The epizootiological occurrence of bluetongue in the central Balkans.

In the last five years, bluetongue has been diagnosed in the following areas of the Mediterranean Basin: Algeria, France, Greece, Italy, Spain, Tunisia and Turkey. In the Balkan Peninsula, the disease has been recorded in Bulgaria (since 1999), Macedonia, Serbia and Montenegro, Croatia, Bosnia and Herzegovina and Albania. Bluetongue arrived in the Balkans from Turkey in 1999. In Serbia and Montenegro, the disease was first diagnosed in July 2001 in the Zubin Potok region of Kosovo. In August, the neighbouring regions of Novi Pazar, Tutin, Rozaje and Leposavic were also affected. The disease was simultaneously observed in eastern Serbia in the regions bordering Bulgaria (city regions of Bosilegrad, Pirot and Knjazevac). The occurrence of the disease and clinical signs indicate that the disease was also present in western Serbia in 2001. During 2002, more extensive serological investigations of animals in some regions of Yugoslavia indicated that the disease had spread towards the north of the country. At the same time, serological evidence also revealed the presence of the disease in the east of Bosnia and Herzegovina. The epizootiological data show that the northernmost point of disease spread in Serbia was the River Sava.

The first bluetongue virus isolation in Yugoslavia.

Catarrhal fever in sheep or bluetongue (BT) has not been recorded in Yugoslavia until recently. During the first incidence of BT disease in Serbia and Montenegro in 2001, the authors conducted field studies on suspected cases of the disease and collected samples for laboratory diagnosis. BT virus (BTV) was isolated and identified as serotype 9 by the Institute for Animal Health in Pirbright, United Kingdom (the Office International des Epizooties BT reference laboratory).

TNF-alpha induces changes in LDH isotype profile following triggering of apoptosis in PBL of non-Hodgkin's lymphomas.

Based on the possibility of tumor necrosis factor (TNF)-alpha to perform multiple and opposite biologic effects, we simultaneously investigated in vitro its effects on intracellular lactate dehydrogenase (LDH)-H and LDH-M isoenzyme activity and morphological characteristics following induction of apoptosis in peripheral blood mononuclear cells (PBMC) of non-Hodgkin's lymphoma patients (NHL) prior to and after the end of applied chemotherapy. TNF-alpha showed a significant increase ( p<0.05) of LDH-H and LDH-M activity in sonified PBMC of healthy controls after 18 h cultures accompanied with an increase of apoptotic index (AI) from 2.3 to 16.2%. Contrary to this, in PBMC of NHL patients prior to therapy TNF-alpha induced a significant decrease ( p<0.05) of LDH-H isotype activity. In patients after administration of chemotherapy, TNF-alpha in a dose of 100 U/ml induced a significant increase ( p<0.05) of LDH-M isotype activity, but not of LDH-H. In the PBMC of NHL patients prior to chemotherapy, TNF-alpha in vitro induced an increase of AI from 2.8 up to 6.8%, while in PBMC of NHL patients after applied chemotherapy AI changed from 7.2 to 14.4%. However, there was no significant difference in the increase of apoptosis in PBMC of NHL patients with high-grade malignancy and high rate response among patients who received first-line therapy, high-dose therapy, or third-line therapy regimens after in vitro TNF-alpha treatment. These results indicated different susceptibilities of PBMC of NHL to TNF-alpha when effects were analyzed by determination of intracellular LDH isotype profile and induction of apoptosis prior to and after administration of therapy in comparison to effects on healthy controls PBMC.

[Glutamate in brain: transmitter and poison]. / Glutamat u mozgu: transmiter i otrov.

Glutamate is a key excitatory neurotransmitter in the central nervous system (CNS). In addition, glutamate has a key role during the brain development, e.g., in synaptogenesis and in adult CNS, glutamate has an important role in learning and memory processes. Glutamate achieves its role through a variety of receptors, ionotropic (NMDA and AMPA) and metabotropic. Large number of glutaminergic synapses combined with wide distribution through the brain makes CNS particularly vulnerable towards uncontrolled release of glutamate such as observed during ischemia and postischemia. It is glutamate--its excitotoxic action--that is often considered as a key player in postichemic brain damage. However, despite the significant role of the neurotransmitter in pathogenesis of ischemic brain damage (in particular in focal brain ischemia), a complexity of metabolic events occuring during and after ischemia must be taken into account, glutamatenot being the solely culprit for the nerovus tissue damage.

Quantitative measurement of cerebral protein synthesis in vivo: theory and methodological considerations.

The true rate of cerebral protein synthesis can be calculated from the ratio of labeled proteins to integrated arterial plasma amino acid specific activity (SA) only when the fraction of amino acid precursor pool dilution is known. In the following, current experimental designs on the measurement of cerebral protein synthesis are discussed and compared to our own approach in which the determination of regional precursor pool dilution by recycled unlabeled leucine is combined with the quantitation of regional cerebral protein synthesis rates. For this purpose, a constant arterial plasma leucine SA level is maintained for 45 min by programmed intravenous infusion which is sufficient for complete equilibrium between tissue leucine pool SAs and plasma free leucine SA. In addition to the regional assessment of the precursor dilution factor, protein radioactivity can be determined in the same tissue sample or in parallel brain sections of the same animal by quantitative autoradiography. It is then possible to calculate the actual rate of protein synthesis using the correct fraction of precursor pool dilution. This renders our approach particularly suitable for the quantitative measurement of regional CPS under pathological conditions.

Ontogenetic differences in energy metabolism and inhibition of protein synthesis in hippocampal slices during in vitro ischemia and 24 h of recovery.

The present study was designed to clarify whether ontogenetic differences in the vulnerability of the brain towards hypoxic-ischemic insults are only caused by the low cerebral energy demand of immature animals or whether there are additional mechanisms, such as protein synthesis (PSR), that may be involved in this phenomenon. We therefore measured tissue levels of adenylates and PSR in hippocampal slices from immature (E40) and mature (E60) guinea pigs fetuses and from adult guinea pigs during in vitro ischemia and 24 h of recovery using a recently modified method. Hippocampal slices were incubated in a temperature controlled flow-through chamber, gassed with 95% O2/5% CO2. In vitro ischemia was induced by transferring slices to a glucose-free artificial cerebrospinal fluid (aCSF) equilibrated with 95% N2/5% CO2. The duration of ischemia ranged from 10 to 40 min. Adenylates were measured by HPLC after extraction with perchloric acid. PSR was evaluated as the incorporation rate of [14C]leucine into proteins. Under control conditions, tissue levels in adenylates did not change, whereas PSR increased slightly in hippocampal slices from mature fetuses and adult animals during a 24-h control incubation period. In slices from immature fetuses ATP levels were only maintained for 2 h. During in vitro ischemia the decline in ATP, total adenylate pool, and adenylate energy charge was much slower in slices from immature fetuses than in slices from mature fetuses or adults. After in vitro ischemia, ATP and the total adenylate pool did not completely recover in mature fetuses and adults, whereas adenylate energy charge almost returned to control values independently of the developmental stage. Two hours after in vitro ischemia PSR was undisturbed in slices from immature fetuses, but severely inhibited in slices from mature fetuses and adults. With ongoing recovery, PSR in mature fetuses returned to control values, while in adults it was still inhibited even 24 h after in vitro ischemia. From these results we conclude that hippocampal slices prepared from mature guinea pig fetuses as well as from adult guinea pigs can be held metabolically stable during long-term incubation using a recently modified technique. However, in slices from immature fetuses a stable energy state could not be maintained for more than 2 h. We further conclude that postischemic disturbances in PSR closely reflect the ontogenetic changes in the vulnerability of the brain to ischemia and that low energy metabolism is certainly not the only cause of the increased vulnerability of the fetal brain to ischemia.

Comparison of in vitro ischemia-induced disturbances in energy metabolism and protein synthesis in the hippocampus of rats and gerbils.

To elucidate whether the high sensitivity of gerbil compared with rat hippocampus to metabolic stress results from tissue-specific or hemodynamic factors, ischemia-induced metabolic disturbances [energy metabolism and protein synthesis rate (PSR)] were studied using the in vitro model of the hippocampal slice preparation. At the end of in vitro ischemia, ATP content was measured in individual slices with HPLC. In other groups of slices, PSR was measured after 120 min of recovery after in vitro ischemia. ATP breakdown was almost identical in rat and gerbil slices at all temperatures (37 degrees C, 34 degrees C, or 31 degrees C) and periods of ischemia (5, 10, or 15 min) studied. In contrast to the identical rate of ATP depletion during ischemia, however, postischemic disturbances in PSR were significantly increased in gerbil slices compared with rat slices and this relationship was stable after different periods of ischemia and at different incubation temperatures. The results illustrate that the pattern of ischemia-induced disturbances observed in vivo can also be reproduced using the in vitro model of hippocampal slice preparation, as evidenced by the postischemic disturbance in PSR. It is concluded that comparison of the extent of metabolic disturbances in gerbil and rat hippocampal slices after transient in vitro ischemia may help to elucidate the mechanisms of ischemic cell damage.

Apoptosis induced by microtubule disrupting drugs in normal murine thymocytes in vitro.

Disruption of cytoplasmic and spindle microtubules by colchicine or nocodazole increases mitotic index, but it also enhances apoptosis in isolated mouse thymocytes; the apoptotic index exceeds 20% after 4 hours of incubation with either drug (5% in controls). Apoptosis was confirmed by DNA fragmentation, and was blocked by calcium chelators and inhibitors of protein synthesis. The apoptotic effect of microtubule disrupting drugs (MDD) was directed to interphase thymocytes and was independent on MDD action on mitotic cells. However, cell death of mitotically arrested cells showed ultrastructural changes similar in many aspects to apoptosis.

Temporal analysis of the upregulation of GluR5 mRNA editing with age: regional evaluation.

The extent of mRNA editing of the kainate receptor subunit GluR5 was evaluated in tissue samples taken from the cerebral cortex, hippocampus and cerebellum of rat brain and in cortical neurons held in tissue culture, by PCR amplification of GluR5 cDNA across the edited base and restriction analysis of the amplification product with Bbv 1. Samples were taken from embryonic brains of rats at day 21 of gestation and from brains 4 days, 25 days and 3 month after birth. Cortical neurons were isolated from the tissue at day 19 of gestation and kept for 2 or 8 days in culture. The extent of editing was sharply upregulated during development in all brain structures studied. In the cortex and hippocampus the extent of editing exhibited already the adult state 4 days after birth. In the cerebellum, in contrast, the extent of editing was still 42 +/- 11.4% 25 days after birth but 82 +/- 6.2% in the adult state. In neurons held in tissue culture for up to 8 days, upregulation of editing did not take place. It is concluded that GluR5 editing is differently regulated in different brain structures and that the developmental changes observed in vivo are blocked when cells are kept in vitro.

Calcium activity and post-ischemic suppression of protein synthesis.

Increase in intracellular calcium concentration is a prominent feature of ischemia and has been considered a major factor in the initiation of ischemic pathology, which involves inhibition of protein synthesis. A reduction of calcium ion activity during and immediately after in vitro ischemia did not prevent inhibition of protein synthesis in hippocampae slices. When slices were overloaded with calcium by NMDA receptor activation or by the calcium ionophore A23187, no significant inhibition of protein synthesis was observed. We conclude that calcium overload plays only a limited role in ischemic inhibition of protein synthesis.

Regional differences in the extent of RNA editing of the glutamate receptor subunits GluR2 and GluR6 in rat brain.

The extent of RNA editing of the glutamate receptor subunits GluR2 and GluR6 was studied by using a newly developed method based on the restriction analysis of the subunit-specific polymerase chain reaction (PCR) product with the enzyme Bbv 1. Total RNA was isolated from following brain regions: cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata and white matter. RNA was transcribed into cDNA, which was used as template for PCR. PCR was run with GluR2- and GluR6-specific primers to amplify a product across the edited region. The PCR products were analysed with the restriction enzyme Bbv 1 and gel electrophoresis of the restriction digest. Bbv 1 recognizes the sequence GCAGC which is identical with the sequence of the PCR product originating from unedited GluR2 or GluR6 mRNA. Thus, this enzyme splits the non-edited PCR product into two fragments while leaving the edited PCR product intact. After electrophoresis of the restriction digest and photographing gels, optical density of bands was quantified with image analysis. For quantification calibration curves were made with PCR products from constructs originating from edited and non-edited GluR6 mRNA. GluR2 mRNA was completely edited in all brain structures studied. Editing of GluR6 mRNA, in contrast, was high in gray matter structures (above 90%) but considerably lower in the pons/medulla oblongata (66%) and white matter (55%). It is, therefore, suggested that editing of GluR2 and GluR6 mRNA is performed by different enzymatic activities. Studying RNA editing of glutamate receptor subunits will extend knowledge about the role of calcium fluxes through non-NMDA glutamate receptor ion channels.

[Cerebral ischemia: ontogenetic differences in energy metabolism and protein biosynthesis]. / Zerebrale Ischämie: Ontogenetische Unterschiede im Energiestoffwechsel und in der Proteinbiosynthese.

OBJECTIVE: Are ontogenetic differences in the resistance of the brain towards ischemia reflected by differences in the postischemic disturbance of cerebral protein synthesis (PS) independently from energy metabolism (EM)? METHODS: We studied hippocampal slices from immature (E60) and mature (E60) fetal guinea pigs as well as from adult guinea pigs. Cerebral EM and PS were measured during and up to 24 h after ischemia. RESULTS: After in vitro ischemia there was no inhibition in cerebral PS in immature fetuses, a transient inhibition in mature fetuses, and a permanent inhibition in adults. During and after in vitro ischemia cerebral EM was hardly disturbed in immature fetuses. No differences in cerebral EM could be observed between mature fetuses and adults. CONCLUSION: Ontogenetic differences in the resistance of the brain towards ischemia are reflected by differences in the postischemic disturbance of cerebral PS. The differences between mature fetuses and adults are independent from cerebral EM.

Protein synthesis and energy metabolism in hippocampal slices during extended (24 hours) recovery following different periods of ischemia.

Hippocampal slices were successfully maintained for 24 hours in vitro in a flow-through chamber by using a modified artificial CSF (amino acids included). Measurement of energy metabolism parameters (adenine nucleotides) and the slice response to KCl-induced depolarization (release of GABA and aspartate) indicated that hippocampal slices were metabolically stable for at least 24 hours. The preparation was used to study recovery of protein synthesis after different periods of in vitro ischemia (5, 10, or 15 min). Protein synthesis inhibition was only partly reversed after 15 min of ischemia, but fully reversible after 5- or 10-min ischemia at 24 hours of recovery. Furthermore, the model was used to study a possible role of glutamate in postischemic inhibition of protein synthesis. Glutamate receptor agonists (glutamate or quinolinic acid) or antagonist (kynurenic acid) were applied during ischemia. Neither treatment affected the late (24 hours) outcome of ischemia, arguing against the critical role of glutamate in ischemic cell damage. The present approach allows use of the hippocampal slice preparation in the study of delayed effects of ischemia of different duration.

NBQX reduces threshold of protein synthesis inhibition in focal ischaemia in rats.

The effect of the AMPA antagonist NBQX on peri-infarct inhibition of cerebral protein synthesis (CPS) was studied in rats subjected to 3 h occlusion of the left middle cerebral artery (MCA). Cerebral blood flow and CPS were measured with double tracer autoradiography and local ATP content was monitored by bioluminescence imaging. In untreated MCA-occluded animals the perfusion threshold of ATP depletion in cerebral cortex was 17 +/- 5 ml 100 g-1 min-1 and that of CPS inhibition was 49 +/- 13 ml 100 g-1 min-1. NBQX treatment (2 x 30 mg kg-1 after vascular occlusion) reduced the perfusion threshold of CPS inhibition to 16 +/- 6 ml 100 g-1 min-1 (p < 0.05) whereas that of ATP depletion was not affected (11 +/- 6 ml 100 g-1 min-1). The NBQX-induced pharmacological improvement of peri-infarct CPS is similar to the previously described amelioration of peri-infarct CPS by MK-801 and may contribute to the reduction of infarct size by this treatment.

Hemeoxygenase expression after reversible ischemia of rat brain.

Heme oxygenase (HO-1) gene expression was studied in the brains of rats subjected to 30 min global cerebral ischemia followed by recirculation of up to 24 h. Total RNA was isolated from the cerebral cortex, striatum and hippocampus and reverse-transcribed into cDNA. cDNA was taken as template for PCR using HO-1-specific primers. We found that, when PCR reactions were run for 22 cycles, the amount of PCR products correlated closely with the amount of cDNA. HO-1 gene expression was sharply increased after cerebral ischemia in all three brain structures studied. In the cortex and striatum, the HO-1 mRNA content increased constantly after cerebral ischemia up to 24 h of recovery, being 8- and 9-fold over control after 24 h of recirculation in the cortex and striatum, respectively. In the hippocampus, HO-1 mRNA levels peaked at 4 h after ischemia (9-fold over control) and declined thereafter to 4.5-fold over control 24 h after ischemia. Assuming that the observed increase in mRNA levels is paralled by increased HO-1 protein synthesis, formation of the products of HO reaction, biliverdin and carbon monoxide, is activated after ischemia. These products may produce different and divergent effects on the recovery from the metabolic stress produced by cerebral ischemia.

Protein synthesis in the hippocampal slice: transient inhibition by glutamate and lasting inhibition by ischemia.

Protein synthesis was measured in hippocampal slices which were exposed to glutamate (1 mM or 10 mM) or which were deprived of glucose and oxygen ('in vitro ischemia') for 15 min. Glutamate at 1 mM, a concentration estimated to occur during in vivo ischemia did not affect protein synthesis. Ten mM glutamate inhibited protein synthesis immediately after exposure (50% of control values) and reduced ATP levels to about 30% of the control. After two hours, slices fully recovered their protein synthesis and energy metabolism. The effect of 10 mM glutamate was not receptor-mediated, as NMDA, AMPA, or metabotropic receptor antagonists failed to block the glutamate effect. Immediately after ischemia, protein synthesis was reduced to 30% of control values, and 2 hours later it was still depressed to one-half of control values. Energy charge, however, recovered completely. Ischemic inhibition of protein synthesis was not reversed by glutamate receptor antagonists. The data indicate that inhibition of protein synthesis in hippocampal slices during ischemia is not glutamate-dependent.

Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat.

1. The structure and function of glutamate receptor subunits GluR2, GluR5, and GluR6 are changed by RNA editing. This reaction produces a base transition in the second transmembrane spanning region. The triplet CAG (coding for glutamine) is changed to CGG (coding for arginine). This transition has a pronounced effect on calcium fluxes through the respective ion channels, because calcium currents decrease with the rate of editing. 2. In the present study the extent of RNA editing of the glutamate receptor subunit GluR5 was studied in different brain regions of control rats using a newly developed analysis system. This system is based on restriction analysis of the polymerase chain reaction (PCR) product, derived from reverse-transcribed mRNA as template, with the enzyme Bbv1. Bbv1 recognizes the sequence of the nonedited receptor subunit around the edited base (sequence GCAGC) but not that of the edited subunit (sequence GCGGC; A edited to G). 3. Total RNA was isolated from the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata, and white matter and reverse transcribed into cDNA. The region across the edited sequence was amplified by PCR using GluR5-specific primers and the cDNA as template. PCR products were cleaned by ethanol precipitation, incubated with Bbv1, and electrophoresed on an agarose gel together with standards. Gels were photographed and the extent of GluR5 mRNA editing was quantified using an image analysis system. A calibration curve was obtained using PCR products amplified from plasmids with edited and nonedited GluR5 as inserts. 4. In the brain of control rats the extent of RNA editing of the GluR5 subunit amounted to 62 +/- 6.0% of total (cortex), 43 +/- 5.3% (striatum), 52 +/- 5.3% (hippocampus), 91 +/- 6.3% (thalamus), 85 +/- 10.2% (hypothalamus), 82 +/- 6.5% (cerebellum), 88 +/- 6.8% (pons/medulla oblongata), and 41 +/- 2.7% (white matter). 5. The extent of RNA editing varied, thus, considerably in different brain regions, being lowest in the white matter and striatum and highest in the thalamus and pons/medulla oblongate. RNA editing of glutamate receptor subunits may play an important role in the control of calcium fluxes through non-N-methyl-D-aspartate receptor channels in different physiological and/or pathological states of the brain.

Developmental changes in the extent of RNA editing of glutamate receptor subunit GluR5 in rat brain.

The extent of RNA editing of the glutamate receptor subunit GluR5 mRNA was evaluated in the cortex, hippocampus and cerebellum of embryonic rat brains, aged 19 days, and adult brains, aged 2-3 months. RNA was isolated and transcribed into cDNA, which was used as template for amplifying a PCR product across the edited region of the GluR5 subunit. Quantification of editing was performed by restriction digest of PCR products with BbvI and image analysis of bands obtained after electrophoresis. The extent of editing was significantly lower in the embryonic state as compared to the adult state. These differences were most pronounced in the cerebellum where the extent of editing amounted to 27 +/- 8% and 78 +/- 2% in the embryonic and adult state, respectively (P < 0.001). Since expression of GluR5 is relatively high in brain areas of neuronal differentiation and synapse formation it is concluded that un-edited GluR5 may play a role in the development of the nervous system.

[The importance of Lyme borreliosis in veterinary medicine]. / Problem i znacaj lajm borelioze u veterinarskoj medicini.

A study of literature concerning Lyme borreliosis related to animals was done. In the research work the epidemiology, pathogenesis, diagnosis and treatment of horses, cattle and dogs affected with Lyme borreliosis have been discussed. The clinical signs of Lyme borreliosis in horses are: chronic weight loss, sporadic lameness, laminitis, low grade fever, swollen joints, muscle tenderness and anterior uvetitis. In addition to these clinical sings, neurological sings such as depression, behavioral changes, dysphagia and encephalitis can be seen in chronic cases. Cattle affected with acute Lyme borreliosis often show fever, stiffness, swollen joints and decreased milk production. Chronic weight loss, laminitis and abortion are also a possible outcome of borreliosis in cattle. An early infection of Lyme borreliosis in dogs may give evidence of inapetenca, lethargy, lyphadenopathy, and an acute onset of stiffness or pain while a recurrent intermittent nonerosive arthritis is a more advanced manifestation of canine Lyme borreliosis. Glomerulonephritis and tubular damage secondary to Borrelia burgdorferi infection have been reported to occur in normally infected dogs. In an endemic area atrioventricular heart block has also been reported. The underlying pathogenesis of Lyme disease is still unknown. The diagnosis of clinical Lyme borreliosis is difficult and it depends on a successful recognition of clinical signs, a history of possible exposures to the infection and on serologic testings. The therapy of Lyme borreliosis in animals is based upon the principal therapy of this disease in human medicine.