F. Nicoletti, Department of Biomedical Sciences, Via Androne n.83, 95124 Catania, Italy. E-mail: email@example.com
In this study, we have evaluated the effects of cyclophosphamide on the development of experimental allergic encephalomyelitis (EAE) in four EAE rodent models: monophasic EAE in Lewis rats, protracted relapsing (PR)-EAE in DA rats, myelin oligodendrocyte protein (MOG)-induced EAE in C57Bl/6 mice and proteolipid protein (PLP)-induced EAE in Swiss/Jackson Laboratory (SJL) mice. Cyclophosphamide, administered either prophylactically or therapeutically, suppressed most strongly the clinical symptoms of PR-EAE in DA rats. Treated rats in this group also exhibited the lowest degree of inflammatory infiltration of the spinal cord, as well as the lowest levels of nuclear factor kappa B, interleukin-12 and interferon-gamma. Cyclophosphamide prophylactically, but not therapeutically, also delayed significantly the onset of EAE in Lewis rats. In contrast, regardless of the treatment regimen used, was unable to influence the clinical course of EAE in either MOG-induced EAE in C57Bl/6 mice or PLP-induced EAE in SJL mice. This heterogeneous pharmacological response to cyclophosphamide suggests that significant immunopathogenic differences exist among these EAE rodent models that must be considered when designing preclinical studies. In addition, the effectiveness of cyclophosphamide in dark Agouti (DA) rats with PR-EAE suggests that this may be a particularly useful model for studying novel therapeutic approaches for refractory and rapidly worsening multiple sclerosis in human patients.
Experimental allergic encephalomyelitis (EAE) is an immunoinflammatory disease of the central nervous system (CNS) that is used widely as a model of multiple sclerosis (MS). EAE can be induced in genetically susceptible rodents by immunization with either whole spinal cord tissue or one of a large number of CNS-derived antigenic myelin proteins. The latter include myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), and are administered usually in adjuvants such as complete Freund's adjuvant (CFA) or pertussis toxin [1,2]. Depending on the immunizing neuroantigen and the rodent strain used, several forms of EAE can be induced, each differing in its clinical, histological and pathogenic features. For example, while immunization of Lewis rats with MBP leads to an immunoinflammatory and non-demyelinating monophasic EAE , Swiss/Jackson Laboratory (SJL) mice and dark Agouti (DA) rats that are immunized, respectively, with PLP and syngeneic whole spinal cord homogenate develop a form of EAE that shows the histological features of demyelination, which is characterized clinically by alternating relapses and remission. In contrast, susceptible mice such as C57Bl/6 that are immunized with myelin oligodendrocyte protein develop a form of progressive demyelinating EAE also characterized by axonal loss [4,5]. These differences indicate that, although the various EAE models each duplicate specific aspects of human MS, none includes all of its features . An understanding of the various histopathological events and pharmacological responses is therefore required to select the most appropriate model to be used for preclinical studies of different forms of human MS [7,8]. For example, whereas Lewis rat EAE mirrors more closely acute and self-remitting immunoinflammatory events of the CNS, relapsing and remitting forms of EAE are models that mimic more closely the corresponding relapsing–remitting (RR) form of MS, and MOG-induced EAE may serve as a preclinical model of progressive MS .
Cyclophosphamide, which is related to nitrogen mustards, is an alkylating agent that interferes with mitosis by binding to DNA. It impairs humoral and cell-mediated immunity by acting on B and T lymphocytes, respectively. Cyclophosphamide is used to treat not only cancer, but also autoimmune diseases such as lupus nephritis, immune-mediated neuropathies and MS [10,11]. Several open-label studies have indicated that cyclophosphamide, either alone or in combination with interferon (IFN)-β, may be effective against the rapidly worsening, treatment-refractory, relapsing–remitting forms of MS [10,12–14]. A large, open-label, retrospective study in patients with primary progressive and secondary progressive MS has also shown a beneficial effect in these patients of 12 monthly pulses of cyclophosphamide, although another study failed to confirm this finding in patients with secondary progressive forms of MS. Despite these conflicting results, as well as the limitations of open-label observations, a general consensus is emerging that cyclophosphamide may be useful in those MS patients with rapidly worsening disease .
The observed variability in the effect of cyclophosphamide against different clinical forms of MS may depend upon the heterogeneous immunopathogenic pathways that underlie the clinical manifestations of the disease. Because the multiple clinical courses of EAE that occur in rodent models are also thought to depend upon heterogeneous encephalitogenic pathways, we evaluated in this study whether, as in human MS, variable pharmacological responses to cyclophosphamide also occur in rodent EAE and which of the models responded most favourably to the drug.
Male Lewis rats weighing between 230 and 270 g, female SJL mice ranging in age from 6 to 7 weeks and female C57BL/6 mice ranging in age from 8 to 10 weeks were purchased from Charles River (Calco, Italy). Female DA rats between 6 and 8 weeks of age, weighing between 130 and 150 g, were purchased from Scanbur AB (Sollentuna, Sweden). The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (D.M. 116192), as well as with European Economic community (EEC) regulations (O.J. of E.C. L 358/1 12/18/1986).
Induction of EAE
PLP-induced EAE in SJL mice. PLP (139–151) was synthesized by Genemed Synthesis (San Francisco, CA, USA). EAE was induced as described previously by . Briefly, mice were immunized with 75 µg PLP emulsified in CFA with 6 mg/ml Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA) to make a 1:1 emulsion. Each mouse received subcutaneous injections of 200 µl emulsion divided among four sites draining into the axillary and inguinal lymph nodes. Pertussis toxin (Calbiochem, Nottingham, UK) was used as a co-adjuvant, and was administered intraperitoneally at a dose of 200 ng/mouse on day 0 and again on day 2.
MOG-induced EAE in C57BL/6 mice. Mice were immunized by injecting subcutaneously into the left flank 0·2 ml of an emulsion composed of 200 µg MOG35-55 peptide (Genemed Synthesis) in CFA (Difco) containing 0·5 mg M. tuberculosis. Immediately after immunization, mice received an intraperitoneal injection of 500 ng pertussis toxin (List Biological Laboratories, Campbell, CA, USA) dissolved in 400 µl buffer (0·5 M NaCl, 0·017% Triton X-100, 0·015 M Tris, pH 7·5).
MBP-induced EAE in Lewis rats. Lewis rats were immunized by subcutaneous injection at the base of the tail with 50 µg guinea pig MBP (Sigma-Aldrich, Milan, Italy) and 2 mg M. tuberculosis strain H37RA (Difco) in Freund's incomplete adjuvant (Difco) to make a 1:1 emulsion in a final volume of 200 µl.
Spinal cord homogenate (SCH)-induced EAE in DA rats. The DA rat was chosen as a rodent species as it is widely accepted as an experimental model. The selected strain has susceptibility to PR-EAE, as documented in our and other laboratories [16–19]. Rats were immunized on day 0 by subcutaneous injection, at the base of the tail, with 0·2 ml of a homogenized emulsion composed of 50 mg whole DA rat spinal cord in CFA and 10 mg/ml M. tuberculosis (both from Difco).
Cyclophosphamide was administered at a dose of 40 mg/kg under either a prophylactic or therapeutic regimen. Cyclophosphamide was dissolved in sterile water and injected intraperitoneally in a final volume of 500 µl. The main prophylactic regimen consisted of a single injection of cyclophosphamide on the seventh day after immunization, prior to the development of clinical signs of the disease. In additional studies, Lewis rats and DA rats were treated prophylactically on both days 0 and 10. For the therapeutic treatment, animals developing chronic and relapsing remitting forms received two doses of cyclophosphamide, separated by a 14-day interval, after they had developed a clinical EAE score of at least 1. The exception to this schedule was the Lewis rat EAE group, in which the animals whose clinical signs had recovered spontaneously by the time that the second injection of cyclophosphamide would have been applied received only a single injection of cyclophosphamide. Control animals were treated with sterile water under the same experimental conditions. An additional group of sham-treated rats was included for comparison.
Animals were observed daily by measuring their body weight and assessing clinical signs of EAE. They were assigned one of the following clinical grades by an observer who was blind to the treatment given: 0, no illness; 1, flaccid tail; 2, moderate paraparesis; 3, severe paraparesis; 4, moribund state; and 5, death. A slightly different EAE scoring was used for MOG-induced EAE: 0·5 = partial tail paralysis; 1 = tail paralysis; 1·5 = tail paralysis + partial unilateral hindlimb paralysis; 2 = tail paralysis + hindlimb weakness or partial hindlimb paralysis; 2·5 = tail paralysis + partial hindlimb paralysis (lowered pelvi); 3 = tail paralysis + complete hindlimb paralysis; 3·5 = tail paralysis + complete hindlimb paralysis + incontinence; 4 = tail paralysis + hindlimb paralysis + weakness or partial paralysis of forelimbs; and 5 = moribund or dead .
A cumulative clinical score was calculated for each mouse by adding the daily scores from the day of onset (score disease ≥ 1) until the end of the experiment. Duration of disease was calculated each day by assigning the animal a score of 0 for a clinical score of 0 and 1 for any higher clinical score. The maximal score reached at the peak of the disease was considered to be the maximal clinical score for each rat.
Histological analyses and detection of spinal cord cytokines and NF-κB levels
One additional study consisting of 36 rats was performed for the measurement of spinal cord cytokines and NF-κB levels. The animals were killed under terminal anaesthesia with isofluorane 12 days after the beginning of treatment and 12 of each group were used for histological analyses and detection of spinal cord cytokines and NF-κB levels. A group of 12 sham-treated rats was included for comparison.
Histological analyses and detection of spinal cord cytokines and NF-κB levels were carried out only in the PR-EAE model of DA rats, as this was the only EAE model that showed an unambiguous clinical benefit from the administration of cyclophosphamide. For the purpose of these studies, the rats were treated under the cyclophosphamide therapeutic regimen when they had exhibited a clinical EAE score greater than 1. They were killed 12 days after treatment.
For histological analysis, half the animals in each of the vehicle- and cyclophosphamide-treated groups were perfused, under isofluorane anaesthesia, through the left ventricle with cold phosphate-buffered saline (PBS) (4°C) for 3–5 min, and then with 4% paraformaldehyde (Sigma-Aldrich) for 10 min. The brain and spinal cord were resected and stored in 4% paraformaldehyde at 4°C. Serial, 4-mm cross-sections were prepared from the spinal cord and brain and were stained with haematoxylin and eosin (H&E) to assess inflammation .
Histopathological parameters were evaluated as described previously [19,22]. The inflammatory index was calculated from the mean number of perivascular inflammatory infiltrates visible in an average of 15 complete spinal cord cross-sections from each animal.
Protein extraction. The proteins were measured as described previously . In brief, spinal cords were harvested from nine non-perfused rats, snap-frozen and pooled. Each pool was homogenized in PBS for 15 min at 4°C, and then enriched with 1 mM sodium orthovanadate, 0·5 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM phenymethylsulphonyl fluoride and protease inhibitor mixture (Sigma). The homogenates were centrifuged at 8000 g at 4°C for 15 min. Protein quantification of supernatants was performed using the Bradford method (Biorad, Hercules, CA, USA).
The amounts of cytokine interleukin (IL)-12 (p40/p70), IFN-γ and IL-4 in the protein lysates were quantified by commercially available enzyme-linked immunosorbent assay (ELISA) kit [R&D Systems (Space Import-Export srl, Milano, Italy) for IL-4 and IFN-γ; Invitrogen (Invitrogen Srl, Milano, Italy) for IL-12], according to the manufacturer's instructions.
NF-κB measurement. As an index of NF-κB activation, NF-κB p65 translocation into the nucleus was measured using a sandwich ELISA, according to the manufacturer's protocol (ActivELISATM; Imgenex, Analitica De Mori, Milan, Italy). In brief, 1 g of spinal cord pooled from three rats of a total of 12 rats from each experimental group was cut into small pieces, washed with cold PBS, homogenized in hypotonic buffer and centrifuged for 10 min at 4400 g. The supernatant was used as a cytoplasmic extract. A 500 µl aliquot of nuclear lysis buffer [dithiothreitol (DTT), protease inhibitor cocktail] was added to the pellet and centrifuged at 8000 g for 10 min at 4°C. This supernatant was used as a nuclear extract. For each sample, containing 0·5–1 mg/ml of protein, plates coated with anti-NF-κB p65 antibody were used to capture the nuclear or cytoplasmic NF-κB p65. The amount of bound NF-κB p65 was detected by adding a secondary antibody followed by an alkaline phosphatase-conjugated antibody. The absorbance value for each well was determined at 405 nm using a microplate reader (Bio-Rad). The ratio of nuclear to cytoplasmic NF-κB p65 was calculated from the respective absorbance values (antibodies nuclear fraction/antibodies cytoplasmic fraction).
The clinical results are shown as mean ± standard deviation (s.d.), which was calculated based on data from two independent experiments. Because the experiments were highly reproducible and with variability lower than 5%, the data are merged and shown as a single study. Statistical analysis for significant differences on clinical scores was performed with Student's t-test for unpaired data for the cytokine and NF-κB levels, with the non-parametric Mann–Whitney U-test for the clinical course of EAE and histopatological parameters, and with the χ2 test for evaluation of the incidence of disease.
PLP-induced EAE in SJL mice
EAE in SJL mice is characterized clinically by an acute onset that occurs 7–14 days after immunization, and which usually progresses from tail flaccidity or hind limb weakness to total hind limb paralysis and moderate forelimb weakness, potentially extending to quadriplegia and even death (score 5) .
Accordingly, in the mice we studied, classical clinical signs began to appear on day 8 after immunization. Variable relapses followed the first attack, with some mice exhibiting remission of clinical signs and up to one or two relapses (Fig. 1a and b, Tables 1 and 2). Neither prophylactic nor therapeutic treatment with cyclophosphamide ameliorated the clinical course of the disease, with mice in both experimental groups attaining scores comparable to those of control animals (Fig. 1a and b, Tables 1 and 2).
Table 1. The effects of cyclophosphamide prophylaxis on clinical readouts.
P < 0·05,
P < 0·01,
P < 0·0001 versus control. EAE, experimental allergic encephalomyelitis; DA, dark Agouti; SJL, Swiss/Jackson Laboratory.
Within 2 weeks after immunization with CFA–MOG emulsion, C57BL/6 mice are known typically to develop a progressively ascending paralysis of the primary progressive form . In the present study, classical clinical signs appeared beginning on day 7 after immunization, and followed a progressive course with intervals of small relapses (Fig. 2a and b, Tables 1 and 2). No significant differences were observed between the clinical scores of control animals and animals treated under either the prophylactic or therapeutic cyclophosphamide regimens (Fig. 2a and b, Tables 1 and 2).
MBP-induced EAE in Lewis rats
The first clinical signs of EAE in the Lewis rat occur typically 10–12 days after immunization, and progress from loss of tail tonicity to hind limb paralysis. Most rats recover 18–20 days after immunization [3,17,26,27].
In the control animals we studied, clinical signs began to appear on day 9 after immunization, with the recovery phase beginning on day 20. Although prophylactic treatment with cyclophosphamide on day 7 delayed the onset of the disease significantly, it failed to influence the cumulative clinical score or the duration of the disease (Fig. 3a, Table 1). The initial beneficial effect of the prophylactic regimen prompted us to carry out an additional study, in which cyclophosphamide was administered 6 h prior to the immunizing challenge and again 10 days after immunization. This dual treatment had the same ability as the single prophylactic injection to delay the onset of the disease, but again failed to ameliorate the overall clinical score (Fig. 3b, Table 1).
When cyclophosphamide was administered under a therapeutic regimen, no significant difference in clinical score was seen between control and treated animals (Fig. 3c, Table 2).
SCH-induced EAE in DA rats
SCH-induced EAE in DA rats is characterized by a severe, protracted, relapsing and demyelinating course, which makes this model a useful in vivo tool for studying the immune-mediated mechanisms involved in the generation of chronicity and demyelination [16,18,19].
As expected, beginning on day 9 after immunization, classical signs of EAE started to appear in rats of all groups. Variable protracted relapses followed the first attack in the vehicle-treated control group, with some animals exhibiting remission of clinical signs and up to one or two relapses. The onset of the disease was delayed in rats treated prophylactically with cyclophosphamide. However, the protective effects of cyclophosphamide appeared to be transient, as these rats subsequently developed PR-EAE with a similar clinical course to that of control rats. No differences in the other clinical parameters, including cumulative score, mean maximal score, duration or cumulative incidence were observed between cyclophosphamide-treated and control rats at the end of the observation period on day 45 (Fig. 4a, Table 1).
In contrast, when cyclophosphamide was administered twice under a prophylactic regimen on day 0 as well as on day 10, we observed a long-lasting protective effect that lasted throughout the observation period until day 45, which led to an almost complete and permanent abrogation of EAE signs (Fig. 4b, Table 1).
Cyclophosphamide was also effective in causing the reversion of clinically active PR-EAE when administered therapeutically to rats with established clinical signs of the disease (Fig. 4c, Table 2). However, following the second therapeutic treatment with cyclophosphamide, administered 14 days after the first, severe relapses occurred in a pattern similar to those seen with rats that had received a single prophylactic injection of cyclophosphamide (Fig. 4d, Table 2).
Histopathological analysis of SCH-induced EAE in DA rats
The histopathological analysis of CNS samples from control rats showed inflammatory infiltration (Fig. 5a and b). In contrast, samples from cyclophosphamide-treated rats showed significantly milder histopathological features, with fewer inflammatory infiltrates (Fig. 5c). Consequently, the histological scores of cyclophosphamide-treated rats were significantly lower than those of their respective controls (Fig. 5a).
Detection of spinal cord cytokines and NF-κB levels of SCH-induced EAE in DA rats
As expected, in comparison to sham-treated rats, SCH from vehicle and cyclophosphamide-treated rats showed higher levels of IFN-γ, IL-12, NF-κB p65 and the NF-κB p65 nuclear-to-cytoplasmic ratio (Figs 6a and b and 7a and b). Cyclophosphamide treatment reduced significantly the spinal cord levels of IFN-γ, tumour necrosis factor (TNF)-α, IL-12 and NF-κB p65, as well as the NF-κB p65 nuclear-to-cytoplasmic ratio. The content of IL-4 in spinal cord homogenates in most samples was below the limit of sensitivity and no significant difference could be observed between the rats treated with cyclophosphamide, those treated with the vehicle and the sham group of animals (Fig. 6c).
Our results show that both under prophylactic and therapeutic regimens, the most powerful suppression of clinical symptoms of EAE by cyclophosphamide was achieved in PR-EAE of DA rats. These rats also exhibited less inflammatory infiltration of the spinal cord and lower levels of NF-κB, IL-12 and IFN-γ than vehicle-treated controls. Cyclophosphamide prophylaxis, but not therapy, also delayed the onset of EAE in Lewis rats significantly. In contrast, regardless of the treatment regimen, cyclophosphamide was unable to influence the clinical course of MOG-induced EAE in C57Bl/6 mice or PLP-induced EAE in SJL mice.
Our results represent the first unambiguous demonstration of the ability of cyclophosphamide prophylaxis to delay or, depending upon treatment regimen, even suppress EAE development permanently in DA rats, as well as its capacity to rapidly revert the course of EAE when administered therapeutically to rats with established disease. When different treatment regimens were evaluated, both the single prophylactic treatment applied on day 7 after immunization nor the standard therapeutic regimen given at the onset of EAE, and again 14 days later, were associated with the reappearance of EAE within 10–14 days after treatment withdrawal. In contrast, administration of cyclophosphamide on a prophylactic regimen, on the day of immunization and 10 days later, led to permanent protection against EAE development. This suggests that cyclophosphamide administration that is timed appropriately with respect to encephalitogenic process may have the capacity to abrogate temporarily or even permanently the demyelinating potential of effector autoreactive lymphocytes. Although the observed rapid reversal of the protective effect suggests that it might be based upon active immunopharmacological suppression of the function of autoreactive cells, the permanent protection against EAE development suggests a mechanism involving either augmented generation or function of regulatory T cells (Tregs) or the deletion of autoreactive effectors. However, the possibility that cyclophosphamide has exerted its beneficial effects by stimulating Treg generation or enhancing their functional activity are difficult to reconcile with the inhibitory effects of this drug on CD4+CD25+forkhead box P3 (FoxP3+) Tregs that has been demonstrated clearly in preclinical model of cancer . As Tregs are thought to play a disease-limiting role in autoimmune pathologies, including MS, suppression of these cells with cyclophosphamide would probably be associated with exacerbation of these pathologies. The different experimental settings considered may account for possible differences of cyclophosphamide on Tregs. For example, Tregs in autoimmune conditions may be more resistant to the inhibitory action of cyclophosphamide than Tregs in cancer models. Consistent with this, doses of cyclophosphamide up to 200 mg/kg are needed to abrogate Treg functions in autoimmune diabetes in the non-obese diabetic mouse (NOD) mouse and accelerate the onset of autoimmune diabetes . Additional studies will be carried out to dissect the mode of action underlying the different degrees and modes of clinical protection afforded by cyclophosphamide in PR-EAE of DA rats, including its effects on the number and functional activity of Tregs. Studies are also in progress to evaluate whether the lack of effects of cyclophosphamide in the mouse models studied may depend upon a higher susceptibility of mice than rats to the inhibitory effects of cyclophosphamide on Tregs.
In addition to benefiting clinically from the therapeutic application of cyclophosphamide, treated rats also exhibited milder histological signs of EAE, with fewer inflammatory infiltrates in spinal cord and brain tissue compared to controls. The spinal cord homogenates from treated rats also contained less activated NF-κB than did those from controls, as well as lower levels of proinflammatory cytokines such as IL-12 and IFN-γ. The local inhibition of production of these proinflammatory cytokines, which have been implicated in the pathogenesis of both EAE and MS, might have contributed to the beneficial effects of cyclophosphamide in the PR-EAE of DA rats. Interestingly, these data resemble closely the inhibition of IL-12 and IFN-γ production observed in the peripheral blood of MS patients treated with cyclophosphamide [30,31]. However, it should be noted that the above finding does not point to any particular mode of action of cyclophosphamide in this setting, as the phenomenon might have been secondary to milder leucocytic infiltration of the spinal cords rather than representing a direct inhibitory action of cyclophosphamide on the production of these cytokines.
We have also shown here that cyclophosphamide prophylaxis was able to delay the onset of monophasic EAE in the Lewis rats, but failed to influence the course of the disease when given therapeutically. Similarly variable effects of cyclophosphamide have been reported previously in the monophasic EAE of the Lewis rat, and have been shown to depend upon treatment variables such as dose and the period of treatment in relation to immunization. For example, a dose of 100–200 (but not 20) mg/kg cyclophosphamide given to convalescent rats led to a reappearance of EAE and also abrogated their acquired resistance to EAE reinduction [32,33]. Other studies have shown that treatment with 40 mg/kg cyclophosphamide 2 days before immunization resulted in a marked potentiation of EAE in Fischer and piebald-viral-Glaxo (PVG) rats, but not in brown Norway (BN) rats or F1 (BN × F) hybrids . Similar data were obtained in albino Oxford (AO) rats by Mostarica-Stojkovic et al.. These findings suggest that cyclophosphamide-sensitive mechanisms of suppression of EAE development exist in Fisher, PVG and AO rats, and that these differ from those of BN rats. They also highlight apparent differences in sensitivity to the immunoregulatory action of cyclophosphamide within the Lewis rat model that appear to depend on the dose of drug and the time of its application in relation to EAE development.
Finally, we noticed no significant effect of either prophylactic or therapeutic treatment with cyclophosphamide on the development of either MOG-induced or PLP induced EAE in C57/Bl6 mice and SJL mice, respectively. This provides the first evidence that these models may be refractory to immunopharmacological manipulation by cyclophosphamide.
Taken together, the different responses of these four preclinical rodent models of EAE to cyclophosphamide provide further evidence that they may depend on heterogeneous immunopathogenic pathways. This may translate into either different clinical manifestations or, as shown here, a differential susceptibility to pharmacological intervention. The favourable response of DA rats with PR-EAE to either prophylactic or therapeutic treatment with cyclophosphamide is of considerable relevance from both a preclinical and clinical perspective as, in light of the beneficial response of severe and rapidly worsening MS to cyclophosphamide , this model may be particularly useful to study novel approaches for the treatment of these forms of disease. The present study warrants additional immunopathogenic and pharmacological studies to obtain additional evidences able of supporting the eventual superiority of PR-EAE in DA rats to other models with regard to its pathogenic similarity and pharmacological predictivity to severe and rapidly worsening forms of MS.