Multiple Sclerosis Articles 1

© 2010

Estrogen and Testosterone Therapies in Multiple Sclerosis

            (Gold and Voskuhl 2009) Download

It has been known for decades that females are more susceptible than men to inflammatory autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis, and psoriasis. In addition, female patients with these diseases experience clinical improvements during pregnancy with a temporary "rebound" exacerbation postpartum. These clinical observations indicate an effect of sex hormones on disease and suggest the potential use of the male hormone testosterone and the pregnancy hormone estriol, respectively, for the treatment of MS. A growing number of studies using the MS animal model experimental autoimmune encephalomyelitis (EAE) support a therapeutic effect of these hormones. Both testosterone and estriol have been found to induce anti-inflammatory as well as neuroprotective effects. Findings from two recent pilot studies of transdermal testosterone in male MS patients and oral estriol in female MS patients are encouraging. In this paper, we review the preclinical and clinical evidence for sex hormone treatments in MS and discuss potential mechanisms of action.

Immune modulation and increased neurotrophic factor production in multiple sclerosis patients treated with testosterone

(Gold, Chalifoux et al. 2008) Download

BACKGROUND: Multiple sclerosis is a chronic inflammatory disease of the central nervous system with a pronounced neurodegenerative component. It has been suggested that novel treatment options are needed that target both aspects of the disease. Evidence from basic and clinical studies suggests that testosterone has an immunomodulatory as well as a potential neuroprotective effect that could be beneficial in MS. METHODS: Ten male MS patients were treated with 10 g of gel containing 100 mg of testosterone in a cross-over design (6 month observation period followed by 12 months of treatment). Blood samples were obtained at three-month intervals during the observation and the treatment period. Isolated blood peripheral mononuclear cells (PBMCs) were used to examine lymphocyte subpopulation composition by flow cytometry and ex vivo protein production of cytokines (IL-2, IFNgamma, TNFalpha, IL-17, IL-10, IL-12p40, TGFbeta1) and growth factors (brain-derived neurotrophic factor BDNF, platelet-derived growth factor PDGF-BB, nerve growth factor NGF, and ciliary neurotrophic factor CNTF). Delayed type hypersensitivity (DTH) skin recall tests were obtained before and during treatment as an in vivo functional immune measure. RESULTS: Testosterone treatment significantly reduced DTH recall responses and induced a shift in peripheral lymphocyte composition by decreasing CD4+ T cell percentage and increasing NK cells. In addition, PBMC production of IL-2 was significantly decreased while TGFbeta1 production was increased. Furthermore, PBMCs obtained during the treatment period produced significantly more BDNF and PDGF-BB. CONCLUSION: These results are consistent with an immunomodulatory effect of testosterone treatment in MS. In addition, increased production of BDNF and PDGF-BB suggests a potential neuroprotective effect. TRIAL REGISTRATION: NCT00405353 http://www.clinicaltrials.gov.

Testosterone treatment in multiple sclerosis: a pilot study

(Sicotte, Giesser et al. 2007) Download

OBJECTIVE: To study the effect of testosterone supplementation on men with multiple sclerosis (MS). DESIGN, SETTING, AND PARTICIPANTS: Men are less susceptible to many autoimmune diseases, including MS. Possible causes for this include sex hormones and/or sex chromosome effects. Testosterone treatment ameliorates experimental allergic encephalomyelitis, an animal model of MS, but the effect of testosterone supplementation on men with MS is not known. Therefore, 10 men with relapsing-remitting MS were studied using a crossover design whereby each patient served as his own control. There was a 6-month pretreatment period followed by a 12-month period of daily treatment with 10 g of the gel containing 100 mg of testosterone. MAIN OUTCOME MEASURES: Clinical measures of disability and cognition (the Multiple Sclerosis Functional Composite and the 7/24 Spatial Recall Test) and monthly magnetic resonance imaging measures of enhancing lesion activity and whole brain volumes. RESULTS: One year of treatment with testosterone gel was associated with improvement in cognitive performance (P = .008) and a slowing of brain atrophy (P <.001). There was no significant effect of testosterone treatment on gadolinium-enhancing lesion numbers (P = .31) or volumes (P = .94). Lean body mass (muscle mass) was increased (P = .02). CONCLUSION: These exploratory findings suggest that testosterone treatment is safe and well tolerated and has potential neuroprotective effects in men with relapsing-remitting MS.

Multiple sclerosis: a complicated picture of autoimmunity

            (McFarland and Martin 2007) Download

Understanding of autoimmune diseases, including multiple sclerosis, has expanded considerably in recent years. New insights have been provided by not only animal models but also studies of patients, often in conjunction with experimental therapies. It is accepted that autoimmune T cells mediate the early steps of new multiple sclerosis lesions, and although uncertainties remain about the specific targets of autoreactive T cells, several studies indicate myelin antigens. Recent findings obtained with both animal models and patients with multiple sclerosis indicate involvement of a T helper cell with a T(H)-17 phenotype, in contrast to previous data indicating that T helper type 1 cells are critical. Evidence has also been presented for CD8(+) and regulatory T cell populations, although their involvement remains to be established. Despite evidence supporting the idea that autoreactive T cells are involved in disease induction, cells of myeloid lineage, antibodies and complement as well as processes intrinsic to the central nervous system seem to determine the effector stages of tissue damage. Careful analysis of the alterations in immune processes should further advance knowledge of the relationship between the inflammatory component of this disease and the more diffuse degeneration of progressive multiple sclerosis.

Sex ratio of multiple sclerosis in Canada: a longitudinal study

            (Orton, Herrera et al. 2006) Download

BACKGROUND: Incidence of multiple sclerosis is thought to be increasing, but this notion has been difficult to substantiate. In a longitudinal population-based dataset of patients with multiple sclerosis obtained over more than three decades, we did not show a difference in time to diagnosis by sex. We reasoned that if a sex-specific change in incidence was occurring, the female to male sex ratio would serve as a surrogate of incidence change. METHODS: Since environmental risk factors seem to act early in life, we calculated sex ratios by birth year in 27 074 Canadian patients with multiple sclerosis identified as part of a longitudinal population-based dataset. FINDINGS: The female to male sex ratio by year of birth has been increasing for at least 50 years and now exceeds 3.2:1 in Canada. Year of birth was a significant predictor for sex ratio (p<0.0001, chi(2)=124.4; rank correlation r=0.84). INTERPRETATION: The substantial increase in the female to male sex ratio in Canada seems to result from a disproportional increase in incidence of multiple sclerosis in women. This rapid change must have environmental origins even if it is associated with a gene-environment interaction, and implies that a large proportion of multiple sclerosis cases may be preventable in situ. Although the reasons why incidence of the disease is increasing are unknown, there are major implications for health-care provision because lifetime costs of multiple sclerosis exceed pound1 million per case in the UK.

The window of therapeutic opportunity in multiple sclerosis

(Coles, Cox et al. 2006) Download

From 1991-2002, we treated 58 patients with multiple sclerosis (MS) using the humanised monoclonal antibody, Campath-1H, which causes prolonged T lymphocyte depletion. Clinical and surrogate markers of inflammation were suppressed. In both the relapsing-remitting (RR) and secondary progressive (SP) stages of the illness, Campath-1H reduced the annual relapse rate (from 2.2 to 0.19 and from 0.7 to 0.001 respectively; both p < 0.001). Remarkably, MRI scans of patients with SP disease, treated with Campath-1H 7 years previously, showed no new lesion formation. However, despite these effects on inflammation, disability was differently affected depending on the phase of the disease. Patients with SPMS showed sustained accumulation of disability due to uncontrolled progression marked by unrelenting cerebral atrophy, attributable to ongoing axonal loss. The rate of cerebral atrophy was greatest in patients with established cerebral atrophy and highest inflammatory lesion burden before treatment (2.3 versus 0.7 ml/year; p = 0.04). In contrast, patients with RR disease showed an impressive reduction in disability at 6 months after Campath-1H (by a mean of 1.2 EDSS points) perhaps owing to a suppression of on-going inflammation in these patients with unusually active disease. In addition, there was a further significant, albeit smaller, mean improvement in disability up to 36 months after treatment. We speculate that this represents the beneficial effects of early rescue of neurons and axons from a toxic inflammatory environment, and that prevention of demyelination will prevent long-term axonal degeneration. These concepts are currently being tested in a controlled trial comparing Campath-1H and IFN-beta in the treatment of drug-naive patients with early, active RR MS.

Dehydroepiandrosterone and ageing

            (Arlt 2004) Download

Dehydroepiandrosterone (DHEA) is the major steroid produced by the adrenal zona reticularis and, in contrast to cortisol and aldosterone, its secretion declines with ageing. This has generated major interest in its putative role as an 'anti-ageing' hormone. However, it is not clear that the age-associated, physiological decline in DHEA secretion represents a harmful deficiency. DHEA exhibits its action mainly by conversion to sex steroids. In addition, DHEA has neurosteroidal properties and may exhibit direct action via specific binding sites on endothelial cells. There is convincing evidence for beneficial effects of DHEA in patients with adrenal insufficiency and future research will hopefully elucidate its role in patients receiving pharmacological glucocorticoid treatment. However, in healthy elderly subjects, current evidence from randomised, controlled trials does not justify the use of DHEA, with no major beneficial effects reported and, in addition, potentially adverse effects on sex steroid-dependent tumour growth need to be considered.

Estriol treatment ameliorates disease in males with experimental autoimmune encephalomyelitis: implications for multiple sclerosis

(Palaszynski, Liu et al. 2004) Download

Estrogen treatment has been found to be protective in experimental autoimmune encephalomyelitis (EAE) and possibly multiple sclerosis (MS). We investigated whether the effect of estrogen treatment is gender-specific. Estrogen receptor (ER) expressions, ERalpha and ERbeta, were found to be equivalent in both genders. EAE disease severity in both females and males was decreased with estriol treatment as compared to placebo. Finally, proinflammatory cytokine production during autoantigen-specific immune responses was decreased with estriol treatment in both females and males. These data support a potential role for estriol treatment for men in addition to women with MS.

Cutting edge: estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment

(Polanczyk, Carson et al. 2004) Download

CD4(+)CD25(+) regulatory T cells are crucial to the maintenance of tolerance in normal individuals. However, the factors regulating this cell population and its function are largely unknown. Estrogen has been shown to protect against the development of autoimmune disease, yet the mechanism is not known. We demonstrate that estrogen (17-beta-estradiol, E2) is capable of augmenting FoxP3 expression in vitro and in vivo. Treatment of naive mice with E2 increased both CD25(+) cell number and FoxP3 expression level. Further, the ability of E2 to protect against autoimmune disease (experimental autoimmune encephalomyelitis) correlated with its ability to up-regulate FoxP3, as both were reduced in estrogen receptor alpha-deficient animals. Finally, E2 treatment and pregnancy induced FoxP3 protein expression to a similar degree, suggesting that high estrogen levels during pregnancy may help to maintain fetal tolerance. In summary, our data suggest E2 promotes tolerance by expanding the regulatory T cell compartment.

Estrogen treatment induces a novel population of regulatory cells, which suppresses experimental autoimmune encephalomyelitis

(Matejuk, Bakke et al. 2004) Download

Multiple sclerosis (MS) is a debilitating neurological disease characterized by a progressive loss of motor and sensory function, eventually leading to paralysis and death. The primary cause of neurological impairment is demyelination of the central nervous system (CNS) caused by an inflammatory autoimmune response. Previous studies have shown that the severity of MS is reduced during pregnancy, suggesting that the increased level of sex hormones may reduce the autoimmune response. Recently, we have shown that estrogen treatment confers protection from experimental autoimmune encephalomyelitis (EAE), which is an animal model for MS. However, the cellular basis of estrogen's action remains unknown. In the current study, we demonstrate that estrogen treatment led to the induction of a novel subpopulation of regulatory cells in spleen and CNS, which also occurs naturally in pregnant mice. These previously uncharacterized cells display a low level expression of CD45 (CD45(dim)) and no detectable expression of many cell surface markers related to TCR signaling, including CD3 and TCR. However, these cells retained expression of VLA-4, an extracellular protein involved in cellular migration. Several lines of evidence suggest that these novel cells, defined as CD45(dim)VLA-4(+) cells, may play a role in the protective effects of estrogen in EAE. Injection of purified CD45(dim)VLA-4(+) cells conferred protection from spontaneous EAE (Sp-EAE). In contrast, injection of CD45(high)VLA-4(+) cells exacerbated the disease course. CD45(dim)VLA-4(+) cells also suppressed antigen-specific proliferation of primed lymphocytes in coculture. A better understanding of how CD45(dim)VLA-4(+) cells suppress the harmful immune response of EAE may help in explaining the induction of immune tolerance during pregnancy and lead to novel therapeutic approaches to combat MS and other autoimmune diseases.

Neuroprotective role of testosterone in the nervous system.

            (Bialek, Zaremba et al. 2004) Download

Testosterone -- the gonadal sex steroid hormone plays an important role in the central nervous system (CNS) development. One of the less known testosterone actions is neuroprotection. There are some evidences supporting the hypothesis that testosterone may act protectively in neurodegenerative disorders, e.g. Alzheimer's disease (AD), mild cognitive impairment (MCI) or depression. Androgens alter also the morphology, survival and axonal regeneration of motor neurons. These hormones accelerate the regeneration of hamster facial nerve and anterior tibialis sciatic nerve in rabbits following crush axotomy. Androgens exert trophic action in laryngeal motor nucleus of Xenopus laevis. Testosterone is linked to an increase in neuron somal size, neuritic growth, plasticity and synaptogenesis in both motoneurons of the spinal nucleus of the bulbocavernosus and several populations of pelvic autonomic neurons. The hormone reduced the extent of spinal cord damage in vitro. There are also evidences against the neuroprotective action of testosterone. Testosterone does not protect against methamphetamine-induced neurotoxicity of the dopaminergic system in mice and does not provide significant neuroprotection against glutamate-induced neurotoxicity. Androgens do not prevent striatal dopamine depletion induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Although the role of testosterone in the CNS is still poorly understood, accumulating evidence suggests that testosterone may create a future treatment for MCI and related cognitive diseases, including dementia and may influence motor neuron regeneration in adulthood. Androgen replacement therapy in selected male populations may hold therapeutic promise for the prevention and/or treatment of age-related disorders associated with neuronal injury.

Androgens and the ageing male

(Swerdloff and Wang 2004) Download

Serum testosterone levels peak in early adulthood in men and fall progressively with age. Since sex hormone binding globulin increases with age, the unbound forms of testosterone (free and bioavailable testosterone) fall more steeply than total testosterone levels. Serum testosterone levels below the normal range for young healthy adult males provide chemical evidence of androgen deficiency independent of the age of the patient. When accompanied by signs or symptoms that are compatible with androgen deficiency, treatment with testosterone should be considered in older men without evidence of prostate or breast cancer. While such therapy for younger hypogonadal men has shown benefit on libido, mood, muscle mass, muscle strength, bone mineral density and haematocrit, similar benefits in older men have not been as adequately assessed. While there is no convincing evidence that testosterone treatment in older men will increase the risk of cardiovascular or prostate cancer, long-term, well-controlled studies are lacking and needed. Treatment options for older men include injectable, transdermal and transbuccal testosterone preparations.

Pregnancy and multiple sclerosis (the PRIMS study): clinical predictors of post-partum relapse

            (Vukusic, Hutchinson et al. 2004) Download

The influence of pregnancy in multiple sclerosis has been a matter of controversy for a long time. The Pregnancy in Multiple Sclerosis (PRIMS) study was the first large prospective study which aimed to assess the possible influence of pregnancy and delivery on the clinical course of multiple sclerosis. We report here the 2-year post-partum follow-up and an analysis of clinical factors which might predict the likelihood of a relapse in the 3 months after delivery. The relapse rate in each trimester up to the end of the second year post-partum was compared with that in the pre-pregnancy year. Clinical predictors of the presence or absence of a post-partum relapse were analysed by logistic regression analysis. Using the best multivariate model, women were classified as having or not having a post-partum relapse predicted, and this was compared with the observed outcome. The results showed that, compared with the pre-pregnancy year, there was a reduction in the relapse rate during pregnancy, most marked in the third trimester, and a marked increase in the first 3 months after delivery. Thereafter, from the second trimester onwards and for the following 21 months, the annualized relapse rate fell slightly but did not differ significantly from the relapse rate recorded in the pre-pregnancy year. Despite the increased risk for the 3 months post-partum, 72% of the women did not experience any relapse during this period. Confirmed disability continued to progress steadily during the study period. Three indices, an increased relapse rate in the pre-pregnancy year, an increased relapse rate during pregnancy and a higher DSS (Kurtzke's Disability Status Scale) score at pregnancy onset, significantly correlated with the occurrence of a post-partum relapse. Neither epidural analgesia nor breast-feeding was predictive. When comparing the predicted and observed status, however, only 72% of the women were correctly classified by the multivariate model. In conclusion, the results for the second year post-partum confirm that the relapse rate remains similar to that of the pre-pregnancy year, after an increase in the first trimester following delivery. Women with greater disease activity in the year before pregnancy and during pregnancy have a higher risk of relapse in the post- partum 3 months. This is, however, not sufficient to identify in advance women with multiple sclerosis who are more likely to relapse, especially for planning therapeutic trials aiming to prevent post-partum relapses.

Immune modulation in multiple sclerosis patients treated with the pregnancy hormone estriol

(Soldan, Alvarez Retuerto et al. 2003) Download

The protective effect of pregnancy on putative Th1-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, is associated with a Th1 to Th2 immune shift during pregnancy. The hormone estriol increases during pregnancy and has been shown to ameliorate experimental autoimmune encephalomyelitis and collagen-induced arthritis. In addition, estrogens induce cytokine changes consistent with a Th1 to Th2 shift when administered in vitro to human immune cells and in vivo to mice. In a pilot trial, oral estriol treatment of relapsing remitting multiple sclerosis patients caused significant decreases in enhancing lesions on brain magnetic resonance imaging. Here, the immunomodulatory effects of oral estriol therapy were assessed. PBMCs collected longitudinally during the trial were stimulated with mitogens, recall Ags, and glatiramer acetate. Cytokine profiles of stimulated PBMCs were determined by intracellular cytokine staining (IL-5, IL-10, IL-12 p40, TNF-alpha, and IFN-gamma) and cytometric bead array (IL-2, IL-4, IL-5, IL-10, TNF-alpha, and IFN-gamma). Significantly increased levels of IL-5 and IL-10 and decreased TNF-alpha were observed in stimulated PBMC isolated during estriol treatment. These changes in cytokines correlated with reductions of enhancing lesions on magnetic resonance imaging in relapsing remitting multiple sclerosis. The increase in IL-5 was primarily due to an increase in CD4(+) and CD8(+) T cells, the increase in IL-10 was primarily due to an increase in CD64(+) monocytes/macrophages with some effect in T cells, while the decrease in TNF-alpha was primarily due to a decrease in CD8(+) T cells. Further study of oral estriol therapy is warranted in Th1-mediated autoimmune diseases with known improvement during pregnancy.

Estrogen as a neuroprotective agent in the treatment of spinal cord injury

            (Sribnick, Wingrave et al. 2003) Download

The following review is a brief discussion about spinal cord injury and the possibility of using estrogen as a neuroprotective agent. There are several pathways by which secondary cell death can occur following spinal cord injury, including infiltration of inflammatory cells, generation of reactive oxygen species, decreases in spinal cord blood flow, and increases in intracellular Ca(2+) levels. This secondary damage leads to apoptotic cell death, and the neuroprotective effects of pharmacologic agents have been investigated using experimentally induced spinal cord injury in animals. Currently, only high-dose methylprednisolone is advocated for the treatment of patients following spinal cord injury. Estrogen has been shown to be neuroprotective in both in vitro and in vivo studies. There are several possible mechanisms of action by which estrogen may attenuate damage following spinal cord injury and improve functional outcome. Estrogen has been shown to have anti-inflammatory properties. Estrogen levels are correlated with an increase in post-traumatic blood flow to injured tissue. Estrogen may also upregulate protein levels of anti-apoptotic Bcl-2 and may attenuate the post-traumatic influx of Ca(2+).

Estrogen attenuates oxidative stress-induced apoptosis in C6 glial cells

(Sur, Sribnick et al. 2003) Download

We examined the mechanism of 17beta-estradiol (estrogen)-mediated inhibition of apoptosis in C6 (rat glioma) cells following exposure to hydrogen peroxide (H(2)O(2)). Cells were preincubated with 4 microM estrogen for 2 h and then exposed to 100 microM H(2)O(2) for 24 h. Exposure to H(2)O(2) caused significant increases in intracellular calcium (Ca(2+)), as determined by fura-2, which was attenuated by preincubation with estrogen. H(2)O(2) and ionomycin caused cell death in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Preincubation with estrogen restored viability in cells exposed to H(2)O(2) but not in cells exposed to ionomycin. Western blot analysis showed an increase in Bax/Bcl-2 ratio, calpain activity, and caspase-3 activity following treatment with H(2)O(2), and estrogen pretreatment decreased levels of all three. Cell morphology, as evaluated by Wright staining, indicated apoptosis in cells treated with H(2)O(2), and pretreatment with estrogen reduced apoptosis. Results from MTT and Wright staining were further supported by the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) assay. These results indicate a role for estrogen in preventing apoptosis in C6 glial cells exposed to H(2)O(2). Our results suggest that estrogen may have a protective role in minimizing glial cell apoptosis in neurological diseases such as demyelinating disease or central nervous system trauma.


References

Arlt, W. (2004). "Dehydroepiandrosterone and ageing." Best Pract Res Clin Endocrinol Metab 18(3): 363-80.

Bialek, M., P. Zaremba, et al. (2004). "Neuroprotective role of testosterone in the nervous system." Pol J Pharmacol 56(5): 509-18.

Coles, A. J., A. Cox, et al. (2006). "The window of therapeutic opportunity in multiple sclerosis: evidence from monoclonal antibody therapy." J Neurol 253(1): 98-108.

Gold, S. M., S. Chalifoux, et al. (2008). "Immune modulation and increased neurotrophic factor production in multiple sclerosis patients treated with testosterone." J Neuroinflammation 5: 32.

Gold, S. M. and R. R. Voskuhl (2009). "Estrogen and testosterone therapies in multiple sclerosis." Prog Brain Res 175: 239-51.

Matejuk, A., A. C. Bakke, et al. (2004). "Estrogen treatment induces a novel population of regulatory cells, which suppresses experimental autoimmune encephalomyelitis." J Neurosci Res 77(1): 119-26.

McFarland, H. F. and R. Martin (2007). "Multiple sclerosis: a complicated picture of autoimmunity." Nat Immunol 8(9): 913-9.

Orton, S. M., B. M. Herrera, et al. (2006). "Sex ratio of multiple sclerosis in Canada: a longitudinal study." Lancet Neurol 5(11): 932-6.

Palaszynski, K. M., H. Liu, et al. (2004). "Estriol treatment ameliorates disease in males with experimental autoimmune encephalomyelitis: implications for multiple sclerosis." J Neuroimmunol 149(1-2): 84-9.

Polanczyk, M. J., B. D. Carson, et al. (2004). "Cutting edge: estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment." J Immunol 173(4): 2227-30.

Sicotte, N. L., B. S. Giesser, et al. (2007). "Testosterone treatment in multiple sclerosis: a pilot study." Arch Neurol 64(5): 683-8.

Soldan, S. S., A. I. Alvarez Retuerto, et al. (2003). "Immune modulation in multiple sclerosis patients treated with the pregnancy hormone estriol." J Immunol 171(11): 6267-74.

Sribnick, E. A., J. M. Wingrave, et al. (2003). "Estrogen as a neuroprotective agent in the treatment of spinal cord injury." Ann N Y Acad Sci 993: 125-33; discussion 159-60.

Sur, P., E. A. Sribnick, et al. (2003). "Estrogen attenuates oxidative stress-induced apoptosis in C6 glial cells." Brain Res 971(2): 178-88.

Swerdloff, R. S. and C. Wang (2004). "Androgens and the ageing male." Best Pract Res Clin Endocrinol Metab 18(3): 349-62.

Vukusic, S., M. Hutchinson, et al. (2004). "Pregnancy and multiple sclerosis (the PRIMS study): clinical predictors of post-partum relapse." Brain 127(Pt 6): 1353-60.