Localization of cytokines in tendinocytes of the superficial digital flexor tendon in the horse

Although inflammatory activation of cytokines have been analyzed in various tissues, there have only been a few and as-yet-inconclusive studies on cytokines in equine tendons. In this study, the localizations of 4 cytokines (IL-1alpha, IL-1beta, TNFalpha and IFNgamma) in tendinocytes of the equine superficial digital flexor tendon (SDFT) were analyzed by the use of an immunohistochemical method. In inflamed tendons positive staining for all 4 cytokines antibodies were detected in endotedinieum cells and vascular epithelial cells. In contrast, negative or trace immunoreactions were obtained in many tendinocytes in the normal tendon. The variation in cellular immune responses depending on the kind of cytokine may reflect the physiological/pathological condition of the SDFT.     

Relationship between Mycobacterium avium subspecies paratuberculosis, IL-1alpha, and TRAF1 in primary bovine monocyte-derived macrophages

Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular pathogen that resides in host macrophage cells. Presently, little is known about how MAP is able to subvert the normal bacteriocidal functions of infected macrophages. Previously, we reported that ileal tissues from MAP infected cattle contained high levels of interleukin-1 alpha (IL-1alpha) and tumor necrosis factor receptor-associated factor 1 (TRAF1), relative to ileal tissues from uninfected cattle. High-level expression of these two proteins could have profound effects on macrophage function, intracellular signaling, and apoptosis. We now demonstrate that high levels of TRAF1 protein are located primarily within macrophages infiltrating areas of MAP infection. We have also utilized cultured bovine monocyte-derived macrophage cells (MDM) either infected with live MAP or stimulated with recombinant IL-1alpha (rIL-1alpha) to determine if there is a relationship between IL-1alpha and TRAF1 expression. These studies have identified a dose dependent increase in TRAF1 protein levels in bovine MDM in response to infection with live MAP or following treatment with rIL-1alpha. Sustained TRAF1 protein expression was dependent upon interaction of rIL-1alpha with it's receptor and rIL-1beta was also able to enhance TRAF1 gene expression. Our results suggest that MAP may use the IL-1-TRAF1 system to enhance TRAF1 protein expression in infected bovine MDM. These novel results provide evidence for a new avenue of research on the effect of MAP and other intracellular pathogens on macrophage signaling and apoptosis.     

Dexamethasone reduces endotoxin-induced tumor necrosis factor activity production in vitro by equine peritoneal macrophages

This study evaluated the effect of dexamethasone on endotoxin-induced production of tumor necrosis factor (TNF) activity in vitro by equine peritoneal macrophages. Peritoneal macrophages from adult horses were cultured in the presence of dexamethasone (1-100 microM) for various time periods (2 hour, 0.5 hour, 0 hour) prior to the addition of endotoxin (5 ng/ml), then the secretion of TNF activity was evaluated. Macrophage supernatant concentrations of TNF activity were estimated by a modified in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI 164 clone 13. An experiment was performed to determine whether dexamethasone interfered with the cytolytic bioassay's ability to detect TNF activity. The endotoxin-induced TNF activity production by equine peritoneal macrophages was significantly reduced by co-incubation with 100 microM dexamethasone, but not by tested concentrations of dexamethasone less than 100 microM. This concentration of dexamethasone greatly exceeds those generally attained by therapeutic use of dexamethasone in horses. Preincubation time did not affect the ability of 100 microM dexamethasone to reduce TNF production by equine macrophages. The quantitation of equine TNF activity by its cytolytic bioassay was not altered by dexamethasone.     

口蹄疫病毒前导蛋白酶抑制宿主先天性免疫应答的机制

口蹄疫是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)引起的一种急性、热性、高度接触传染性动物疫病.口蹄疫病毒有多种机制对抗宿主的先天性免疫应答,在这个过程中病毒的前导蛋白酶(L^pro)发挥了关键作用.L^pro可切断宿主细胞帽子依赖性的蛋白翻译,抑制干扰素蛋白的合成;L^pro通过破坏核转录因子-κB (NF-κB)的完整性或减少干扰素调节因子3/7(IRF3/7)的表达,从而抑制IFN mRNA的产生;L^pro还会参与维甲酸诱导基因Ⅰ(RIG-Ⅰ)、TANK结合激酶1(TBK1)、TNF受体相关因子3(TRAF3)和TRAF6的去泛素化,从而影响Ⅰ型干扰素信号通路的活化.     

Leptin, tumor necrosis factor-alpha (TNF), and CD14 in ovine adipose tissue and changes in circulating TNF in lean and fat sheep

Four studies were designed to determine whether 1) tumor necrosis factor-alpha (TNF) and the Lipopolysaccharide (LPS) binding ligand, CD14, are produced by sheep adipose tissue; 2) nutritional reserves and/or short-term fasting affect circulating concentrations of TNF; 3) there is a relationship between TNF and metabolic factors in sheep; and 4) inflammation alters circulating concentrations of leptin. In Exp. 1 and 2, ewes were assigned, based on ultrasonic assessments of last-rib subcutaneous fat measurements to fat (fat thickness > 1 cm; mean = 1.52 +/- 0.03 cm) or thin (fat thickness < 1 cm; mean = 0.25 +/- 0.03 cm) groups. Fat and thin ewes were assigned to fed or fasted groups for a total of four groups (fed-fat; fasted-fat; fed-thin; fasted-thin). Fed-ewes had ad libitum access to feed, and fasted-ewes were prohibited feed 48 h before initiation of sample collection. In Exp. 1, subcutaneous fat samples were collected from just above the last rib for detection of TNF and CD14 mRNA, and immunoreactivity. Tumor necrosis factor-alpha-like immunoreactivity in adipocytes was sparse, more pronounced in cells in fed-ewes than fasted-ewes, and localized to membranes between adjacent cells in nucleated regions. Immunoreactivity for CD14 was minimally observed but present in adipocytes and widely expressed in infiltrating monocytes and epithelial vascular cells. Leptin was detected in adipocytes. In Exp. 2, plasma samples collected every 6 h for 24 h were analyzed for plasma concentrations of TNF. Fat ewes had greater plasma concentrations of TNF than thin ewes (P = 0.039). In Exp. 3, wethers were injected i.v. with interleukin-1beta or TNF. Blood samples were collected every 15 min for 8 h following injection. Plasma concentration of leptin was not affected by treatment (P > 0.39). In Exp. 4, wethers were injected with LPS. Blood samples were collected every 15 min for 8 h following injection. Plasma concentration of leptin was not altered by LPS (P > 0.20). These results provide evidence: 1) of TNF-like immunoreactivity within fat tissue; 2) that elements within fatty tissues have CD14 that may allow adipocyte function to be directly affected by LPS; 3) that plasma concentrations of leptin are not altered by LPS treatment; and 4) that circulating concentrations of TNF are elevated with obesity in sheep.     

鸡TRAF6和TIFA蛋白的序列分析与相互作用验证

旨在对鸡肿瘤坏死因子受体相关因子6（tumor necrosis factor（TNF）receptor-associated factor 6,TRAF6）和肿瘤坏死因子受体相关因子相互作用的具有叉形头相关结构域蛋白（TNF receptor associated factor（TRAF）-interacting protein with a forkhead-associated（FHA）domain,TIFA）进行序列分析,并进行蛋白质相互作用验证。本研究首先以鸡胚成纤维细胞提取的总RNA反转录产物为模板扩增鸡TRAF6和TIFA基因的CDS区,分别构建重组真核表达载体pCMV-HA-TRAF6和pEGFP-C1-TIFA;运用生物信息学软件ProtParam、SOPMA、SWISS-MODEL、MegAlign和PSORTⅡ分别对鸡TRAF6和TIFA蛋白理化性质、二级和三级结构、氨基酸同源性、功能结构域保守性和亚细胞定位进行分析。将重组真核表达载体共转染细胞,通过荧光共定位和免疫共沉淀（Co-IP）试验验证鸡TRAF6和TIFA蛋白之间的相互作用。结果,成功扩增鸡TRAF6和TIFA基因,并构建其重组真核表达载体pCMV-HA-TRAF6和pEGFP-C1-TIFA。序列分析结果显示,鸡TRAF6和TIFA基因CDS区分别为1 638和564 bp,共编码545和187个氨基酸,分子量约为62和22 ku。蛋白质二级和三级结构分析结果表明,鸡TRAF6和TIFA蛋白均以无规则卷曲和α螺旋为主。氨基酸同源性分析结果发现,鸡与人和其他哺乳动物TRAF6和TIFA蛋白的同源性分别为76.4%~80.3%和49.7%~53.3%,而与非洲爪蟾的同源性分别为69.4%和49.7%,并且鸡TRAF6和TIFA蛋白的功能结构域存在多个氨基酸位点变异。亚细胞定位预测结果表明,鸡TRAF6和TIFA蛋白主要定位在细胞核。荧光共定位和Co-IP试验结果显示,鸡TRAF6和TIFA蛋白在细胞核中具有共定位,并且存在相互作用。本研究结果显示,鸡与人和其他哺乳动物以及非洲爪蟾TRAF6和TIFA蛋白的同源性及功能结构域保守性均较低,但鸡TRAF6和TIFA蛋白能在细胞核中发生相互作用,这可为进一步研究鸡TRAF6和TIFA蛋白相互作用调控鸡相关病毒复制的作用机制奠定基础。     

Expressions of tumor necrosis factor-α, its receptor I,II and receptor-associated factor 2 in the porcine corpus luteum during the estrous cycle and early pregnancy

We examined the gene and protein levels of tumor necrosis factor (TNF)-α, its receptors (types I and II, designated TNF-RI and TNF-RII, respectively), TNF receptor-associated factor 2 (TRAF2) and morphological features in the porcine corpus luteum (CL), on Days 13 and 17 (Day 0 = the last day of estrus) of the estrous cycle or of early pregnancy. Gene expression levels of TNF-α, TNF-RI, TNF-RII and TRAF2 were unaffected by the day or reproductive status. TNF-α concentration was significantly higher in the CL on Day 17 of pregnancy than on Day 13 of pregnancy and on day 17 of the estrous cycle. The TNF-RI protein level was significantly higher in the CL on Days 13 and 17 of pregnancy than those of the estrous cycle, significantly increasing on Day 17 compared with those on Day 13 in pregnancy. In relation to TNF-RII protein levels, although there were no change during pregnancy, there was a tendency (P?=?0.0524) to up-regulate as pregnancy proceeded. In estrous cycle, TNF-RII protein levels decreased significantly as luteolysis proceeded. TRAF2 protein level was significantly higher in the CL on Days 13 and 17 of pregnancy than during estrous. There were few apoptotic bodies in the CL between Days 13 and 17 of pregnancy than during esrous. There were few apoptotic bodies in the CL between Days 13 and 17 of pregnancy. The number of apoptotic bodies was much greater than the CL on Day 17 of the estrous than those of pregnancy. Thus, the TNF-α and TNF-RI and TNF-RII pathways including the TRAF2 protein, known to control of cell differentiation, tissue renewal and apoptosis, might participate in maintaining the porcine CL during early pregnancy.     

Investigation of mRNA expression of tumor necrosis factor-alpha, interleukin-1beta, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin.

OBJECTIVE: To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin- (IL)-1beta from cultured equine smooth muscle cells (SMC). SAMPLE POPULATION: Segments of palmar digital artery harvested from 6 clinically normal adult horses. PROCEDURE: Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 microg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-alpha, and IL-1beta was performed, using isolated total cellular RNA. RESULTS: Although no message was detected for IL-1beta or TNF-alpha in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1beta and TNF-alpha mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis.     

An anti-human recombinant tumor necrosis factor alpha (TNF alpha) monoclonal antibody recognizes an epitope in feline TNF alpha

It is likely that the murine response to human recombinant TNF alpha (hrTNF alpha) may generate antibodies (Ab) to epitopes present in TNF alpha from other species. Here, we demonstrate that F5 anti-hrTNF alpha monoclonal antibody (mAb) recognizes feline TNF alpha while E8 anti-hrTNF alpha mAb failed to do so. In order to demonstrate that E8 and F5 mAb recognize different epitopes in the hrTNF alpha molecule, a constant concentration of E8 and variable concentrations of F5 were incubated with solid phase bound hrTNF alpha. Binding of E8 and F5 to hrTNF alpha was determined with anti-mu and gamma chain specific Ab. F5 bound equally to hrTNF alpha in the presence or absence of E8 and the same amount of E8 bound to hrTNF alpha, in spite of the presence of F5. When using the E8 and F5 mAb for capturing the TNF alpha from the equine, canine, feline and bovine species, in supernatants of an ex vivo lipopolysaccharide (LPS)-stimulated whole blood cell culture, we only detected the feline TNF alpha by F5 mAb (p = 0.001). By a cytotoxic assay on L929 fibroblasts, we indeed demonstrated the feline TNF alpha production after the LPS stimulus. In an inhibition assay, the human and feline cytokines competed for F5, although the inhibition of native human TNF alpha binding to F5 was significant but only about 20% (p = 0.001). In conclusion, most likely the F5 anti-hrTNF alpha mAb recognizes an epitope in feline TNF alpha. Its immunomodulatory potential in the feline model remains to be studied.     

Changes in Third Carpal Bone Articular Cartilage After Synovectomy in Normal and Inflamed Joints

Objective —To determine if arthroscopic synovectomy in normal and inflamed joints had temporal or site-related effects on articular cartilage.
Study Design —Alterations in equine third carpal bone articular cartilage were studied at two time periods: groups 1 and 2 (6 weeks) and groups 3 and 4 (2 weeks) after synovectomy in normal (groups 2 and 4) and inflamed carpi (groups 1 and 3).
Animal Population —16 carpi from eight horses.
Methods —Biochemical and biomechanical properties of dorsal and palmar articular cartilage were determined by radiolabeling, proteoglycan (PG) extraction, chromatography, electrophoresis, and indentation testing.
Results —Synovectomy in inflamed joints produced the greatest concentration of newly synthesized PG in articular cartilage by 2 weeks. Synovectomy in normal joints produced significantly greater newly synthesized PG in articular cartilage by 6 weeks. Endogenous PG was only significantly greater in inflamed joints after 6 weeks. Dorsal sites had greater newly synthesized and endogenous PG in some groups. Chromatographic profiles of newly synthesized PG demonstrated early and late PG peaks. Electrophoresis of late PG peak showed a toluidine blue-positive band that comigrated with human A1D1 PG monomer in the two groups with the most newly synthesized PG. This band was reactive with monoclonal antibody 1C6 specific for the hyaluronic acid-binding region of aggrecan. For the material properties evaluated, only Poisson's ratio was significantly decreased between groups as a function of time (6 weeks < 2 weeks), and this was most pronounced in the thicker dorsal sites.
Conclusions —Synovectomy in inflamed joints produced site-specific, significantly greater responses in articular cartilage as compared with synovectomy in normal joints.
Clinical Relevance —Synovectomy may not be beneficial to the articular cartilage in inflamed joints.     

Molecular study on chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor-associated factor-5

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the tumor necrosis factor receptor (TNFR) superfamily. In this study, we cloned and characterized two genes that encode chicken TNFR-II and TRAF5. The initial cDNA fragments were obtained by suppressive subtractive hybridization (SSH) of chicken spleen cells with or without lipopolysaccharide stimulation (Salmonella typhimurium SL1181 (RE-mutant)). The results showed that chicken TNFR-II is 1518 bp in length with an open reading frame (ORF) of 1386 bp having 31% homology with human TNFR-II. Expression analysis of chicken TNFR-II revealed that it is highly expressed in the spleen and bursa of Fabricius. The chicken cell lines IN24, MSB1 and 1104B express TNFR-II abundantly. The time course analysis of expression in spleen, bursa of Fabricius and IN24 cell line showed that TNFR-II is maximally expressed at 6 h after stimulation in bursa of Fabricius and after 8 h stimulation in the IN24 cell line. With regard to TRAF5, the complete sequence was 1936 bp in length with an ORF of 1671 bp that showed 71.3% homology with human TRAF5. Expression analysis showed that, among the tissues examined, TRAF5 was strongly expressed in spleen and bursa of Fabricius, while among the cell lines examined, it was maximally expressed in IN24. Thus, both genes were expressed in the same tissues and cell line among examined materials. These results suggest that chicken TNFR-II may interact with TRAF5 adaptor protein to complete its signal transduction pathway.     

Prekallikrein deficiency in a family of miniature horses

Two sibling miniature horses, a male and a female, had a normal 1-stage prothrombin time and a prolonged activated-partial thromboplastin time (APTT). The addition of as little as 5% of a normal equine plasma pool to the plasma samples of both horses shortened their prolonged APTT to within normal limits. Coagulation factor analysis revealed deficiencies in factor XII (12 and 13 U/dl, control population 77 to 128 U/dl), when determined with a feline factor XII-deficient plasma substrate, but normal concentrations (119 and 96 U/dl) when a human factor XII-deficient plasma substrate was used. Deficiencies of another factor, prekallikrein, were detected with a human prekallikrein-deficient plasma substrate (16 and 6 U/dl, control population 70 to 173 U/dl). Other intrinsic coagulation factors were present in normal concentrations. The APTT was measured with plasma from the 2 horses after various incubation periods (1 to 15 minutes) with a contact activator before the addition of Ca ions. With incubation times of greater than or equal to 10 minutes, the APTT of both horses were essentially the same as that of the normal equine plasma pool. Several family members of the 2 prekallikrein-deficient miniature horses appeared to be heterozygous carriers of the prekallikrein deficiency.     

Tumor necrosis factor activity in serum from neonatal foals with presumed septicemia.

A study was performed to determine prevalence of tumor necrosis factor (TNF) activity in serum of equine neonates with presumed sepsis and to determine correlation between serum TNF activity and severity and outcome of disease. Twenty foals less than 21 days old were considered suitable for inclusion in this study by satisfying clinical and laboratory criteria suggestive of septicemia. At admission, blood samples were collected from all foals for determination of serum TNF activity, then clinical course and outcome of disease were recorded. Thirty-one clinically normal foals less than 21 days old served as controls for serum TNF activity. Serum TNF activity was estimated by use of an in vitro cytotoxicity bioassay and WEHI 164 clone-13 murine fibrosarcoma cells. Of the 20 foals with presumed sepsis, 5 had high serum TNF activity. Mean heart rate (P less than 0.005), mucosal petechial hemorrhages (P = 0.06), and death rate (P = 0.06) were greater in the group of foals with high serum TNF activity. These foals also had a lower mean neutrophil count (P less than 0.001), greater band-to-segmented neutrophil ratio (P less than 0.0001), and more prevalent neutrophil toxic changes (P = 0.07) than did foals without serum TNF activity (P = 0.02). Joint swelling was more prevalent in foals without serum TNF activity. Results of the study indicate that serum TNF activity is correlated with clinical criteria of sepsis in equine neonates. An association was apparent between disease severity and serum TNF activity in this group of foals with presumed septicemia.     

Mycoplasma ovipneumoniae induces inflammatory response in sheep airway epithelial cells via a MyD88-dependent TLR signaling pathway

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a bacterium that specifically infects sheep and goat and causes ovine infectious pleuropneumonia. In an effort to understand the pathogen–host interaction between the M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory response using a primary air–liquid interface (ALI) epithelial culture model generated from bronchial epithelial cells of Ningxia Tan sheep (Ovis aries). The ALI culture of sheep bronchial epithelial cells showed a fully differentiated epithelium comprising distinct epithelial types, including the basal, ciliated and goblet cells. Exposure of ALI cultures to M. ovipneumoniae led to increased expression of Toll-like receptors (TLRs), and components of the myeloid differentiation factor 88 (MyD88)-dependent TLR signaling pathway, including the MyD88, TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinases (IRAKs) and nuclear factor-kappa B (NF-κB), as well as subsequent pro-inflammatory cytokines in the epithelial cells. Of interest, infection with M. ovipneumoniae failed to induce the expression of TANK-binding kinase 1 (TBK1), TRAF3 and interferon regulatory factor 3 (IRF3), key components of the MyD88-independent signaling pathway. These results suggest that the MyD88-dependent TLR pathway may play a crucial role in sheep airway epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI culture system may be a reliable model for investigating pathogen–host interactions between M. ovipneumoniae and airway epithelial cells.     

Immunohistochemical study of angiogenesis and angiogenic factors in equine granulosa cell tumours

The first part of our study (Müller et al., 2009) characterized angiogenesis in the equine cycling ovary through histomorphological and immunohistochemical examinations (vascular endothelial growth factors A and B [VEGF A, VEGF B], vascular endothelial growth factor receptors 1 and 2 [VEGF-R1, VEGF-R2], vascular angiopoietins 1 and 2 [Ang1, Ang2], angiopoietin receptor [Tie2], and von Willebrand Factor). Since angiogenesis plays an important role in development and growth of numerous tumours, the second part of our study involved a similar examination of 70 equine granulosa cell tumours (GCTt). The results of the second study were compared with those of the normal equine ovary. Certain similarities in the expression pattern could be detected between normal, cyclical ovaries (Müller et al., 2009) and GCTt. The immunoreactivity of granulosa cells and Leydig-like cells in GCTt resembles granulosa cells and luteinized thecal cells in periovulatory cycling ovaries. The neoplastic cells support circulation, supply and growth of GCTt by contributing to angiogenesis.     

Non-steroidal anti-inflammatory drugs in equine orthopaedics

Orthopaedic disorders are commonly encountered in equine veterinary medicine, and non-steroidal anti-inflammatory drugs (NSAIDs) play an important role in the management of many equine orthopaedic disorders. There are multiple NSAIDs available for use in horses, including both non-selective and selective NSAIDS, and the body of literature evaluating the efficacy of these medications, their effects on normal and inflamed musculoskeletal tissues, and their side effects is broad. This review aims to summarise the current literature on the use of NSAIDs for equine orthopaedic disorders and examines new and future avenues for the management of inflammation in equine orthopaedics.     

早期断奶对仔猪血清肿瘤坏死因子和白细胞介素-2含量的影响

本文旨在研究仔猪血清肿瘤坏死因子(TNF)和白细胞介素-2(IL-2)含量的变化规律,以及早期断奶、免疫球蛋白和乳铁蛋白对TNF和IL-2含量的影响。试验选用约克夏仔猪,试验1分别在1、7、14、21和28日龄选取2头仔猪;试验2仔猪分别在14、18和35日龄断奶;试验3选取16头仔猪,分为4个处理,每个处理4个重复;试验4选取32头仔猪,分为4个处理,每个处理8个重复。结果表明:(1)血清TNF(P<0.01)、IL-2(P<0.05)含量从1日龄到7日龄显著降低,14日龄又上升,并维持一定水平。(2)与35日龄断奶仔猪相比,早期断奶仔猪断奶14和35 d血清IL-2含量显著降低(P<0.05),14日龄断奶仔猪血清TNF含量也表现出降低的趋势。(3)饲喂血浆蛋白粉和特异性免疫卵黄日粮仔猪血清TNF含量显著提高(P<0.05)。(4)饲喂2、10和20 mg/kg乳铁蛋白仔猪血清TNF、IL-2含量均表现出提高的趋势。由此得出结论,早期断奶降低了仔猪血清IL-2含量,饲喂血浆蛋白粉和特异性免疫卵黄日粮可以提高血清TNF含量。     

Autologous conditioned serum: the comparative cytokine profiles of two commercial methods (IRAP and IRAP II) using equine blood

Reasons for performing study: Osteoarthritis (OA) is one of the most prevalent and debilitating conditions affecting the horse. Autologous conditioned serum (ACS), commercially available as IRAP and IRAP II, is a recently developed treatment for OA in which plasma is prepared from venous blood by incubation with glass beads for 24 h. This product has been shown to increase anti‐inflammatory cytokines and growth factors in human blood. However, data for equine ACS preparations are lacking. Objectives: To characterise the protein profiles produced by commercially available ACS systems in equine blood. Methods: Blood was drawn from 5 horses into 6 groups: red top vacutainer (control), IRAP and IRAP II, with and without heparin. Samples were collected 1 or 24 h post draw and analysed for IL‐1ra, IL‐10, IGF‐1, TGF‐β, TNF‐α and IL‐1β using ELISAs. Results: Twenty‐four hour IRAP and IRAP II samples contained significantly higher levels of all cytokines relative to 1 h serum controls. At 24 h, IRAP II contained significantly higher levels of IL‐1ra and IRAP contained significantly higher levels of TNF‐α, compared to 24 h controls. In addition, TGF‐β, IL‐10 and IL‐1β in IRAP and IRAP II sera were similar to 24 h serum controls. The addition of heparin significantly reduced levels of IGF‐1, TNF‐α and TGF‐β, and significantly elevated levels of IL‐1ra. Conclusions: The cytokine profile that IRAP II produced is modestly better than IRAP. Incubation of whole blood in glass tubes stimulated cytokine synthesis, although not as efficiently as IRAP II. Potential relevance: Although high levels of IL‐1ra were found in ACS, elevation of other factors suggests these cytokines play a previously understated role in clinical improvements. Because ACS has been shown to alleviate clinical symptoms of OA, the present study suggests that factors other than IL‐1ra alone might be involved in its clinical efficacy. Species‐dependent elevations of cytokines warrant further investigation and optimisation of the systems appears to be necessary based on the differences between human and equine blood.