other cells was negligible. The relative number of CFU-E colonies over time is significantly decreased in untreated CBA mice (**Figure 15D**), which Abs are completely inactive in the hydrolysis of MBP, MOG, and DNA (**Figure 12**). In addition, maximal activities of Abzs (**Figure 12**) correlate with maximal increase of CFU-E cells in treated CBA mice (**Figure 15**). Moreover, maximal increase in the activities of Abzs in MOG-treated EAE mice at 10–20 days (**Figure 12**) correlates with a sharp increase in the relative amount of these cells at this time (**Figure 15C**). The totality of our data suggests that it is the CFU-E cells may be early progenitors of cells producing catalytic antibodies.

On the one hand, in the debut of various autoimmune diseases usually occurs production of auto-antibodies to different autoantigens and abzyme with different catalytic activities. On the other hand, the repertoires of abzymes with different enzymatic activities and autoantibodies to various autoantigens are constantly expanding. This is indicative of the fact that the basis of all autoimmune diseases may be to some extent similar.

#### **7. Comparison of stem cell differentiation in SLE and EAE mice**

C57BL/6 and MRL-lpr–lpr are two various mouse models spontaneously developing two different autoimmune diseases, EAE and SLE, respectively. Stimulation of EAE pathology development usually occurs after immunization of C57BL/6 mice with MOG [112, 113], while SLE after treatment of MRL-lpr–lpr mice with DNA [22–24]. It was interesting to understand a possible difference or similarity in the changing of the differentiation profiles of stem cells of the bone marrow in the case of these models comparing with those for the control CBA mice before and after their treatment with MOG and with DNA. **Figure 16** demonstrates the relative levels BFU-E, CFU-E, CFU-GM, and CFU-GEMM units (%) at the beginning (zero time), spontaneous changes and after mice treatment with MOG (57BL/6) and DNA (MRL-lpr–lpr) at 40 days of the experiments. One can see that the relative content of BFU-E colonies (%) constantly decrease in autoimmune EAE and SLE mice at transition from zero time to spon‐ taneous development of these diseases (40 days) and acceleration of their development by treatment with MOG and DNA at 40 days (**Figure 16A**).

**Figure 16.** Change in the relative percent (sum of four types of colonies was taken for 100%) of BFU-E (A), CFU-E (B), CFU-GM (C), and BFU-GEMM (D) types of colonies in comparison with zero time of the experiments (first group of values) in the case of spontaneous development of EAE and SLE by respectively C57BL/6 and MRL-lpr–lpr mice after 40 days and changes of differentiation profile of HSCs in CBA mice after 40 days (second group of values); third group of values corresponds to relative amount of the colonies after 40 days in the case C57BL/6 and CBA mice treated with MOG [110, 111] and MRL-lpr–lpr mice immunized with DNA [22–24].

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 135

Non-autoimmune CBA mice demonstrate at 40 days a decrease in the percent of BFU-E, when their treatment with MOG leads to remarkable increase in the relative number of these cells. Very similar regularities in the changes of the relative content of CFU-E (**Figure 16B**) and CFU-GM (**Figure 16C**) are observed for EAE and SLE mice, and they are directly opposite than that for the CBA mice. There is a remarkable but not essentially important difference in the curves corresponding relative number of CFU-GEMM colonies for EAE and SLE mice, but they are very different to that for CBA mice (**Figure 16D**). Thus, it is obvious that over time some changes in the profile of stem cell differentiation can occur in the case of non-autoimmune and autoimmune mice. However, these changes are very different or even opposite for nonautoimmune and autoimmune mice during their growth (CBA) or spontaneous development of ADs (SLE and EAE) as well as after immunization of mice with different specific stimulators of autoimmune processes. Thus, one can suppose that SLE and EAE pathologies in two different autoimmune lines of mice on overall demonstrate very similar regularities of change in differentiation profiles of bone marrow stem cells. Interestingly, at 40 days corresponding to spontaneous development of SLE in MRL-lpr/lpr and EAE in 57BL/6 mice demonstrating high activities of different Abzs, there is a strong increase in CFU-E colonies (**Figure 16B**). At the same time, very strong reduction in the relative number of only CFU-E colonies is observed in the case of CBA mice showing catalytically inactive antibodies (**Figure 16B**). This again is indicative of the fact that CFU-E can be precursors of cells producing Abzs in different organs. In this connection, it should be mentioned that IgGs from CSF of patients with MS 30- to 50 fold more active in the hydrolysis of MBP, DNA, and oligosaccharides comparing with Abs from sera of the same patients [110, 111]. It means, that even CSFs of patients with MS contain specific cells producing these catalytically active Abs.
