A prospective study of serum amyloid A in relation to plasma administration in neonatal foals.

SAA is a commonly used biomarker for measuring acute inflammation in equine practice, and the administration of prophylactic plasma to foals is a routine practice in large breeding farms. Despite this, limited information is available on the values of SAA in healthy or sick neonatal foals following this common procedure. A prospective study was conducted with 31 foals from a veterinary hospital in Texas in one year. Enrolled foals were part of a foaling program, where a prophylactic hyperimmunized plasma was administered 12 h after birth. Blood was collected for SAA measurements at birth and at 12 h (pre-plasma), 13 h (post-plasma), 24 h, 48 h, 72 h, and 96 h. Eight of the foals were clinically ill prior to plasma administration, and 23 foals were clinically normal. The mean SAA of all foals at birth was 1 µg/mL, increased to 11 µg/mL at 12 h (pre-plasma), and at 13 h (post-plasma) was 155 µg/mL. At 13 h, 65% of normal foals and 63% of sick foals had an SAA value >100 µg/mL. Transient but substantial increases in SAA following prophylactic plasma administration were frequently observed in this study. Veterinarians evaluating neonatal foals for clinical disease in the field should be cognizant of the timing of blood sampling in relation to plasma administration.

Pharmacokinetics of a single dose of oclacitinib maleate as a top dress in adult horses.

The objective of this study was to determine the pharmacokinetic parameters of oclacitinib maleate as a top dress given to adult horses. Six adult horses with a mean weight of 528 kg were administered a single dose of 0.5 mg/kg oclacitinib maleate. Blood was collected prior to drug administration and at 15 min, 30 min, 45 min, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after treatment. Oclacitinib maleate plasma concentrations were measured by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were found best to fit a one-compartment model. Mean Cmax was 486 ng/ml (range 423-549 ng/ml), and Tmax was estimated to be 1.7 h (range 0.3-3.1 h). The estimated T1/2 was 7.5-8 h.

Pharmacokinetics of dipyrone in horses: A multi-dose, dose escalation study.

Dipyrone is a non-opioid, nonsteroidal anti-inflammatory drug with antipyretic and analgesic properties commonly used in horses. Dipyrone is rapidly hydrolyzed to the primary active metabolite 4-methylaminoantipyrine (4-MAA). The purpose of this study was to determine the pharmacokinetic profile of 4-MAA following repeated and escalating doses of intravenously administered dipyrone. Twenty-six horses were randomly allocated to five treatment groups (one placebo group and four dipyrone groups [30 mg/kg q8h, 30 mg/kg q12h, 60 mg/kg q8h, and 90 mg/kg q12h]) and treated for nine consecutive days. Blood was collected at predetermined timepoints, and plasma was analyzed for 4-MAA concentrations with a validated LC/MS/MS method. Following a single dose, there was a linear correlation to the maximum concentration (Cmax ) achieved. There was a disproportionate increase in the minimum concentration (Cmin ) of 4-MAA with accumulation occurring at higher doses or more frequent dosing intervals. Significant differences were noted in 4-MAA Cmax , half-life, and area under the curve during the dosing interval (AUCtau ) when dipyrone was administered at 30 mg/kg q12h versus q8h. Adverse effects attributed to drug administration were not noted.

A retrospective study of bronchoalveolar lavage fluid analysis in barrel racing horses with exercise-induced pulmonary hemorrhage and asthma in Texas from 2016 to 2018.

Exercise-induced pulmonary hemorrhage (EIPH) and asthma in barrel racing horses is a common disease across the United States. Limited information is available on non-infectious respiratory diseases in this population, the interaction between these two diseases, and the occurrence of both EIPH and asthma in the horse. The purpose of this study was to evaluate the bronchoalveolar lavage (BAL) fluid cytological results of barrel racing horses with EIPH, asthma, or both. A retrospective study was conducted using the medical records of horses that presented with cough and decreased athletic performance and BAL results that met the criteria for inclusion. Data from 95 horses were included from a private practice referral hospital in Texas. No statistical difference was found in the frequency of neutrophilia, eosinophilia, or mastocytosis between diagnoses of EIPH, asthma, or concurrent diagnoses of EIPH and asthma. Bronchoalveolar lavage of horses suspected of EIPH is warranted to fully characterize the noninfectious respiratory disease of barrel racing horses.

Randomized blinded controlled trial of dipyrone as a treatment for pyrexia in horses.

OBJECTIVE To evaluate the effectiveness and safety of dipyrone to control pyrexia in horses with naturally occurring disease under field conditions. ANIMALS 138 horses with pyrexia and various infections evaluated at 14 veterinary sites in 12 states. PROCEDURES In the first (effectiveness) phase of this 2-phase study, horses were randomly assigned 3:1 to receive 1 dose of dipyrone (30 mg/kg [13.6 mg/lb], IV) or an equivalent amount of placebo. Effectiveness was defined as a decrease in rectal temperature ≥ 1.1°C (2°F), compared with the pretreatment value, or a rectal temperature of ≤ 38.3°C (101.0°F) 6 hours after treatment administration. Horses deemed to have an appropriate reduction in rectal temperature (regardless of treatment group) by 6 hours were immediately entered into the safety phase of the study, in which dipyrone was administered IV at 30 mg/kg between 0 and 8 times up to every 8 hours on an as-needed basis, as determined by the clinical investigators. Horses were monitored throughout for adverse events. RESULTS A significantly greater proportion of dipyrone-treated horses (76/99 [77%]) had an effective treatment response than did placebo-treated horses (6/31 [19%]). Posttreatment adverse events were mild and transient. No differences in types or prevalence of gastrointestinal adverse events were evident between treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE Dipyrone was effective in controlling pyrexia by 6 hours after IV administration of a single 30-mg/kg dose in a large proportion of treated horses. Adverse effects were minimal.

Leukocyte-Reduced Platelet-Rich Plasma Normalizes Matrix Metabolism in Torn Human Rotator Cuff Tendons.

BACKGROUND: The optimal platelet-rich plasma (PRP) for treatment of supraspinatus tendinopathy has not been determined. PURPOSE: To evaluate the effect of low- versus high-leukocyte concentrated PRP products on catabolic and anabolic mediators of matrix metabolism in diseased rotator cuff tendons. STUDY DESIGN: Controlled laboratory study. METHODS: Diseased supraspinatus tendons were treated with PRP made by use of 2 commercial systems: Arthrex Autologous Conditioned Plasma Double Syringe System (L(lo) PRP) and Biomet GPS III Mini Platelet Concentrate System (L(hi) PRP). Tendon explants were placed in 6-well plates and cultured in L(lo) PRP, L(hi) PRP, or control media (Dulbecco's Modified Eagle Medium + 10% fetal bovine serum) for 96 hours. Tendons were processed for hematoxylin-eosin histologic results and were scored with the modified Bonar scale. Group 1 tendons were defined as moderate tendinopathy (Bonar score <3); group 2 tendons were assessed as severely affected (Bonar score = 3). Transforming growth factor ß-1 (TGFß-1), interleukin-1ß (IL-1ß), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and matrix metalloproteinase-9 (MMP-9) concentrations in PRP media were measured by use of enzyme-linked immunosorbent assay after 96 hours of culture with diseased tendon. Tendon messenger RNA expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), MMP-9, MMP-13, and IL-1ß was measured with real-time quantitative polymerase chain reaction. RESULTS: Leukocytes and platelets were significantly more concentrated in L(hi) PRP compared with L(lo) PRP. Increased IL-1ß was present in L(hi) PRP after culture with group 1 tendons. IL-6 was increased in L(hi) PRP after culture with group 2 tendons. Both TGFß-1 and MMP-9 were increased in L(hi) PRP after culture with either tendon group. In L(lo) PRP cultures, IL-1Ra:IL-1ß in PRP used as media and COL1A1:COL3A1 gene expression were increased for group 1 tendon cultures. Gene expression of MMP-9 and IL-1ß was increased in group 2 tendons cultured in L(lo) PRP. There was no significant difference in the expression of MMP-13 or COMP in either group of tendons cultured in L(lo) PRP or L(hi) PRP. CONCLUSION: L(lo) PRP promotes normal collagen matrix synthesis and decreases cytokines associated with matrix degradation and inflammation to a greater extent than does L(hi) PRP in moderately degenerative tendons. In severely degenerative tendons, neither PRP preparation enhanced matrix synthesis. CLINICAL RELEVANCE: L(lo) PRP may promote healing in moderately degenerative rotator cuff tendons.

The anti-inflammatory and matrix restorative mechanisms of platelet-rich plasma in osteoarthritis.

BACKGROUND: Intra-articular (IA) treatment with platelet-rich plasma (PRP) for osteoarthritis (OA) results in improved patient-reported pain and function scores. PURPOSE: To measure the effects of PRP and high molecular weight hyaluronan (HA) on the expression of anabolic and catabolic genes and on the secretion of nociceptive and inflammatory mediators from OA cartilage and synoviocytes. STUDY DESIGN: Controlled laboratory study. METHODS: Synovium and cartilage harvested from patients undergoing total knee arthroplasty were co-cultured with media of PRP or HA. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß were measured in the media by enzyme-linked immunosorbent assay. Hyaluronan synthase-2 (HAS-2), matrix metalloproteinase-1 (MMP-1), MMP-13, and TNF-α genes were measured in synoviocytes by reverse transcription polymerase chain reaction (RT-PCR). Collagen type I α1 (COL1A1), COL2A1, aggrecan (ACAN), and MMP-13 gene expression were measured in cartilage by quantitative RT-PCR. RESULTS: Media TNF-α concentration was decreased in PRP and HA compared with control cultures (PRP = 6.94 pg/mL, HA = 6.39 pg/mL, control = 9.70 pg/mL; P ≤ .05). Media IL-6 concentration was decreased in HA compared with PRP and control (HA = 5027 pg/mL, PRP = 5899 pg/mL, control = 5613 pg/mL; P ≤ .05). Media IL-1ß was below detectable concentrations (<0.1 pg/mL) in all samples. Synoviocyte MMP-13 expression was decreased in PRP compared with HA and control (PRP = 10.1, HA = 12.8, control = 13.5; P ≤ .05). Synoviocyte HAS-2 expression was increased in PRP compared with HA and control (PRP = 12.1, HA = 9.8, control = 8.7; P ≤ .05). Cartilage ACAN expression was increased in PRP compared with HA, but neither was different from control (PRP = 8.8, HA = 7.7, control = 7.6; P ≤ .05); COL1A1 expression was increased in HA compared with PRP, but neither was different from control (PRP = 14.9, HA = 13.5, control = 12.9; P ≤ .05). Neither platelet nor leukocyte concentration had a significant effect on outcome measurements (gene or protein expression data) in cartilage or synoviocytes (P > .05). CONCLUSION: Both PRP and HA treatments of OA joint tissues result in decreased catabolism, but PRP treatment also resulted in a significant reduction of MMP-13, an increase in HAS-2 expression in synoviocytes, and an increase in cartilage synthetic activity compared with HA. These results indicate that PRP acts to stimulate endogenous HA production and decrease cartilage catabolism. Platelet-rich plasma showed similar effects as HA in the suppression of inflammatory mediator concentration and expression of their genes in synoviocytes and cartilage. CLINICAL RELEVANCE: The antinociceptive and anti-inflammatory activities of PRP support its use in OA joints to reduce pain and modulate the disease process. This study supports further clinical investigations of IA PRP for the treatment of OA.

Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.

BACKGROUND: Platelet-rich plasma (PRP) is used for the treatment of tendinopathy. There are numerous PRP preparations, and the optimal combination of platelets and leukocytes is not known. HYPOTHESIS: Within leukocyte-reduced PRP (lrPRP), there is a plateau effect of platelet concentration, with increasing platelet concentrations being detrimental to extracellular matrix synthesis. STUDY DESIGN: Controlled laboratory study. METHODS: Different formulations of lrPRP with respect to the platelet:leukocyte ratio were generated from venous blood of 8 horses. Explants of the superficial digital flexor tendon were cultured in lrPRP products for 96 hours. Platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), and interleukin-1ß (IL-1ß) concentrations were determined in the media by enzyme-linked immunosorbent assay. Gene expression in tendon tissue for collagen type I and III (COL1A1 and COL3A1, respectively), matrix metalloproteinase-3 and -13 (MMP-3 and MMP-13, respectively), cartilage oligomeric matrix protein (COMP), and IL-1ß was determined. Data were divided into 3 groups of lrPRP based on the ratio of platelets:leukocytes and evaluated to determine the effect of platelet concentration. RESULTS: Complete blood counts verified leukocyte reduction and platelet enrichment in all PRP preparations. In the lrPRP preparation, the anabolic growth factors PDGF-BB and TGF-ß1 were increased with increasing platelet concentrations, and the catabolic cytokine IL-1ß was decreased with increasing platelet concentrations. Increasing the platelet concentration resulted in a significant reduction in COL1A1 and COL3A1 synthesis in tendons. CONCLUSION: Increasing the platelet concentration within lrPRP preparations results in the delivery of more anabolic growth factors and less proinflammatory cytokines, but the biological effect on tendons is diminished metabolism as indicated by a decrease in the synthesis of both COL1A1 and COL3A1. Together, this information suggests that minimizing leukocytes in PRP is more important than maximizing platelet numbers with respect to decreasing inflammation and enhancing matrix gene synthesis. CLINICAL RELEVANCE: This study suggests that reducing leukocytes to minimize catabolic signaling appears to be more important than increasing platelets in an effort to maximize anabolic signaling. Further, a maximum biological threshold of benefit was demonstrated with regard to the number of platelets beyond which further increases in platelet concentration did not result in further anabolic upregulation. In vivo investigations documenting the use of platelets for the treatment of tendinopathy are justified as well as further in vitro characterization of the ideal PRP product for the treatment of tendinopathy and other musculoskeletal applications.

Platelet-rich plasma: a milieu of bioactive factors.

Platelet concentrates such as platelet-rich plasma (PRP) have gained popularity in sports medicine and orthopaedics to promote accelerated physiologic healing and return to function. Each PRP product varies depending on patient factors and the system used to generate it. Blood from some patients may fail to make PRP, and most clinicians use PRP without performing cell counts on either the blood or the preparation to confirm that the solution is truly PRP. Components in this milieu have bioactive functions that affect musculoskeletal tissue regeneration and healing. Platelets are activated by collagen or other molecules and release growth factors from alpha granules. Additional substances are released from dense bodies and lysosomes. Soluble proteins also present in PRP function in hemostasis, whereas others serve as biomarkers of musculoskeletal injury. Electrolytes and soluble plasma hormones are required for cellular signaling and regulation. Leukocytes and erythrocytes are present in PRP and function in inflammation, immunity, and additional cellular signaling pathways. This article supports the emerging paradigm that more than just platelets are playing a role in clinical responses to PRP. Depending on the specific constituents of a PRP preparation, the clinical use can theoretically be matched to the pathology being treated in an effort to improve clinical efficacy.

Growth factor and catabolic cytokine concentrations are influenced by the cellular composition of platelet-rich plasma.

BACKGROUND: Previous studies of bioactive molecules in platelet-rich plasma (PRP) have documented growth factor concentrations that promote tissue healing. However, the effects of leukocytes and inflammatory molecules in PRP have not been defined. HYPOTHESIS: The hypothesis for this study was that the concentration of growth factors and catabolic cytokines would be dependent on the cellular composition of PRP. STUDY DESIGN: Controlled laboratory study. METHODS: Platelet-rich plasma was made from 11 human volunteers using 2 commercial systems: Arthrex ACP (Autologous Conditioned Plasma) Double Syringe System (PRP-1), which concentrates platelets and minimizes leukocytes, and Biomet GPS III Mini Platelet Concentrate System (PRP-2), which concentrates both platelets and leukocytes. Transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor-AB (PDGF-AB), matrix metalloproteinase-9 (MMP-9), and interleukin-1ß (IL-1ß) were measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: The PRP-1 system consisted of concentrated platelets (1.99×) and diminished leukocytes (0.13×) compared with blood, while PRP-2 contained concentrated platelets (4.69×) and leukocytes (4.26×) compared with blood. Growth factors were significantly increased in PRP-2 compared with PRP-1 (TGF-ß1: PRP-2 = 89 ng/mL, PRP-1 = 20 ng/mL, P < .05; PDGF-AB: PRP-2 = 22 ng/mL, PRP-1 = 6.4 ng/mL, P < .05). The PRP-1 system did not have a higher concentration of PDGF-AB compared with whole blood. Catabolic cytokines were significantly increased in PRP-2 compared with PRP-1 (MMP-9: PRP-2 = 222 ng/mL, PRP-1 = 40 ng/mL, P < .05; IL-1ß: PRP-2 = 3.67 pg/mL, PRP-1 = 0.31 pg/mL, P < .05). Significant, positive correlations were found between TGF-ß1 and platelets (r(2) = .75, P < .001), PDGF-AB and platelets (r(2) = .60, P < .001), MMP-9 and neutrophils (r(2) = .37, P < .001), IL-1ß and neutrophils (r(2) = .73, P < .001), and IL-1ß and monocytes (r(2) = .75, P < .001). CONCLUSION: Growth factor and catabolic cytokine concentrations were influenced by the cellular composition of PRP. Platelets increased anabolic signaling and, in contrast, leukocytes increased catabolic signaling molecules. Platelet-rich plasma products should be analyzed for content of platelets and leukocytes as both can influence the biologic effects of PRP. CLINICAL RELEVANCE: Depending on the clinical application, preparations of PRP should be considered based on their ability to concentrate platelets and leukocytes with sensitivity to pathologic conditions that will benefit most from increased platelet or reduced leukocyte concentration.

Differences in oocyte development and estradiol sensitivity among mouse strains.

Mouse oocytes develop in clusters of interconnected cells called germline cysts. Shortly after birth, the majority of cysts break apart and primordial follicles form, consisting of one oocyte surrounded by granulosa cells. Concurrently, oocyte number is reduced by two-thirds. Exposure of neonatal females to estrogenic compounds causes multiple oocyte follicles that are likely germline cysts that did not break down. Supporting this idea, estrogen disrupts cyst breakdown and may regulate normal oocyte development. Previously, the CD-1 strain was used to study cyst breakdown and oocyte survival, but it is unknown if there are differences in these processes in other mouse strains. It is also unknown if there are variations in estrogen sensitivity during oocyte development. Here, we examined neonatal oocyte development in FVB, C57BL/6, and F2 hybrid (Oct4-GFP) strains, and compared them with the CD-1 strain. We found variability in oocyte development among the four strains. We also investigated estrogen sensitivity differences, and found that C57BL/6 ovaries are more sensitive to estradiol than CD-1, FVB, or Oct4-GFP ovaries. Insight into differences in oocyte development will facilitate comparison of mice generated on different genetic backgrounds. Understanding variations in estrogen sensitivity will lead to better understanding of the risks of environmental estrogen exposure in humans.