The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway
Richard M. Lawn, David P. Wade, Michael R. Garvin, Xingbo Wang, Karen Schwartz, J. Gordon Porter, Jeffrey J. Seilhamer, Ashley M. Vaughan, John F. OramAbstract | Full text | PDF (Page R25)
The ABC1 transporter was identified as the defect in Tangier disease by a combined strategy of gene expression microarray analysis, genetic mapping, and biochemical studies. Patients with Tangier disease have a defect in cellular cholesterol removal, which results in near zero plasma levels of HDL and in massive tissue deposition of cholesteryl esters. Blocking the expression or activity of ABC1 reduces apolipoprotein-mediated lipid efflux from cultured cells, and increasing expression of ABC1 enhances it. ABC1 expression is induced by cholesterol loading and cAMP treatment and is reduced upon subsequent cholesterol removal by apolipoproteins. The protein is incorporated into the plasma membrane in proportion to its level of expression. Different mutations were detected in the ABC1 gene of 3 unrelated patients. Thus, ABC1 has the properties of a key protein in the cellular lipid removal pathway, as emphasized by the consequences of its defect in patients with Tangier disease.
Th2 cells and GATA-3 in asthma: new insights into the regulation of airway inflammation
Chemokines and asthma: redundancy of function or a coordinated effort?
Airway remodeling in asthma
The interaction of nitric oxide, bradykinin, and the angiotensin II type 2 receptor: lessons learned from transgenic mice
ABC1: connecting yellow tonsils, neuropathy, and very low HDL
Two of the usual suspects, platelet-activating factor and its receptor, implicated in acute lung injury
Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation
David A. Randolph, Robin Stephens, Cynthia J.L. Carruthers, David D. ChaplinAbstract | Full text | PDF (Page 1021)
We have studied the actions of helper T lymphocyte-1 and -2 (Th1 and Th2) cells in an acute model of eosinophilic airway inflammation by infusing chicken ovalbumin-specific (OVA-specific) Th1 cells, Th2 cells, or both into unsensitized mice and challenging the mice with an OVA aerosol. OVA challenge after infusion of Th1 cells alone resulted in airway inflammation with lymphocytes and monocytes. Challenge after the infusion of Th2 cells alone resulted in minimal inflammation. In contrast, when Th1 and Th2 cells were transferred together, they cooperated to promote a robust eosinophil-predominant inflammatory response. Th1 cells alone were readily recruited to the airways after challenge, but in the absence of Th1 cells, Th2 cells did not accumulate in the airways. When transferred together, both Th1 and Th2 cells, as well as endogenous eosinophils, were effectively recruited. This recruitment was correlated with increased VCAM-1 expression in the medium- and large-sized vessels of the lung and could be inhibited by treating the mice with neutralizing antibodies to TNF-α or VCAM-1. These data indicate that Th2 cells require signals in addition to antigen for their effective recruitment to the airways. Th1 cells can provide these signals.
Analysis of the human thymic perivascular space during aging
Kristina G. Flores, Jie Li, Gregory D. Sempowski, Barton F. Haynes, Laura P. HaleAbstract | Full text | PDF (Page 1031)
The perivascular space (PVS) of human thymus increases in volume during aging as thymopoiesis declines. Understanding the composition of the PVS is therefore vital to understanding mechanisms of thymic atrophy. We have analyzed 87 normal and 31 myasthenia gravis (MG) thymus tissues from patients ranging in age from newborn to 78 years, using immunohistologic and molecular assays. We confirmed that although thymic epithelial space (TES) volume decreases progressively with age, thymopoiesis with active T-cell receptor gene rearrangement continued normally within the TES into late life. Hematopoietic cells present in the adult PVS include T cells, B cells, and monocytes. Eosinophils are prominent in PVS of infants 2 years of age or younger. In the normal adult and the MG thymus, the PVS includes mature single-positive (CD1a– and CD4+ or CD8+) T lymphocytes that express CD45RO, and contains clusters of T cells expressing the TIA-1 cytotoxic granule antigen, suggesting a peripheral origin. PBMCs bind in vitro to MECA-79+ high endothelial venules present in the PVS, suggesting a mechanism for the recruitment of peripheral cells to thymic PVS. Therefore, in both normal subjects and MG patients, thymic PVS may be a compartment of the peripheral immune system that is not directly involved in thymopoiesis.
Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells
François Mach, Alain Sauty, Albert S. Iarossi, Galina K. Sukhova, Kuldeep Neote, Peter Libby, Andrew D. LusterAbstract | Full text | PDF (Page 1041)
Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-γ, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-γ–inducible CXC chemokines — IFN-inducible protein 10 (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α chemoattractant (I-TAC) — by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MØ) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MØ, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1β, TNF-α, and CD40 ligand potentiated IP-10 expression from IFN-γ–stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-γ induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis.
Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice
Jane K. Howard, Graham M. Lord, Giuseppe Matarese, Silvia Vendetti, Mohammad A. Ghatei, Mary A. Ritter, Robert I. Lechler, Stephen R. BloomAbstract | Full text | PDF (Page 1051)
Thymic atrophy is a prominent feature of malnutrition. Forty-eight hours’ starvation of normal mice reduced the total thymocyte count to 13% of that observed in freely fed controls, predominantly because of a diminution in the cortical CD4+CD8+ thymocyte subpopulation. Prevention of the fasting-induced fall in the level of the adipocyte-derived hormone leptin by administering exogenous recombinant leptin protected mice from these starvation-induced thymic changes. The ob/ob mouse, which is unable to produce functional leptin because of a mutation in the obese gene, has impaired cellular immunity together with a marked reduction in the size and cellularity of the thymus. We found that ob/ob mice had a high level of thymocyte apoptosis resulting in a ratio of CD4+CD8+ (cortical) to CD4–CD8– (precursor) thymocytes that was 4-fold lower than that observed in wild-type mice. Peripheral administration of recombinant leptin to ob/ob mice reduced thymocyte apoptosis and substantially increased both thymic cellularity and the CD4+CD8+/CD4–CD8– ratio. In contrast, a comparable weight loss in pair-fed PBS-treated ob/ob mice had no impact on thymocyte number. In vitro, leptin protected thymocytes from dexamethasone-induced apoptosis. These data indicate that reduced circulating leptin concentrations are pivotal in the pathogenesis of starvation-induced lymphoid atrophy.
Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells
Nicola J. Jordan, George Kolios, Stewart E. Abbot, Michael A. Sinai, Darren A. Thompson, Kalliopi Petraki, John WestwickAbstract | Full text | PDF (Page 1061)
In addition to their role as regulators of leukocyte migration and activation, chemokines and their receptors also function in angiogenesis, growth regulation, and HIV-1 pathogenesis — effects that involve the action of chemokines on nonhematopoietic cells.To determine whether chemokine receptors are expressed in human colonic epithelium, HT-29 cells were examined by RT-PCR for the expression of the chemokine receptors for lymphotactin, fractalkine, CCR1-10, and CXCR1-5. The only receptor consistently detected was CXCR4 (fusin/LESTR), although HT-29 cells did not express mRNA for its ligand, stromal cell–derived factor (SDF-1α). Flow cytometric analysis with anti-CXCR4 antibody indicated that the CXCR4 protein was expressed on the surface of roughly half of HT-29 cells. CXCR4 was also expressed in colonic epithelial cells in vivo as shown by immunohistochemistry on biopsies from normal and inflamed human colonic mucosa. The mRNA for SDF-1α and other CC and CXC chemokines was present in normal colonic biopsies. The CXCR4 receptor in HT-29 cells was functionally coupled, as demonstrated by the elevation in [Ca2+ ]i, which occurred in response to 25 nM SDF-1α and by the SDF-1α–induced upregulation of ICAM-1 mRNA. Sodium butyrate downregulated CXCR4 expression and induced differentiation of HT-29 cells, suggesting a role for CXCR4 in maintenance and renewal of the colonic epithelium. This receptor, which also serves as a coreceptor for HIV, may mediate viral infection of colonic epithelial cells.
Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice
Takahide Nagase, Satoshi Ishii, Kazuhiko Kume, Naonori Uozumi, Takashi Izumi, Yasuyoshi Ouchi, Takao ShimizuAbstract | Full text | PDF (Page 1071)
Adult respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and the molecular mechanisms underlying it are poorly understood. Acid aspiration–induced lung injury is one of the most common causes of ARDS, characterized by an increase in lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration, and respiratory failure. Here, we investigated the role of platelet-activating factor (PAF) and the PAF receptor (PAFR) gene in a murine model of acid aspiration–induced lung injury. Overexpression of the PAFR gene in transgenic mice enhanced lung injury, pulmonary edema, and deterioration of gas exchange caused by HCl aspiration. Conversely, mice carrying a targeted disruption of the PAFR gene experienced significantly less acid-induced injury, edema, and respiratory failure. Nevertheless, the efficiency of PMN sequestration in response to acid aspiration was unaffected by differences in PAFR expression level. The current observations suggest that PAF is involved in the pathogenesis of acute lung injury caused by acid aspiration. Thus, inhibition of this pathway might provide a novel therapeutic approach to acute lung injury, for which no specific pharmaceutical agents are currently available.
Adenoviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy
Thierry Bordet, Henning Schmalbruch, Brigitte Pettmann, Albert Hagege, Laetitia Castelnau-Ptakhine, Axel Kahn, Georg HaaseAbstract | Full text | PDF (Page 1077)
Cardiotrophin-1 (CT-1), an IL-6–related cytokine, causes hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types, including motoneurons. Here, we analyzed systemic CT-1 effects in progressive motor neuronopathy (pmn) mice that suffer from progressive motoneuronal degeneration, muscle paralysis, and premature death. Administration of an adenoviral CT-1 vector to newborn pmn mice leads to sustained CT-1 expression in the injected muscles and bloodstream, prolonged survival of animals, and improved motor functions. CT-1–treated pmn mice showed a significantly reduced degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. The terminal innervation of skeletal muscle, grossly disturbed in untreated pmn mice, was almost completely preserved in CT-1–treated pmn mice. The remarkable neuroprotection conferred by CT-1 might become clinically relevant if CT-1 side effects, including cardiotoxicity, could be circumvented by a more targeted delivery of this cytokine to the nervous system.
Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance
Elizabeth J. Parks, Ronald M. Krauss, Mark P. Christiansen, Richard A. Neese, Marc K. HellersteinAbstract | Full text | PDF (Page 1087)
Low-fat, high-carbohydrate (LF/HC) diets commonly elevate plasma triglyceride (TG) concentrations, but the kinetic mechanisms responsible for this effect remain uncertain. Subjects with low TG (normolipidemic [NL]) and those with moderately elevated TG (hypertriglyceridemic [HTG]) were studied on both a control and an LF/HC diet. We measured VLDL particle and TG transport rates, plasma nonesterified fatty acid (NEFA) flux, and sources of fatty acids used for the assembly of VLDL-TG. The LF/HC diet resulted in a 60% elevation in TG, a 37% reduction in VLDL-TG clearance, and an 18% reduction in whole-body fat oxidation, but no significant change in VLDL-apo B or VLDL-TG secretion rates. Significant elevations in fasting apo B-48 concentrations were observed on the LF/HC in HTG subjects. In both groups, fasting de novo lipogenesis was low regardless of diet. The NEFA pool contributed the great majority of fatty acids to VLDL-TG in NL subjects on both diets, whereas in HTG subjects, the contribution of NEFA was somewhat lower overall and was reduced further in individuals on the LF/HC diet. Between 13% and 29% of VLDL-TG fatty acids remained unaccounted for by the sum of de novo lipogenesis and plasma NEFA input in HTG subjects. We conclude that (a) whole-food LF/HC diets reduce VLDL-TG clearance and do not increase VLDL-TG secretion or de novo lipogenesis; (b) sources of fatty acids for assembly of VLDL-TG differ between HTG and NL subjects and are further affected by diet composition; (c) the presence of chylomicron remnants in the fasting state on LF/HC diets may contribute to elevated TG levels by competing for VLDL-TG lipolysis and by providing a source of fatty acids for hepatic VLDL-TG synthesis; and (d) the assembly, production, and clearance of elevated plasma VLDL-TG in response to LF/HC diets therefore differ from those for elevated TG on higher-fat diets.
Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis–like lesions
Christian Vestergaard, Hiroyuki Yoneyama, Masako Murai, Kohichiro Nakamura, Kunihiko Tamaki, Yuya Terashima, Toshio Imai, Osamu Yoshie, Tatsuro Irimura, Hitoshi Mizutani, Kouji MatsushimaAbstract | Full text | PDF (Page 1097)
We have examined the expression of chemokines and their receptors in the atopic dermatitis–like (AD-like) lesions of NC/Nga mice. Such lesions develop when the mice are kept in conventional conditions, but not when they are kept isolated from specific pathogens. The thymus- and activation-regulated chemokine TARC is unexpectedly highly expressed in the basal epidermis of 14-week-old mice with lesions, whereas it is not expressed in the skin without lesions. Production of TARC by keratinocytes was confirmed by culturing murine keratinocytic cell line cells (PAM212) with TNF-α, IFN-γ, or IL-1β. Expression of another Th2 chemokine, macrophage-derived chemokine (MDC), was observed in the skin from mice kept in both conventional and pathogen-free conditions, but expression of MDC was increased severalfold in the skin with lesions. The cellular origin of MDC was identified to be dermal dendritic cells. Infiltration of the skin by IL-4–producing T cells and mast cells, and the increase of CCR4 mRNA in the skin, coincided with the development of AD lesions. These observations indicate that TARC and MDC actively participate in the pathogenesis of AD-like lesions in NC/Nga mice and that these Th2 chemokines could be novel targets for intervention therapy of AD in humans.
Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa
Michael D. Levitt, Julie Furne, John Springfield, Fabrizis Suarez, Eugene DeMasterAbstract | Full text | PDF (Page 1107)
Colonic bacteria liberate large quantities of the highly toxic gases hydrogen sulfide (H2S) and methanethiol (CH3SH). The colonic mucosa presumably has an efficient means of detoxifying these compounds, which is thought to occur through methylation of H2S to CH3SH and CH3SH to dimethylsulfide (CH3SCH3). We investigated this detoxification pathway by incubating rat cecal mucosal homogenates with gas containing H2S, CH3SH, or CH3SCH3. Neither CH3SH nor CH3SCH3 was produced during H2S catabolism, whereas catabolism of CH3SH liberated H2S but not CH3SCH3. Thus, H2S and CH3SH are not detoxified by methylation to CH3SCH3. Rather, CH3SH is demethylated to H2S, and H2S is converted to nonvolatile metabolites. HPLC analysis of the homogenate showed the metabolite to be primarily thiosulfate. Analysis of cecal venous blood obtained after intracecal instillation of H235S revealed that virtually all absorbed H2S had been oxidized to thiosulfate. The oxidation rate of H2S by colonic mucosa was 10,000 times greater than the reported methylation rate. Conversion to thiosulfate appears to be the mechanism whereby the cecal mucosa protects itself from the injurious effects of H2S and CH3SH, and defects in this detoxification possibly could play a role in colonic diseases such as ulcerative colitis.
Mutations in Igα (CD79a) result in a complete block in B-cell development
Yoshiyuki Minegishi, Elaine Coustan-Smith, Lisa Rapalus, Fügen Ersoy, Dario Campana, Mary Ellen ConleyAbstract | Full text | PDF (Page 1115)
Mutations in Btk, μ heavy chain, or the surrogate light chain account for 85–90% of patients with early onset hypogammaglobulinemia and absent B cells. The nature of the defect in the remaining patients is unknown. We screened 25 such patients for mutations in genes encoding components of the pre–B-cell receptor (pre-BCR) complex. A 2-year-old girl was found to have a homozygous splice defect in Igα, a transmembrane protein that forms part of the Igα/Igβ signal-transduction module of the pre-BCR. Studies in mice suggest that the Igβ component of the pre-BCR influences V-DJ rearrangement before cell-surface expression of μ heavy chain. To determine whether Igα plays a similar role, we compared B-cell development in an Igα-deficient patient with that seen in a μ heavy chain–deficient patient. By immunofluorescence, both patients had a complete block in B-cell development at the pro-B to pre-B transition; both patients also had an equivalent number and diversity of rearranged V-DJ sequences. These results indicate that mutations in Igα can be a cause of agammaglobulinemia. Furthermore, they suggest that Igα does not play a critical role in B-cell development until it is expressed, along with μ heavy chain, as part of the pre-BCR.
CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression
Laurie E. Cohen, Yukiko Hashimoto, Kerstin Zanger, Fredric Wondisford, Sally RadovickAbstract | Full text | PDF (Page 1123)
Hypothalamic growth hormone–releasing hormone (GHRH) stimulates growth hormone (GH) gene expression in anterior pituitary somatotrophs by binding to the GHRH receptor, a G-protein–coupled transmembrane receptor, and by mediating a cAMP-mediated protein kinase A (PKA) signal-transduction pathway. Two nonclassical cAMP-response element motifs (CGTCA) are located at nucleotides –187/–183 (distal cAMP-response element; dCRE) and –99/–95 (proximal cAMP-response element; pCRE) of the human GH promoter and are required for cAMP responsiveness, along with the pituitary-specific transcription factor Pit-1 (official nomenclature, POU1F1). Although a role for cAMP-response element binding protein (CREB) in GH stimulation by PKA has been suggested, it is unclear how the effect may be mediated. CREB binding protein (CBP) is a nuclear cofactor named for its ability to bind CREB. However, CBP also binds other nuclear proteins. We determined that CBP interacts with Pit-1 and is a cofactor for Pit-1–dependent activation of the human GH promoter. This pathway appears to be independent of CREB, with CPB being the likely target of phosphorylation by PKA.
Renal cytochrome P450 ω-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride
Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of ω-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [14C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10–100 μM), an NO donor, inhibited renal microsomal conversion of [14C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 μM), the cell-permeable analogue of cGMP, did not affect conversion of [14C]AA. Inhibition of NOS with Nω-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [14C]AA to HETE and greatly increased the expression of ω-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical ω-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [14C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased ω-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.
Killing of Streptococcus pneumoniae by capsular polysaccharide–specific polymeric IgA, complement, and phagocytes
Edward N. Janoff, Claudine Fasching, Jan M. Orenstein, Jeffrey B. Rubins, Nancy L. Opstad, Agustin P. DalmassoAbstract | Full text | PDF (Page 1139)
The role of IgA in the control of invasive mucosal pathogens such as Streptococcus pneumoniae is poorly understood. We demonstrate that human pneumococcal capsular polysaccharide–specific IgA initiated dose-dependent killing of S. pneumoniae with complement and phagocytes. The majority of specific IgA in serum was of the polymeric form (pIgA), and the efficiency of pIgA-initiated killing exceeded that of monomeric IgA–initiated killing. In the absence of complement, specific IgA induced minimal bacterial adherence, uptake, and killing. Killing of S. pneumoniae by resting phagocytes with immune IgA required complement, predominantly via the C2-independent alternative pathway, which requires factor B, but not calcium. Both S. pneumoniae–bound IgA and complement were involved, as demonstrated by a 50% decrease in killing with blocking of Fcα receptor (CD89) and CR1/CR3 (CD35/CD11b). However, IgA-mediated killing by phagocytes could be reproduced in the absence of opsonic complement by pre-activating phagocytes with the inflammatory products C5a and TNF-α. Thus, S. pneumoniae capsule–specific IgA may show distinct roles in effecting clearance of S. pneumoniae in the presence or absence of inflammation. These data suggest mechanisms whereby pIgA may serve to control pneumococcal infections locally and upon the pathogen’s entry into the bloodstream.
Temporal adjustment of the juxtaglomerular apparatus during sustained inhibition of proximal reabsorption
Scott C. Thomson, Sebastian Bachmann, Magdalena Bostanjoglo, Carolyn A. Ecelbarger, Orjan W. Peterson, Doron Schwartz, Dingjiu Bao, Roland C. BlantzAbstract | Full text | PDF (Page 1149)
Tubuloglomerular feedback (TGF) stabilizes nephron function by causing changes in single-nephron GFR (SNGFR) to compensate for changes in late proximal flow (VLP). TGF responds within seconds and reacts over a narrow range of VLP that surrounds normal VLP. To accommodate sustained increases in VLP, TGF must reset around the new flow. We studied TGF resetting by inhibiting proximal reabsorption with benzolamide (BNZ; administered repeatedly over a 24-hour period) in Wistar-Froemter rats. BNZ acutely activates TGF, thereby reducing SNGFR. Micropuncture was performed 6–10 hours after the fourth BNZ dose, when diuresis had subsided. BNZ caused glomerular hyperfiltration, which was prevented with inhibitors of macula densa nitric oxide synthase (NOS). Because of hyperfiltration, BNZ increased VLP and distal flow, but did not affect the basal TGF stimulus (early distal salt concentration). BNZ slightly blunted normalized maximum TGF response and the basal state of TGF activation. BNZ sensitized SNGFR to reduction by S-methyl-thiocitrulline (SMTC) and caused the maximum TGF response to be strengthened by SMTC. Sensitization to type I NOS (NOS-I) blockers correlated with increased macula densa NOS-I immunoreactivity. Tubular transport measurements confirmed that BNZ affected TGF within the juxtaglomerular apparatus. During reduced proximal reabsorption, TGF resets to accommodate increased flow and SNGFR through a mechanism involving macula densa NOS.