Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA.

OBJECTIVE: To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. DESIGN: A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3'-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion. RESULTS: We identified 990 known miRNAs and 1621 potential novel miRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate miRNAs were analysed further, of which six remained after northern blot analysis. Three novel miRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin). CONCLUSION: Deep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis (OA). Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man.

NK cells and NKT cells collaborate in host protection from methylcholanthrene-induced fibrosarcoma.

NK1.1(+) V(alpha)14J(alpha)281(+) (NKT) cells can be induced by IL-12 therapy to mediate tumor rejection; however, methylcholanthrene (MCA)-induced fibrosarcoma is the only tumor model described where NKT cells play a natural role in controlling tumor initiation. From our previous study in C57BL/6 mice it remained unclear whether NK cells were also involved in this natural response. Herein, to discriminate the function of NK and NKT cells, we have evaluated fibrosarcoma development in mice deficient in NKT cells, but not NK cells, and mice deficient in NK cells, but not NKT cells. The results indicate that both NK cells and NKT cells are essential and collaborate in natural host immunity against MCA-induced sarcoma. In contrast, sarcoma incidence and growth rate were reduced using IL-12 therapy, this effect was mediated in the absence of T cells (including NKT cells), but not NK cells.

Differential tumor surveillance by natural killer (NK) and NKT cells.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jalpha281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

NKT cells are phenotypically and functionally diverse.

NK1.1(+)alpha betaTCR(+) (NKT) cells have several important roles including tumor rejection and prevention of autoimmune disease. Although both CD4(+) and CD4(-)CD8(-) double-negative (DN) subsets of NKT cells have been identified, they are usually described as one population. Here, we show that NKT cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets (CD4(+), DN and CD8(+)) are differentially distributed in a tissue-specific fashion. CD8(+) NKT cells are present in all tissues but the thymus, and are highly enriched for CD8alpha(+)beta(-) cells. These subsets differ in their expression of a range of cell surface molecules (Vbeta8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL-4 and IFN-gamma, with splenic NKT cell subsets producing lower levels than thymic NKT cells. Developmentally, most CD4(+) and DN NKT cells are thymus dependent, in contrast to CD8(+) NKT cells, and are also present amongst recent thymic emigrants in spleen and liver. TCR Jalpha281-deficient mice show a dramatic deficiency in thymic NKT cells, whereas a significant NKT cell population (enriched for the DN and CD8(+) subsets) is still present in the periphery. Taken together, this study reveals a far greater level of complexity within the NKT cell population than previously recognized.

Enzyme-linked immunosorbent assay for thrombin-antithrombin III complexes in horses.

OBJECTIVES: To adapt and characterize a human ELISA kit to quantify thrombin-antithrombin III (TAT) complexes in horses, and to evaluate TAT as a marker for hypercoagulation in horses. ANIMALS: 29 clinically normal horses used as controls, and 4 ill horses used to evaluate assay for known causes of hypercoagulation. PROCEDURE: A commercially available human sandwich-type ELISA kit with 2 antibodies against human thrombin and antithrombin III that bind selectively to their corresponding TAT antigenic sites was used. Equine TAT standards were made from purified equine thrombin and antithrombin III. Proteins diluted in a phosphate-buffered saline solution containing 0.1% Tween and 1 U of heparin/ ml were used to establish standard curves. Reference intervals for TAT concentration in citrated equine plasma, and intra- and interassay coefficients of variation were determined. RESULTS: Mean +/- SD values were 3.95 +/- 1.93 micrograms/L, with median of 3.18 micrograms/L and range of 1.95 to 9.03 micrograms/ L. One horse with cecal perforation had TAT concentration of 174.30 micrograms/L, and a horse infused IV with endotoxin had TAT concentration of 62.98 micrograms/L 12 hours after infusion. CONCLUSIONS: The data suggest that human TAT ELISA kits can be used to measure TAT concentration in citrated equine plasma, and that TAT is a marker for hypercoagulation in horses. CLINICAL RELEVANCE: Assays for equine TAT many help to further characterize the hypercoagulable state in horses.

Hemostatic indices in healthy foals from birth to one month of age.

Hemostatic indices were determined in 45 healthy light breed foals, from birth to 1 month of age, and in 20 healthy adult (> 2 years of age) light breed horses. Blood samples were obtained from each foal at 4 ages: 1) < 24 hours, 2) 4-7 days, 3) 10-14 days, and 4) 25-30 days. The following hemostatic indices were determined: platelet count; prothrombin and activated partial thromboplastin times; activity concentrations of protein C, antithrombin III, plasminogen, alpha-2 antiplasmin, tissue plasminogen activator, and plasminogen activator inhibitor-1; plasma protein C antigen and fibrinogen concentrations; and serum fibrin degradation products concentration. Prothrombin and activated partial thromboplastin times were significantly longer at birth than in older foals. The plasma concentrations of the following were significantly lower at birth than in older foals: antithrombin III, plasminogen and tissue plasminogen activator activities, protein C antigen, and fibrinogen. Concentrations of the following were significantly higher at birth than in older foals: protein C and plasminogen activator inhibitor-1 activities and fibrin degradation products. These results indicate that hemostatic indices of neonatal foals differ significantly from those of older foals and adults. With the exceptions of antithrombin III and tissue plasminogen activator activities, all hemostatic indices measured in foals at 1 month of age were equivalent to adult values.

Equine tumor necrosis factor alpha: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and development of a sensitive enzyme-linked immunosorbent assay.

We describe the production and purification of recombinant equine tumor necrosis factor alpha (rETNF alpha), generation and characterization of murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNF alpha, and development of a sensitive enzyme-linked immunosorbent assay (ELISA). Genomic-derived DNA sequences encoding mature ETNF alpha were reconstructed by the polymerase chain reaction (PCR) and oligonucleotide-directed mutagenesis and were cloned into the vector pFLAG-1 for expression in Escherichia coli. rETNF alpha was purified by anti-FLAG immunoaffinity chromatography and then used as immunogen for production of murine Mabs and rabbit Pabs. Three Mabs (6H4, 9B10, and 12F6) were obtained from one fusion. All three Mabs recognized rETNF alpha on western blots. Mabs 6H4 and 9B10 recognized similar epitopes on rENTF alpha and neutralized both rETNF alpha and native ETNF alpha (nETNF alpha) in a WEHI cell cytotoxicity assay. A sensitive ELISA was developed using Mab 6H4 and biotin-labeled rabbit Pabs. The ELISA was shown to detect levels of ENTF alpha as low as 100 pg/ml and was used to demonstrate the induction of ETNF alpha in horses with experimental endotoxemia. The rETNF alpha, antibodies, and ELISA developed in this report should be useful tools for studies of TNF-mediated diseases in horses.

Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro.

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (IL-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours' incubation and were frozen at -70 C until assayed for IL-6 activity. Supernatant IL-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on IL-6 for survival. Results indicated that equine peritoneal macrophages produce IL-6 in vitro and that supernatant medium IL-6 activity was significantly (P less than 0.05) increased by exposure to endotoxin. Significant (P less than 0.05) time and treatment effects on macrophage IL-6 production were apparent. The IL-6 activity peaked at 6 or 12 hours' incubation, then remained high through 24 hours' incubation, regardless of endotoxin exposure. Medium IL-6 activity during 3 and 6 hours' incubation was significantly (P less than 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak IL-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium IL-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.

Effect of experimentally induced endotoxemia on serum interleukin-6 activity in horses.

A study was conducted to determine whether serum interleukin-6 (IL-6) activity increased in horses during experimentally induced endotoxemia and whether serum IL-6 activity correlated to changes in clinical or laboratory data. Six clinically normal horses were given endotoxin IV (30 ng/kg of body weight) in 0.9% NaCl solution over 1 hour. Five of these and 1 additional horse served as controls and were given only 0.9% NaCl solution. Venous blood, for determination of serum IL-6 activity and WBC count, was collected before and at various times through 8 hours after the start of endotoxin or NaCl infusion. Rectal temperature and heart and respiratory rates were recorded throughout the study period. Serum IL-6 activity was determined by bioassay of proliferation of the B13.29 clone B.9 hybridoma cell line. From 1.5 through 5 hours after start of the infusion, serum IL-6 activity was significantly (P less than 0.05) increased in horses given endotoxin. Mean peak serum IL-6 activity was observed between 3 and 4 hours. In response to endotoxin infusion, horses became lethargic, tachycardic, and febrile. Leukopenia developed by 1 hour, followed by leukocytosis at 8 hours. Significant (P less than 0.05) positive association and linear correlation were apparent between mean serum IL-6 activity and mean rectal temperature in the group of horses that were given endotoxin. Changes from baseline were not evident in any of the clinical or laboratory values in horses given only NaCl solution.

Reduced endotoxin-induced production of tumor necrosis factor activity by equine peritoneal macrophages exposed to the dual inhibitor of arachidonic acid metabolism, SK & F 86002.

The purpose of this study was to determine if a structurally novel dual inhibitor of arachidonic acid metabolism, SK & F 86002, would inhibit the endotoxin-induced production of tumor necrosis factor (TNF) activity by equine peritoneal macrophages. Equine peritoneal macrophages were variously pretreated for 0, 0.5 and 2 h with SK & F 86002 at 10(-9) to 10(-4) molar final concentrations or were left untreated. Then, the macrophages were cultured in vitro in the presence of endotoxin (5 ng/mL). Supernatant media were collected after 4 h and stored at -70 degrees C until assayed for TNF activity and immunoreactive thromboxane B2 (iTxB2). Macrophage supernatant TNF activities were estimated by an in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI 164 clone 13. Concentrations of iTxB2 were quantitated by radioimmunoassay. Coincubation of macrophages with SK & F 86002 significantly decreased the subsequent supernatant TNF activity. Concentrations of SK & F 86002 from 10(-7) to 10(-4) molar effectively reduced TNF production when added to macrophages 0 and 0.5 h prior to endotoxin. After 2 h of preincubation, SK & F 86002 significantly reduced supernatant TNF activity at 10(-5) and 10(-4) M concentrations. Supernatant concentrations of iTxB2 were reduced when SK & F 86002 was added at 10(-6) to 10(-4) M concentrations, 0 and 0.5 h prior to endotoxin, and at all concentrations (10(-9) to 10(-4)) when preincubated with macrophages for 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum tumor necrosis factor activity in horses with colic attributable to gastrointestinal tract disease.

Over a 24-month period, serum tumor necrosis factor (TNF) activity was determined in 289 horses with colic attributable to gastrointestinal tract disease. Serum TNF activity was quantitated by use of a modified in vitro cytotoxicity bioassay, using WEHI 164 clone-13 murine fibrosarcoma cells. Causes for colic, determined by clinical and laboratory evaluation, exploratory celiotomy, or necropsy included: gastrointestinal tract rupture (GTR); ileal impaction; small intestinal strangulating obstruction (SIO); proximal enteritis (PE); transient small intestinal distention; large-colon displacement; large-colon volvulus; large-colon impaction; colitis; small-colon obstruction; peritonitis; and unknown. Each diagnosis was placed into 1 of 3 lesion categories: inflammatory disorders (GTR, PE, colitis, peritonitis); strangulating intestinal obstruction (SIO, large-colon volvulus); and nonstrangulating intestinal obstruction (ileal impaction, transient small intestinal distension, large-colon displacement, large-colon impaction, small-colon obstruction, unknown). The prevalence of high serum TNF activity and/or mortality were evaluated. Differences were tested at significance level of P less than 0.05. Approximately 20% of the 289 horses has serum TNF activity greater than that found in clinically normal horses (greater than 2.5 U/ml). Twenty-three horses (8%) had marked increase in serum TNF activity (greater than or equal to 10 U/ml) which was more prevalent among horses with SIO and PE than in horses of other diagnostic groups, except those with GTR. Mortality and marked increase in serum TNF activity were greater in horses with intestinal inflammatory disorders or strangulating intestinal obstruction than in horses with nonstrangulating intestinal obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Influence of dietary aluminum and phosphorus on zinc metabolism in dairy calves.

The metabolism of a single oral zinc-65 dose was studied in young dairy calves fed two concentrations of added A1 (0 and .20% A1) and two concentrations of added P (0 and .22% P) for 7 wk. The four treatments were 1) normal P-low A1, 2) low P-low A1, 3) normal P-high A1 and 4) low P-high A1. The basal diet (low P-low AL) contained, by analysis, .132% P, .74% Ca, .021% A1 and 59 ppm Zn. Zinc-65 absorption was greater (66.5 vs 63.2% of dose, P less than .10) with the low-P diet; added A1 reduced (P less than .05) 65Zn absorption. Calves fed low-P diets had higher (P less than .10) concentrations of 65Zn in liver, kidney, spleen, heart, small intestine and testicle than those fed normal-P diets. Zinc-65 was reduced (P less than .10) in pancreas, heart, testicle and muscle of calves fed high A1. Iron was increased in liver and kidney (P less than .10), Zn (P less than .10) and Mn (P less than .01) were increased in liver, but Fe in small intestine and Cu in muscle and tibia shaft were decreased (P less than .10) in calves fed the low-P diets compared to those fed adequate-P diets. High A1 reduced (P less than .10) Cu in small intestine and tibia shaft. The results suggest that zinc metabolism may be moderately affected in calves fed either low-P or high-A1 diets.

Correlation of clinical and laboratory data with serum tumor necrosis factor activity in horses with experimentally induced endotoxemia.

Serum tumor necrosis factor (TNF) activity was quantitated in 8 horses given an IV infusion of endotoxin (0.03 micrograms of lipopolysaccharide/kg of body weight, from Escherichia coli 055:B5) in 0.9% NaCl solution over 1 hour. Serum TNF activity was likewise measured in 6 horses given only 0.9% sterile NaCl solution at the same rate. The duration of serum TNF activity was determined, and serum TNF activity was correlated with clinical and laboratory changes during the induced endotoxemia. Horses had no serum TNF activity prior to endotoxin administration, but geometric mean serum TNF activity was significantly higher from 1 to 4 hours after the start of the infusion. In response to endotoxin, horses seemed depressed, had signs of mild to moderate abdominal pain, developed tachycardia and fever, and had leukopenia followed by leukocytosis. Association between serum TNF activity and temperature, heart rate, attitude abnormality score, and WBC count of horses given endotoxin was significant. Serum TNF activity had a significant positive linear correlation with attitude abnormality and heart rate and a negative linear correlation with the WBC count during endotoxemia. Geometric mean serum TNF activity peaked approximately 1.5 hours prior to mean peak fever, and these data were significantly correlated. Results of this study suggest that TNF is an important mediator of endotoxemia in horses.

Effects of dietary aluminum and phosphorus on magnesium metabolism in dairy calves.

The metabolism of Mg was studied in young dairy calves fed two levels of added Al (0 and .20% Al) and two levels of added P (0 and .22% P) for 7 wk. The four treatments were 1) normal P-low Al, 2) low P-low Al, 3) normal P-high Al and 4) low P-high Al. The basal diet (low P-low Al) contained, by analysis, .132% P, .021% Al and .17% Mg. Added Al did not affect (P greater than .10) serum Mg. An Al x P interaction on bone Mg was detected (P less than .01). Magnesium was reduced in tibia shaft (.34 vs .44%) and in tibia joint (.43 vs .53%) in calves fed high Al in the presence of normal dietary P, but Mg was not reduced in the calves fed low-P diets. Apparent absorption of Mg was reduced by approximately five-fold (.18 g/d vs -.84 g/d, P less than .01); urinary Mg excretion was reduced 31% (1.12 g/d vs .77 g/d, P less than .01); and Mg retention declined 41% (-95 g/d vs -1.61 g/d, P less than .01) in calves fed added A1. Compared with calves fed low-P diets, calves fed normal levels of P had a higher Mg concentration in tibia shaft (P less than .01) and tibia joint (P less than .05). The data indicate that supplemental Al may adversely affect Mg metabolism in calves.

Influence of high dietary aluminum on performance and phosphorus bioavailability in dairy calves.

Sixteen male intact Holstein calves averaging 72 kg and 64 d of age were used to study the effects of high dietary Al on calf performance and P bioavailability. The main effects were two concentrations of added aluminum (0 and .20% Al) and two of added P (0 and .22% P). The basal diet contained, by analysis, .132% P, .74% Ca, and .021% Al. The calves were assigned to four treatment groups balanced according to body weight. The four treatments were 1) normal P, low Al; 2) low P, low Al; 3) low P, high Al; and 4) normal P, high Al. Calved had ad libitum access to their respective diets for 7 wk. Metabolism of a single oral 32P dose was determined during wk 6. The adverse effects of high dietary Al include a 17% reduction in feed intake and a 47% reduction in body weight gains. Alkaline phosphatase and plasma glutamic oxaloacetate transaminase activities increased in calves receiving the high Al diets. A negative balance of P and Ca was noted in the calves fed high concentrations of Al. Apparent absorption of 32P was reduced (37%) in calved fed diets high in Al (44% of dose vs. 69%). Urinary excretion of 32P was not affected by dietary Al concentrations. Calves fed the low P (deficient) diet showed significant reductions in feed intake, weight gain, serum inorganic P, bone ash, and P content of bone. Dietary P did not significantly affect 32P absorption. Adding .20% dietary Al severely affects P metabolism and performance of young growing calves.

Intrachiasmal hemorrhage: a cause of delayed post-traumatic blindness.

A 49-year-old man presented with blurred vision 9 days following frontal head trauma. Visual loss progressed to bilateral blindness. Magnetic resonance imaging revealed hemorrhage and swelling within the optic nerves and chiasm. Indirect trauma to the anterior visual pathways may cause delayed blindness due to hemorrhage and edema within these structures.

Enhancement by retinyl acetate of hormone-induced mammary tumorigenesis in female GR/A mice.

Feeding of retinyl acetate (82 mg/kg ration) for 13-16 weeks to estrone- and progesterone-treated nulliparous and multiparous inbred GR/A mice resulted in a substantial increase in the incidence of mammary carcinomas. Mammary carcinoma incidence in nulliparous control and retinoid-fed mice in experiment #1 was 22/65 (34%) and 37/65 (57%) (P less than 0.05), respectively; in experiment #2, 27/48 (56%) and 37/48 (77%) (P less than 0.05), respectively. Mammary carcinoma incidence in multiparous control and retinoid-fed mice in experiment #1 was 13/30 (43%) and 23/30 (77%) (P less than 0.05), respectively; in experiment #2, 19/19 (100%) and 19/19 (100%), respectively. The purported chemopreventive activities of retinyl acetate in murine mammary tumorigenesis were not demonstrated in this study; indeed, the vitamin A analog appeared to enhance this oncogenic process in the steroid hormone-treated GR mouse mammary cancer model.