Phylogeny and evolution of infectious bursal disease virus circulating in Turkish broiler flocks.

The emergence of new infectious bursal disease virus (IBDV) variants can threaten poultry health and production all over the world causing significant economic losses. Therefore, this study was performed to determine IBDV molecular epidemilogy, VP2 gene variation, and corresponding pathological lesions in IBDV infected chickens in Turkey. For this, 1855 bursa of Fabricius samples were collected from 371 vaccinated broiler flocks. Atrophia and haemorrhages were seen in the bursa Fabricius of very virulent IBDV (vvIBDV) infected chickens. Partial VP2 gene was sequenced and phylogenetic, recombination, and evolutionary analyses were performed. 1548 (83.5%) out of 1855 of bursa of Fabricius samples were IBDV positive and 1525 of those could be sequenced. The recombination analysis did not detect occurrence of any recombination event among the Turkish strains. Among 1525 sequenced samples, 1380 of them were found to be classical strains. Among 1380 classical strains, 1317 were similar to IBDV 2512, 11 to Faragher 52/70, 40 to 228 E, and 12 to Lukert strain. Out of 1525 reverse transcriptase ploymerase chain reaction positive samples, 144 of them were found to be similar to vvIBDV-VP2 gene reported to GenBank previously. The phylogenetic tree performed on a broad sequence dataset demonstrated grouping of vvIBDV Turkish strains in three different clusters, including sequences collected also from Iraq and Kuwait (Cluster 1), Indian (Cluster 2), and a distinct Turkish-only cluster (Cluster 3). The evolutionary rate estimation on branches/clades including Turkish strain mirrored the expected one for RNA viruses and no significant differences were found among different considered branches. In conclusion, results of this study indicate that vvIBDV strains similar to those circulating in various countries in the Middle East are present and undergoing evolution in chickens from Turkish broiler flocks. This point needs to be taken into account in planning adequate control strategies.

Interactions of metronidazole with other medicines: a brief review.

Metronidazole, a medicine discovered in the late 1950 s is mainly an antibiotic active against anaerobes and protozoa. There are numerous side effects of metronidazole. Some interactions with other medicines enhance its activity, and some cause the decrease in activity and plasma concentration. A short review of its side effects, mechanisms of action and interactions with other medicines is provided. As resistance towards metronidazole occurred, these mechanisms are described as well. The data presented in the review were selected on the basis of relevancy, citation and age of the used references.

IL-17 and glutamate excitotoxicity in the pathogenesis of multiple sclerosis.

Immunoinflammatory-mediated demyelination, the main pathological feature of multiple sclerosis (MS), is regularly accompanied by neurodegenerative processes, mostly in the form of axonal degeneration, which could be initiated by glutamate excitotoxicity. In the current study, the relationship between Th17-mediated inflammatory and excitotoxic events was investigated during an active phase of MS. Cerebrospinal fluid (CSF) of patients with MS and control subjects was collected, and IL-17A and glutamate levels were determined. IL-17A level was significantly higher in patients with MS; whereas no statistically significant changes in glutamate concentrations were found. There was a direct correlation between IL-17A and glutamate levels; IL-17A levels were also associated with the neutrophil expansion in CSF and blood-brain barrier disruption. However, IL-17A level and the number of neutrophils tended to fall with disease duration. The results suggest that Th17 cells might enhance and use glutamate excitotoxicity as an effector mechanism in the MS pathogenesis. Furthermore, Th17 immune response, as well as neutrophils, could be more important for MS onset rather than further disease development and progression, what could explain why some MS clinical trials, targeting Th17 cells in the later stage of the disease, failed to provide any clinical benefit.

Response of monocyte-derived dendritic cells to rapidly solidified nickel-titanium ribbons with shape memory properties.

Ni-Ti Shape Memory Alloys (SMAs) have attracted considerable attention as biomaterials for medical devices. However, the biocompatibility of Ni-Ti SMAs is often unsatisfactory due to their poor surface structure. Here we prepared Rapidly Solidified (RS) Ni-Ti SMA ribbons by melt-spinning and their surface was characterised by Auger-electron spectroscopy, X-ray photoelectron spectrometry and scanning electron microscopy. The biocompatibility of the produced ribbons and their immunomodulatory properties were studied on human monocyte-derived dendritic cells (MoDCs). We showed that melt-spinning of Ni-Ti SMAs can form a thin homogenous oxide layer, which improves their corrosion resistance and subsequent toxicity to MoDCs. Ni-Ti RS ribbons stimulated the maturation of MoDCs, as detected by changes in the cells' morphology and increased expression of HLA-DR, CD86, CD40 and CD83 molecules. However, Ni-Ti RS ribbons enhanced the tolerogenic properties of immature MoDCs, which produced higher levels of IL-10 and IL-27, driving the differentiation of IL-10- and TGF-ß-producing CD4+T cells. On the other hand, in the presence of lipopolysaccharide, an important pro-inflammatory biomolecule, Ni-Ti RS ribbons enhanced the allostimulatory and Th1 polarising capacity of MoDCs, whereas the production of Th2 and Th17 cytokines was down-regulated. In conclusion, Ni-Ti RS ribbons possess substantial immunomodulatory properties on MoDCs. These findings might be clinically relevant, because implanted Ni-Ti SMA devices can induce both desired and adverse effects on the immune system, depending on the microenvironmental stimuli.

Mechanical properties and tissue reinforcement of polypropylene grafts used for pelvic floor repair--an experimental study.

PURPOSE: Although grafts can be made from the same materials, the final weave of the product may play a role in the ultimate outcomes or complications. The aim of this experimental study is to evaluate and compare the mechanical properties of the meshes and their influence on biological characteristics as well. METHODS: Full-thickness abdominal wall defect with respect to the peritoneum was primary repaired in 144 male Wister rats using the overlay technique. Monofilament, multifilament and coated polypropylene grafts were used for the repairs. Graft shrinkage, thickness and tensile strength of the explants were analysed after 3 and 6 weeks. RESULTS: Monofilament grafts showed the highest shrinkage level of 11.97-18% during the investigated period. The thickness of monofilament explants increased gradually, with a maximum of 25.35% after 6 weeks. Multifilament and collagen explants did swell more rapidly in the third week, decreasing thereafter to 19.63-23.04% above the pre-implantation values after 6 weeks. The tensile strength showed a gradual increase over the course of the experiment in all groups. CONCLUSION: After graft stabilisation, all samples had similar strength, regardless of the significant differences in the pre-implantation values of graft strength.

Inter-observer agreement of standard joint counts in early rheumatoid arthritis: a comparison with grey scale ultrasonography--a preliminary study.

OBJECTIVES: The aims of the present study were to assess the inter-observer agreement of standard joint count and to compare clinical examination with grey scale ultrasonography (US) findings in patients with early rheumatoid arthritis (RA). METHODS: The study was conducted on 44 RA patients with a disease duration of <2 yrs. Clinical evaluation was performed independently by two rheumatologists for detection of tenderness in 44 joints and swelling in 42 joints. All patients underwent US assessment by a rheumatologist experienced in this method and blinded to the clinical findings. Joint inflammation was detected by US when synovial fluid and/or synovial hypertrophy was identified using OMERACT preliminary definitions. The inter-observer reliability was calculated by overall agreement (percentage of observed exact agreement) and kappa (kappa)-statistics. The reliability of US was calculated in 12 RA patients. RESULTS: There was fair to moderate inter-observer agreement on individual joint counts for either tenderness or joint swelling apart from the glenohumeral joint. US detected a higher number of inflamed joints than did clinical examination. The mean (+/-S.D.) US joint count for joint inflammation was 19.1 (+/-4.1), while the mean (+/-S.D.) number of swollen joints was 12.6 (+/-3.6), with a significant difference of P = 0.01. CONCLUSIONS: Our results provide evidence in favour of the hypothesis that clinical examination is far from optimal for assessing joint inflammation in patients with early RA. Furthermore, this study suggests that US can considerably improve the detection of signs of joint inflammation both in terms of sensitivity and reliability.

Immunological studies in Balkan Endemic Nephropathy.

AIM: To re-examine serum complement and immunoglobulin levels in patients with Balkan Endemic Nephropathy (BEN) in the early stage of the disease; to study autoantibodies (anti-thyroid, anti-smooth muscle, anti-mitochondrial, anti-parietal, anti-nuclear, and anti-DNA) in these patients; and, finally, to re-asses the immunohistology in kidney biopsies from BEN patients. A review of the immunological studies in BEN will be presented. METHODS: Forty-five BEN patients in the early stage of disease, from the South Morava Region, were included in this study. Fifty-five apparently healthy controls, matched for age and sex, from the neighbouring control villages were selected. Serum complement and immunoglobulin levels were determined by the Immunochemistry system of Beckman. Auto-antibodies were detected in the sera by the indirect fluorescence method or by radio-immunoassay (anti-single stranded DNA). Immunofluorescent microscopy was used for kidney samples treated with FITC conjugated antisera for IgG, IgM, IgA, C3, C1q, C4 and fibrin/fibrinogen. RESULTS: Serum C3 was decreased in the sera of patients with BEN compared to healthy controls (p < 0.001), serum IgM was also decreased (p < 0.05). Anti-thyroid antibodies were detected in 5/45 BEN patients, anti-parietal in 7/45 patients and ANA in 2 BEN patients. No anti-smooth muscle, anti-mitochondrial, or anti-DNA antibodies were detected in any BEN patient. Immunofluorescent studies of 10 kidney biopsy specimens showed rare, unspecific, focal, mesangial deposits of C3 and IgM in some glomeruli, and rare deposits, mostly of C3, in tubuli and extraglomerular vessels. CONCLUSION: Humoral immune mechanisms do not appear to play a pathogenetic role in BEN. A few studies on cell-mediated immunity in BEN were performed, and further studies are needed on patients in the early stage of the disease.

Dietary curcumin does not protect kidney in glycerol-induced acute renal failure.

Generation of reactive oxygen species significantly contribute to the pathogenesis of renal injury induced by myoglobin release. The present study was performed to investigate the effects of dietary curcumin, a natural antioxidant isolated from plant Curcuma longa, in an experimental model of myoglobinuric acute renal failure. Rats received curcumin at an oral dose of 100mg/kg/day for 30 days. Renal injury was induced with injection of hypertonic glycerol (10 ml/kg 50% solution) in hind limb muscle with blood urea of 57.8+/-7.2 vs. 7.72+/-1.03 mmol/l and serum creatinine of 444.4+/-61.3 vs. 51.8+/-10.6 micromol/l, in glycerol-induced acute renal failure (ARF) vs. control rats, respectively. After 48 h rats were sacrificed and thiobarbituric acid reactive substance (TBARS), glutathione, carbonyl content and kidney cortex brush border peptidase activities were determined in serum, kidney and liver. Rats that received curcumin in addition to glycerol had significantly lower TBARS in serum but not in kidney and liver. Carbonyl content in kidney and liver was significantly elevated in curcumin and glycerol treated rats and improved in animals treated with curcumin and glycerol together. The activities of kidney cortex enzymes, aminopeptidase N, angiotensinase A and dipeptidyl peptidase IV, were reduced in glycerol as well as in curcumin treated rats. The results obtained in this study provided additional evidence that despite its limited antioxidant activity curcumin did not protect kidney in myoglobinuric model of ARF.

Repair of bone tissue affected by osteoporosis with hydroxyapatite-poly-L-lactide (HAp-PLLA) with and without blood plasma.

The aim of this study is to examine the reparatory ability of the synthetic biomaterial hydroxyapatite-poly-L-lactide (HAp-PLLA), the replacement of alveolar ridge, and rehabilitation of bone defects caused by osteoporosis, in an experimental group of animals. The experiments are performed on syngeneic Sprague Dawley rats. Osteoporosis is induced by glucocorticoids in rats during a 12-week period. After this, the experimental group of animals is divided into five subgroups. An artificial defect is made in the alveolar bone on the left side of the mandible. In one group of animals, the defect is left to heal by itself, while in other groups, pure HAp-PLLA or one mixed with plasma is implanted. The best results are achieved by the implantation of the HAp-PLLA composite biomaterial mixed with autologous plasma. Formation of a new mandibular bone is seen, growing intensely, leading to rapid osteogenesis.

Acidity constants of cefetamet, cefotaxime and ceftriaxone; the effect of the substituent at C3 position.

Ionization constants of three cephalosporin antibiotics, cefetamet (CEF), cefotaxime (CFX) and ceftriaxone (CFTR) are determined using pH-potentiometric titrations at I=0.1 M (NaCl) and t=25 degrees C. Cefetamet and cefotaxime have three ionization groups: carboxylic, amide and aminothiazole. Besides those three, ceftriaxone possesses an hydroxytriazinone group as new and additional ionization center. In acid medium two overlapping acid-base processes are occurring with acidity constants being: pK1 2.93 (COOH) and pK2 3.07 (aminothiazole) for cefetamet, and pK1 2.21 (COOH) and pK2 3.15 (aminothiazole) for cefotaxime. In the case of ceftriaxone the situation is even more complicated, three overlapping processes coexist with pK1 2.37 (COOH), pK2 3.03 (aminothiazole) and pK3 4.21 (hydroxytriazinone). Protolysis of amide group is happening in the alkaline medium as completely separated process from those in acid medium. The acidity constants which correspond to amide group are pK3 10.65 (CEF), pK3 10.87 (CFX) and pK4 10.74 (CFTR). The influence of the C3 substituent on the dissociation process of the neighboring ionization group, particularly carboxylic group, was considered. The differences in acidity of CEF, CFX and CFTR (pK1: 2.93, 2.21 and 2.37, respectively) are likely to be caused by the stereoelectronic properties of substituents in the beta-position to the carboxylic group due to the combined inductive, hyperconjugative and resonance effects.

Biological evaluation of hydroxyapatite/poly-L-lactide (HAp/PLLA) composite biomaterials with poly-L-lactide of different molecular weights intraperitoneally implanted into mice.

Histopathologic analysis of the tissue with HAp/PLLA implants was made and the leukocyte formula and chemiluminescence response of peritoneal phagocytes 2, 7 and 12 weeks after intraperitoneal implantation studied. Implants were made of HAp/PLLA biocomposites with PLLA molecular weights of 50000 (HAp/PLLA(50)) and 430000 g/mol (HAp/PLLA(430)) and of crushed devitalized femur bone of a young Wistar rat. Leukocyte formula and chemiluminescence of peritoneal phagocytes showed no systemic inflammatory response. The studied implants caused locally weak inflammatory reaction. The resorption of implants ranges in intensity (polymer resorption, i.e. disappearance rate), from the highest with the bone implants, low with HAp/PLLA(50), to the lowest with the HAp/PLLA(430) implants. Good resorption of the biocomposites and its mutual ingrowth with connective tissue prove their good biocompatibility.

Genetic analysis of Newcastle disease virus strains isolated in Bosnia-Herzegovina, Croatia, Slovenia and Yugoslavia, reveals the presence of only a single genotype, V, between 1979 and 2002.

Newcastle disease (ND) epizootics in some European countries after the World War II were caused by ND virus (NDV) of multiple genotypes (IV-VIIa) occurring sequentially and/or simultaneously. This study was carried out to characterise the genetic composition of NDV strains during the outbreaks in the territory of the former Yugoslavia in order to enhance our understanding of the relationships of past epizootics in Europe. Sixty-eight NDV strains isolated between 1979 and 2002 were analysed by restriction enzyme digestion and partial sequencing of the fusion protein gene. All isolates were placed in genotype V, an exotic type, that was introduced to western Europe in 1970. Residue substitution analysis has allowed the recognition of four genetic variants, Vb1-Vb4, and the tracing of their movements. Vb1, a dominant variant in Bulgaria from the late 1970s, was also wide spread in the former Yugoslavia throughout the period under investigation. Vb2, a variant occurring in the neighbouring countries in the early 1970s could be the founder of the epidemic in Yugoslavia and it was present up to the late 1980s. Variants Vb3 and Vb4 could be found only after 1987. In conclusion, the ND outbreaks in Yugoslavia were part of the epizootic wave due to genotype V viruses that started in western Europe in 1970 and became endemic in the region. Inter-country transmission occurred for all variants, and Vb3 and Vb4 might have evolved during the endemic period.

Nephroprotective effects of pentoxifylline in experimental myoglobinuric acute renal failure.

The nephroprotective effects of pentoxifylline, a methylxantine, were studied in glycerol-induced acute renal failure. Glycerol treated rats exhibited collecting duct and medullary ascending limb dilation and casts, with focal tubular damage, confined mainly to the superficial cortex. In the interstitium focal mononuclear infiltration was observed. In some glomeruli there was swelling of mesangial spaces and mesangial cells. Pentoxifylline injected to glycerol pretreated rats exerted a protective effect. Only few groups of proximal tubules in the subcapsulary region of renal cortex showed necrosis and tubulorhexis. There were not leukocyte infiltrations or vascular congestion. Morphometric analysis showed increased surface area fraction of tubular lumen in rats treated with glycerol (p < 0.01) compared to those in controls. Intratubular cast formations in rats treated with glycerol alone were significantly higher than in rats given pentoxifylline in addition to glycerol. Kidney cortex ectopeptidases (APA, APN and DPP IV) were not significantly changed after glycerol administration. Serum creatinine and blood urea were markedly increased in glycerol treated rats, however, pentoxifylline reduced significantly their levels. This study in glycerol-induced acute renal failure showed a marked renal morphologic and functional protection by pentoxifylline.

[Use of fibrin glue in the prevention of leakage in pancreatico-jejunal anastomoses]. / Primena fibrinskog lepka u prevenciji popustanja pankreasno-jejunalne anastomoze.

The aim of this study was to test the protective effects of fibrin sealing on the pancreatico-jejunostomy (PJA), the high-risk anastomosis following pancreas head resection. Experimental study was performed on the mongrel dogs, divided in two groups (20 animals each): Experimental group-with end to end "dunking" PJA, protected by temporary occlusion of the pancreatic duct with fibrin sealant/Tissucol/Immuno Ag/, while control group was without any protective procedure. The animals were followed 5 months in order to study: protective effects of such procedure on the PJA quantified with the percent of anastomotic leakage, effects of the exocrine secretion and effects the endocrine function Results: PJA leakage occurred in 13.33% in control group. No leakage was registered in experimental group. Biochemical, histological and electron microscopic study showed slight transitory elevation of amylase levels. Fibrin glue plug was dissolved and pancreatic juice output was reestablished 12th days postoperatively. Long term follow-up showed no damages of the endocrine and exocrine pancreas. Pancreatic duct occlusion with fibrin glue appeared to be an useful method in the prevention of pancreatico-jejunostomy leakage, without negative effects on the exocrine and endocrine pancreas.

Immune status assessment by abundance of IFN-alpha and IFN-gamma mRNA in chicken blood.

Avian diseases, including such viral infection as infectious bursal disease, infectious anemia, and Marek's disease, often cause immunosuppression, leading to more severe infection, problems with secondary infection, and inadequate responses to vaccination. Immunosuppression thus causes serious economic losses in commercial poultry production. To date, methods for assessing immune status have been too slow to be of practical help. Reasoning that immunosuppression should be reflected by reduced production of interferons (IFN) in response to a viral antigen, we have developed competitive nucleic acid hybridization microtiter plate assays for chicken IFN-alpha (ChIFN-alpha) and ChIFN-gamma mRNA. To evaluate the assay, chickens were challenged with inactivated Newcastle disease virus (iNDV). Whole blood samples were collected at various times subsequently and preserved with a cationic detergent. Later, total RNA was extracted, and mRNA for both ChIFN-alpha and ChIFN-gamma was measured. Both rose from undetectable levels to reach a peak by 4 h, remained high for about 3 days, and fell to undetectable levels by day 5. Results were similar in chickens aged between 1 and 28 days. In later experiments, blood was collected 4 h after viral challenge. When chickens were immunosuppressed by administering 4-5 mg cyclophosphamide (CY) daily for 3 days and challenged with iNDV, they transcribed less ChIFN-alpha and ChIFN-gamma mRNA, and their antibody response was impaired. Our results suggest that suspected immunosuppression in a commercial flock could be assessed within 2-3 days by challenging birds with iNDV and measuring the amounts of ChIFN-alpha and ChIFN-gamma mRNA in blood obtained 2-4 h later.

A study of HAp/PLLA composite as a substitute for bone powder, using FT-IR spectroscopy.

Chemically synthesized hydroxyapatite/poly-L-lactide (HAp/PLLA) composite biomaterial was studied in vivo. The biocomposite was implanted into Balb/c Singen mice and after 1 and 3 weeks removed from their organisms and analyzed by the FT-IR spectroscopy. After 1 week of testing in vivo the implanted sample gave a spectrum in which absorption bands arising from newly formed functional groups of amine and peptide can be seen. After 3 weeks, a spectrum with pronounced absorption bands at 3420 and 1650cm(-1) assigned to newly generated collagen, a component of the extracellular connective-tissue matrix, was registered. Also, decrease of the intensity absorption band at 1760cm(-1) originating from the C=O group of PLLA indicates bioresorption of the PLLA used. Analysis of the microstructure of the sample surface by scanning electron microscopy before and after implantation revealed bioresorption of the PLLA polymer phase and generation of collagen fibers at the sites of implanted bioresorptive PLLA. A mixture of autologous bone powder and HAp/PLLA biocomposite was also examined. After implantation, the same final products as in the case of HAp/PLLA composite biomaterial used alone were found.

Does captopril change oxidative stress in puromycin aminonucleoside nephropathy?

Puromycin aminonucleoside (PAN) nephropathy in rats has been induced by the intraperitoneal injections of PAN. One group of animals which received PAN has been treated simultaneously with captopril (angiotensine converting enzyme-ACE-inhibitor) with the aim to test whether continuing treatment with captopril along with PAN injections would be able to modulate the toxic effects of PAN. The third group of rats was given only captopril. Morphological changes in the kidney were evaluated by scanning electron microscopy that showed the loss of podocyte foot processes in the kidney of PAN treated animals but also in the kidney of captopril treated ones as well as in the animals treated with both drugs simultaneously. Reduced glutathione content, catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), xanthine oxidase activities as well as lipid peroxides were investigated in rat blood and kidney. Captopril given alone produced a significant decrease of plasma lipid peroxides, but it did not show any significant effect on investigated antioxidative factor levels neither in blood nor in the kidney. PAN given alone produced a significant depletion of plasma lipid peroxides, kidney catalase and erythrocyte GSH-Px activity as well as a significant increase of plasma catalase and erythrocyte SOD activity. Treatment of animals with both drugs simultaneously resulted in a significant increase of erythrocyte SOD activity and a significant decrease of plasma lipid peroxides, erythrocyte GSH-Px and kidney SOD activities. Kidney xanthine oxidase activity showed a significant increase in both PAN and PAN plus captopril treated animals in comparison with the values of captopril treated rats. These data suggest that PAN changes the antioxidative factor pattern in rat blood and kidney. Contrary to our expectations that captopril may protect the toxic effects of PAN it only to a certain extent modifies these effects showing protective effect only on tissue catalase activity.

Pulmonary blast injury increases nitric oxide production, disturbs arginine metabolism, and alters the plasma free amino acid pool in rabbits during the early posttraumatic period.

Plasma nitrate + nitrite (nitrates), as final NO products, and free amino acid pool (FAAP) characteristics, as indicators of protein/amino acid metabolism, were analyzed in the early (30 min) period following blast injury. The experiments were performed on 27 rabbits subjected to pulmonary blast injury (experimental group) or not exposed to overpressure (controls). We report that pulmonary blast injury (PBI) induces prompt NO overproduction within a very early period. Increased arginine utilization via NO synthase, presumably associated with its cleavage by arginase, leads to the depletion of the arginine level in arterial plasma 30 min following PBI. Impaired balance between arginine utilization and release/resynthesis from endogenous sources causes disturbed nutritional status and urea cycle activity. Early identification and appropriate management of the changes in amino acid metabolism should be included in the evaluation of patients with blast injury. Furthermore, the results suggest that depleted arterial levels of arginine and NO overproduction may be helpful in diagnosis and prognosis of blast injury.