Mary Ann Fletcher

Mary Ann Fletcher, Ph.D., is the first Schemel Professor for Neuro Immune Medicine at the Institute for Neuro Immune Medicine, Nova Southeastern University, Fort Lauderdale, Florida, USA. Previously, she spent 40 years at the University of Miami Miller School of Medicine as a Professor of Medicine, Microbiology/Immunology and Psychology. She holds two U.S. Patents for developing tests to determine if a person has mononucleosis.

Education

 * Bachelor of Science in Microbiology from Texas Tech University
 * Master of Science in Immunology and Virology from University of Texas Medical School
 * Doctor of Philosophy in Immunochemistry from Baylor University
 * Postdoctoral research at Northwestern University in Chicago.

Chronic Fatigue Syndrome Advisory Committee
Dr. Fletcher was a voting member of the Chronic Fatigue Syndrome Advisory Committee, for the U.S. Department of Health and Human Services, from June 13, 2012 to December 13, 2016.

Open Letter to The Lancet
Two open letters to the editor of The Lancet urged the editor to commission a fully independent review of the PACE trial, which the journal had published in 2011. In 2016, Dr. Fletcher, along with 41 colleagues in the ME/CFS field, signed the second letter.
 * 10 February 2016, An open letter to The Lancet, again - Virology blog

Notable studies

 * 2017, Telephone-administered versus live group cognitive behavioral stress management for adults with CFS"Abstract - Objective: Chronic fatigue syndrome (CFS) symptoms have been shown to be exacerbated by stress and ameliorated by group-based psychosocial interventions such as cognitive behavioral stress management (CBSM). Still, patients may have difficulty attending face-to-face groups. This study compared the effects of a telephone-delivered (T-CBSM) vs a live (L-CBSM) group on perceived stress and symptomology in adults with CFS. Methods: Intervention data from 100 patients with CFS (mean age 50years; 90% female) participating in T-CBSM (N=56) or L-CBSM (N=44) in previously conducted randomized clinical trials were obtained. Perceived Stress Scale (PSS) and the Centers for Disease Control and Prevention symptom checklist scores were compared with repeated measures analyses of variance in adjusted and unadjusted analyses. RESULTS: Participants across groups showed no differences in most demographic and illness variables at study entry and had similar session attendance. Both conditions showed significant reductions in PSS scores, with L-CBSM showing a large effect (partial ε2=0.16) and T-CBSM a medium effect (partial ε2=0.095). For CFS symptom frequency and severity scores, L-CBSM reported large effect size improvements (partial ε2=0.19-0.23), while T-CBSM showed no significant changes over time. Conclusions: Two different formats for delivering group-based CBSM-live and telephone-showed reductions in perceived stress among patients with CFS. However, only the live format was associated with physical symptom improvements, with specific effects on post-exertional malaise, chills, fever, and restful sleep. The added value of the live group format is discussed, along with implications for future technology-facilitated group interventions in this population."
 * 2016, Poor sleep quality is associated with greater circulating pro-inflammatory cytokines and severity and frequency of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) symptoms in women"Abstract: Objective - Poor sleep quality has been linked to inflammatory processes and worse disease outcomes in the context of many chronic illnesses, but less is known in conditions such as chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME). This study examines the relationships between sleep quality, pro-inflammatory cytokines, and CFS/ME symptoms. Methods - Sixty women diagnosed with CFS/ME were assessed using the Pittsburgh Sleep Quality Index (PSQI), Fatigue Symptom Inventory (FSI) and Center for Disease Control and Prevention (CDC)-based CFS/ME symptom questionnaires. Circulating plasma pro-inflammatory cytokine levels were measured by ELISA. Multiple regression analyses examined associations between sleep, cytokines and symptoms, controlling for age, education, and body mass index. Results - Poor sleep quality (PSQI global score) was associated with greater pro-inflammatory cytokine levels: interleukin-1β (IL-1β) (β = 0.258, p = 0.043), IL-6 (β = 0.281, p = 0.033), and tumor necrosis factor-alpha (TNF-α) (β = 0.263, p = 0.044). Worse sleep quality related to greater fatigue severity (β = 0.395, p = 0.003) and fatigue-related interference with daily activities (β = 0.464, p < 0.001), and more severe and frequent CDC-defined core CFS/ME symptoms (β = 0.499, p < 0.001, and β = 0.556, p < 0.001, respectively). Conclusions - Results underscore the importance of managing sleep-related difficulties in this patient population. Further research is needed to identify the etiology of sleep disruptions in CFS/ME and mechanistic factors linking sleep quality to symptom severity and inflammatory processes."
 * 2016, Tracking post-infectious fatigue in clinic using routine Lab tests "Abstract: Background: While biomarkers for chronic fatigue syndrome (CFS) are beginning to emerge they typically require a highly specialized clinical laboratory. We hypothesized that subsets of commonly measured laboratory markers used in combination could support the diagnosis of post-infectious CFS (PI-CFS) in adolescents following infectious mononucleosis (IM) and help determine who might develop persistence of symptoms. Methods: Routine clinical laboratory markers were collected prospectively in 301 mono-spot positive adolescents, 4 % of whom developed CFS (n = 13). At 6, 12, and 24 months post-diagnosis with IM, 59 standard tests were performed including metabolic profiling, liver enzyme panel, hormone profiles, complete blood count (CBC), differential white blood count (WBC), salivary cortisol, and urinalysis....Results: Lower ACTH levels at 6 months post-IM diagnosis were highly predictive of CFS (AUC p = 0.02). ACTH levels in CFS overlapped with healthy controls at 12 months, but again showed a trend towards a deficiency at 24 months. Conversely, estradiol levels depart significantly from normal at 12 months only to recover at 24 months (AUC p = 0.02). Finally, relative neutrophil count showed a significant departure from normal at 24 months in CFS (AUC p = 0.01). Expression of these markers evolved differently over time between groups. Conclusions: Preliminary results suggest that serial assessment of stress and sex hormones as well as the relative proportion of innate immune cells measured using standard clinical laboratory tests may support the diagnosis of PI-CFS in adolescents with IM.'"
 * 2016, Illness progression in chronic fatigue syndrome: a shifting immune baseline
 * 2015, Perceived Fatigue Interference and Depressed Mood: Comparison of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients with Fatigued Breast Cancer Survivors (Full Text)
 * 2015, Findings from a clinical and laboratory database developed for discovery of pathogenic mechanisms in myalgic encephalomyelitis/chronic fatigue syndrome. Abstract
 * 2014, Stress management skills, cortisol awakening response, and post-exertional malaise in Chronic Fatigue Syndrome
 * 2012, Biomarkers for chronic fatigue. "Abstract: Fatigue that persists for 6 months or more is termed chronic fatigue. Chronic fatigue (CF) in combination with a minimum of 4 of 8 symptoms and the absence of diseases that could explain these symptoms, constitute the case definition for chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME). Inflammation, immune system activation, autonomic dysfunction, impaired functioning in the hypothalamic-pituitary-adrenal axis, and neuroendocrine dysregulation have all been suggested as root causes of fatigue. The identification of objective markers consistently associated with CFS/ME is an important goal in relation to diagnosis and treatment, as the current case definitions are based entirely on physical signs and symptoms. This review is focused on the recent literature related to biomarkers for fatigue associated with CFS/ME and, for comparison, those associated with other diseases. These markers are distributed across several of the body’s core regulatory systems. A complex construct of symptoms emerges from alterations and/or dysfunctions in the nervous, endocrine and immune systems. We propose that new insight will depend on our ability to develop and deploy an integrative profiling of CFS/ME pathogenesis at the molecular level. Until such a molecular signature is obtained efforts to develop effective treatments will continue to be severely limited."
 * 2012, Cytokine expression profiles of immune imbalance in post-mononucleosis chronic fatigue
 * 2010, A Formal Analysis of Cytokine Networks in Chronic Fatigue Syndrome Full Text
 * 2010, Plasma neuropeptide Y: a biomarker for symptom severity in chronic fatigue syndrome
 * 2009, Plasma cytokines in women with chronic fatigue syndrome (FULL TEXT) "Abstract - Background: Chronic Fatigue Syndrome (CFS) studies from our laboratory and others have described cytokine abnormalities. Other studies reported no difference between CFS and controls. However, methodologies varied widely and few studies measured more than 4 or 5 cytokines. Multiplex technology permits the determination of cytokines for a large panel of cytokines simultaneously with high sensitivity and with only 30 ul of plasma per sample. No widely accepted laboratory test or marker is available for the diagnosis or prognosis of CFS. This study screened plasma factors to identify circulating biomarkers associated with CFS. Methods: Cytokines were measured in plasma from female CFS cases and female healthy controls. Multiplex technology provided profiles of 16 plasma factors including the pro -inflammatory cytokines: tumor necrosis factor alpha (TNFalpha), lymphotoxin alpha (LTalpha), interleukin (IL) - IL-Ialpha, IL-1beta, IL-6; TH1 cytokines: interferon gamma (IFNgamma), IL-12p70, IL-2, IL-15; TH2: IL-4, IL-5; TH17 cytokines, IL-17 and IL-23; anti-inflammatory cytokines IL-10, IL-13; the inflammatory mediator and neutrophil attracting chemokine IL-8 (CXCL8). Analysis by receiver operating characteristic (ROC) curve assessed the biomarker potential of each cytokine. Results: The following cytokines were elevated in CFS compared to controls: LTalpha, IL-1alpha, IL-1beta, IL-4, IL-5, IL-6 and IL-12. The following cytokines were decreased in CFS: IL-8, IL-13 and IL-15. The following cytokines were not different: TNFalpha, IFNgamma, IL-2, IL-10, IL-23 and IL-17. Applying (ROC) curve analyses, areas under the curves (AUC) for IL-5 (0. 84), LTalpha (0.77), IL-4 (0.77), IL-12 (0.76) indicated good biomarker potential. The AUC of IL-6 (0.73), IL-15 (0.73), IL-8 (0.69), IL-13 (0.68) IL-1alpha (0.62), IL-1beta (0.62) showed fair potential as biomarkers. Conclusion: Cytokine abnormalities are common in CFS. In this study, 10 of 16 cytokines examined showed good to fair promise as biomarkers. However, the cytokine changes observed are likely to more indicative of immune activation and inflammation, rather than specific for CFS. As such, they are targets for herapeutic strategies. Newer techniques allow evaluation of large panels of cytokines in a cost effective fashion."
 * 2008, The associations between basal salivary cortisol levels and illness symptomatology in chronic fatigue syndrome. (Full text) "Abstract: Hypocortisolism has been reported in chronic fatigue syndrome (CFS), with the significance of this finding to disease etiology unclear. This study examined cortisol levels and their relationships with symptoms in a group of 108 individuals with CFS. CFS symptoms examined included fatigue, pain, sleep difficulties, neurocognitive functioning, and psychiatric status. Alterations in cortisol levels were examined by calculation of mean daily cortisol, while temporal variation in cortisol function was examined by means of a regression slope. Additionally, deviation from expected cortisol diurnal pattern was determined via clinical judgment. Results indicated that fatigue and pain were associated with salivary cortisol levels. In particular, variance from the expected pattern of cortisol was associated with increased levels of fatigue. The implications of these findings are discussed."
 * 2008, Evidence for T-helper 2 shift and association with illness parameters in chronic fatigue syndrome (CFS)"Abstract: Few immunological markers have been consistently reported in CFS. However, a shift to a T-helper 2 (Th2) type immune response has been hypothesized for individuals with CFS. The current study investigated whether individuals with CFS who exhibited a stronger shift towards a Th2 type of immune response would also exhibit more severe symptoms, poorer neurocognitive functioning, and poorer physical and psychosocial functioning. The current investigation measured the percentage of Th1-like and Th2-like memory cells using cell surface flow cytometry in 114 individuals with CFS. The associations between the ratio of Th1 and Th2 memory cells and various illness parameters measures were then examined, including symptom severity, psychiatric functioning, neurocognitive functioning, salivary cortisol levels, and chronic pain status. Results indicated that individuals who exhibited a more extreme shift towards a Th2 immune response also exhibited poorer sleep and high levels of basal salivary cortisol. The implications of these findings are discussed."
 * 2007, Baseline Cortisol Levels Predict Treatment Outcomes in Chronic Fatigue Syndrome Nonpharmacologic Clinical Trial"Abstract - Objective: Understanding how nonpharmacologic interventions differentially affect the subgroups of patients with chronic fatigue syndrome (CFS) might provide insights into the pathophysiology of this illness. In this exploratory study, baseline measures of normal versus abnormal cortisol were compared on a variety of immune markers and other self-report measures. Normal versus abnormal cortisol ratings were used as predictors in a nurse-delivered nonpharmacologic intervention. Methods: Participants diagnosed with CFS were assigned to 6-month nonpharmacologic interventions. Individuals were classified as having abnormal or normal cortisol levels on the basis of scores over the five testing times. Cortisol levels were considered abnormal if they continued to rise, were flat, or were at abnormally low over time. Results: Across interventions, those with abnormal cortisol at the baseline appeared not to improve over time, whereas those with normal baseline cortisol evidenced improvements on a number of immunologic and self-report measures. Conclusion: It appears that, in subgroups of individuals with CFS, baseline cortisol markers are associated with outcome trajectories for nonpharmacologic treatment trials. The implications of these findings are discussed.'"
 * 2002, Effects of Benzalkonium Salts on G-protein-Mediated Processes and Surface Membranes: Relevance to Microbial- and Chemical-Induced Diseases"Abstract - Benzalkonium salts comprise a group of positively charged surface-active alkylamine biocides with the general formula alkyldi-methylbenzylammonium chloride or bromide. They interact with guanine nucleotide triphosphate-binding proteins (G proteins), thereby affecting signal transduction in a variety of cell types and processes. The present report reviews the known and potential basic science research and clinical applications and manifestations of benzalkonium salts. Benzalkonium salts have antiproliferative effects on a variety of cells (including T cells) through G-protein-dependent pathways, affect cytokine gene expression (downregulate tumor necrosis factor expression), and are also effective bactericidal, fungicidal, and virucidal agents with multisite (direct and immunologically-mediated) inhibitory activity against many pathogens, including the human immunodeficiency virus (HIV), papillomavirus, and herpesviruses. Therefore, benzalkonium salts not only appear to be effective as disinfectants and spermicides but may also prove useful in the prevention and treatment of several diseases, particularly those linked to viruses and originating at the skin or mucosal surface. The untoward effects of benzalkonium salts are also discussed as a paradigm for chemical-induced diseases."
 * 2002, The Paul-Bunnell Heterophile Antibody Determinant in Epstein-Barr Virus-Associated Disease"Abstract - Reactivation of latent herpes viruses (notably Epstein-Barr virus, human herpesvirus-6) is commonly seen in chronic fatigue syndrome and it is believed to contribute to symptom perpetuation. Epstein-Barr virus (EBV), which was first isolated by Epstein, Barr and Achong (1964) from a cultured Burkitts's lymphoma lymphoblast cell line, is the etiological agent for infectious mononucleosis (IM), polyclonal and oligoclonal lymphomas associated with primary and acquired immunodeficiencies, and the complications of X-linked lymphoproliferative syndrome (XLP) (Cantani and Mastrantoni, 1989; Englund, 1988; Ernberg et al., 1990; Jones and Straus, 1987; Okano et al., 1988; Purtilo, 1987; Purtilo et al., 1981; Rowe et al., 1986; Saemundsen et al., 1981) and nasopharyngeal cancer (Pearson et al., 1984). Furthermore, people who have had IM have higher rates of subsequent development of malignant lymphoproliferative disorders (Abo et al., 1982; Snydman et al., 1982) and Hodgkins's disease (Green et al., 1979; Mueller, 1987; Poppema et al., 1985; Weiss et al., 1989), while patients with XLP have a higher incidence of non-Hodgkins's malignant lymphoma (Harrington et al., 1987). The precise role of EBV in these diseases or in CFS is not well understood. Nonetheless, it is known that EBV infection triggers the formation of heterophile antibodies that, for many decades, have formed the basis for serologic diagnosis of IM. In this review, we discuss the discovery, species variation, and structure of the erythrocyte membrane-associated Paul-Bunnell (PB) heterophile antibody determinant, its implications to IM diagnosis, and its potential contribution to defective immune surveillance, such as that seen in chronic fatigue syndrome."
 * 2001, Cytokine and Other Immunologic Markers in Chronic Fatigue Syndrome and Their Relation to Neuropsychological Factors
 * 2000, Comparative Analysis of Lymphocytes in Lymph Nodes and Peripheral Blood of Patients with Chronic Fatigue Syndrome"Abstract - Blood and lymph node samples were obtained from patients with chronic fatigue syndrome (CFS) who had volunteered to undergo a lymph node biopsy while participating in a phase 1 clinical trial of a novel immunomodulatory therapy. The surface marker phenotypes of the peripheral blood and lymph node samples were examined using four-color flow cytometry and compared to published proportions of cells in peripheral blood and lymph nodes from control individuals. While a greater proportion of T lymphocytes from both lymph nodes and peripheral blood of control subjects are immunologically “naive” (CD45RA+), the proportions of lymphocytes with a “memory” phenotype predominate in lymph nodes and peripheral blood of CFS patients. CFS has been proposed to be a disease of autoimmune etiology and in this respect it is interesting to note that decreased proportions of CD45RA+ T (“naive”) cells are also seen in the peripheral blood of patients with autoimmune diseases."
 * 2000, Clinical and Immunologic Effects of Autologous Lymph Node Cell Transplant in Chronic Fatigue Syndrome"Abstract - An open labeled, phase 1, safety and feasibility study using lymph node extraction, ex vivo lymph node cell expansion, followed by autologous cell reinfusion was evaluated as a potential immunomodulatory treatment strategy in patients with chronic fatigue syndrome (CFS). The experimental therapy utilized the cells of the lymph node, activated and grown in culture with defined media, interleukin-2 (IL-2) and anti-CD3 to activate and enhance cellular immunological functions. This procedure was designed to change the cytokine pattern of the lymph node lymphocytes to favor expression of T-helper (Th)l-type over Th2-type cytokines. The mixed population of ex vivo immune-enhanced cells were reinfused into the donor, who was carefully monitored for adverse events and possible clinical benefit. There were no adverse events. There were significant improvements in clinical status in association with a significant decrease in Th2-type cytokine production."
 * 2000, Immunotherapy of Chronic Fatigue Syndrome: Therapeutic Interventions Aimed at Modulating the Th1/Th2 Cytokine Expression Balance"Abstract - Based on the postulates of viral and autoimmune etiologies of CFS, several interventions have been designed and tested by different research groups around the world, including the United States, Sweden, United Kingdom, Italy, and Japan. This review addresses those interventions aimed at altering the balance of certain cytokines, the mediators of immune responses. Patients with CFS who show evidence of activation of the immune system have poor immune cell function and a predominance of what is called a T-helper (Th)2-type cytokine response when their lymphocytes are activated. A Th2-type response, which is characterized by production of cytokines such as interleukin (IL)−4, −5, and −10, favors the function of B lymphocytes, the cellular factories of immunoglobulins. A predominance of a Th2-type response is therefore consistent with pathologies, such as autoimmunity and atopy, which are based on inappropriate production of immunoglobulins. Many of the CFS therapies discussed decrease the Th2-type predominance seen at baseline in CFS patients, thereby allowing a greater predominance of a Thl-type response, which favors the function of macrophages and natural killer cells. The function of the latter cells, which have the natural ability of directly destroying invading microbes and cancer cells, is defective in untreated CFS patients. Typical Thl-type cytokines include IL-2 and interferon-gamma, and some of the therapies induce their production. The interventions discussed in this review cover a wide spectrum of therapeutic tools ranging from lymph node cell immunotherapy, herbal products, and small molecules to vaccines. Despite the controversies on the etiology of CFS, immunotherapy research is useful and necessary."
 * 2000, Chronic Fatigue Syndrome: Evidence Supporting the Hypothesis of a Behaviorally-Activated Neuromodulator of Fatigue Abstract - Chronic Fatigue Syndrome (CFS) is a disorder characterized by a prolonged, debilitating fatigue of unknown etiology. In addition, patients with CFS frequently report enhanced fatigue symptoms following even mild physical exertion, and their tolerance for physical exercise is limited relative to healthy individuals. The physiological mechanisms underlying the excessive fatigue and weakness common to this disorder remain an issue of scientific debate. Collectively, the available data suggest that fatigue in CFS is not due to any neuromuscular dysfunction, per se, but possibly is caused or influenced by some centrally acting mediator that is released during behavioral activities that require physical or mental exertion. In addition to persistent fatigue, there is growing evidence that many CFS patients exhibit alterations in hypothalamic-pituitary-adrenal (HPA) axis and autonomic function, including the inability to maintain the blood pressure response to orthostatic challenge. When an individual engages in mental or physical behavioral activation, there is a release of numerous centrally acting neuromodulators, some of which have been postulated to influence fatigue. This paper examines the evidence supporting a common pathway through which these centrally-mediated psychological and autonomic abnormalities may be linked. It is hypothesized that as a consequence of behavioral activation there is an abnormality in neuromodulator release or action in individuals with CFS, and that this abnormal neuromodulator activity results in increased fatigue. Furthermore, it is postulated that the CNS initiates a counter-regulatory mechanism to reduce the activity of those systems responsible for the production of the neuromodulator; and that the consequence of this counter-regulatory maneuver is the prevailing dysregulation of the autonomic and HPA axes and other dysfunctional cardiovascular and immunological sequelae.
 * 1998, Interleukin-6 and Disease: Two Case Reports that Point to the Usefulness of Measuring Cytokine Levels in Clinical Settings
 * 1996, Interindividual Immune Status Variation Patterns in Patients with Chronic Fatigue Syndrome: Association with Gender and the Tumor Necrosis Factor System Abstract - Changes in soluble immune mediator levels in association with the chronic fatigue syndrome (CFS) usually occur within normal ranges and are apparent mainly as changes in the skewness of population distributions. The latter finding undermines the usefulness of cytokine levels as clinical tools at the individual level as has been seen in sepsis syndrome where a similar overlap occurs. Nonetheless, changes in cytokine levels at the population level can contribute to an understanding of the disease process. For example, we reported previously that significant proportions of CFS patients showed elevated serum levels of either soluble tumor necrosis factor-receptor I (sTNF-RI, sCD120a) or TNF-a as compared to controls. The latter results could reflect different disease processes or extremes of a common disease process. Using sera collected over a five-year period, we have now studied an extended cohort of 108 CFS patients and our results are consistent with a common graded disease process. When we assessed the effect of gender on the distributions of serum levels of immune mediators, levels of sTNF-RI, sTNF-RII (sCD 12Ob), sIL-6R (sCDl26), and sICAM-1 were found to be consistently higher among males than females and among CFS patients as compared to controls regardless of gender. Moreover, differences in soluble immune mediator levels between CFS and control individuals were more clearly defined when restricting the analysis to the female gender. These observations are consistent with endocrine influences on immunological changes.
 * 1995, Relationships of Cognitive Difficulties to Immune Measures, Depression and Illness Burden in Chronic Fatigue Syndrome - Abstract
 * 1995, Physical symptoms of chronic fatigue syndrome are exacerbated by the stress of Hurricane Andrew
 * 1995, Dysregulated Expression of Soluble Immune Mediator Receptors in a Subset of Patients with Chronic Fatigue Syndrome: Cross-Sectional Categorization of Patients by Immune Status - Abstract
 * 1994, Dysregulated expression of tumor necrosis factor in chronic fatigue syndrome: interrelations with cellular sources and patterns of soluble immune mediator expression"Abstract: Among a group of 70 individuals who met the criteria established by the Centers for Disease Control and Prevention (Atlanta) for chronic fatigue syndrome (CFS), 12%-28% had serum levels exceeding 95% of control values for tumor necrosis factor (TNF) alpha, TNF-beta, interleukin (IL) 1 alpha, IL-2, soluble IL-2 receptor (sIL-2R), or neopterin; overall, 60% of patients had elevated levels of one or more of the nine soluble immune mediators tested. Nevertheless, only the distributions for circulating levels of TNF-alpha and TNF-beta differed significantly in the two populations. In patients with CFS--but not in controls--serum levels of TNF-alpha, IL-1 alpha, IL-4, and sIL-2R correlated significantly with one another and (in the 10 cases analyzed) with relative amounts (as compared to beta-globin or beta-actin) of the only mRNAs detectable by reverse transcriptase-coupled polymerase chain reaction in peripheral-blood mononuclear cells: TNF-beta, unspliced and spliced; IL-1 beta, lymphocyte fraction; and IL-6 (in order of appearance). These findings point to polycellular activation and may be relevant to the etiology and nosology of CFS."