Effects of Topical Treatment of Foot Rot in Sheep Using Ozonated Olive Ointment.

INTRODUCTION: Foot rot in small ruminants is highly contagious, causes severe lameness, and impairs fertility and wool and meat production. It is usually treated with parenteral antibiotics, with attendant antibiotic resistance risk, and with bactericidal footbaths, potentially harmful to humans and the environment. An alternative treatment in sheep is proposed based on repeated topical ozonated ointment application. Its effectiveness and safety were evaluated by estimation of acute-phase response, biochemical indicators of organic damage, and antioxidant/oxidant balance (AOB). MATERIAL AND METHODS: The study was conducted on ten sheep with Egerton scale 2-3 lesions. Ozone application was repeated every day for seven days. Blood was drawn first (T0) after foot cleaning and before ozonation, then (T1) seven days after the first ozone application, and finally (T2) four days after the last application. RESULTS: High clinical effectiveness was observed, with total recovery by 28 days from the start of treatment. A significant increase in antiradical activity was noted on the basis of a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay from 1.16 ± 0.04 µmolTe/mL at T0 to 1.23 ± 0.03 µmolTe/mL at T1, with a slight decrease in oxidative stress. Calculated on the basis of antiradical capacity, AOB was higher at T1 (130 ± 19%) and decreased to 110 ± 16% at T2. Calculated on the basis of reducing power, it was 169 ± 22% at T1 and 131 ± 17% at T2. CONCLUSION: These results indicated that the AOB is efficient enough to prevent oxidative organ injury and the applied doses of ozone are safe for animals.

The Effect of Neutrophil-Derived Products on the Function of Leukocytes Obtained after Titanium Implantation in the Ovine Model.

Titanium (Ti) is currently the most common biomaterial used for orthopedic implants; however, these implants may cause deleterious immune response. To investigate the possible mechanisms involved in excessive inflammation, we assessed the activity of neutrophils and monocyte-derived macrophages (MDMs) during the insertion of the Ti implant in a sheep model. The study was conducted on 12 sheep, 4 of which were control animals and 8 were in the experimental group with inserted Ti implant. Neutrophil secretory response was estimated at two time points T0 before surgery and T1 1 h after implantation and was based on the release of enzymes from neutrophil granules and reactive oxygen and nitrogen species (RONS) generation. MDM function was evaluated 5 months after implantation, on the basis of RONS generation arginase activity and morphological changes. Moreover, the influence of some autologous neutrophil derived products, namely, antimicrobial neutrophil extract (ANE) and neutrophil degranulation products (DGP) on leukocytes was estimated. Our study revealed that Ti implant insertion did not cause any adverse effects up to 5 months after surgical procedure. Stimulation of neutrophil cultures with ANE decreased the enzyme release as well as superoxide generation. Treatment of MDM with ANE diminished superoxide and NO generation and increased arginase activity. On the other hand, MDM stimulated with DGP showed elevated superoxide and NO generation as well as decreased arginase activity. To summarize, ANE exerted an anti-inflammatory and pro-resolving effect on studied leukocytes, whereas DGP acted as pro-inflammatory.

Long-term Interactions of Circulating Neutrophils with Titanium Implants, the Role of Platelets in Regulation of Leukocyte Function.

Despite the fact that different biomaterials are widely used in many biomedical applications, they can still cause side effects. Therefore, our aim was to assess neutrophil activity during the inflammatory phase of the repair process and long-term interactions between circulating neutrophils and Titanium (Ti) implants. Additionally, neutrophil in vitro response after stimulation by the extract of antimicrobial peptides (AMP extract), pentoxifylline (PTX) and some platelet-rich (L-PRP and PURE PRP) and platelet-poor (PPP) concentrates were tested. The study was conducted on eight sheep after Ti implant insertion into the tibia and revealed that the Ti implant did not cause any side effects during the course of experiment. After addition of L-PRP into neutrophils, culture activity of these cells significantly increased (p < 0.01), whereas treatment with AMP extract, PURE PRP, PPP or PTX caused decrease in neutrophil enzymatic response (on the basis of elastase, myeloperoxidase and alkaline phosphatase release) and free radical generation. These effects were observed in neutrophils isolated during the inflammatory phase as well as 4 and 10 months after implantation. Obtained results will be useful in regulation of inflammatory response during implantation of biomaterial and create possibility to modulate the cells response towards pro- or anti-inflammatory to reduce host tissue damage.

Application of Natural Neutrophil Products for Stimulation of Monocyte-Derived Macrophages Obtained before and after Osteochondral or Bone Injury.

We evaluated the use of some neutrophil products, namely; autologous rabbit antimicrobial neutrophil extract (rANE), heterologous porcine antimicrobial neutrophil extract (pANE), neutrophil degranulation products (DGP) and neutrophil microvesicles (MVs) for stimulation of monocyte-derived macrophages (MDMs) to improve healing. Two animal models were evaluated; the rabbit model for autologous osteochondral transplantation (OT) with application of rabbit ANE, DGP or MVs for MDMs stimulation, and the ovine model of the insertion of a Ti implant with the use of porcine ANE, and ovine DGP or MVs for MDMs stimulation. Macrophage activity was assessed on the basis of free radical generation and arginase activity. We estimated that DGP acted in a pro-inflammatory way both on rabbit and ovine MDMs. On the other hand, MVs acted as anti-inflammatory stimulator on MDMs in both experiments. The response to ANE depended on origin of extract (autologous or heterologous). Macrophages from rabbits before and after OT stimulated with autologous extract generated lower amount of NO and superoxide, especially after transplantation. In the ovine model of Ti implant insertion, heterologous ANE evoked increased macrophage pro-inflammatory activity. Our study revealed that these neutrophil products could regulate activity of macrophages, polarizing them into pro-or anti-inflammatory phenotypes that could enhance bone and osteochondral tissue healing.

Prospects and Applications of Natural Blood-Derived Products in Regenerative Medicine.

Currently, there are a number of therapeutic schemes used for the treatment of various types of musculoskeletal disorders. However, despite the use of new treatment options, therapeutic failure remains common due to impaired and delayed healing, or implant rejection. Faced with this challenge, in recent years regenerative medicine started looking for alternative solutions that could additionally support tissue regeneration. This review aims to outline the functions and possible clinical applications of, and future hopes associated with, using autologous or heterologous products such as antimicrobial peptides (AMPs), microvesicles (MVs), and neutrophil degranulation products (DGP) obtained from circulating neutrophils. Moreover, different interactions between neutrophils and platelets are described. Certain products released from neutrophils are critical for interactions between different immune cells to ensure adequate tissue repair. By acting directly and indirectly on host cells, these neutrophil-derived products can modulate the body's inflammatory responses in various ways. The development of new formulations based on these products and their clinically proven success would give hope for significant progress in regenerative therapy in human and veterinary medicine.