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.

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.

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.

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.

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.

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.

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.

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.

Adenosine stimulates 5'-nucleotidase activity in rat mesangial cells via A2 receptors.

Because A2 adenosine receptor activation stimulates adenylate cyclase and cyclic AMP induces 5'-nucleotidase expression in rat mesangial cells, we examined the effect of adenosine and its analogs on 5'-nucleotidase activity in these cells. A2 adenosine receptors were characterized using [3H]5'-N-ethylcarboxamidoadenosine (NECA) as a tracer. There was a single group of receptor sites with a KD value of 0.53 microM and a number of sites of 1,317 fmol/mg. [3H]NECA binding was inhibited preferentially by A2 adenosine analogs and antagonists. Similarly, the order of potency for cAMP stimulation was in favour of A2 adenosine analogs. Rat mesangial cells expressed surface 5'-nucleotidase activity. Exposure of cells for 48 h to adenosine analogs showed that at low concentrations A2 analogs stimulated 5'-nucleotidase activity. These results indicate that adenosine upregulates activity of 5'-nucleotidase, the enzyme responsible for its local formation, via A2 receptor stimulation and increase in cAMP production.

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.

Characterization of plasma magnesium concentration and oxidative stress following graded traumatic brain injury in humans.

Plasma magnesium, calcium, and oxidative status were investigated in 31 male casualties with traumatic brain injury (TBI) during a 7-day posttraumatic period. The study group consisted of eight patients with mild closed head injury (Glasgow Coma Scale score [GCS] of 13-15), 10 patients with extensive penetrating head injury (GCS 4-6), and 13 patients with blast injuries but without direct head trauma. The latter group was included since previous experimental and clinical data have confirmed the development of indirect brain trauma in patients with blast injuries. Patients with multiple injuries were not included. Significant declines in plasma divalent cations were found in GCS 4-6 patients immediately after TBI and persisting for the entire 7-day study period. Similar changes in magnesium, but not calcium, were present in the GCS 13-15 and the blast injury groups, but only up until day 3 after injury. Alterations in lipid peroxidation products and superoxide anions were also observed following TBI. Increased lipid peroxidation was noted in all three groups over the entire posttraumatic period while increases in superoxide anion generation occurred transiently immediately following TBI. Thereafter, in the GCS 13-15 and blast injury groups, superoxide anions subsequently normalized, whereas in extensive head injury (GCS 4-6), superoxide anion generation significantly declined. A negative correlation between magnesium balance and oxidative stress was observed in all patients immediately after injury persisting in GCS 4-6 patients to the end of the observation period. Our findings suggest an interrelationship between magnesium changes and blood oxidants/antioxidants after TBI, which could be of both diagnostic and prognostic value in patients with neurotrauma.

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.

Peripheral nerve injury and immune response in rat.

The effect of peripheral nerve axotomy on cellular and humoral immunity after bovine serum albumin (BSA) sensitization was examined. Arthus and delayed hypersensitivity skin reactions and anti-BSA antibody production in 7, 14 and 21 days after immunization were significantly increased after axotomy of n. saphenus, n. tibialis and n. fibularis. The results indicate that peripheral nerves are also involved in the regulation of immune homeostasis from the place of peripheral immunization to or out the central nervous system (CNS).

Partial lecithin:cholesterol acyltransferase (LCAT) deficiency in Balkan endemic nephropathy.

The role of lipid abnormalities has been also implicated in the progression of renal diseases. The search for lipid abnormalities in Balkan endemic nephropathy (BEN) has roused sporadic interest and has not been fully elucidated. This study was performed in 54 healthy subjects from the families affected with BEN (group A), 18 members from non-affected families living in the same location (group B), and 25 control subjects (group C). Lipid profiles and lecithin:cholesterol acyltransferase (LCAT) were determined in each subject. The most striking distinction between the groups was that of the LCAT activity, which was abnormally low in group A (39 +/- 2), significantly different (P less than 0.0001) from that of the other groups. Thirty individuals from group A were those accounting for the low LCAT activity (A1). This group had a significantly lower total cholesterol and free cholesterol than all of the other subjects. The entire group A subjects had a significantly lower percentage of free cholesterol than the other two groups. There was no significant difference in HDL cholesterol between any of the groups, but group A1 had significantly higher HDL than group C (P less than 0.04). What emerges from our study is that a certain proportion of subjects from BEN families have a peculiar form of lipid abnormalities associated with an abnormal LCAT activity. At present we have no explanation for these findings. We believe that these changes may have an important role in the pathogenesis of BEN.

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.

Effect of glomerular cells interaction with macrophages on surface 5'-nucleotidase activity.

Interaction of cultured rat mesangial cells with macrophages induces a marked increase in surface 5'-nucleotidase activity of these mesangial cells. A significant increase in surface 5'-nucleotidase was also observed upon interaction of rat fibroblast with macrophages, both in cocultures and after treatment of fibroblasts with macrophage conditioned medium. Interaction of glomerular epithelial cells in primary culture with macrophages had no effect on surface 5'-nucleotidase activity. No significant effect on enzyme activity was observed after interaction of mesangial-epithelial cells, mesangial cells-fibroblasts, and epithelial cells-fibroblasts.

Effects of growth factors on the enzymes of purine metabolism in culture of regenerating rat liver cells.

Hepatocytes are affected by many cytokines and growth factors during liver regeneration. In regenerating rat liver cells cultures, liver cell growth factor (LCGF), hepatic stimulator substance (HSS), interleukin-1 beta (IL-1 beta), as well as their combination, were tested for their ability to activate the enzymes involved in purine metabolism. The enzymes tested were 5' nucleotidase, AMP deaminase, adenosine deaminase and xanthine oxidase. The cytokines alone or in combination, activated 5' nucleotidase and adenosine deaminase. Activity of AMP deaminase was stimulated by IL-1 beta associated with LCGF, HSS and IL-1 beta. Xanthine oxidase was stimulated by IL-1 beta but not with HSS and LCGF. Associated with IL-1 beta these two substances decreased its activity. A novel approach to the understanding of the mechanisms involved in the regulation of purine metabolism during liver regeneration, is proposed.

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.

Alterations in magnesium and oxidative status during chronic emotional stress.

Magnesium and oxidative status were investigated in young volunteers exposed to chronic stress (political intolerance, awareness of potential military attacks, permanent stand-by duty and reduced holidays more than 10 years) or subchronic stress consisting of everyday mortal danger in military actions lasting more than 3 months. Significant decreases in plasma ionized Mg2+, total Mg and ionized Ca2+ concentrations were found in both groups. Similarly, both study groups exhibited oxidative stress as assessed by increased plasma superoxide anions and malondialdehyde and modified antioxidant defense. There were no significant differences between the two stress groups. A negative correlation between magnesium balance and oxidative stress was observed suggesting that the same etiological factor (chronic stress) initiate decreases in both free and total magnesium concentrations and simultaneously increase oxidative stress intensity. These findings support the need for magnesium supplementation with antioxidant vitamins for people living in conditions of chronic stress.

Quantitative analysis of the renal changes in Balkan endemic nephropathy.

The volume densities of the cortical interstitium (CI), glomeruli and proximal tubular epithelium have been investigated on a sample involving 18 patients suffering from Balkan endemic nephropathy (BEN), classified into three groups with regard to the clearance of 99mTc-DTPA. In comparison with the control group, the evolution of the disease is marked by a significant increase of the CI volume (p<0.001), as well as a significant reduction of glomerular (p<0.01) and tubular volume densities (p<0.001). The most intensive changes of CI and glomerular volumes are characteristic of the initial stage of the disease when glomerular filtration (GF) has shown no signs of deterioration yet. On the other hand, a significant reduction of the tubular epithelium volume density is characteristic of the advanced stages. The specified changes, particularly those taking place at the level of interstitium bear the key responsibility for the BEN progression. However, a number of links constituting the chain of BEN morphogenesis remained insufficiently clarified. This urges for a precise quantification of all histological changes taking place in different stages of the disease, starting from the earliest, in order to get a better insight into the order and dynamics of their occurrence.