Clinical results of an inactivated anti-brucella vaccine in combination with immunomodulators.

AIM: The aim of the study was to obtain a vaccine against animal brucellosis having high immunogenic properties by carrying an evaluation of the effectiveness of split-conjugated animal brucellosis vaccine combined with fosprenil and polypeptide C as a molecular immunomodulatory adjuvant according to the results of serological studies of the blood of animals: Agglutination reaction, complement fixation, and rose Bengal sample. MATERIALS AND METHODS: Eighteen calves of Holstein Friesians breed, aged 5 months, with a living weight of 100-150 kg, were divided into three groups of six animals each. All animals were healthy and they received a prophylactic vaccination against brucellosis. The dry split-conjugated vaccine against brucellosis in animals was dissolved in saline and for this purpose, 10 ml of saline was poured into the vaccine vial. Then the content was mixed, and afterward 1 ml was used per animal. Fosprenil was used at the rate of 1 kg of animal weight: 100 kg (calf weight) was multiplied by 0.05 (dose/1 kg of animal weight); 5 ml of fosprenil was obtained, which was collected into disposable syringes and intramuscularly sterilely injected into the croup area.Calves in the first group (control) were intramuscularly injected with the vaccine at a dose of 1.0 ml, and fosprenil at a dose of 5.0 ml was administered intramuscularly once to the croup area. Animals from the second group were subcutaneously immunized by the vaccine with polypeptide C at a dose of 1.0 ml. Polypeptide C is a solution that was poured into a vial with a vaccine at a dose of 10.0 ml, the content was mixed, and then calves were injected subcutaneously into the middle third of the neck in 1 ml (10 doses in a vial).Immunization of calves in the third group was carried out with a vaccine, diluted with an isotonic sodium chloride solution of 0.9%, at a dose of 1.0 ml subcutaneously once. At the 14th, 30th, and 90th days after vaccination, a blood sampling was taken for serological tests: Agglutination test, complement fixation test, and rose Bengal test. RESULTS: After conducting serological studies, it was noted that split-conjugated vaccine against animal brucellosis using fosprenil forms antibodies in large titers and they persist for a longer time in the body of animals compared to the other tested vaccine: The first combination with the immunomodulatory polypeptide C and the vaccine only on the physiological solution. CONCLUSION: The developed complex of split-conjugated vaccine against brucellosis in animals enhances the humoral immune response of the organism against brucellosis and improves the protection of animals against the disease when it is used with the immunomodulatory fosprenil. In the future, we want to expand the use of the resulting complex in the fight against brucellosis on a larger population and to study the change in cellular immunity after the introduction of the resulting complex on an animal organism.

Epizootiological study on spatiotemporal clusters of Schmallenberg virus and Lumpy skin diseases: The case of Russia.

AIM: The submitted article attempts to highlight and specify the development of Schmallenberg virus (SBV) and lumpy skin disease (LSD) in cartographic illustrations, as well as to assess the epizootic situation of these diseases in the world, especially in Russia. MATERIALS AND METHODS: Outbreaks (samples were collected from clinically healthy as well as suspected animals in infected areas) were confirmed and reported to the World Organization for Animal Health by veterinary officials representing countries in different geographical regions in the world. The reports showed that ELISA and polymerase chain reaction were used to identify SBV and LSD, taking into account number of infected, dead, and susceptible animals in infection foci since their first registration including in Russia. Once conventional statistical population (arrange data according to the main goal by regions, infected, and dead animals) was defined, a model was installed. A geo-information system, QuickMAP, was used to clarify the disease distribution map, and through the illustrations, analysis values were obtained. RESULTS: Using information clusters of some epizootological criteria in various territories has demonstrated 1.302 focus of infection of SBV, of which 63.22% were registered in Europe and 36.78% in Russia. The seroprevalence in Russia was about 7.92% of the examined animals. According to the morbidity structure, the causative agent mainly affected cattle (64.76%), small ruminants (33.68%), and goats (1.56%). A global assessment of the effectiveness of primary epizootic diagnosis by practicing veterinarians was 63.19%, i.e., of 100 suspicion reports of SBV, 63.19 cases are confirmed by laboratory methods. A detailed assessment of the types of animals affected by the disease showed that it was easily diagnosed in sheep (70.38%), cattle (60.4%), and goats (48.57%), respectively. In the wild animal species, a significant prevalence was recorded as- 54.5%. In 2016, 1.209 foci of LSD were registered in the world, with 20.548 heads of cattle affected, while 8.5% of them identified in Russia (in 2017, the figure was 7.5%). Different maps had been generated in QuickMAP. Cluster analysis of the infected livestock in different regions in Russia showed that, in 2016, the Chechen Republic, Krasnodar, and Volgograd regions were, respectively, severely, moderately, and mildly affected. In 2017, the situation changed and Saratov, Orenburg regions, and Bashkiria were severely affected. However, the number of outbreaks decreased by 84.81% by contribution to the previous year. Eritrea, Namibia, and South Africa were leading in a cluster of most infected areas in 2017. CONCLUSION: Infectious diseases do not know borders. The emergence of SBV and LSD in the territory of the Russian Federation has followed the most common general dynamics of transborder diseases without ignoring details. The epizootic risk from wild animals and favorable climatic conditions is critical to fight against transmission of these diseases in Russia.

Experimental Model of Intervertebral Disk Mediated Postoperative Epidural Fibrosis.

BACKGROUND: Postoperative epidural fibrosis (EF) after lumbar discectomy is the most common and at the same time controversial issue. PURPOSE: The etiology and pathogenesis creates a lot of discussion and selection of methods of treatment and prevention continues. METHODS: LIV laminectomy with dura mater (DM) exposition was done in 24 rats, and then, 0.3 ml of elements of suspension of autologous intervertebral disk was implicated on DM. As autologous intervertebral disk, we used the intervertebral disk from amputated tail. In all the animals, incisions were closed with 3/0 Vicryl. EF was examined. Fibroblast cell density was calculated in each field at ×40 magnification: Grade 1 - fewer than 100 fibroblasts in each field; Grade 2 - 100-150 fibroblasts in each field; Grade 3 - more than 150 fibroblasts in each field. RESULTS: Based on histological results, we confirmed our model of experiment. On the 30th day of evaluation, there were significant histological evidences of postoperative epidural adhesions in experimental animals, which included the obliteration of epidural space, the presence of adhesions in the dura and nerve roots, the restructuring of the yellow ligament, bone sclerosis, excessive appearance of fibrous tissue around the autologous intervertebral disk tissue that applied on the DM. CONCLUSION: In our work, we describe a new experimental model, where the elements of autologous intervertebral disk play the role of inflammation trigger, which cause postoperative scar and EF.

The usage of phage mini-antibodies as a means of detecting ferritin concentration in animal blood serum.

Mini-antibodies that have specific ferritin response have been produced for the first time using sheep's phage libraries (Griffin.1, Medical Research Council, Cambridge, UK). Produced phage antibodies were used for the first time for the development of diagnostic test kits for ferritin detection in the blood of cattle. The immunodot assay with secondary biospecific labeling is suggested as means of ferritin detection in cow blood serum (antiferritin phage antibodies and rabbit antiphage antibodies conjugated with different labels). Сolloidal gold, gold nanoshells, and horse reddish peroxidase used as labels have shown a similar response while detecting concentration of ferritin (0.2 mg/mL). It is shown that the method of solid-phase immunoassay with a visual view of the results allows determination of the minimum concentration of ferritin in the blood of cows at 0.225 g/mL.

Spatially pathogenic forms of tau detected in Alzheimer's disease brain tissue by fluorescence lifetime-based Förster resonance energy transfer.

In tauopathies including Alzheimer's disease (AD) tau molecules have lost their normal spatial distance to each other and appear in oligomeric or aggregated forms. Conventional immunostaining methods allow detection of abnormally phosphorylated or conformationally altered aggregated tau proteins, but fail to visualize oligomeric forms of tau. Here we show that tau molecules that lost their normal spatial localization can be detected on a subcellular level in postmortem central nervous system (CNS) tissue sections of AD patients by fluorescence lifetime-based Förster resonance energy transfer (FRET). Paraffin sections were co-immunostained with two tau-specific monoclonal antibodies recognizing the same epitope, but labeled with distinct fluorescence dyes suitable for spatial resolution at a nanometer scale by lifetime-based FRET. A FRET signal was detected in neuritic plaques and neurofibrillary tangles of CNS tissue sections of AD patients, showing associated tau proteins typically reflecting either fibrillary, oligomeric or aggregated tau. The 'pretangle-like' structures within the neuronal perikarya did not contain spatially pathogenic forms of tau accordingly to this method. Data demonstrate that fluorescence lifetime-based FRET can be applied to human brain tissue sections to detect pathogenic forms of tau molecules that lost their normal spatial distance.

Signal regulatory protein-beta1: a microglial modulator of phagocytosis in Alzheimer's disease.

The signal regulatory protein-beta1 (SIRPbeta1) is a DAP12-associated transmembrane receptor expressed in a subset of hematopoietic cells. Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Here, we found that SIRPbeta1 was up-regulated and acted as a phagocytic receptor on microglia in amyloid precursor protein J20 (APP/J20) transgenic mice and in Alzheimer's disease (AD) patients. Interferon (IFN)-gamma and IFN-beta stimulated gene transcription of SIRPbeta1 in cultured microglia. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase-2. Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta (Abeta). Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells. Thus, SIRPbeta1 is a novel IFN-induced microglial receptor that supports clearance of neural debris and Abeta aggregates by stimulating phagocytosis.

Accumulation of tau induced in neurites by microglial proinflammatory mediators.

Aggregated fibrillary microtubule-associated protein tau is the major component of neurofibrillary tangles in Alzheimer's disease. The exact molecular mechanism of tau aggregation is unknown. Microglial cell activation and migration toward amyloid-beta plaques precede the appearance of dysmorphic neurites and formation of neurofibrillary tangles. Here, we analyzed the accumulation of tau at a distance range of expected spontaneous aggregation by fluorescence lifetime-based Förster resonance energy transfer in cultured primary murine neurons cotransfected with the human tau gene tagged to the green fluorescent protein variants Citrine (tau-Citrine) and Cerulean (tau-Cerulean). No spontaneous accumulation of cotransfected tau-Citrine and tau-Cerulean was detected in untreated neurons. Coculture of neurons with activated microglia induced aggregation of tau in neurites. Treatment of neurons with tumor necrosis factor-alpha (TNF-alpha) stimulated reactive oxygen species generation and resulted in the accumulation of tau-Citrine and tau-Cerulean in neurites, which was inhibited by neutralization of TNF and the free radical inhibitor 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox). These data demonstrate that activated microglia and the microglial-derived proinflammatory cytokine TNF can induce accumulation of the aggregation-prone tau molecules in neurites via reactive oxygen species.

Capillary cerebral amyloid angiopathy is associated with vessel occlusion and cerebral blood flow disturbances.

The role of cerebral amyloid angiopathy (CAA) in the pathogenesis of Alzheimer's disease (AD) is not fully understood. Here, we studied whether CAA is associated with alterations in microvascularisation in transgenic mouse models and in the human brain. APP23 mice at 25-26 months of age exhibited severe CAA in thalamic vessels whereas APP51/16 mice did not. Wild-type littermates were free of CAA. We found CAA-related capillary occlusion within the thalamus of APP23 mice but not in APP51/16 and wild-type mice. Magnetic resonance angiography (MRA) showed blood flow alterations in the thalamic vessels of APP23 mice. CAA-related capillary occlusion in the branches of the thalamoperforating arteries of APP23 mice, thereby, corresponded to the occurrence of blood flow disturbances. Similarly, CAA-related capillary occlusion was observed in the human occipital cortex of AD cases but less frequently in controls. These results indicate that capillary CAA can result in capillary occlusion and is associated with cerebral blood flow disturbances providing an additional mechanism for toxic effects of the amyloid beta-protein in AD.

Staging of neurofibrillary pathology in Alzheimer's disease: a study of the BrainNet Europe Consortium.

It has been recognized that molecular classifications will form the basis for neuropathological diagnostic work in the future. Consequently, in order to reach a diagnosis of Alzheimer's disease (AD), the presence of hyperphosphorylated tau (HP-tau) and beta-amyloid protein in brain tissue must be unequivocal. In addition, the stepwise progression of pathology needs to be assessed. This paper deals exclusively with the regional assessment of AD-related HP-tau pathology. The objective was to provide straightforward instructions to aid in the assessment of AD-related immunohistochemically (IHC) detected HP-tau pathology and to test the concordance of assessments made by 25 independent evaluators. The assessment of progression in 7-microm-thick sections was based on assessment of IHC labeled HP-tau immunoreactive neuropil threads (NTs). Our results indicate that good agreement can be reached when the lesions are substantial, i.e., the lesions have reached isocortical structures (stage V-VI absolute agreement 91%), whereas when only mild subtle lesions were present the agreement was poorer (I-II absolute agreement 50%). Thus, in a research setting when the extent of lesions is mild, it is strongly recommended that the assessment of lesions should be carried out by at least two independent observers.

Assessment of alpha-synuclein pathology: a study of the BrainNet Europe Consortium.

To determine the reliability of assessment of alpha-synuclein-immunoreactive (alphaS-IR) structures by neuropathologists, 28 evaluators from 17 centers of BrainNet Europe examined current methods and reproducibility of alphaS-IR evaluation using a tissue microarray (TMA) technique. Tissue microarray blocks were constructed of samples from the participating centers that contained alphaS-IR structures. Slides from these blocks were stained in each center and assessed for neuronal perikaryal inclusions, neurites, and glial cytoplasmic inclusions. The study was performed in 2 phases. First, the TMA slides were stained with the antibody of the center's choice. In this phase, 59% of the sections were of good or acceptable quality, and 4 of 9 antibodies used performed consistently. Differences in interpretation and categorization of alphaS-IR structures, however, led to differing results between the laboratories. Prior to the second phase, the neuropathologists participated in a training session on the evaluation of alphaS-IR structures. Based on the results of the first phase, selected antibodies using designated antigen retrieval methods were then applied to TMA slides in the second phase. When the designated methods of both staining and evaluation were applied, all 26 subsequently stained TMA sections evaluated were of good/acceptable quality, and a high level of concordance in the assessment of the presence or absence of specific alphaS-IR structures was achieved. A semiquantitative assessment of alphaS-IR neuronal perikaryal inclusions yielded agreements ranging from 49% to 82%, with best concordance in cortical core samples. These results suggest that rigorous methodology and dichotomized assessment (i.e. determining the presence or absence of alphaS-IR) should be applied, and that semiquantitative assessment can be recommended only for the cortical samples. Moreover, the study demonstrates that there are limitations in the scoring of alphaS-IR structures.

Management of acute subdural hematomas in infants: intrathecal infusion streptokinase for clot lysis combined with subdural to subgaleal shunt.

OBJECT: Subdural hematomas (SDHs) in full-term infants have the potential to cause death or lifelong disability. We report management and outcomes of eight cases of newborn with large SDH treated by streptokinase (SK) lavage and drainage. MATERIALS AND METHODS: Between 2003 and 2006, eight infants with large acute SDH with focal or diffuse hypodensity showing signs and symptoms of neurological deterioration were treated by drainage and subdural SK lavage. There were eight full-term infants, five boys and three girls, with ages between 10 days and 2 months. Head injuries were shaken baby syndrome in three cases, fall from height in three cases, caused by traffic accident in one case, and reportedly not due to trauma in one case. In all patients, SDHs were unilateral. We used a new surgical approach, SDH evacuation, involving the subdural instillation of SK for lysis and after drainage of acute SDH in infants. Follow-up in the series ranged from 1 to 42 months (average 30 months). There was no mortality in this series, neither in the early postoperative period nor in the follow-up period. Five patients of this series lead a normal life; two children were mildly neurodevelopmentally delayed. CONCLUSION: Subdural infusion of SK followed by drainage may be as safe and effective for treatment of acute SDHs in infants as other reported methods.

Association of the glutathione S-transferase omega-1 Ala140Asp polymorphism with cerebrovascular atherosclerosis and plaque-associated interleukin-1 alpha expression.

BACKGROUND AND PURPOSE: Glutathione S-transferase omega-1 is a multifunctional enzyme. The Asp/Asp genotype of the Ala140Asp polymorphism of the GSTO1 gene has been alleged to increase the risk of vascular dementia. The objective of this study is to address the question of whether common vessel disorders known to cause vascular dementia are modified in their severity by this polymorphism. METHODS: The severity and expansion of atherosclerosis in the circle of Willis vessels, cerebral small vessel disease, and cerebral amyloid angiopathy were studied in a sample of 79 autopsy cases. Genotyping of the GSTO1 Ala140Asp polymorphism as well as immunohistochemistry for glutathione S-transferase omega-1 was performed. RESULTS: Carriers of the GSTO1 Asp/Asp genotype presented with more severe and widespread atherosclerosis than noncarriers. However, there was no effect on small vessel disease expansion and cerebral amyloid angiopathy severity. Immunohistochemically, we detected interleukin-1 alpha expressing macrophages in the lipid core of atherosclerosis plaques exhibiting glutathione S-transferase omega-1-positive material. GSTO1 Asp/Asp carriers showed larger areas of atherosclerosis plaques containing interleukin-1 alpha-positive material than carriers of the GSTO1 Ala-allele. CONCLUSIONS: The GSTO1 Asp/Asp genotype presumably modulates the severity and expansion of atherosclerosis in the circle of Willis. The cellular colocalization of glutathione S-transferase omega-1 and interleukin-1 alpha suggests a functional interaction between both proteins which in part might explain the function of glutathione S-transferase omega-1 in the pathogenesis of cerebral atherosclerosis.

Expression of alpha2-macroglobulin, neutrophil elastase, and interleukin-1alpha differs in early-stage and late-stage atherosclerotic lesions in the arteries of the circle of Willis.

Different types of atherosclerotic (AS) lesions can be distinguished histologically and represent different stages of AS plaque development. Late-stage lesions more frequently develop complications such as plaque rupture and thrombosis with vessel occlusion than early AS lesions. To clarify whether protective, destructive, and inflammatory proteins are differentially expressed in early-stage and late-stage AS plaques we examined the proteinase inhibitor alpha(2)-macroglobulin (A2M), the neutrophil elastase (NE)-an enzyme degrading elastin and collagen fibers-and the proinflammatory protein interleukin-1alpha (IL-1alpha) in all types of AS plaques in the arteries of the circle of Willis from 78 human autopsy cases of both genders (61-91 years of age). Paraffin sections of AS plaques were immunostained with antibodies directed against A2M, NE and IL-1alpha. In initial AS lesions A2M was found, whereas NE and IL-1alpha were absent. NE and IL-1alpha became detectable as soon as a significant number of macrophages occurred within AS lesions. With increasing histopathological type of AS lesions, a marked increase of the area of the plaque exhibiting NE and IL-1alpha was observed. The area which exhibits A2M in AS plaques, on the other hand, did not vary significantly between the different stages. Thus, our results indicate a disproportionately high increase of the destructive enzyme NE and the proinflammatory protein IL-1alpha in relation to A2M with the progression of the grade of AS lesions pointing to the transgression of the protective capacity of A2M by NE and IL-1alpha in late-stage plaques. Therefore, our findings support the hypothesis that NE-induced tissue damage in late-stage AS plaques contributes to the development of plaque rupture and subsequent thrombosis.

Occurrence and co-localization of amyloid beta-protein and apolipoprotein E in perivascular drainage channels of wild-type and APP-transgenic mice.

The deposition of the amyloid beta-protein (Abeta) is a hallmark of Alzheimer's disease (AD). One reason for Abeta-accumulation and deposition in the brain may be an altered drainage along perivascular channels. Extracellular fluid is drained from the brain towards the cervical lymph nodes via perivascular channels. The perivascular space around cerebral arteries is the morphological correlative of these drainage channels. Here, we show that Abeta is immunohistochemically detectable within the perivascular space of 25 months old wild-type and amyloid precursor protein (APP)-transgenic mice harboring the Swedish double mutation driven by a neuron specific promoter. Only small amounts of Abeta can be detected immunohistochemically in the perivascular space of wild-type mice. Cerebrovascular and parenchymal Abeta-deposits were absent. In APP-transgenic mice, large amounts of Abeta were found in the perivascular drainage channels accompanied with cerebrovascular and parenchymal Abeta-deposition. The apolipoprotein E (apoE) immunostaining within the perivascular channels did not vary between wild-type and APP-transgenic mice. Almost 100% of the area that represents the perivascular space was stained with an antibody directed against apoE. Here, Abeta co-localized with apoE indicating an involvement of apoE in the perivascular clearance of Abeta. Fibrillar congophilic amyloid was not seen in wild-type mice. In APP-transgenic animals, congophilic fibrillar amyloid material was seen in the wall of cerebral blood vessels but not in the perivascular space. In conclusion, our results suggest that non-fibrillar forms of Abeta are drained along perivascular channels and that apoE is presumably involved in this clearance mechanism. Overloading such a clearance mechanism in APP-transgenic mice appears to result in insufficient Abeta-clearance, increased Abeta-levels in the brain and the perivascular drainage channels, and finally in Abeta-deposition. In so doing, our results strengthen the hypothesis that an alteration of perivascular drainage supports Abeta-deposition and the development of AD.

Unloading kinesin transported cargoes from the tubulin track via the inflammatory c-Jun N-terminal kinase pathway.

Axonal transport of mitochondria and synaptic vesicle precursors via kinesin motor proteins is essential to keep integrity of axons and synapses. Disturbance of axonal transport is an early sign of neuroinflammatory and neurodegenerative diseases. Treatment of cultured neurons by the inflammatory cytokine tumor necrosis factor-alpha (TNF) stimulated phosphorylation of c-Jun N-terminal kinase (JNK) in neurites. TNF treatment induced dissociation of the heavy chain kinesin family-5B (KIF5B) protein from tubulin in axons but not cell bodies as determined by lifetime-based Förster resonance energy transfer (FRET) analysis. Dissociation of KIF5B from tubulin after TNF treatment was dependent on JNK activity. Furthermore, TNF inhibited axonal transport of mitochondria and synaptophysin by reducing the mobile fraction via JNK. Thus, TNF produced by activated glial cells in inflammatory or degenerative neurological diseases acts on neurites by acting on the kinesin-tubulin complex and inhibits axonal mitochondria and synaptophysin transport via JNK.