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Currently, the main limitation for the use of adult differentiated chondrocytes in cell-based therapy and tissue engineering for the repair of articular cartilage is the difficulty of maintaining their state of differentiation during cell expansion. The adult articular cartilage has no direct blood supply, and local oxygen concentrations range from 5%-10% at the surface near the synovial fluid to less than 1% in the deep layer. Low oxygen tension is currently considered an important environmental condition for chondrocytes, and hypoxia has been explored as a signal potentially promoting differentiation and matrix deposition. In the present study, hypoxia and PL supplementation were studied to maintain differentiation in adult articular chondrocytes. Freshly isolated equine articular chondrocytes were grown in monolayer culture at a low seeding density (condition favoring proliferation and dedifferentiation) and in alginate beads (3D culture condition maintaining chondrocyte differentiation) both in normoxic and hypoxic conditions and in various conditions of supplementation or deprivation (fetal bovine serum [FBS]- and PL-free; 10% FBS; 5% PL; 10% PL). Results demonstrated that hypoxia is a micro-environmental condition that reduces chondrocyte dedifferentiation or maintains differentiation during in vitro expansion, as shown by the sustained expression of differentiation markers (COL2, ACAN, SOX9, HIF1a) and the reduction of dedifferentiation marker expression (COL1, RUNX2). In association with hypoxia, PL supplementation demonstrated a positive effect on chondrocyte differentiation in association with hypoxia. This promising result should be confirmed in other conditions of chondrocyte differentiation before proposing PL as a complete alternative to xenogenic serum for the expansion of articular chondrocytes.

Simple Summary Recently, mesenchymal stromal cells have been proposed as a valid approach for treating osteoarthritis diseases, mainly due to the secretion of soluble factors and extracellular vesicles known as secretome. In this paper, an injectable freeze-dried pharmaceutical powder containing canine mesenchymal stromal cells-secretome (Lyosecretome) has been formulated, and in vitro potency experiments were conducted. Furthermore, the Lyosecretome was applied in dogs affected by osteoarthritis to assess its safety. Results indicated that intra-articular injection of allogeneic Lyosecretome is safe and does not induce an adverse response, thus paving the way for future studies regarding the efficacy. Abstract In recent years, mesenchymal stromal cells (MSCs) have shown promise as a therapy in treating musculoskeletal diseases, and it is currently believed that their therapeutic effect is mainly related to the release of proteins and extracellular vesicles (EVs), known as secretome. In this work, three batches of canine MSC-secretome were prepared by standardized processes according to the current standard ISO9001 and formulated as a freeze-dried powder named Lyosecretome. The final products were characterized in protein and lipid content, EV size distribution and tested to ensure the microbiological safety required for intraarticular injection. Lyosecretome induced the proliferation of adipose tissue-derived canine MSCs, tenocytes, and chondrocytes in a dose-dependent manner and showed anti-elastase activity, reaching 85% of inhibitory activity at a 20 mg/mL concentration. Finally, to evaluate the safety of the preparation, three patients affected by bilateral knee or elbow osteoarthritis were treated with two intra-articular injections (t = 0 and t = 40 days) of the allogeneic Lyosecretome (20 mg corresponding 2 × 106 cell equivalents) resuspended in hyaluronic acid in one joint and placebo (mannitol resuspended in hyaluronic acid) in the other joint. To establish the safety of the treatment, the follow-up included a questionnaire addressed to the owner and orthopaedic examinations to assess lameness grade, pain score, functional disability score and range of motion up to day 80 post-treatment. Overall, the collected data suggest that intra-articular injection of allogeneic Lyosecretome is safe and does not induce a clinically significant local or systemic adverse response.

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties, (ii) extracellular vesicle morphology, size distribution, and surface biomarker, (iii) protein and lipid content, (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma, and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases

Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.

Infections caused by the opportunistic yeast

Infections caused by the opportunistic yeast Candida albicans are one of the major life threats for hospitalized and immunocompromised patients, as a result of antibiotic and long-term antifungal treatment abuse. Odorant binding proteins can be considered interesting candidates to develop systems able to reduce the proliferation and virulence of this yeast, because of their intrinsic antimicrobial properties and complexation capabilities toward farnesol, the major quorum sensing molecule of Candida albicans. In the present study, a hybrid system characterized by a superparamagnetic iron oxide core functionalized with bovine odorant binding protein (bOBP) was successfully developed. The nanoparticles were designed to be suitable for magnetic protein delivery to inflamed areas of the body. The inorganic superparamagnetic core was characterized by an average diameter of 6.5 ± 1.1 nm and a spherical shape. Nanoparticles were functionalized by using 11-phosphonoundecanoic acid as spacer and linked to bOBP via amide bonds, resulting in a concentration level of 26.0 ± 1.2 mg bOBP/g SPIONs. Finally, both the biocompatibility of the developed hybrid system and the fungistatic activity against Candida albicans by submicromolar OBP levels were demonstrated by in vitro experiments.

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products.

In vitro studies have demonstrated that platelet lysate (PL) can serve as an alternative to platelet-rich plasma (PRP) to sustain chondrocyte proliferation and production of extracellular matrix components in chondrocytes. The present study aimed to evaluate the direct effects of PL on equine articular chondrocytes in vitro in order to provide a rationale for in vivo use of PL. An in vitro cell proliferation and de-differentiation model was used: primary articular chondrocytes isolated from horse articular cartilage were cultured at low density under adherent conditions to promote cell proliferation. Chondrocytes were cultured in serum-free medium, 10% foetal bovine serum (FBS) supplemented medium, or in the presence of alginate beads containing 5%, 10% and 20% PL. Cell proliferation and gene expression of relevant chondrocyte differentiation markers were investigated. The proliferative capacity of cultured chondrocytes, was sustained more effectively at certain concentrations of PL as compared to that with FBS. In addition, as opposed to FBS, PL, particularly at percentages of 5% and 10%, could maintain the gene expression pattern of relevant chondrocyte differentiation markers. In particular, 5% PL supplementation showed the best compromise between chondrocyte proliferation capacity and maintenance of differentiation. The results of the present study provide a rationale for using PL as an alternative to FBS for in vitro expansion of chondrocytes for matrix-assisted chondrocyte implantation, construction of 3D scaffolds for tissue engineering, and treatment of damaged articular cartilage.

Keratoma is a nonmalignant horse tumor that grows in the space between the horn of the hoof and the distal phalanx. Keratoma causes lameness in the horse, and surgical excision is the treatment of choice. Four horses underwent removal of a keratoma by complete hoof wall resection. The remaining wound was treated with platelet-rich plasma (PRP) combined with a sterile three-dimensional polylactic acid scaffold. The PRP was applied at 3, 6, 9, 12, 15, and 18 days postoperatively. The surgical site was cleaned with gauzes and swabs soaked in Ringer’s lactate solution before applying PRP and the foot bandage. Healthy granulation tissue developed at 6–21 days postoperatively. The hoof wall defect was completely filled with new hoof wall within 6–8 months after surgery. All horses returned to their previous exercise level, and no recurrence of lameness was reported by the owner.

In recent years, nutritional supplements from different sources have been widely considered to support medical treatments in patients affected by chronic hepatopathies. Their potential therapeutic benefit has been recognized, but some evidence of safety issues has been reported. Recently it has been hypothesized that the liver could produce various of bioactive factors to maintain organ homeostasis and promote tissue healing. Thus, liver-specific preparations containing bioactive factors could provide a suitable substrate for in vitro study of liver tissue maintenance/healing, as a prospective regenerative medicine approach. Furthermore, they could represent a dietary supplement or nutraceutical for adjuvant therapies when correctly prepared and formulated. This work aims to provide data about the safety and biological activity of a freeze-dried porcine liver preparation. The lyophilized powder obtained from the whole organ has been tested in term of in vitro cell cytotoxicity (MTT assay) and proliferation assays (bromo-deoxyuridine incorporation and direct cell count) in two different cell types: human hepatoma HepG2 cell line and adipose-tissue-derived canine mesenchymal stromal cells (At-MSCs). At concentration levels between 100 to 500 µg/mL, the lyophilized liver powder stimulated mitochondrial metabolism as assessed by MTT assay (p ≤ 0.001 for HepG2 and for At-MSCs) and induced an increase in bromo-deoxyuridine incorporation in both cell types (p ≤ 0.01 for HepG2 and p < 0.001 for At-MSCs). In addition, direct cell count demonstrated a higher proliferative activity in treated At-MSCs (p < 0.001). Although preliminary, these data suggest that the whole-liver powder is noncytotoxic in vitro and may represent a stimulus to cell metabolism and proliferation. Further studies are needed to detect the bioactive components of the supplement and characterize in deeper detail the cellular pathways that they can modulate.

The bacterium Pseudomonas aeruginosa (PA) and the yeast Candida albicans (CA) are pathogens that cohabit the mucosa of the respiratory tracts of animals and humans. Their virulence is largely determined by chemical communication driven by quorum sensing systems (QS), and the cross perception of their quorum sensing molecules (QSM) can modulate the prevalence of one microorganism over the other. Aiming to investigate whether some of the protein components dissolved in the mucus layering the respiratory mucosa might interfere with virulence and cross-communication of these, and eventually other microorganisms, ligand binding assays were carried out to test the scavenging potential of the bovine and porcine forms of the Lipocalin odorant binding protein (OBP) for several QSMs (farnesol, and acylhomoserine lactones), and for pyocyanin, a toxin produced by PA. In addition, the direct antimicrobial activity of the OBPs was tested by time kill assay (TKA) against CA, PA and other bacteria and yeasts. The positivity of all the ligand binding assays and the antimicrobial activity determined for CA, and for some of the other microorganisms tested, let hypothesize that vertebrate OBPs might behave as humoral components of innate immunity, active against pathogenic bacteria and fungi. In addition, TKAs with mutants of bovine OBP with structural properties different from those of the native form, and with OBP forms tagged with histidines at the amino terminal, provided information about the mechanisms responsible of their antimicrobial activity and suggested possible applications of the OBPs as alternative or co-adjuvants to antibiotic therapeutic treatments.

Key Clinical Message Extensive full-thickness skin wounds are quite common in domestic animals. In these report, following the failure of reconstructive surgery, adipose tissue-derived mesenchymal stem cells, and platelet-rich plasma were successfully used in a dog to improve speed and quality of skin tissue healing, avoiding suffering, and debilitating effects.

Abnormalities of the canine prostate gland, in particular, benign hyperplasia and cysts, are frequent in dogs over 5 years of age. Treatment strategies for dogs with prostatic cysts include ultrasound-guided percutaneous drainage with alcohol sclerotherapy, reduction surgery, and omentalization. The aim of this study was to evaluate the use of platelet-rich plasma (PRP) for the treatment of prostatic cysts in dogs. Ten mixed breed adult male dogs were included in the study. Percutaneous drainage was performed prior to ultrasound-guided PRP injection with a 21G spinal needle. The PRP dose contained half of the solution drained from the cyst. Follow-ups were performed 6, 12, 24, and 60 d after treatment. Ten prostatic cysts were successfully treated with PRP after the ultrasound-guided drainage of the cystic cavities. No side effects were observed during the procedures. After the introduction of PRP, cysts detected by ultrasound appeared hypoechoic. On the sixth day, the hypoechoic appearance was maintained and margins were still identifiable. At 24-day and 60-day follow-ups, cysts were no longer detectable by ultrasound. One of the ten dogs had to be euthanized because of the presence of a malignant mesenchymal neoplasm of the oral cavity (fibroblastic sarcoma) and histological examination of serial sections of the prostate gland was performed. Histologic evaluation 1 year after treatment showed a regression of the cystic lesion injected with PRP. The therapy, which uses PRP injection in combination with ultrasound-guided drainage, can be considered a safe and effective strategy for the treatment of prostatic cysts in dogs.Les anomalies de la prostate chez le chien, plus particulièrement l’hyperplasie bénigne et les kystes, sont fréquentes chez les chiens âgés de 5 ans ou plus. Les stratégies de traitement pour les chiens avec des kystes prostatiques incluent le drainage transcutané par échographie guidée avec sclérothérapie à l’alcool, chirurgie de réduction, et omentalisation. Le but de la présente étude était d’évaluer l’utilisation de plasma enrichi de plaquettes (PEP) pour le traitement de kystes prostatiques chez des chiens. Dix chiens mâles adultes de race mélangée ont été inclus dans l’étude. Le drainage transcutané a été effectué avant l’injection par échographie guidée de PEP avec une aiguille spinale de 21G. La dose de PEP contenait la moitié de la solution drainée du kyste. Des suivis ont été faits à 6, 12, 24, et 60 j suivant le traitement. Dix kystes prostatiques ont été traités avec succès avec le PEP suite au drainage des cavités kystiques par échographie guidée. Aucun effet secondaire ne fut observé durant les procédures. Après l’introduction du PEP, les kystes détestés par échographie apparaissaient hypoéchogène. Au sixième jour, l’apparence hypoéchogène était maintenue et les bordures étaient toujours identifiables. Lors des suivis aux jours 24 et 60, les kystes n’étaient plus détectables par échographie. Un des dix chiens dû être euthanasié à cause de la présence d’un néoplasme mésenchymateux malin de la cavité orale (sarcome fibroblastique) et l’examen histologique de sections sériées de la prostate a été effectué. L’évaluation histologique 1 an après le traitement a montré une régression de la lésion kystique injectée avec du PEP. La thérapie, qui utilise l’injection de PEP en combinaison avec un drainage par échographie guidée, peut être considérée comme une stratégie sécuritaire et efficace pour le traitement de kystes prostatiques chez le chien(Traduit par Docteur Serge Messier).

During corpus luteum formation, impressive biological events take place to guarantee the transition from original follicular to luteal cells and to support required massive angiogenesis. It has been demonstrated that these phenomena resemble those essential for wound healing. After ovulation, blood vessels release their content in the antral cavity and coagulation takes place. Involvement of platelets in corpus luteum growth has been hypothesized both in human and in rat. On this basis, using platelet lysate (PL), a blood derivative with a higher platelet concentration, we aimed to assess a potential involvement of platelets in swine granulosa cell luteinization and on new blood vessel growth. Our results demonstrate, for the first time in the swine, that platelets could be directly involved in granulosa cell physiological luteinization, since the treatment with PL shifted steroid production from estradiol 17β to progesterone. Moreover, PL stimulated angiogenesis. Nitric oxide could be involved in these effects. These results are important to clarify complex intrafollicular molecular machinery. A better understanding of these mechanisms can be useful to develop more focused therapeutic strategies to contrast sow infertility. In addition, since the pig represents a model for translational studies, collected data could be of interest for human medicine because reproductive pathologies such as Polycystic Ovary Syndrome (PCOS) and endometriosis are often accompanied by platelet dysfunctions.

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29+, CD44+, CD73+, CD90+, CD34-, CD45- and MHC-II- with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90+, CD73+, CD105+, CD44+, CD13+, CD29+, Oct-4+ gene and CD31- and CD45- expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures.

Contents In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT-PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue-derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue-derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue-derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow-derived MSC in dog.

Odorant binding protein (OBP) is a multi-functional scavenger for small hydrophobic molecules dissolved in the mucus lining the nasal epithelia of mammals, characterized by broad ligand binding specificity towards a large number of structurally unrelated natural and synthetic molecules of different chemical classes. Here, we demonstrate for the first time the application of OBP as the active element of an innovative filtering matrix for the removal of environmental pollutants such as triazine herbicides from water samples. The filtering device, obtained by coupling histidine-tagged bovine OBP to a nickel nitrilotriacetic acid (Ni-NTA) agarose resin, was characterized in terms of retention capacity for the herbicides atrazine, simazine, and propazine. Analysis of these herbicides at trace levels with solid-phase microextraction followed by gas chromatography-mass spectrometry using the selected ion monitoring mode proved the capabilities of the proposed device for the decontamination of surface and groundwater samples in the 0.2-2,300 μg/L concentration range, obtaining a reduction in the triazine content greater than 97 %, thus suggesting its possible use for the potabilization of water.

Equine mesenchymal stem cells (MSC) are of particular interest both for basic research and for the therapeutic approach to musculoskeletal diseases in the horse. Their multilineage differentiation potential gives them the capability to contribute to the repair of tendon, ligament and bone damage. MSCs are also considered a promising therapeutic aid in allogeneic cell transplantation, since they show low immunogenicity and immunomodulating functions.Adipose tissue-derived adult equine stem cells (AdMSC) can be isolated, expanded in vitro and then inoculated into the damaged tissue, eventually in the presence of a biological scaffold. Here we report our preliminary experience with adipose-derived mesenchymal stem cells in allogeneic cell-therapy of tendonitis in the horse. MSCs, derived from visceral adipose tissue, were grown in the presence of autologous platelet lysate and characterized for their differentiation and growth potential. Expanded AdMSC were inoculated into the damaged tendon after their dispersion in activated platelet-rich plasma (PRP), a biological scaffold that plays an important role in maintaining cells in defect sites and contributes to tissue healing. Fourteen out of sixteen treated horses showed a functional recovery and were able to return to their normal activity.

Del Bue, M., Ricco, S., Conti, V., Merli, E., Ramoni, R. and Grolli, S., 2007. Platelet lysate promotes in vitro proliferation of equine mesenchymal stem cells and tenocytes. Veterinary Research Communications, 31(Suppl. 1), 289–292

Platelet concentrates are widely used in mammalian regenerative medicine to improve tissue healing. Chelonians (Testudines) would benefit from the application of thrombocyte preparations to regenerate damaged tissues, since traumatic injuries are leading causes of morbidity and mortality for both wild-living and domesticated animals. The aim of this study was to establish a protocol that optimized the recovery of the thrombocytes from blood samples and to show the efficacy of thrombocyte-enriched plasma in chelonians. Peripheral blood samples were obtained from Testudo spp. (n = 12) and Trachemys scripta elegans (n = 10). Blood cells were fractionated by sodium diatrizoate-sodium polysucrose density gradient using a two-step centrifugation protocol. Thrombocytes and leukocytes were isolated and resuspended to obtain thrombocyte-leucocyte rich plasma (TLRP). The mean recovery of leukocytes and thrombocytes was 48.9% (±4.0 SEM, n = 22) of the whole blood cell content. No statistically significant difference was observed between blood samples collected from different turtle species. The ability of TLRP to form a gel was evaluated by adding variable concentrations of calcium gluconate at room temperature and at 37°C. A reliable and consistent clotting of the TLRP was obtained in glass tubes and dishes by adding 5-20% v/v of a 100 mg/ml solution of calcium gluconate. Furthermore, in order to test the clinical efficacy of TLRP, a preliminary evaluation was performed on four turtles (Testudo spp.) with traumatic injuries. In all the four animals, a successful clinical outcome was observed. The results demonstrated that a thrombocyte-enriched plasma, comparable to mammalian platelet rich plasma, can be prepared from chelonian blood samples. Furthermore, although the low number of cases presented does not allow definitive conclusions from a clinical point of view, their outcome suggests that TLRP application could be further investigated to improve the healing process of both soft and hard tissue injuries in chelonians.

A small column containing 2 mM CH-Sepharose 4B-immobilized trypsin was connected to a flow injection device equipped for potentiometric measurements (0.01–2 mM protons) and for post-column analysis by spectrophotometry and capillary electrophoresis (CE). The device was engaged with N α -benzoyl- l -arginine pNO 2 -anilide (BAPNA), β -lactoglobulin ( β -Lac) and peptides of V8-protease predigested β -Lac. At a given flow rate, the reaction with BAPNA or β -Lac (below 2 mM) produced about 1 proton per substrate molecule in each sample (linear relation to substrate amount); with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalents per substrate molecule (hyperbolic dependence). Final experiments demonstrated that the reactor gave a correct estimate of available lysine in peptides of β -Lac modified with 5-nitrosalicylaldehyde. The data could be predicted by a kinetic model describing the reactor performance in `single turnover' conditions. The interplay between resident time and the non-catalytic amount of trypsin prevented each enzyme molecule from recycling as well as each substrate molecule (containing one or more cleavage sites) from encountering the enzyme more than once. In conclusion, both from the experimental and the theoretical point of view, this work permitted the analysis of trypsin behaviour in some extreme working conditions and indicates how to modulate the performance of an endoprotease-based reactor. A brief discussion on potential applications in protein mapping and tagging and in the quantitative analysis of protein bioavailability by means of a biosensorial strategy is also described.

Excessive meat tenderization in cured raw Parma ham has recently been correlated with abnormal levels of cathepsin B in freshly slaughtered thigh meat. We have developed a visual assay employing the substrate Z-Arg-Arg-NNapOMe for the quantitative detection of active cathepsin B levels in pork thigh muscle homogenates. The work was based on a kinetic characterization, in steady state condition, of pig muscle cathepsin B with several peptidyl chromophoric substrate analogs. This assay can easily and safely be performed by non-specialized personnel directly in the slaughterhouse or in the factory, for an early quality evaluation of thighs selected for Parma ham production. Our characterization has further indicated that the catalytic properties of porcine muscle cathepsin B and those of isoforms from other animal and plant species are practically identical. This is particularly evident in the commercially available bovine spleen isoform, which was employed as a model enzyme in most of the experiments.

Atrazine is one of the most widely employed herbicides. Due to its environmental persistence, it can be detected in ground and water thus becoming the subject of a serious concern because of its potential endocrine disrupting activity. In particular, several in vitro and in vivo studies point out adverse effects on reproduction. However, these data were mainly collected in the male, while studies on females are lacking. Present work was therefore set up on swine ovarian granulosa cells to investigate the effect of atrazine on steroidogenesis and proliferation. Moreover, since vessel growth is fundamental for reproductive function, we evaluated the herbicide's effect on two of the main angiogenesis signaling molecules, VEGF and NO. Our data show that atrazine markedly interferes with steroidogenesis while it does not modify cell proliferation; in addition, the herbicide has also been found to affect the production of the examined angiogenesis molecules. Collectively, these results indicate for the first time a potential negative effect of atrazine on ovarian functions in the swine species.

Background Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated. Results Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to neo gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck. Conclusion This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.

The X-ray structure of bovine Odorant Binding Protein (bOBP) revealed its association as a domain swapped dimer. bOBP, devoid of any cysteines, contrasts with other mammalian OBPs, which are monomeric and possess at least one disulfide bridge. We have produced a mutant of bOBP in which a glycine residue was inserted after position 121. This mutation yielded a monomeric bOBP-121Gly+ in which domain swapping has been reverted. Here, we have subsequently introduced two mutations, Trp64Cys and His155Cys, in view to stabilize the putative monomer with a disulfide bridge. We have determined the crystal structure of this triple mutant at 1.65 A resolution. The mutant protein is monomeric, stabilized by a disulfide bridge between Trp64Cys and His155Cys, with a backbone superimposable to that of native bOBP, with the exception of the hinge and of the 10 residues at the C-terminus. bOBP triple mutant binds 1-amino-anthracene, 1-octen-3-ol (bOBP co-purified ligand) and other ligands with microM Kd values comparable to those of the swapped dimer.

The detection of hazard exposure is a current priority, including the detection of traces of explosive molecules in different environments like luggage storage rooms and public places, and is becoming a major requirement for homeland security. In the present study we carried out a preliminary investigation on the binding capacities of four forms of the lipocalin odorant-binding protein (OBP) for the detection of explosive components such as diphenylamine, dimethyl-phthalate, resorcinol and dinitrotoluene. The experimental results, showing that OBP binds these compounds with affinity constants ranging between 80 nM and 10.6 mM, indicate that this protein can be used as a probe for the realization of a biosensor to sense explosive compounds.

Odorant binding proteins (OBP) are soluble lipocalins produced in large amounts in the nasal mucosa of several mammalian species. Although OBPs can bind a large variety of odorous compounds, direct and exclusive involvement of these proteins in olfactory perception has not been clearly demonstrated. This study investigated the binding properties and chemical resistance of OBP to the chemically reactive lipid peroxidation end-product 4-hydroxy-2-nonenal (HNE), in an attempt to establish a functional relationship between this protein and the molecular mechanisms combating free radical cellular damage. Experiments were carried out on recombinant porcine and bovine OBPs and results showed that both forms were able to bind HNE with affinities comparable with those of typical OBP ligands (K(d) = 4.9 and 9.0 microm for porcine and bovine OBP, respectively). Furthermore, OBP functionality, as determined by measuring the binding of the fluorescent ligand 1-aminoanthracene, was partially lost only when incubating HNE levels and exposure time to HNE exceeded physiological values in nasal mucosa. Finally, preliminary experiments in a simplified model resembling nasal epithelium showed that extracellular OBP can preserve the viability of an epithelial cell line derived from bovine turbinates exposed to toxic amounts of the aldehyde. These results suggest that OBP, which is expressed at millimolar levels, might reduce HNE toxicity by removing from the nasal mucus a significant fraction of the aldehyde that is produced as a consequence of direct exposure to the oxygen present in inhaled air.

Bovine odorant-binding protein (bOBP) is a dimeric lipocalin present in large amounts in the respiratory and olfactory nasal mucosa. The structure of bOBP refined at 2.0-A resolution revealed an elongated volume of electron density inside each buried cavity, indicating the presence of one (or several) naturally occurring copurified ligand(s) (Tegoni et al. (1996) Nat. Struct. Biol. 3, 863-867; Bianchet et al. (1996) Nat. Struct. Biol. 3, 934-939). In the present work, by combining mass spectrometry, x-ray crystallography (1.8-A resolution), and fluorescence, it has been unambiguously established that natural bOBP contains the racemic form of 1-octen-3-ol. This volatile substance is a typical component of bovine breath and in general of odorous body emanations of humans and animals. The compound 1-octen-3-ol is also an extremely potent olfactory attractant for many insect species, including some parasite vectors like Anopheles (Plasmodium) or Glossina (Trypanosoma). For the first time, a function can be assigned to an OBP, with a possible role of bOBP in the ecological relationships between bovine and insect species.

Different types of cAMP dependent regulatory subunits have been characterized in the mammalian brain: RI alpha and beta, RII alpha and beta. The subcellular distribution of RI subunits has been examined in the rat brain. Partial amino acid sequencing of tryptic fragments from the Triton insoluble pellet of the rat brain shows that cAMP dependent regulatory subunits type RI alpha are found in this fraction. Immunohistochemistry shows that Triton-insoluble RI subunits are concentrated to form clusters and this distribution is distinct from RII subunits. Immunohistochemistry and fluorescent cAMP labeling show that the clusters bind fluorescent cAMP analogues. These results suggest that the high local concentration of RI subunits can modulate cAMP distribution among different cellular compartments.

An easy and rapid detection of hazardous compounds is crucial for making on-the-spot irreversible decisions at airport security gates, luggage storage rooms, and other crowded public places, such as stadia, concert halls, etc. In the present study we carried out a preliminary investigation into the possibility of utilizing as advanced nano-biosensors a mutant form of the bovine odorant-binding protein (bOBP) immobilized onto carbon nanotubes. In particular, after immobilization of the protein on the carbon nanotubes we developed a competitive resonance energy transfer (RET) assay between the protein tryptophan residues located at the positions 17 and 133 (W17 and W133) and the 1-amino-anthracene (AMA), a molecule that fits in the binding site of bOBP. The bOBP–AMA complex emitted light in the visible region upon excitation of the Trp donors. However, the addition of an odorant molecule to the bOBP–AMA complex displaced AMA from the binding site making the carbon nanotubes colorless. The results presented in this work are very promising for the realization of a color on/ color off b-OBP-based biosensor for the initial indication of hazardous compounds in the environment.

Overstrain tendonitis are common pathologies in the sport horses. Therapeutic approaches to tendon healing do not always result in a satisfactory anatomical and functional repair, and healed tendon is often characterized by functional impairment and high risk of reinjury. Recently, mesenchymal stem cells (MSCs) and platelet rich plasma (PRP) have been proposed as novel therapeutic treatments to improve the tendon repair process. MSCs are multipotent, easy to culture and being originated from adult donors do not pose ethical issues. To date, autologous MSCs have been investigated mainly in the treatment of large bone defects, cardiovascular diseases, osteogenesis imperfecta and orthopaedic injuries both in human and veterinary medicine. The clinical applications in which autologous MSCs can be used are limited because patient-specific tissue collection and cell expansion require time. For clinical applications in which MSCs should be used right away, it would be more practical to use cells collected from a donor, expanded in vitro and banked to be readily available when needed. However, there are concerns over the safety and the efficacy of allogeneic MSCs. The safety and efficacy of a therapy based on the use of allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) associated to platelet rich plasma (PRP) were evaluated in 19 horses affected by acute or subacute overstrain superficial digital flexor tendonitis (SDFT). The application of allogeneic ASCs neither raised clinical sign of acute or chronic adverse tissue reactions, nor the formation of abnormal tissue in the long-term. After a follow-up of 24 months, 89.5% horses returned to their previous level of competition, while the reinjury rate was 10.5%, comparable to those recently reported for SDFT treated with autologous bone marrow derived MSCs. This study suggests that the association between allogeneic ASCs and PRP can be considered a safe and effective strategy for the treatment of SDF tendonitis in the horse.