Immunomodulatory agents are substances that can modify or regulate one or more immune functions in horses. These agents can either enhance or suppress the immune response, depending on the desired outcome. In equine medicine, immunomodulatory therapies may be employed to manage various conditions, including autoimmune disorders, allergies, and infections. This topic encompasses research on the mechanisms of action, therapeutic applications, and effects of different immunomodulatory agents in horses. The page aggregates peer-reviewed studies and scholarly articles that investigate the efficacy, safety, and clinical implications of using immunomodulatory treatments in equine health management.
Romantsov MG, Galimzianov KhM, Lokteva OM, Kovalenko AL, Stepanov AV.Search for drugs efficient in prophylaxis and treatment of dangerous infections (especially arboviral ones) is rather actual, since no specific therapy is available. Many-year investigations of interferon inductors showed that they had immunomodulating, antiviral and antiinflammatory effects and were low toxic. The present study demonstrated that the protective effect was the following: Venezuelan equine encephalitis (VEE)--cycloferon > amixin = ridostin, Rift Valley fever (RVF)--cycloferon > amixin > ridostin, predator pox (PP)--cycloferon > amixin = ridostin, that was obvious tha...
Woodward EM, Christoffersen M, Campos J, Horohov DW, Scoggin KE, Squires E, Troedsson MH.The first objective of this study was to evaluate intrauterine nitric oxide (NO) and endometrial inducible NO synthase (iNOS) in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) within 24 h after breeding. Mares susceptible (n = 6) or resistant (n = 6) to PBIE were inseminated over five cycles, and uterine secretions and endometrial biopsies were collected before and 2, 6, 12 and 24 h after insemination. Uterine secretions were analysed for NO and biopsies were analyzed for iNOS expression. A second experiment evaluated the effect of treatment with dexame...
Ma G, Feineis S, Osterrieder N, Van de Walle GR.Major histocompatibility complex class I (MHC-I) molecules play an important role in host immunity to infection by presenting antigenic peptides to cytotoxic T lymphocytes (CTLs), which recognize and destroy virus-infected cells. Members of the Herpesviridae have developed multiple mechanisms to avoid CTL recognition by virtue of downregulation of MHC-I on the cell surface. We report here on an immunomodulatory protein involved in this process, pUL56, which is encoded by ORF1 of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus. We show that EHV-1 pUL56 is a phosphorylated early protein w...
Klier J, May A, Fuchs S, Schillinger U, Plank C, Winter G, Gehlen H, Coester C.Recurrent airway obstruction (RAO) in horses has become a common problem in stabled horses in industrialized countries and deserves new therapeutic strategies. CpG-oligodeoxynucleotides (CpG-ODNs) were developed as effective immunostimulating agents to induce a Th2/Th1 shift. These agents showed a beneficial therapeutic effect in allergic diseases with predominant Th2 immunoresponse. CpG-ODN delivery by gelatin nanoparticles (GNPs) resulted in enhanced cellular uptake in murine and human in vitro studies and was a starting point for the present trial. The aim of this study was to identify an o...
Alcott CJ, Sponseller BA, Wong DM, Davis JL, Soliman AM, Wang C, Hsu W.Ketamine has immunomodulating effects both in vitro and in vivo during experimental endotoxemia in humans, rodents, and dogs. Objective: Subanesthetic doses of ketamine will attenuate the clinical and immunologic responses to experimental endotoxemia in horses. Methods: Nineteen healthy mares of various breeds. Methods: Experimental study. Horses were randomized into 2 groups: ketamine-treated horses (KET; n = 9) and saline-treated horses (SAL; n = 10). Both groups received 30 ng/kg of lipopolysaccharide (LPS, Escherichia coli, O55:B5) 1 hour after the start of a continuous rate infusion (CRI)...
Strasser A, Kühnel H, Velde K, Dadak A.Genotoxic DNA damage due to inhalation anaesthesia has been demonstrated in human lymphocytes. In order to evaluate anaesthesia-associated changes in cell-mediated immunity on the basis of a potential DNA damage as a health risk in horses, single cell gel electrophoresis and lymphocyte proliferation assay were performed on equine lymphocytes which were obtained before, during and after regular castration under inhalation anaesthetic. No significant lymphocytic DNA damage due to isoflurane anaesthesia was observed, whereas lymphocyte proliferative reactivity and lymphocyte counts decreased sign...
LeBlanc MM.Ascending placentitis is a common cause of premature birth, abortion and delivery of compromised, ill foals. Recent experimental models have investigated diagnostic procedures and treatment strategies in an attempt to improve live foal rate. Diagnostics such as transrectal and transabdominal ultrasonography are used to evaluate foetal well-being and placental separation, while measurement of plasma progestins or oestrogen identifies a stressed or hypoxic foetus. Treatment is directed at stopping spread of infection, maintaining uterine quiescence and blocking production of pro-inflammatory cyt...
Neuhauser S, Palm F, Ambuehl F, Möstl E, Schwendenwein I, Aurich C.Mares with compromised pregnancies are often treated with altrenogest to prevent abortion. However, there is only limited information about effects on the foal when altrenogest treatment is continued during final maturation of the fetus. Objective: To determine effects of altrenogest treatment during late gestation in mares on maturity, haematology changes, adrenocortical function and serum electrolytes in their newborn foals. Methods: Six mares were treated with altrenogest (0.088 mg/kg bwt) once daily from Day 280 of pregnancy until foaling and 7 mares served as controls. Results: Foals born...
Sun WC, Moore JN, Hurley DJ, Vandenplas ML, Fortes B, Thompson R, Linden J.Adenosine is an endogenous nucleoside that has potent receptor-mediated immunomodulatory effects on macrophage/monocyte function. In this study, we determined the effects of an adenosine A(2A) receptor agonist, ATL313, on the expression of mRNAs for four pro-inflammatory mediators, IL-1beta, IL-8, COX-2, and TNF-alpha, and the mRNA and protein for the anti-inflammatory cytokine, IL-10 in equine monocytes incubated with lipopolysaccharide (LPS). The results indicate that ATL313 significantly reduces LPS-induced expression of COX-2 and TNF-alpha, enhances the expression of IL-10 and IL-8, but do...
LeBlanc MM, Causey RC.Endometritis, a major cause of mare infertility arising from failure to remove bacteria, spermatozoa and inflammatory exudate post-breeding, is often undiagnosed. Defects in genital anatomy, myometrial contractions, lymphatic drainage, mucociliary clearance, cervical function, plus vascular degeneration and inflamm-ageing underlie susceptibility to endometritis. Diagnosis is made through detecting uterine fluid, vaginitis, vaginal discharge, short inter-oestrous intervals, inflammatory uterine cytology and positive uterine culture. However, these signs may be absent in subclinical cases. Hyper...
Fumuso EA, Aguilar J, Giguère S, Rivulgo M, Wade J, Rogan D.Our objective was to characterize immune parameters in susceptible (SM) and resistant (RM) mares, with and without artificial insemination (AI) and immunomodulation. Eight RM and eight SM were selected based on their reproductive history and functional tests. Both groups of mares were evaluated during three consecutive cycles: Cycle 1, untreated cycle (control); Cycle 2, AI with dead semen; Cycle 3, AI with dead semen and immunomodulation. Endometrial biopsies were taken during the three cycles as follows: Cycle 1--at estrus, when follicles > or =35mm and at diestrus (7+/-1 days after ovulatio...
Kolm G, Knapp E, Wagner R, Klein D.Lactoferrin (LF), a glycogen of the transferrin family with anti-bacterial and immunomodulatory properties, is expressed in various secretions and tissues. Cutaneous LF serves as a mast cell stabilising compound, modulates T cell activity and is found during IgE-mediated late phase reactions at allergen challenged sites. Culicoides hypersensitivity (CHS) in horses is a common IgE-mediated allergic dermatitis, characterised by an early and late phase cutaneous reaction upon allergen challenge. The aim of the study presented here was to examine whether LF mRNA expression in skin biopsies from ho...
Fumuso E, Giguère S, Wade J, Rogan D, Videla-Dorna I, Bowden RA.Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial ...
Davis EG, Rush BR, Blecha F.Immunomodulation with Propionibacterium acnes is used for prophylaxis of respiratory disease or in horses suffering from chronic pulmonary inflammation; however, the mechanism for this response is poorly understood. Semiquantitative reverse transcriptase-polymerase chain reaction assays were used to evaluate gene expression of interleukin (IL)-2, IL-10, interferon (IFN)-gamma, and NK-lysin in healthy horses treated with P. acnes. Findings in the study indicated that horses treated with a P. acnes-based immunomodulator exhibited increased IFN-gamma and NK-lysin gene expression in peripheral blo...
Kim HY, Mott J, Zhi N, Tajima T, Rikihisa Y.Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A. phagocytophila or by allowing feeding by infected ticks. The p44 genes encoding the major outer membrane protein P44s of A. phagocytophila were detected...
Hamm D, Willeford KO, White G, Reed SM, Hamm J.Suppurative lower airway disease is a common debilitating disease in performance horses and, while rarely fatal, is often recalcitrant to conventional therapy. A variety of treatments have been used to combat this condition and we conducted two types of studies to determine if caprine serum fraction--immunomodulator (CSFI), a nonspecific immunomodulator, improved recovery from lower respiratory disease. Two dose response studies were performed to ascertain the efficacy of CSFI. Horses were maintained daily on conventional antibiotic therapy. Respiratory tract exudate, nasal discharge, dyspnoea...
Knottenbelt DC, Kelly DF.OBJECTIVE: Diagnosis and management of periorbital sarcoids in horses is a significant clinical challenge for the practicing veterinary surgeon and pathologist. The purpose of this study was to investigate the response of various types of sarcoids to different therapeutic methods. Animals studied Medical records of 445 clinical patients. Procedures This paper retrospectively examines the clinical and histological features of periorbital sarcoids and the treatment of 445 cases. Treatment by surgical excision, cryosurgery, Bacillus-Calmette-Gaérin (BCG) immunomodulation, topical cytotoxic appli...
Rush BR, Flaminio MJ.Nonspecific immunomodulators are substances that induce non-antigen-specific enhancement of the body's native or acquired defense mechanisms. Immunomodulant preparations are most often used for treatment of chronic, viral, or bacterial infection with evidence of secondary immunosuppression. The proposed mechanism of action of these products is macrophage activation and subsequent release of cytokines that enhance humoral and cell-mediated immunity. In equine medicine, nonspecific immunostimulant products are used for prevention and treatment of infectious respiratory disease and treatment of s...
Ziebell KL, Steinmann H, Kretzdorn D, Schlapp T, Failing K, Schmeer N.The efficacy of an immunomodulator, Baypamun N, was tested in 4-10-month-old horses which were exposed to stress by weaning, transport and commingling with yearlings from different breeders (crowding). Verum (n = 26) and placebo animals (n = 27) received three intramuscular injections of the investigational preparations (days 0, 2, 9) starting at the day of commingling in one stable. The incidence of acute respiratory disease was high during the first 4 weeks after commingling. Approximately 50% of all horses showed seroconversion due to field infection by EHV1 and EHV4 during the observation ...
Stitz L, Bilzer T, Richt JA, Rott R.Borna disease represents a unique model of a virus-induced immunological disease of the brain. Naturally occurring in horses and sheep, the mechanisms of pathogenesis have been studied in experimental animals, namely in the rat. Many investigations have revealed that the infection of the natural hosts principally follows the same pathogenic pathways as observed in rats, leading to a severe encephalomyelitis. This affliction of the central nervous system results in severe neurological disorders that again, are fully comparable in laboratory animals to those in the natural and the different expe...
Mayr A.Among all infectious diseases affecting horses, respiratory disease pose the greatest threat to horses kept in stables, horses used for breeding and race horses. Here a distinction should be made between the so-called monocausal infectious diseases (so-called Henle-Koch postulates) and multicausal infectious diseases which are the result of the synergistic interaction of different processes, that alone do not lead to disease. There is no clearcut distinction between the two groups. The most important monocausal respiratory infections of horses are caused by equine influenza virus (subtypes 1 a...
Eyre P, Hanna CJ, Wells PW, McBeath DG.This article reviews anti-inflammatory and antihypersensitivity drugs under these 4 headings: Functional or physiological antagonists; Selective pharmacological inhibitors; Broad spectrum anti-inflammatory drugs; Miscellaneous inhibitors. The compounds considered include sympathomimetic amines, anticholinergic drugs, antihistamine drugs, tryptamine antagonists and dopamine antagonists, glucocorticosteroids and non-steroidal anti-inflammatory drugs, disodium cromoglycate and diethylcarbamazine citrate. The relationship of the pharmacological actions of these compounds is considered in the conte...
Leal Reis I, Lopes B, Sousa P, Sousa AC, Branquinho MV, Caseiro AR, Rêma A, Briote I, Mendonça CM, Santos JM, Atayde LM, Alvites RD, Maurício AC.Horses are high-performance athletes prone to sportive injuries such as tendonitis and desmitis. The formation of fibrous tissue in tendon repair remains a challenge to overcome. This impels regenerative medicine to develop innovative therapies that enhance regeneration, retrieving original tissue properties. Multipotent Mesenchymal Stem/Stromal Cells (MSCs) have been successfully used to develop therapeutic products, as they secrete a variety of bioactive molecules that play a pivotal role in tissue regeneration. These factors are released in culture media for producing a conditioned medium (...
Terpeluk ER, Schäfer J, Finkler-Schade C, Schuberth HJ.Feed supplements supporting animal welfare and performance are becoming increasingly important. Immunomodulatory effects of such products have been observed in many species. The aim of this study was to analyze whether food supplementation with a fermentation product (SCFP) affects the occurrence of foal diarrhea in early life, and whether the SCFP feeding has an impact on the immediate response to a parenteral vaccination at the age of 6-9 months. Eleven foals received the SCFP (OLI) and eleven foals were fed a placebo (PLA) for 29 days. Growth, diarrhea, and diarrhea severity were observed ...
Barot D, Usimaki A, Linardi RL, Arensberg CM, Ortved KF.Quantify the concentration of α2-macroglobulin (A2M), immunomodulatory cytokines, and TGF-β1 factors in 4 commercially available autologous blood-based products including conditioned A2M (CA2M; Alpha2EQ; Astaria Global), autologous protein solution (APS; Pro-Stride; Zoetis), platelet-rich plasma (PRP; Restigen; Zoetis), and autologous conditioned plasma (ACP; Arthrex ACP). We hypothesized that CA2M would have higher concentrations of A2M and lower concentrations of cytokines and growth factors compared to APS, PRP, and ACP. Unassigned: Blood was obtained from 6 healthy, adult horses and proc...
Mayr A.Among all infectious diseases affecting horses, respiratory disease pose the greatest threat to horses kept in stables, horses used for breeding and race horses. Here a distinction should be made between the so-called monocausal infectious diseases (so-called Henle-Koch postulates) and multicausal infectious diseases which are the result of the synergistic interaction of different processes, that alone do not lead to disease. There is no clearcut distinction between the two groups. The most important monocausal respiratory infections of horses are caused by equine influenza virus (subtypes 1 a...
Fedorka CE, Murase H, Loux SC, Loynachan AT, Walker OF, Squires EL, Ball BA, Troedsson MHT.Maintaining yearly foal production is important for the economic success of the broodmare, and this requires breeding to occur as quickly postpartum as possible. The initial postpartum estrus occurs within 5-20 days postpartum, whereas the uterus is still undergoing repair from tissue alterations during pregnancy and parturition, a process known as involution. Attempts have been made to hasten this process, but with minimal success. Mycobacterium cell wall fraction (MCWF) is an immunomodulator that has been shown to reduce bacterial growth and alter aspects of the immune response to breeding,...
Hamm D, Willeford KO, White G, Reed SM, Hamm J.Suppurative lower airway disease is a common debilitating disease in performance horses and, while rarely fatal, is often recalcitrant to conventional therapy. A variety of treatments have been used to combat this condition and we conducted two types of studies to determine if caprine serum fraction--immunomodulator (CSFI), a nonspecific immunomodulator, improved recovery from lower respiratory disease. Two dose response studies were performed to ascertain the efficacy of CSFI. Horses were maintained daily on conventional antibiotic therapy. Respiratory tract exudate, nasal discharge, dyspnoea...
Berghaus LJ, Cathcart J, Berghaus RD, Ryan C, Toribio RE, Hart KA.Rhodococcus equi (R. equi), a pneumonia-causing intracellular bacterium, results in significant morbidity and mortality in young foals, while healthy adult horses rarely develop disease. Survival and replication within alveolar macrophages (AMφ) are the hallmarks of R. equi's pathogenicity. The vitamin D receptor (VDR) and its ligand, the active vitamin D metabolite 1,25(OH)2D, are important in immune responses to intracellular bacteria. The vitamin D/VDR pathway regulates the downstream production of cytokines in infected human AMφ. The immunomodulatory role of the vitamin D/VDR pathway in ...
Battistin L, Moya LFA, Ferreira LVO, Braz AMM, Carvalho M, Golim MA, Amorim RM.Cell-based therapy using mesenchymal stem cells (MSCs) shows promise for treating several diseases due to their anti-inflammatory and immunomodulatory properties. To enhance the therapeutic potential of MSCs, in vitro priming strategies have been explored. Cannabidiol (CBD), a non-psychoactive compound derived from cannabis, may influence MSC proliferation, differentiation, and immunomodulatory properties. This study evaluates the immunomodulatory potential of equine adipose tissue-derived MSCs (EqAT-MSCs) primed with a CBD-rich cannabis extract. EqAT-MSCs (P3) were primed with CBD concentrati...
Rostang A, Desjardins I, Espana B, Panzuti P, Berny P, Prouillac C, Pin D.This study aimed to investigate both the pharmacokinetic behavior and tolerance of methotrexate (MTX) in horses to design a specific dosing regimen as a new immunomodulatory drug for long-term treatment. To determine the primary plasma pharmacokinetic variables after single intravenous, subcutaneous or oral administration, six horses were administered 0.3 mg/kg MTX in a crossover design study. After a 10-week washout, MTX was administered subcutaneously to three of the six previously treated horses at a dose of 0.3 mg/kg once per week for 3 months. In both studies, MTX and metabolite concen...
Romantsov MG, Galimzianov KhM, Lokteva OM, Kovalenko AL, Stepanov AV.Search for drugs efficient in prophylaxis and treatment of dangerous infections (especially arboviral ones) is rather actual, since no specific therapy is available. Many-year investigations of interferon inductors showed that they had immunomodulating, antiviral and antiinflammatory effects and were low toxic. The present study demonstrated that the protective effect was the following: Venezuelan equine encephalitis (VEE)--cycloferon > amixin = ridostin, Rift Valley fever (RVF)--cycloferon > amixin > ridostin, predator pox (PP)--cycloferon > amixin = ridostin, that was obvious tha...
Bello K, Lorch G, Papich MG, Kim K, Toribio RE, Yan L, Xie Z, Hill K, Phelps MA.Additional immunomodulatory treatment is needed for the management of immune-mediated disease in horses. Mycophenolate mofetil (MMF) is an immunomodulatory agent used in human and veterinary medicine for the prevention of graft rejection and the management of autoimmune diseases. Few studies exist investigating the pharmacokinetics of MMF in horses. The aim of this study was to evaluate the pharmacokinetics of a single dose of MMF in healthy horses in the fed vs. fasted state. Six healthy Standardbred mares were administered MMF 10 mg/kg by a nasogastric (NG) tube in a fed and fasted state. ...
Giraldo A, Koch TG, Madan P, Lepage S, Monteith G, Alizadeh AH, Tran A, Mortagy N, Koenig JB.There is a knowledge gap regarding the effect of extracorporeal shockwave treatment (ESWT) on the stress response and immunomodulatory and anti-inflammatory properties of equine umbilical cord blood mesenchymal stromal cells (CB-MSCs). The objective of this study was to investigate the presence of cellular oxidative stress, inflammatory response, and production of growth factors in CB-MSCs after treatment with ESWT. We hypothesized that CB-MSCs treated with ESWT will experience higher levels of cellular stress and increased production of anti-inflammatory cytokines and growth factors compared ...
Al-Khaldi KO, Al-Ruzaiqi KH, Babji AS, Lim SJ, Sadiq MB, Yimer N.Exercise-induced oxidative stress and inflammation adversely affect the health and performance of athletic horses. This study aimed to evaluate the safety of edible bird's nest (EBN) supplementation and its potential anti-inflammatory and immunomodulatory effects in Arabian race stallions during exercise. Key objectives included assessing EBN's impact on hepatic and renal function, hematological profiles, and sialic acid (SA) levels. Unassigned: Two experiments were conducted on 18 Arabian stallions. In Experiment 1, six healthy stallions were divided into control (n = 2) and EBN-supplemented ...
Cequier A, Serrano MB, Soler-Monsó MT, Bernad E, Vázquez FJ, Vitoria A, Fuente S, Zaragoza P, Romero A, Rodellar C, Barrachina L.The allogeneic administration of equine mesenchymal stem/stromal cells (MSCs) has numerous advantages over autologous therapy, but their interactions with the patient's immune system need to be further elucidated. These interactions can be influenced by factors such as the compatibility between donor-receptor for the major histocompatibility complex (MHC) and by the MHC expression levels, which can change under different conditions like inflammatory exposure and chondrogeneic differentiation. In this study, we evaluated the local immune response induced by chondrogeneically differentiated (MSC...
de Oliveira Ferreira LV, Maia L, Barberini DJ, Takahira RK, de Vasconcelos Machado VM, Machado GF, de Melo GD, Amorim RM.Mesenchymal stem cells (MSCs) have emerged as a promising alternative for treating neurological disorders due to their neuroprotective, neuroregenerative, immunomodulatory, anti-inflammatory, and anti-apoptotic properties. The use of allogeneic MSCs offers advantages such as the selection of specific cells and their immediate availability. This study aimed to evaluate the safety and feasibility of intrathecal transplantation of allogeneic equine bone marrow-derived MSCs (EqBM-MSCs) in healthy horses. Ten healthy horses (five-12 years old) were randomly divided into two groups. In the control g...
Salinas-Varas C, Espinosa G, Muñoz-Caro T, Conejeros I, Gärtner U, Fey K, Arnhold S, Taubert A, Hermosilla C.Neutrophil extracellular trap (NET) are thin and long web-like structures composed of DNA and antimicrobial proteins released by activated polymorphonuclear neutrophils (PMN) as part of the innate immune response. Adipose-derived stem cells (ADSCs) represent an accessible, abundant and minimal invasive source of mesenchymal stem cells (MSCs), with high regenerative potential, immunomodulatory and anti-inflammatory properties. Although recognized immunomodulatory properties of ADSCs, their interaction with PMN and their role on NET formation remains poorly characterized. The present study aimed...
Graide H, Duysens J, Frank T, Mouithys-Mickalad A, Niesten A, Sandersen C, Ceusters J, Serteyn D.Musculoskeletal pathologies present challenges in athletic horses, often leading to functional impairment. The slow or limited regenerative capacity of bone, joint, and tendon/ligament injuries, coupled with the limitations of conventional treatments, highlights the need for innovative therapies such as ortho-biologics and mesenchymal stem/stroma cells. Traditional 2D cell culture systems with fetal bovine serum (FBS) fail to replicate the complexity of the in vivo environment, whereas 3D cultures more accurately mimic native tissue architecture and cell-cell interactions. This study describes...
Pfannkuche H, Maus J, Engel KM, Schiller J, Schneider MR.Skin sebaceous glands (SGs) synthesize and secrete sebum, a mixture of lipids and cellular debris that defends the external body surface against physicochemical challenges. Recent data define the SG as a dynamic entity with potential functions beyond skin protection, including immunomodulatory actions and the regulation of energy metabolism. We postulate that the SG also has important, unrecognized roles in physiological and pathological processes in domestic animals. Conversely, data derived from domestic animals may have translational relevance for humans. This review article summarizes SG s...