The Role of Antimicrobial Neutrophil Extract in Modification of the Inflammatory Response During Osteochondral Autograft and Allograft Transplantation in Rabbits.

Osteochondral autograft or allograft transplantation is one of the surgical options for the management of large cartilage defects; however, the mechanisms of cartilage healing after this procedure, especially the immunological mechanisms, are not fully understood. The present study examined whether a grafting procedure changed the in-vitro responses of neutrophils and monocyte-derived macrophages (MDMs). Additionally, antimicrobial neutrophil extract (ANE) was assessed for its ability to modulate excessive cellular responses during and after implantation. The neutrophil secretory response was tested by measuring enzyme release and free radical generation, while the MDM response was evaluated by assessing morphological and functional changes of the cells after polarization. Osteochondral implantation evoked a transient secretory response by circulating neutrophils, but MDMs were not activated postoperatively. ANE from rabbit blood may be considered as a modulator of the inflammatory response because of its influence on neutrophils and MDMs. Inhibition of the neutrophil secretory response prevents complications that may arise following excessive activity of these cells. Stimulation of MDMs with ANE induces formation of a partial anti-inflammatory phenotype with enhanced regenerative properties.

Extrathoracic negative pressure ameliorates lung injury during mechanical ventilation in experimental pigs.

Mechanical ventilation (MV) is a supportive and life-saving therapy, however, it can cause ventilator-induced lung injury as a common complication. Thus, recruitment manoeuvres (RM) are applied to open the collapsed alveoli to ensure sufficient alveolar surface area for gas exchange. In the light of the fact that positive pressure ventilation is currently the standard treat- ment for improving pulmonary function, extrathoracic negative pressure is considered as an alter- native form of respiratory support. The aim of this study was to estimate the proinflammatory and oxidative response during MV and lung injury as well as the response after RM. All studied parameters were assessed at the following time points: T1-spontaneous breathing, T2- MV, T3- lung injury, T4 -RM. During MV (T2) elastase, MPO, ALP release, nitrite and superoxide generation significantly increased, whereas in later measurements a decrease in these values was noted. The MDA plasma concentration significantly (p⟨0.05) increased at T2, reaching a level of 13.30±0.87 nmol/ml; at other time points the values obtained were similar to the baseline value of 9.94±0.94 nmol/ml, whereas a gradual decrease in SOD activity at time T2-T4 points in comparison with the baseline value was found. During the study both neutrophil activity and oxi- dative stress indicate exacerbated response after MV and lung injury by bronchoalveolar lavage; however, extrathoracic negative pressure system as the MR ameliorates damaging changes which could further lead to serious lung injury.

Transport induced inflammatory responses in horses.

Deleterious response to road transport is an important problem in equine practice. It determines different physiological, immunological and metabolic changes which lead to increased susceptibility to several disorders such as pneumonia, diarrhea, colics, laminitis, injuries and rhabdomyolisis. The aim of our study was to look for possible relationships between transportation of female young and older horses over a long and short distance and an inflammatory state reflected by an increase of acute phase protein concentration, oxidative stress and muscle injury. The study was conducted on 24 cold-blooded female horses divided into four groups. Six fillies aged 6-18 months and six mares aged 10-12 years were transported over the distance of about 550 km, six fillies aged 6-18 months and six mares aged 10-12 years were transported over the distance of about 50 km. Plasma and serum were obtained from blood samples taken before transportation (T0), immediately after transportation (T1) and at an abattoir during slaughter (T2). In these samples fibrinogen, MDA, AST and CK were assessed. Fibrinogen increased in all studied groups especially in fillies after long distance transportation, where it reached 205±7.07 mg/dl before transportation, 625±35.35 mg/dl after transportation, and 790±14.14 mg/dl during slaughter. MDA concentrations rose after transportation and reached the maximal level during slaughter. CK activity was more elevated after short transportation in younger horses, whereas initial activity of AST was higher in older horses. We estimated that intensified responses from acute phase, oxidative stress and muscle injury parameters indicated an inflammatory state.

Proliferative and oxidative response of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats exposed to ketogenic diet.

Ketogenic diet (KD) is considered in the context of its anti-epileptic effects, but its influence on liver dysfunction has not been elucidated yet. The study was aimed to investigate the activity of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats fed with KD, in respect of NO and superoxide generation by these cells as well as their proliferative activity in vitro. We also sought to characterize the plasma FFA profiles in control and ketogenic rats. Hep and HSC were isolated by the collagenase perfusion method and separated by the Percoll gradient centrifugation. After the 4th, 8th and 12th day of incubation, the media were collected for further analysis. NO generation increased within the time of incubation both in Hep and HSC isolated from KD-rats. In HSC group NO production raised significantly from 2.65 ± 0.07 µM/10(6) cells on 4th day of incubation to 5.49 ± 1.2 µM/10(6) cells on 12th day of incubation. In respect to O2⁻· generation experimental Hep and HSC provide considerably higher quantities of this free radical until 12th day of incubation (2.5 ± 0.07 and 3.2 ± 0.3 nM/10(6) cells, respectively). Although KD exerts anti-proliferative effect on hepatocytes, in respect to HSC it intensifies their proliferative activity. Furthermore, as we estimated on the basis of NO and O2⁻. generation both Hep and HSC exposed to KD are the source of free radicals.

Effect of leukotoxin of Mannheimia haemolytica and LPS of E. coli on secretory response of bovine neutrophils in vitro.

To evaluate the role of leukotoxin (LKT) of Mannheimia haemolytica and lipopolysaccharide (LPS) of E. coli 055:B5 in pathogenesis of bovine respiratory disease (BRD) we investigated their in vitro effects on cultured bovine neutrophils. Functional parameters of neutrophils including degranulation, generation of superoxide, and nitric oxide were distorted in response to both toxins. The most essential reaction of neutrophils was found in respect to release of elastase after addition of LKT as well as LPS at concentration of 300 microg/ml. Moreover, we observed an increased release of myeloperoxidase (MPO) and alkaline phosphatase (ALK-P) from polymorphonuclear cells (PMN) after addition of LKT and LPS. We also found enhanced superoxide generation by bovine neutrophils after exposure to different concentrations of LKT and LPS. In cultures of PMN treated with LKT, concentration of nitrite increased with growing concentrations of LKT. Lower values of nitrite were obtained in cultures exposed to LPS. Partial lysis of PMN, determined by LDH (lactate dehydrogenase) leakage, started at concentration of 300 microg/ml for both toxins, meanwhile LKT concentration above 300 microg/ml was lethal. Our study has revealed that neutrophils in response to both toxins exaggerate release of analysed substances, which participate in worsening the course of the disease and play a role in lung injury during BRD. Toxins introduced to the cultural medium stimulate release of studied constituents from neutrophils by combined activation and lysis of neutrophils.

Assessment of neutrophil components as markers of lung injury in the course of bovine respiratory tract infections.

To define the role of activated neutrophils in lung injury during bovine respiratory tract infections (BRTI) their in vitro function was investigated. As a means to achieve this goal the comparison of secretory action between neutrophils from the BRTI group and control was made on the basis of elastase, myeloperoxidase (MPO), alkaline phosphatase (ALK-P) release, and nitric oxide production. We noted that there is an interdependence between secretory response of neutrophils and clinical severity of BRTI. The release of elastase was greater in the BRTI group than in the control group (49.17+/-4.41 versus 46.43+/-4.95% of the total content). Neutrophils from infected heifers exhibited a significantly (p<0.05) higher value of MPO release than from healthy heifers and reached 39.23+/-10.18 versus 25.54+/-8.41% of the total content. ALK-P containing granules released significantly (p<0.001) more enzyme in the group with BRTI than in the control group (22.42+/-6.27 versus 13.74+/-2.01% of the total enzyme content). The level of nitrite accumulation rose in the culture of cells isolated from heifers with BRTI from 4+/-0.53 microM after 0.5h to 6.9+/-0.52 microM after 72 h. Our data suggest that during BRTI the increase of neutrophil secretory action results in augmentation of enzyme release including elastase, MPO and ALK-P, and the nitrite production. During an excessive secretory response of neutrophils all these factors contribute to lung injury and worsen the course of a disease and might be recognised as markers of lung injury. Moreover, such a destructive action of neutrophils must be taken into account during the introduction of new methods of BRTI treatment.