Effects of topical treatment of cannabidiol extract in a unique manuka factor 5 manuka honey carrier on second intention wound healing on equine distal limb wounds: a preliminary study.

OBJECTIVE: Evaluate the effect of topical 1% cannabidiol on second intention wound healing in distal limb wounds of horses. DESIGN: Experimental. ANIMALS: Six Standardbred horses. METHODS: A total of five 2.5 cm × 2.5 cm full thickness skin wounds were created on the dorsomedial aspect of the metacarpi of 6 horses. Wounds were contaminated with faeces on the day of wound creation. Each wound was then assigned to a treatment group; compounded 1% cannabidiol in unique manuka factor (UMF) 5 manuka honey, UMF 5 manuka honey, UMF 20 manuka honey or saline. Each treatment was applied topically daily for a total of 42 days. Legs were bandaged and bandages were changed, daily, for 13 days postoperatively. Digital photographs of each wound were taken on day 1 then weekly for 6 weeks. Wound size, daily healing rate and total time to healing were recorded and compared statistically. RESULTS: Irrespective of the treatment, wounds did not retract as expected in the first 7 days after wound creation. There was no difference in wound area, daily healing rate, days to complete healing between treatment groups. CONCLUSIONS: This preliminary study failed to demonstrate any difference in wound healing variables between treatment groups in this model of second intention wound healing. This was unexpected due to the established effects of UMF 20 manuka honey on wound healing using the same model. This may be due to systemic effects of cannabidiol and study design. Further research into the use of cannabidiol in equine wounds is warranted.

Pilot study to quantify the time to clear dexamethasone from plasma and urine of adult horses following a single nebulisation.

OBJECTIVE: To quantify the time to clear dexamethasone from plasma and urine of horses following a single nebulisation. DESIGN: Experimental using six Standardbred mares. METHODS: Dexamethasone sodium phosphate (0.04 mg/kg) diluted in 0.9% sodium chloride was administered as an aerosol using a Flexineb E2® nebuliser. Blood samples (0, 2, 4, 6, 8, 10, 12, 24, 32, 48, 72 and 96 h) and urine samples (0, 1, 4, 8, 24, 32, 48, 72 and 96 h) were collected for analysis using liquid chromatography mass spectrometry. RESULTS: Maximum plasma concentrations (tmax ) were reached by the earliest detection point (2 h) after nebulisation (0.6-1.8 ng/mL), but was no longer detectable at 48 h. However, in one horse 0.1 ng/mL was found at 96 h after three consecutive readings of 0 ng/mL. The tmax in urine was reached by the earliest collection point (1 h) after nebulisation (3.2-23.8 ng/mL), but was no longer present in urine at 72 h in five horses, while detectable levels (0.1 ng/mL) were still present at 96 h in one horse. CONCLUSIONS: A single dose of 0.04 mg/kg of DSP administered as an aerosol through a FlexinebE2® mask was no longer detectable in blood at 48 h in six horses tested, but one horse returned a reading of 0.1 ng/mL at 96 h after having no detectable levels. Dexamethasone was not detectable in urine at 72 h in five horses but was detectable at a low concentration (0.1 ng/mL) at 96 h in one horse.

Comparison of the effects of topical application of UMF20 and UMF5 manuka honey with a generic multifloral honey on wound healing variables in an uncontaminated surgical equine distal limb wound model.

OBJECTIVE: To compare the effect of application of manuka honey with unique manuka factor (UMF) 5 or 20 with a generic multifloral honey on equine wound healing variables. METHODS: Two full-thickness skin wounds (2.5 × 2.5 cm) were created on the metatarsus of both hindlimbs of eight Standardbred horses. The wounds on each horse were assigned to 1 of 4 treatments: UMF20 (UMF20) and UMF5 (UMF5) manuka honey; generic multifloral honey (GH); and a saline control. Bandages were changed daily for 12 days, after which treatment was stopped and the bandages were removed. Wound area was measured on day 1, then weekly until day 42. Overall wound healing rate (cm2 /day) and time to complete healing were recorded. RESULTS: There was no difference in wound area for any of the treatments on any measurement day except for day 21, where the mean wound area for wounds treated with UMF20 was smaller than the mean wound area for the UMF5-treated wounds (P = 0.031). There was no difference in mean (± SE) overall healing rate (cm2 /day) among the treatment groups. There were differences in mean (± SE) days to complete healing. Wounds treated with UMF20 healed faster than wounds treated with GH (P = 0.02) and control wounds (P = 0.01). CONCLUSIONS: Treatment of wounds with UMF20 reduced overall wound healing time compared with wounds treated with GH and control wounds. However, using this model the difference in the overall time to complete healing was small.

Effect of activated protein C in second intention healing of equine distal limb wounds: a preliminary study.

OBJECTIVE: To investigate the effect of activated protein C (APC) on second intention healing of distal limb wounds in horses. METHODS: In this experimental study of eight Standardbred geldings, six full-thickness skin wounds (2 × 1.5 cm) were created on one metacarpus (biopsy limb) and five similar wounds were created on the contralateral metacarpus (photographed limb). Three wounds on the biopsy limb were treated topically with 190 µg APC on days 1, 3, 6 and 9, while the remaining three wounds were untreated (control). One treated and one control wound were biopsied on days 4, 7 and 11 for histopathology. Wounds on the photographed limb were treated with either 66% Manuka honey gel, a commercial antibiotic ointment (bacitracin-neomycin-polymixin B ointment; BNP) or petrolatum daily throughout healing, treated on days 1,3,6 and 9 with 190 µg APC or left untreated. These wounds were digitally photographed and the wound area measured on day 1, then weekly until day 49. Overall time to healing was recorded. RESULTS: There was no effect of APC on wound size, the rate of healing or the overall time to heal. However, compared with control wounds, histological scoring demonstrated enhanced epithelialisation (day 4) and angiogenesis (day 11). Wound healing variables for wounds treated with APC, Manuka honey gel and control wounds were not different and the variables for wounds treated with BNP and petrolatum demonstrated delayed healing. CONCLUSION: The improvements in histological scores in APC-treated wounds suggest further study into the effect of APC on second intention wound healing in horses is warranted.

Survey of the therapeutic approach and efficacy of pentosan polysulfate for the prevention and treatment of equine osteoarthritis in veterinary practice in Australia.

OBJECTIVE: To survey veterinary practitioners in Australia on how they administer pentosan polysulfate (PPS) to horses and their perceptions of the efficacy of PPS for: the prevention and treatment of osteoarthritis (OA), the treatment of OA when PPS is combined with other drugs, and the efficacy of PPS compared with other disease-modifying osteoarthritic drugs. DESIGN: Practitioners were contacted by email, which contained a link to an online survey. RESULTS: A total of 76 responses (34.5%) to the survey were received. Respondents most commonly used PPS as prophylactic therapy prior to competition (80.3%). As a prophylactic agent, PPS was considered by 48.2% of respondents to have high efficacy. The most common dose regimen for prevention and treatment of OA was 3 mg/kg, intramuscularly, once weekly for 4 weeks followed by monthly injections. Most respondents (78%) combined PPS with other drugs for treatment of OA. Intra-articular corticosteroids and hyaluronate (HA) was the most common drug combination used with PPS. PPS was preferred as a prophylactic agent when compared with HA (88.7% vs 11.3%). For treating OA, 83% of respondents considered a combination of PPS, HA and glucosamine to be more efficacious than PPS alone. However, the most common reason not to use this combination was cost (79.1%). CONCLUSION: All respondents used PPS for prophylaxis and/or treatment of OA despite limited published scientific evidence proving its efficacy in horses. Further research is necessary to provide evidence of the clinical efficacy of PPS for the prevention and treatment of OA in horses.

Translocation of an unusual cAMP receptor to the nucleus during development of Dictyostelium discoideum.

cAMP has been implicated in the control of the expression of developmental genes in Dictyostelium discoideum. To determine the potential role of cAMP receptors as regulators of gene expression, we have used immunocytochemical and immunoblotting techniques to reveal the subcellular localization of a cAMP binding protein CABP1. Most of the CABP1 antigen in early developing cells is localized near the cell periphery, with a small amount found in the nucleus. The level of CABP1 in the nucleus increases approximately 30-fold during development. Moreover, immunofluorescence studies showed that CABP1 can also be detected on the cell surface. Binding of anti-CABP1 to intact cells followed by reaction with 125I-labeled secondary antibody revealed that the cell-surface CABP1 activity peaks during aggregation and culmination. In addition, several proteins related to CABP1 are found mainly in the nuclear fraction of developing cells. The possible role of these proteins in the regulation of developmental gene activity is discussed.

Expression of an altered cAMP binding protein by rapid-developing strains of Dictyostelium discoideum.

Immunoblotting with a monoclonal antibody raised against a novel cAMP binding protein termed CABP1 revealed that the molecular weights of the two CABP1 subunits are altered in certain strains of Dictyostelium discoideum. Cell-free translation followed by immunoprecipitation showed that the altered CABP1 polypeptides are derived from primary translation products. In addition, the affinity of the altered CABP1 for cAMP is much higher than the wild-type form. Morphologically, these strains are indistinguishable from other wild-type strains except that their developmental phase is considerably shorter. The rapid developers also exhibit a precocious appearance of CABP1. These results indicate a good correlation between an altered CABP1 and rapid development.

Identification of multiple cyclic AMP-binding proteins in developing Dictyostelium discoideum cells.

We have purified two cAMP-binding proteins from developing Dictyostelium discoideum cells, which we designate as CABP-1 and CABP-2. Purified CABP-1 consists of two polypeptides of Mr 41,000 and 36,000, which we refer to as CABP-1A and CABP-1B, respectively. Although CABP-1 exhibited specificity for cAMP, it was not labeled at a detectable level when mixed with 8-azidoadenosine 3':5'-monophosphate (8-N3[3H]cAMP). Unlike CABP-1, CABP-2 was labeled efficiently with 8-N3[3H]cAMP. Purified CABP-2 has a molecular weight of 41,000 and an isoelectric point of 5.8-6.0. The physical and biochemical properties of CABP-2 suggest that it is the regulatory subunit of cAMP-dependent protein kinase described by others (de Gunzburg, J., Part, D., Guiso, N., and Veron, M. (1984) Biochemistry 23, 3805-3812; Majerfeld, J. H., Leichtling, B. H., Maligeni, J. A., Spitz, E., and Rickenberg, H. V. (1984) J. Biol. Chem. 259, 654-661). Although CABP-1A and CABP-2 have the same molecular weight, they appear to be encoded by different genes. Two-dimensional gel electrophoresis revealed that the two polypeptides had different isoelectric points. Moreover, monoclonal antibodies raised against CABP-1 did not cross-react with CABP-2. Also, in vitro translation followed by immunoprecipitation showed that these two polypeptides were derived from primary translation products. Our finding of a novel cAMP-binding protein, CABP-1, suggests that cAMP-dependent protein kinase may not be the only intracellular regulator mediating the effects of cAMP in developing D. discoideum cells.

Cell-type-specific actin mRNA populations in dictyostelium discoideum.

We have analyzed actin mRNA sequences present in the terminal stages of the development of Dictyostelium discoideum. Four different actin mRNA sequences were detected in migrating pseudoplasmodia. Nucleotide sequence analysis of primer-extension products derived from the four mRNA sequences showed that they each encoded an actin protein with the same eight N-terminal amino acids and that they did not derive from transcription of any previously characterized actin gene. Preculmination pseudoplasmodia of Dictyostelium contain two distinct populations of committed cells, termed prespore and prestalk cells. We show that prestalk cells contain all four of the actin mRNA sequences found in pseudoplasmodia, while prespore cells contain only three of the sequences, and mature spores contain only two. Thus there is a differential loss of actin mRNA sequences during spore-cell differentiation in Dictyostelium.

Differential expression of the members of the discoidin I multigene family during growth and development of Dictyostelium discoideum.

Discoidin I and II are lectins synthesized during the aggregation of Dictyostelium discoideum amoebae which may play a role in cellular cohesion. Discoidin I was thought to consist of two major polypeptides, but we show that there are three. The N-terminal amino acid sequence of the polypeptides has been predicted by determining part of the nucleotide sequence of their respective mRNAs. We obtained the nucleotide sequences by reverse transcription of the mRNAs, using as primers, fragments derived from the coding region of two cloned discoidin I sequences, and utilizing cross hybridization to the various mRNA species and differences in the length of their 5' noncoding regions to isolate fragments for DNA sequencing. We used primer extension to measure the relative concentration of the three major discoidin I mRNA sequences. We show that during development changes in the abundance of all three mRNA sequences occur coordinately. In cells growing in nutrient broth, however, only two of the three major discoidin I mRNA sequences accumulate, and if such cells are grown to a very high density, both sequences disappear. These results indicate that the coordination of discoidin I gene expression is not obligatory and that the members of this multigene family may differ in the mode of their induction during normal development.

A change in the rate of transcription of a eukaryotic gene in response to cyclic AMP.

The plasmid pDd812 contains the DNA copy of an mRNA sequence from Dictyostelium discoideum that undergoes first an increase and then a decrease in concentration during the first few hours of differentiation. We have recently shown that the mRNA sequence complementary to pDd812 encodes discoidin I, a developmentally regulated lectin that may play a role in cellular cohesion. By using pDd812 as a hybridization probe, we found that addition of cyclic AMP during the first few hours of development inhibited the accumulation of discoidin I mRNA. By measuring the rate of transcription in isolated nuclei, we showed that, at least in part, this inhibition results from a rapid and specific reduction in the rate of transcription of the discoidin I gene. Addition of cyclic AMP during the first few hours of development inhibited the accumulation of discoidin I mRNA. By measuring the rate of transcription in isolated nuclei, we showed that, at least in part, this inhibition results from a rapid and specific reduction in the rate of transcription of the discoidin I gene. Addition of cyclic AMP during the first few hours of development inhibited the accumulation of discoidin I mRNA. By measuring the rate of transcription in isolated nuclei, we showed that, at least in part, this inhibition results from a rapid and specific reduction in the rate of transcription of the discoidin I gene. Addition of high external concentrations of cAMP is known to increase the intracellular concentration to a level normally found later in development. This natural increase in cAMP concentration occurs at the time during development when transcription of the discoidin I gene ceases. We suggest, therefore, that changes in the intracellular concentration of cAMP act at the level of transcription to control gene expression during development. This hypothesis is supported by our observation that several poly(A)+RNA sequences that normally accumulate after transcription of the discoidin I gene has ceased are synthesized prematurely in cells exposed to exogenous cAMP.