Analysis of free cisplatin in microdialysates and plasma ultrafiltrate by liquid chromatography-tandem mass spectrometry.

Cisplatin is a potent cytotoxic agent used in the treatment of various malignancies and exerts its antitumor effect through malignant cell DNA damage and apoptosis induction. Evaluation of systemic delivery of cisplatin is important in optimization of cisplatin treatment. However, accurate quantification of systemic cisplatin is challenging due to its various forms in circulation. This study aimed to develop a sensitive (LOQ < 0.1 µg/mL) and precise Ultra Performance Liquid Chromatography (UPLC) - Tandem Mass Spectrometry (MS/MS) method for quantifying free cisplatin in microdialysates and plasma. Furthermore the aim was to compare free cisplatin concentrations measured in standard plasma samples with those obtained from intravenous microdialysis catheters in a porcine model. The method developed utilizes dichloro(ethylenediamine)platinum(II) as an internal standard that co-elutes with cisplatin, ensuring precise correction for ion suppression/enhancement effects. The method was validated, demonstrating linearity up to 100 µg/mL and good intermediate precision (CV% < 6 %) in the range of 1.0-100 µg/mL, with an LOQ of 0.03 µg/mL. The pharmacokinetic parameters (AUC0-last, Cmax, T1/2, and Tmax) showed no significant differences between the two sampling methods. This validated LC-MS/MS method provides a reliable tool for quantifying systemic free cisplatin concentrations, facilitating future systemic and local pharmacokinetic evaluations for optimization of cisplatin-based cancer treatments.

Allogeneic adipose tissue-derived mesenchymal stem cells in combination with platelet rich plasma are safe and effective in the therapy of superficial digital flexor tendonitis in the horse.

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.

Equine adipose-tissue derived mesenchymal stem cells and platelet concentrates: their association in vitro and in vivo.

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.

Incomplete humeral condylar fracture in two English Pointer dogs.

Incomplete humeral condylar fracture was diagnosed by means of radiology, CT, scintigraphy, arthroscopy and bone biopsy in two English Pointer dogs. In both cases an acute thoracic limb lameness, unrelated to a known episode of major trauma, was observed. Incomplete humeral condylar fracture, mainly described in the Spaniel breeds, has been recently diagnosed in Labrador retrievers, Rottweiler, German Wachtel and other breeds. The pathogenesis of the condition is still unknown, but incomplete ossification of the humeral condyle and mechanical stress, alone or associated, have to be considered. However, our clinical and histopathological data lead us to believe that in Pointers, high performance dogs, the mechanical stress can assume a critical ethiopathogenetic role.

Cyclin D1 expression in pre-cancerous and cancerous lesions of the canine mammary gland.

Overexpression of cyclin D1, the regulatory subunit of cyclin-dependent kinases (cdk4 and cdk6) involved in cell cycle control, has often been found in breast cancer and other types of human cancer. Increased expression, or stability, of cyclin D1 molecules may cause sufficient cdk4 activation to produce retinoblastoma protein phosphorylation independently of mitogenic signals; this results in commitment of cells to the G1 phase at mitosis. In the present study, cyclin D1 expression was investigated in pre-cancerous and cancerous lesions of the canine mammary gland by a complex experimental approach, which included Western blot and immunohistochemical analysis of cyclin D1 and the related molecular system. Furthermore, to define relationships between cell growth and expression of cyclin D1, proliferative activity was studied by the AgNOR technique. The study provided the following information. Cyclin D1 overexpression was largely independent of the type of proliferative anomaly. Indeed, cyclin D1 was expressed in 60% of the pre-cancerous lesions and in 44% of cancerous lesions. Mitotic activity and cyclin D1 expression were related: mammary lesions that expressed cyclin D1 showed a high proliferative ratio, the opposite being true of cyclin D1-negative cell populations. This study may contribute to the establishment of an animal model for anti-cancer research based on cyclin D1 suppression or cdk inactivation, or both.

A simplified technique for diagnostic and surgical arthroscopy of the shoulder joint in the dog.

A modified technique is presented for surgical and diagnostic arthroscopy of the shoulder joint in the dog. The technique involves access to the joint through two points only; one was created in place of the drainage needle-cannula, which was replaced with a portal, while the second was located more caudally compared with previous techniques. Using a changing guide rod system the two portals are completely interchangeable in order to perform easier arthroscopic surgery either in the cranial or caudal aspect of the joint. The presence of only one portal caudal to the lateral collateral ligament allows more freedom of movement and avoids interference between the arthroscope and the instruments. The modified procedure was performed on 33 joints affected by osteochondritis dissecans or tenoligament diseases and facilitated straightforward diagnostic examinations, and simple and rapid surgical procedures.

Extra-articular absorbable suture stabilization of coxofemoral luxation in dogs.

OBJECTIVE: To evaluate the effectiveness of an extra-articular surgical technique using absorbable suture material for the stabilization of traumatic coxofemoral luxation in dogs. STUDY DESIGN: Prospective, clinical study. ANIMALS: Fourteen client-owned dogs with recent and long-standing traumatic coxofemoral luxation (13 craniodorsal and 1 ventral). METHODS: Coxofemoral luxations were surgically reduced and maintained in place with an extra-articular iliofemoral multifilamentous absorbable suture (3 to 6 strands of 2 USP Polyglactin 910). No external support was employed, and all the dogs were encouraged to use the affected limb postoperatively. The average time of clinical and radiographic follow-up was 11.6 +/- 6.3 months (from 2 to 22 months). RESULTS: During the follow-up period, no reluxations occurred and no complications associated with the surgical technique were identified. The dogs started bearing weight from 1 to 10 days after the surgery (mean, 4.3 +/- 2.9 days) and the period of lameness ranged from 7 to 30 days (20 +/- 8.6 days). At the final clinical examination, the dogs did not demonstrate any lameness or pain during passive flexo-extension movements, and there was no significant limitation of the range of motion. CONCLUSION: Extra-articular stabilization with multifilamentous absorbable sutures is a simple, effective method of treatment for acute and chronic coxofemoral luxation. The absorbable material used is strong enough to maintain articular stability during the period of scar tissue formation even in large-breed dogs. CLINICAL RELEVANCE: Absorbable sutures avoid the possible complications related to the use of nonabsorbable material and seem to be sufficient to maintain articular stability during the capsular healing process.

The inhibitory effects of interleukin-6 on synaptic plasticity in the rat hippocampus are associated with an inhibition of mitogen-activated protein kinase ERK.

Several cytokines have short-term effects on synaptic transmission and plasticity that are thought to be mediated by the activation of intracellular protein kinases. We have studied the effects of interleukin-6 (IL-6) on the expression of paired pulse facilitation (PPF), posttetanic potentiation (PTP), and long-term potentiation (LTP) in the CA1 region of the hippocampus as well as on the activation of the signal transducer and activator of transcription-3 (STAT3), the mitogen-activated protein kinase ERK (MAPK/ERK), and the stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK). IL-6 induced a marked and dose-dependent decrease in the expression of PTP and LTP that could be counteracted by the simultaneous treatment with the tyrosine kinase inhibitor lavendustin A (LavA) but did not significantly affect PPF. The IL-6-induced inhibition of PTP and LTP was accompanied by a simulation of STAT3 tyrosine phosphorylation and an inhibition of MAPK/ERK dual phosphorylation, in the absence of changes in the state of activation of SAPK/JNK. Both effects of IL-6 on STAT3 and MAPK/ERK activation were effectively counteracted by LavA treatment. The results indicate the tyrosine kinases and MAPK/ERK are involved in hippocampal synaptic plasticity and may represent preferential intracellular targets for the actions of IL-6 in the adult nervous system.

Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection.

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

Glutathione inhibits HIV replication by acting at late stages of the virus life cycle.

We investigated the effect of glutathione on the replication of human immunodeficiency virus (HIV) in chronically infected macrophages, a known reservoir of the virus in the body. We found that exogenous GSH strongly suppresses the production of p24gag protein as well as the virus infectivity. This is related to a dramatic decrease in both budding and release of virus particles from chronically infected cells (either macrophages or lymphocytes), together with a selective decrease in the expression of gp120, the major envelope glycoprotein, rich in intrachain disulfide bonds and thus potentially sensitive to the effect of a reducing agent such as GSH. Overall data suggest that GSH can interfere with late stages of virus replication. This would be in agreement with data obtained in cells exposed to herpesvirus type 1 (a DNA virus) or to Sendai (an RNA virus), showing that the suppression of virus replication by GSH is related to the selective inhibition of envelope glycoproteins. These results suggest a potential role of GSH in combination with other antivirals in the treatment of virus-related diseases.

[Tension-free laparoscopic hernioplasty]. / Ernioplastica "tension free" per via laparoscopica.

Hernia recurrence after traditional "open" hernioplasty is an observed event. The tension undergone by anatomical structures of the area is believed to be responsible in large part for these failures. Thus, techniques involving "tension free" hernioplasty have been developed, some of which involve laparoscopic access. Here an experience is reported regarding laparoscopic hernioplasty carried out to repair groin hernias, first of all on an animal model (pigs: 7 cases) then in the clinical field. The technique chosen was the endo-peritoneal positioning of a PTFE prosthesis. The results revealed several advantages over the traditional methods, basically the possibility of reinforcing the entire inguinal floor at the same time as repairing the hernia, and the decrease in groin area discomfort and less time off from work for the patient.

[Experimental intestinal laparoscopic resection]. / Resezione intestinale laparoscopica sperimentale: note di tecnica.

At present, there is a debate in the literature regarding the possibility of extending the laparoscopic approach to intestinal resection. Some techniques have been developed, but certain imperfections and problems remain. This experimental contribution suggests answers to some technical problems arising in the course of a small-bowel laparoscopic resection on a pig.

nprR1 and nprR2 regulatory regions for neutral protease expression in Bacillus subtilis.

The gene coding for the Bacillus subtilis extracellular neutral protease was isolated from strain BGSC 1A341, an overproducer carrying the nprR2 region, and from strain 168, a normal producer with the nprR1 sequence. The sequence of about 600 nucleotides upstream from the start codon of the protease gene was determined for both strains. The two regions are highly homologous except for a stretch of 66 base pairs close to the promoter region, which is absent in the BGSC 1A341 gene. Northern blot analysis of the in vivo RNAs indicated that the different levels of enzyme secreted by the two strains were due to different amounts of transcripts that accumulated in the cells. Furthermore, at the end of exponential growth, the amount of transcript increased dramatically in the overproducer strain but remained approximately constant in the normal producer strain. The start point(s) for transcription, however, as determined by S1 nuclease mapping of the in vivo transcripts, appeared to be the same for both genes.