Distribution of TNF receptors and TNF receptor-associated intracellular signaling factors on equine tendinocytes in vitro

Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-R-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-kappaB), and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTs of thoroughbred horses. The tendinocytes were treated with 10 ng/ml equine TNFalpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF -R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFalpha-treated cells (inflamed condition). Intense TRAF2 and NF-kappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in vitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.     

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.     

Alteration in the apoptosis process of rat esophageal epithelium with hyperproliferation of indigenous bacteria under a physiological condition

The apoptosis process in rat esophageal epithelium was investigated using enzyme-immunohistochemistry and transmission electron microscopy. As a result, Fas and Fas-L were expressed in the epithelial cell membrane and cytoplasm from the stratum spinosum (SS) to the stratum granulosum (SG). No TNF-R1 show immunopositivity in the cell membranes. TNF-α and caspase-8 were not observed in any layer. Caspase-10, cleaved caspase-3, XIAP and DNase-1 were found in the epithelial cytoplasm from the SS to the SG, whereas Bid, Apaf-1 and cleaved caspase-9 were detected only in the SG. Cytochrome c was observed as cytoplasmic granular positivity from the stratum basale (SB) and altered into homogeneous immunopositivity in the SG. Bcl-2 and Bcl-X immunopositivity was detected in cytoplasm from the SB to the SG. Immunoreactions of Bak in the cytoplasm and Bax beneath the cell membrane were observed from the upper portion of the SS with increasing intensity toward the SG. In the sites with the hyperproliferation of indigenous bacteria, TNF-R1, TNF-α and caspase-8 were detected in the SG and the immunopositive intensities of Bid, Apaf-1 and cleaved caspase-9 were altered to be strong. Prominently swollen cells and decreased mitochondria were ultrastructurally confirmed in the uppermost layers of stratum corneum. These findings suggest that the Fas-Fas-L-interaction initially induces apoptosis through a mitochondria-independent pathway and secondarily through a mitochondria-dependent pathway, leading to eventual epithelial cell death in the rat esophageal epithelium. The bacterial stimuli probably enhance the mitochondria-dependent pathway through the TNF-R1-TNF-α interaction.     

白头翁汤通过抑制TLR4-ERK1/2信号通路缓解LPS诱导的微血管内皮细胞炎性反应

近年来,中药复方抗炎机制的研究不断深入,白头翁汤复方（Pulsatilla decoction,PD）作为经典的清热解毒中药方剂常用于预防和治疗细菌性腹泻。然而其抗炎机制和靶细胞研究仍然不明确,本课题以大鼠肠黏膜微血管内皮细胞（RIMVECs）为模式细胞,旨在研究白头翁汤对LPS诱导的RIMVECs炎症反应的调控作用。利用LPS刺激RIMVECs,通过荧光定量PCR（RT-PCR）、蛋白免疫印迹（Western blot）的方法检测白头翁汤对LPS刺激后RIMVECs的炎性信号通路TLR4-ERK1/2信号通路关键蛋白TLR4、TRAF6、ERK的mRNA及蛋白表达。进一步采用酶联免疫吸附方法（ELISA）检测白头翁汤对LPS刺激后的炎性因子IL-6、IL-8、IL-1β、TNF-α的分泌情况。结果表明：白头翁汤可以显著降低LPS诱导的TLR4、TRAF6、ERK的mRNA水平及蛋白表达并降低了LPS诱导的细胞下游炎性因子的分泌。白头翁汤通过抑制TLR4-ERK1/2信号通路缓解LPS所诱导的RIMVECs炎性反应,发挥抗炎作用。     

Cloning and expression of equine insulin-like growth factor binding proteins in normal equine tendon

OBJECTIVES: To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons. ANIMALS: 7 horses. PROCEDURE: Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB). RESULTS: Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected byWLB in normal tendon and markedly increased in damaged tendons. CONCLUSIONS AND CLINICAL RELEVANCE: Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs.     

蓝舌病毒NS4基因真核表达对IFN-β信号通路基因mRNA表达的影响

通过构建蓝舌病毒(BTV)NS4基因真核表达载体pcDNA3.1-NS4-eGFP,转染HEK-293T细胞,利用Western blot及荧光显微镜分析NS4蛋白的表达与亚细胞定位特征;pcDNA3.1-NS4-eGFP转染的HEK-293T细胞添加20 HAU/mL仙台病毒(SeV)刺激后,qRT-PCR法分析NS4基因表达对SeV诱导的上游识别基因RIG-Ⅰ、MDA5、VISA、TBK1、IKKε、IRF3、TRAF3、TRAF6、IRF9、干扰素基因(IFN-α、IFN-β)以及干扰素刺激基因ISG15和USP18的mRNA表达水平的影响。在HEK-293T细胞内转染pcDNA3.1-NS4-eGFP质粒24 h后,分别添加20 HAU/mL SeV刺激24,48 h,qRT-PCR结果表明,细胞内表达NS4-EGFP后,RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15及IFN-β基因mRNA表达极显著下降,随着SeV诱导时间的延长,VISA、TBK1、IKKε、USP18基因mRNA表达差异呈不显著趋势。本研究成功构建BTV NS4基因真核表达载体pcDNA3.1-NS4-eGFP,NS4-EGFP融合蛋白在HEK-293T细胞中主要分布于细胞核周围及细胞核内。BTV NS4基因在HEK-293T细胞内的表达显著下调SeV诱导的IFN信号通路相关基因RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15和IFN-β的表达,为进一步探究NS4基因在BTV拮抗宿主细胞免疫应答中的机制奠定基础。     

Cloning and expression of type III collagen in normal and injured tendons of horses

OBJECTIVE: To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses. ANIMALS: 14 healthy adult horses. PROCEDURE: The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides were assessedby gel electrophresis. RESULTS: Type III collagen mRNA expression and protein content increased immediately after injury and remained increased. Type III collagen was localized to the endotenon in normal tendon and in injured tendon at 1 week. At 8 and 24 weeks, expression became more widely distributed throughout the tendon parenchyma. Injured tendon contained 6 times more type I than type III collagen mRNA. Quantities of type III collagen protein were maximal in the first 4 weeks after injury (approx 33%) and then began to decrease. CONCLUSIONS AND CLINICAL RELEVANCE: Type III collagen expression is increased initially in endotenon and subsequently in parenchyma of healing tendon; however, type III remains the minor collagen throughout the healing process. The role of type III collagen in tendon healing is not fully elucidated.     

Evaluation of cyclooxygenase protein expression in traumatized versus normal tissues from eastern box turtles (Terrapene carolina carolina)

This pilot study was designed to determine whether cyclooxygenase (COX)-1, COX-2, or both are expressed in normal turtle tissues and whether level of expression changes when tissue becomes inflamed. Five eastern box turtles, Terrapene carolina carolina, that either died or were euthanatized due to disease or injuries were used for this work. Tissues were obtained from the five turtles. Western blot analysis was used to evaluate tissues for COX-1 and COX-2 proteins. Densiometric analysis was used to compare Western blot bands within each turtle. COX-1 and COX-2 were found in the liver, kidney, grossly normal muscle, and grossly traumatized (inflamed) muscle of all study turtles. In all cases, COX-1 and COX-2 proteins were increased in traumatized muscle over grossly normal nontraumatized muscle. The highest levels of COX-1 and COX-2 proteins were found in kidney and liver. There was no statistical difference between the amount of COX-1 protein in liver and kidney, but traumatized muscle compared with grossly normal muscle had significantly greater COX-1 but not COX 2 protein concentrations. There was no statistical difference between the amount of COX-2 protein in liver and kidney. Traumatized muscle expressed nonstatistically significant greater amounts of COX-2 compared with grossly normal muscle. COX-1 and COX-2 proteins are expressed in turtle tissues, and both isoforms are upregulated during inflammation of muscle tissue. Traditional nonsteroidal anti-inflammatory drugs (NSAIDs) that block both COX isoforms might be more efficacious than COX-2-selective drugs. This work suggests that NSAIDs should be evaluated for potential liver and kidney toxicity in turtles.     

重组结核分枝杆菌CFP10蛋白对A549细胞TLR信号途径介导的炎症反应的影响

本研究旨在检测重组结核分枝杆菌10 ku培养滤液蛋白（culture filtrate protein 10,CFP10）对肺泡Ⅱ型上皮细胞系A549细胞Toll样受体（TLRs）信号途径及其介导的炎症反应的影响。PCR扩增cfp10目的基因片段,构建重组质粒载体pCzn1-CFP10。将重组质粒pCzn1-CFP10转入BL21（DE3）大肠杆菌感受态细胞,IPTG诱导表达重组蛋白CFP10（rCFP10）并鉴定,纯化rCFP10,去除内毒素及脱盐备用。设置对照组,利用MTT检测rCFP10对A549细胞的存活率的影响;通过qRT-PCR、Western blot及ELISA等技术,分别在转录和翻译水平检测rCFP10对A549细胞TLRs信号途径关键分子及其下游炎症因子表达的影响。结果显示,成功构建重组表达载体pCzn1-CFP10并表达纯化出高纯度的rCFP10。MTT结果显示,随着rCFP10浓度增加和处理时间延长,A549细胞存活率显著降低。与空白对照组相比,rCFP10分别从转录和翻译水平显著（P<0.05）或极显著（P<0.01）上调A549细胞中TLRs途径关键分子TLR2、TLR4、MyD88、TRAF6和NF-κB p65及下游炎症因子IL-6、TNF-α的表达水平。rCFP10蛋白可以通过激活A549细胞TLRs受体信号途径促进细胞炎症因子的分泌,这将为进一步加深理解结核病发病机制提供理论依据。     

The effects of the Recombinant Mycobacterium Tuberculosis CFP10 Protein on the TLR Signal-mediated Inflammatory Responses in A549 Cells

The purpose of this study was to investigate the effects of recombinant 10 kDa culture filtrate protein (CFP10) of Mycobacterium tuberculosis on the inflammatory responses mediated by Toll-like receptor (TLR) signaling in A549 cells. The recombinant plasmid pCzn1-CFP10 was obtained by amplifying the cfp10 gene fragment using PCR and cloning it into the prokaryotic expression vector pCzn1. The obtained recombinant plasmid pCzn1-CFP10 was transformed into Escherichia coli BL21(DE3) and induced by IPTG to express the recombinant protein CFP10 (rCFP10). The purified rCFP10 protein was preserved after endotoxin was removed and desalted before use. The effect of rCFP10 treatment on the survival rate of A549 cells was detected by MTT assay. A549 cells were treated with rCFP10 to detect changes of key molecules of TLR signaling pathway and downstream inflammatory factors in A549 cells by qRT-PCR,Western blot and ELISA. The results showed that the recombinant expression vector pCzn1-CFP10 was successfully constructed and the high-purity rCFP10 protein was expressed and purified in this study. MTT assay showed that rCFP10 could inhibit the survival rate of A549 cells in a time- and concentration-dependent manner. In addition, rCFP10 could significantly (P<0.05) up-regulate the key molecules of TLR pathways TLR2, TLR4, MyD88, TRAF6, NF-κBp65 and downstream inflammatory cytokines IL-6 and TNF-α in A549 cells as compared to the control group. The recombinant Mycobacterium tuberculosis protein CFP10 could promote the secretion of cytokines by activating TLR receptor signaling pathway in A549 cells, which will provide a theoretical basis for further understanding of the pathogenesis of Mycobacterium tuberculosis.     

Toll-like receptor signaling is changed in ovine lymph node during early pregnancy

Toll-like receptors (TLRs) participate in regulation of adaptive immune responses, and lymph nodes play key roles in the initiation of immune responses. There is a tolerance to the allogenic fetus during pregnancy, but it is unclear that expression of TLR signaling is in ovine lymph node during early pregnancy. In this study, lymph nodes were sampled from day 16 of nonpregnant ewes and days 13, 16, and 25 of pregnant ewes, and the expressions of TLR family (TLR2, TLR3, TLR4, TLR5 and TLR9), adaptor proteins, including myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), and interleukin-1-receptor-associated kinase 1 (IRAK1), were analyzed through real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry analysis. The results showed that mRNA and protein levels of TLR2, TLR3, TLR4, TRAF6, and MyD88 were upregulated in the maternal lymph node, but TLR5, TLR9, and IRAK1 were downregulated during early pregnancy. In addition, MyD88 protein was located in the subcapsular sinus and lymph sinuses. Therefore, it is suggested that early pregnancy induces changes in TLR signaling in maternal lymph node, which may be involved in regulation of maternal immune responses in sheep.     

Nucleotide structure of equine platelet-derived growth factor-A and -B and expression in horses with induced acute tendinitis

OBJECTIVE: To characterize the nucleotide sequence of equine platelet-derived growth factor (PDGF)-A and -B and analyze temporal expression of these genes in equine tendon after induced tendinitis injury. Animals-18 mature horses. PROCEDURES: Genes for equine PDGF-A and -B were reverse transcribed and sequenced from synovial tissue mRNA obtained from a 3-year-old horse. Collagenase-induced lesions were created in the tensile region of the superficial digital flexor tendon in 14 horses; 3 horses served as uninjured control animals. Tendons were harvested and total RNA was isolated from experimental horses 1, 2, 4, 8, and 24 weeks after collagenase injection. Temporal gene expression for PDGF-A and -B was determined by use of quantitative PCR analysis. RESULTS: Equine PDGF-A shared 83.8% sequence and 87.5% peptide homology with human PDGF-A, with a discrepancy of 70 bp from the human sequence. Equine PDGF-B was similar in length to the human gene, sharing 90.3% and 91.7% nucleotide and peptide identity, respectively. Expression of PDGF-A mRNA in collagenase-induced tendinitis lesions was unchanged, compared with expression for normal control tendon, and remained steady throughout the 24-week study. Expression of PDGF-B mRNA decreased over time, and the expression at 24 weeks was significantly reduced, compared with expression in normal and acutely injured tendon. CONCLUSIONS AND CLINICAL RELEVANCE: Injured tendon mounts a minimal constitutive PDGF-A or -B mRNA response. Serial exogenous treatment with either PDGF isoform within the first 2 to 4 weeks after tendon injury may bolster the meager PDGF paracrine-autocrine intrinsic response to injury.     

BCG诱导RAW264.7细胞脂肪酸氧化对细胞自噬和促炎因子表达的调控作用

旨在探究脂肪酸氧化（fatty acid oxidation,FAO）对BCG介导的RAW264.7细胞自噬和促炎因子表达的调控作用。用BODIPY染色和游离脂肪酸定量试剂盒检测BCG感染后RAW264.7细胞中脂滴聚集情况以及脂肪酸含量;Western blot检测BCG感染对肉毒碱棕榈酰基转移酶1A （CPT-1A）表达的影响;Etomoxir （100 μmol·L^-1）预处理细胞2 h后,BCG感染细胞6 h,检测RAW264.7细胞中BCG存留量,并用Western blot方法检测自噬相关蛋白（Beclin1、LC3-II）和溶酶体蛋白（Rab7）的表达情况;用免疫荧光方法和mRFP-GFP-LC3荧光双标腺病毒分别检测自噬小体聚集和自噬流;荧光定量PCR和ELISA分别检测促炎因子IL-1β、IL-6和TNF-α mRNA表达情况以及在细胞培养上清中的含量。结果显示,BCG感染促进RAW264.7细胞中脂滴聚集和CPT-1A的表达,而游离脂肪酸含量降低;Etomoxir预处理抑制了细胞中BCG存活,并上调了Beclin1、LC3-II和Rab7表达,且细胞中出现大量自噬小体聚集,自噬流增强,却抑制了促炎因子IL-1β、IL-6和TNF-α mRNA表达与分泌。综上表明,抑制FAO可促进BCG感染诱导的RAW264.7细胞自噬,并抑制BCG感染引起的炎症反应。     

Expressions of estrogen receptors in the bovine corpus luteum: cyclic changes and effects of prostaglandin F2alpha and cytokines

Estrogen (E) exerts its function by binding to two intracellular estrogen receptors, ERalpha and ERbeta. Although ERs have been reported to be expressed in the bovine corpus luteum (CL), the mechanisms that control ER expression in the bovine CL are not fully understood. To determine the possible regulatory mechanisms of ERalpha and ERbeta that meditate distinct E functions, we examined 1) the changes in the protein expressions of ERs in the CL throughout the luteal phase and 2) the effects of prostaglandin (PG) F2alpha, tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) on the expressions of ERs in cultured bovine luteal cells. Western blot analyses revealed that ERalpha and ERbeta proteins were expressed throughout the luteal phase. The ERalpha protein level was high at the early luteal (Days 2-3 after ovulation) and mid-luteal stages (Days 8-12) and was extremely low at the regressed luteal stage (Days 19-21). The ERbeta protein level increased from the early to developing luteal stage, remained at the same level at the mid-luteal stage and decreased thereafter. The ratio of ERbeta to ERalpha was higher in the regressed stage than in the other stages. Luteal cells obtained from mid-stage CLs (Days 8-12) were incubated with PGF2alpha (0.01-1 microM), TNFalpha (0.0145-1.45 nM) or IFNgamma (0.0125-1.25 nM) for 24 h. PGF2alpha and TNFalpha inhibited ERa and ERbeta mRNA expressions. IFNgamma suppressed ERbeta mRNA expression but did not affect the expression of ERalpha mRNA. However, the ERalpha and ERbeta protein levels were not affected by any of the above treatments. These data indicate that PGF2alpha, TNFalpha and IFNgamma regulate ERalpha and ERbeta mRNA expressions in bovine luteal cells. Moreover, the changes in the ERbeta/ERalpha ratio throughout the luteal phase suggest that ERalpha is associated with luteal maintenance. Therefore, a dramatic decrease in ERalpha at the regressed luteal stage could result in progression of structural luteolysis in the bovine CL.     

Tumor necrosis factor-alpha as a possible auto-/paracrine factor affecting estrous cycle in the cat uterus

Tumor Necrosis Factor-alpha (TNFalpha) is a pleiotrophic cytokine, affects either normal or tumor cells, and influences cellular differentiation. TNFalpha role in female reproduction has been proven to be mediated through an influence on prostaglandin (PGs) synthesis and output. To evaluate the possible role of TNFalpha in an auto-/paracrine regulation in the cat uterus, mRNA expression coding for TNFalpha and its receptors (TNFR1 and TNFR2), and TNFalpha protein content at different stages of the estrous cycle were investigated. Additionally, TNFalpha involvement in PG secretion at different stages of the estrous cycle was investigated by in vitro tissue culture. Gene expressions coding for TNFalpha and TNFR1 were the highest at diestrus (P < 0.05). TNFalpha protein expression was the lowest at interestrus (P < 0.05). Nevertheless, TNFR2 was not affected by the estrous stage. TNFalpha at a dose of 1 ng/ml significantly increased PGF2alpha secretion at estrus (P < 0.01) and PGE2 secretion at diestrus (P < 0.001) after 12h incubation. Overall findings indicate that TNFalpha locally produced in the cat's uterus, stimulates PG secretion in an estrous cycle-related manner.     

Enhanced production of IL-1beta and IL-6 following endotoxin challenge in rats with dietary magnesium deficiency

Serum IL-1beta, IL-6 and TNFalpha were not detected in control and Mg-deficient rats. These three cytokine levels in serum were increased after endotoxin challenge (1 mg/kg., i.p.), and the increase of IL-1beta and IL-6, but not TNFalpha, was significantly larger in Mg-deficient rats than in controls. Levels of mRNA for IL-1beta, IL-6 and TNFalpha in alveolar macrophages showed a tendency to decrease during Mg deficiency, but the levels of IL-1beta and TNFalpha mRNAs after endotoxin challenge were higher in Mg-deficient rats than in controls. These results suggest that the increased synthesis of cytokines by alveolar macrophages might contribute, in part, to high sensitivity to endotoxin during Mg deficiency.     

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.     

Collagen Types in Healing Rabbit Tendons: A Biochemical Assessment

Eight repaired and eight contralateral control superficial digital flexor tendons were collected from rabbits (2–6 kg) 1, 4, and 8 weeks after surgery. Acid soluble and pepsin solubilized collagen fractions were prepared and separated using SDS-PAGE to determine if healing tendons contain detectable amounts of Type III collagen. No biochemical differences between normal and healing tendon collagen content could be detected. Type I collagen was the predominant collagen species found in all 88 samples examined. Alpha chains indicative of Type III collagen were detected in control and experimental tendons of five animals. Alpha chains suggestive of Type IV collagen were detected in control and experimental tendons of eight rabbits.