Inhibition Of Function Research Articles

AAV Vector Mediated Delivery of NG2 Function Neutralizing Antibody and Neurotrophin NT-3 Improves Synaptic Transmission, Locomotion, and Urinary Tract Function after Spinal Cord Contusion Injury in Adult Rats.

NG2 is a structurally unique transmembrane chondroitin sulfate proteoglycan (CSPG). Its role in damaged spinal cord is dual. NG2 is considered one of key inhibitory factors restricting axonal growth following spinal injury. Additionally, we have recently detected its novel function as a blocker of axonal conduction. Some studies, however, indicate the importance of NG2 presence in the formation of synaptic contacts. We hypothesized that the optimal treatment would be neutralization of inhibitory functions of NG2 without its physical removal. Acute intraspinal injections of anti-NG2 monoclonal antibodies reportedly prevented an acute block of axonal conduction by exogenous NG2. For prolonged delivery of NG2 function neutralizing antibody, we have developed a novel gene therapy: adeno-associated vector (AAV) construct expressing recombinant single-chain variable fragment anti-NG2 antibody (AAV-NG2Ab). We examined effects of AAV-NG2Ab alone or in combination with neurotrophin NT-3 in adult female rats with thoracic T10 contusion injuries. A battery of behavioral tests was used to evaluate locomotor function. In vivo single-cell electrophysiology was used to evaluate synaptic transmission. Lower urinary tract function was assessed during the survival period using metabolic chambers. Terminal cystometry, with acquisition of external urethral sphincter activity and bladder pressure, was used to evaluate bladder function. Both the AAV-NG2Ab and AAV-NG2Ab combined with AAV-NT3 treatment groups demonstrated significant improvements in transmission, locomotion, and bladder function compared with the control (AAV-GFP) group. These functional improvements associated with improved remyelination and plasticity of 5-HT fibers. The best results were observed in the group that received combinational AAV-NG2Ab+AAV-NT3 treatment.SIGNIFICANCE STATEMENT We recently demonstrated beneficial, but transient, effects of neutralization of the NG2 proteoglycan using monoclonal antibodies delivered intrathecally via osmotic mini-pumps after spinal cord injury. Currently, we have developed a novel gene therapy tool for prolonged and clinically relevant delivery of a recombinant single-chain variable fragment anti-NG2 antibody: AAV-rh10 serotype expressing scFv-NG2 (AAV-NG2Ab). Here, we examined effects of AAV-NG2Ab combined with transgene delivery of Neurotrophin-3 (AAV-NT3) in adult rats with thoracic contusion injuries. The AAV-NG2Ab and AAV-NG2Ab+AAV-NT3 treatment groups demonstrated significant improvements of locomotor function and lower urinary tract function. Beneficial effects of this novel gene therapy on locomotion and bladder function associated with improved transmission to motoneurons and plasticity of axons in damaged spinal cord.

N-glycosylation on Asn-57 is required for the correct HAI-2 protein folding and protease inhibitory activity.

Hepatocyte growth factor activator inhibitor (HAI)-2 is an integral membrane Kunitz-type serine protease inhibitor that regulates the proteolysis of matriptase and prostasin in a cell-type selective manner. The cell-type selective nature of HAI-2 function depends largely on whether the inhibitor and potential target enzymes are targeted to locations in close proximity. The N-glycan moiety of HAI-2 can function as a subcellular targeting signal. HAI-2 is synthesized with 1 of 2 different N-glycan modifications: one of oligomannose-type, which largely remains in the endoplasmic reticulum/GA, and another of complex-type, which is targeted toward the apical surface in vesicle-like structures, and could function as an inhibitor of matriptase and prostasin. HAI-2 contains 2 putative N-glycosylation sites, Asn-57 and Asn-94, point mutations of which were generated and characterized in this study. The protein expression profile of the HAI-2 mutants indicates that Asn-57, and not Asn-94, is responsible for the N-glycosylation of both HAI-2 species, suggesting that the form with oligomannose-type N-glycan is the precursor of the form with complex-type N-glycan. Unexpectedly, the vast majority of non-glycosylated HAI-2 is synthesized into multiple disulfide-linked oligomers, which lack protease inhibitory function, likely due to distorted conformations caused by the disarrayed disulfide linkages. Although forced expression of HAI-2 in HAI-2 knockout cells artificially enhances HAI-2 oligomerization, disulfide-linked HAI-2 oligomers can also be observed in unmodified cells. These results suggest that N-glycosylation on Asn-57 is required for folding into a functional HAI-2 with full protease suppressive activity and correct subcellular targeting signal.

MRNA vaccines against SARS-CoV-2 induce comparably low long-term IgG Fc galactosylation and sialylation levels but increasing long-term IgG4 responses compared to an adenovirus-based vaccine.

The new types of mRNA-containing lipid nanoparticle vaccines BNT162b2 and mRNA-1273 and the adenovirus-based vaccine AZD1222 were developed against SARS-CoV-2 and code for its spike (S) protein. Several studies have investigated short-term antibody (Ab) responses after vaccination. However, the impact of these new vaccine formats with unclear effects on the long-term Ab response - including isotype, subclass, and their type of Fc glycosylation - is less explored. Here, we analyzed anti-S Ab responses in blood serum and the saliva of SARS-CoV-2 naïve and non-hospitalized pre-infected subjects upon two vaccinations with different mRNA- and adenovirus-based vaccine combinations up to day 270. We show that the initially high mRNA vaccine-induced blood and salivary anti-S IgG levels, particularly IgG1, markedly decrease over time and approach the lower levels induced with the adenovirus-based vaccine. All three vaccines induced, contrary to the short-term anti-S IgG1 response with high sialylation and galactosylation levels, a long-term anti-S IgG1 response that was characterized by low sialylation and galactosylation with the latter being even below the corresponding total IgG1 galactosylation level. Instead, the mRNA, but not the adenovirus-based vaccines induced long-term IgG4 responses - the IgG subclass with inhibitory effector functions. Furthermore, salivary anti-S IgA levels were lower and decreased faster in naïve as compared to pre-infected vaccinees. Predictively, age correlated with lower long-term anti-S IgG titers for the mRNA vaccines. Furthermore, higher total IgG1 galactosylation, sialylation, and bisection levels correlated with higher long-term anti-S IgG1 sialylation, galactosylation, and bisection levels, respectively, for all vaccine combinations. In summary, the study suggests a comparable "adjuvant" potential of the newly developed vaccines on the anti-S IgG Fc glycosylation, as reflected in relatively low long-term anti-S IgG1 galactosylation levels generated by the long-lived plasma cell pool, whose induction might be driven by a recently described TH1-driven B cell response for all three vaccines. Instead, repeated immunization of naïve individuals with the mRNA vaccines increased the proportion of the IgG4 subclass over time which might influence the long-term Ab effector functions. Taken together, these data shed light on these novel vaccine formats and might have potential implications for their long-term efficacy.

Identification of Phenolics Profile in Freeze-Dried Apple Peel and Their Bioactivities during In Vitro Digestion and Colonic Fermentation.

Freeze-dried apple peel powder (Fd-APP) was subjected to in vitro digestion and colonic fermentation to evaluate the variations in its phenolic composition, bioactivities (antioxidant activity, α-amylase, and α-glucosidase inhibition), and fecal metabolic outputs. A total of 88 phenolics were tentatively identified, of which 51 phenolic compounds were quantitated in Fd-APP sample extracts before digestion, and 34 were released during subsequent phases of digestion. Among these, phenolic acids showed the highest bio accessibility index (BI) of 68%, followed by flavonoids (63%) and anthocyanins (52%). The inhibitory functions of Fd-APP extract against α-amylase and α-glucosidase pre- and post-digestion were moderate and ranged from 41.88 to 44.08% and 35.23 to 41.13%, respectively. Additionally, the antioxidant activities revealed a significant (p ≤ 0.05) decline during the in vitro digestion. However, the colonic fermentation stage presented different products where the intact parent phenolic compounds present in Fd-APP were utilized by gut microbes and produced various phenolic metabolites such as 3- hydroxyphenyl acetic acid (3-HPAA), ferulic acid (FA), 3-(4-hydroxyphenyl) propionic acid (3,4 HPPA) and 4- hydroxybenzoic acid (4-HBA). Furthermore, colonic fermentation of Fd-APP accelerated the production of short-chain fatty acids (SCFAs), with acetic acid being the most prevalent (97.53 ± 9.09 mM). The decrease in pH of fermentation media to 4.3 significantly (p ≤ 0.05) enhanced counts of Bifidobacterium (10.27 log CFU/mL), which demonstrated the potential prebiotic effects of Fd-APP. These findings indicated that the consumption of apple peel as a constituent of novel functional foods may support and protect the intestinal microbiota and consequently promote human health.

Soluble Signal Inhibitory Receptor on Leukocytes-1 Is Released from Activated Neutrophils by Proteinase 3 Cleavage.

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes that dampens antimicrobial functions. We previously showed that sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared with blood neutrophils and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in patients with RSV bronchiolitis and hospitalized patients with COVID-19, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared with plasma of patients with RSV bronchiolitis and in serum of hospitalized patients with COVID-19 compared with control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage and that this diminishes the ability to inhibit neutrophilic reactive oxygen species production via SIRL-1. Finally, we found that SIRL-1 shedding is prevented by proteinase 3 inhibition and by extracellular adherence protein from Staphylococcus aureus. Notably, we recently showed that SIRL-1 is activated by PSMα3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.

Correlation between Chronic Pain with BDNF Levels, Histophatology of Hippocampus, and Spatial Memory in Wistar Rats

BACKGROUND: Pain is defined as an unpleasant sensory and emotional experience associated with actural or potential tissue damage. Cognitive impairment can occur due to various processes in the brain, one of which resulted from chronic pain. Brain-derived neurotrophic factor (BDNF) is a neurotropin that plays a role in mediating disinhibition in the excitability of the motor cortex of the brain and inhibitory function in descending pain pathways. Chronic pain of periodontitis causes systemic inflammation that activates microglia resulting in degeneration of CA1 pyramidal neurons in the hippocampus and affects cognitive function, especially spatial memory. AIM: The objectives of this study were to determine the correlation between BDNF levels, spatial memory, and histopathology of hippocampus on periodontitis Wistar rats. METHODS: This observational prospective study was conducted between January and April 2022 at Laboratory Negeri Semarang University. Frontal inferior teeth ligation of Wistar rats was carried out to induce periodontitis for 4 weeks. Chronic pain was assessed using Rat Grimace Scale. Morris water maze (MWM) adaptation was applied for 4 weeks and then BDNF levels, spatial memory, and histopathology of hippocampus were investigated. T-test independent and spearman correlation test were used to data analysis. RESULTS: Among 12 rats, the mean of BDNF levels, spatial memory, and histopathology of hippocampus score were 7.57 ng/mL, 65.08 second, and 3, respectively. There were significant relationships between BDNF levels (p = 0.028), spatial memory (p = 0.001), and histopathology of hippocampus score (p = 0.017) on Wistar rats with chronic pain. Strong correlation (r = −0.721, r2 = 0.52, p = 0.004) between BDNF levels and spatial memory, and moderate correlation (r = −0.597, p = 0.02) between BDNF levels and histopathology of hippocampus score was obtained. There was no significant correlation between spatial memory and histopathology of hippocampus score. CONCLUSIONS: There were significant differences between BDNF levels, spatial memory, and histopathology of hippocampus on periodontitis Wistar rats.

Effects of Physical Activity on Inhibitory Function in Children with Attention Deficit Hyperactivity Disorder: A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis aimed to systematically evaluate the effect of physical activity (PA) on inhibitory function in children with ADHD. Experimental studies on the effect of PA on the inhibitory function of children with ADHD were retrieved. The data were obtained from PubMed, The Cochrane Library, Web of Science, EBSCO (MEDLINE, APA Psyclnfo, ERIC), Embase, Scopus, and ProQuest. The search period was from the date of inception of the respective databases to 4 May 2022, and Reviewer Manager software (version 5.3) was used for analysis. Eleven articles and 713 samples were included in the meta-analysis. Results revealed that PA can significantly improve the inhibitory function of children with ADHD (SMD = 0.78, 95% CI: 0.45−1.10, p < 0.001). Subgroup analysis showed that the effectiveness of PA as an intervention in improving the inhibitory function of children with ADHD was moderated by the frequency, intensity, duration, type, and length of intervention. Based on the findings, PA can effectively improve interference suppression inhibitory function in children with ADHD. Longitudinal open-skill exercise for 60 min or more, two times/week has the best effect on improving inhibitory function in children with ADHD.

Tight Junction Protein 1 Suppresses Kidney Renal Clear Cell Carcinoma Cells Proliferation by Inducing Autophagy.

TJP1, an adaptor protein of the adhesive barrier, has been found to exhibit distinct oncogenic or tumor suppressor functions in a cell-type dependent manner. However, the role of TJP1 in kidney renal clear cell carcinoma (KIRC) remains to be explored. The results showed a marked down-regulation of TJP1 in KIRC tissues compared to normal tissues. Low expression of TJP1 was significantly associated with high grade and poor prognosis in KIRC. Autophagosome aggregation and LC3 II conversion demonstrated that TJP1 may induce autophagy signaling in 786-O and OS-RC-2 cells. Knockdown of TJP1 led to a decrease in the expression of autophagy-related genes, such as BECN1, ATG3, and ATG7. Consistently, TJP1 expression showed a significant positive correlation with these autophagy-related genes in KIRC patients. Furthermore, the overall survival analysis of KIRC patients based on the expression of autophagy-related genes revealed that most of these genes were associated with a good prognosis. TJP1 overexpression significantly suppressed cell proliferation and tumor growth in 786-O cells, whereas the addition of an autophagy inhibitor diminished its inhibitory function. Taken together, these results suggest that TJP1 serves as a favorable prognostic marker and induces autophagy to suppress cell proliferation and tumor growth in KIRC.

Poremećaj pažnje sa hiperaktivnošću udružen sa tikovima - prikaz slučaja

Attention deficit hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder in children and adolescents, with estimated global prevalence ranging between 5% and 12%. The frequency of ADHD comorbid with tics is significantly higher than expected. Children diagnosed with ADHD are much more likely to have chronic tics compared to those without ADHD. When ADHD occurs alongside tic disorders, treatment can be challenging. Through this case report, we aim to shed light on the unique challenges and potential treatment strategies for this comorbidity. A nine-year-old boy was admitted to the Department of Psychiatry due to hyperactivity, poor concentration, attention, and motor tics such as frequent blinking, grimacing, and head nodding. He was admitted for additional diagnostic procedures and possible initiation of differential psychopharmacotherapy. Psychological testing revealed that his overall intellectual achievement was average, but there was a discrepancy between verbal and manipulative abilities. During hospitalization, observation, comprehensive psychological testing, and the use of tests (SNAP-IV borderline scores) did not directly indicate the presence of hyperkinetic disorder. Therefore, a recommendation was made for a six-month follow-up and continuation of speech therapy and defectology treatment. Meanwhile, he was followed up by a psychiatrist, and based on psychodiagnostic assessments, attention deficit with hyperactivity was diagnosed. After ruling out contraindications, methylphenidate therapy was initiated at a dose of 18mg, titrated to 36mg after two months when a satisfactory therapeutic response was achieved. The introduction of methylphenidate had no significant effect on tics but did not worsen them either. Behavioral disorders and functional impairments associated with ADHD have negative impacts on academic, social, and family aspects. Unlike individuals facing only with Tourette's syndrome (TS), those with both TS and ADHD have more pronounced difficulties in areas such as planning, working memory, inhibitory function, and visual attention. Clinicians treating patients with tics and ADHD may consider using methylphenidate to address ADHD symptoms. However, it is important for clinicians to discuss this issue with caregivers when prescribing the medication and carefully monitor patients due to precautions listed in the drug information.

Fabrication of Nanoscale Arrays to Study the Effect of Ligand Arrangement on Inhibitory Signaling in NK Cells.

Molecular scale nanopatterns of bioactive molecules have been used to study the effect of transmembrane receptor arrangement on a variety of cell types, including immune cells and their immune response in particular. However, state-of-the-art fabrication approaches have thus far enabled the production of patterns with control over one receptor type only. Herein, we describe a protocol to fabricate arrays for the molecular scale control of the segregation between activating and inhibitory receptors in NK cells. We used this platform to study how ligand segregation regulates NK cell inhibitory signaling and function. The arrays are based on patterns of nanodots of two metals, selectively functionalized with activating and inhibitory ligands. Due to the versatility of our functionalization approach, this protocol can be applied to configurate virtually any combination of extracellular ligands into controlled multifunctional arrays.

Does inhibitory (dys)function account for involuntary autobiographical memory and déjà vu experience?

External cues and internal configuration states are the likely instigators of involuntary autobiographical memories (IAMs) and déjà vu experience. Indeed, Barzykowski and Moulin discuss relevant neuroscientific evidence in this direction. A complementary line of enquiry and evidence is the study of inhibition and its role in memory retrieval, and particularly how its (dys)function may contribute to IAMs and déjà vu.

GABA has a presynaptic inhibitory effect at Lumbricus terrestris body wall muscle synapses.

Earthworm body wall muscle synapses have been suggested to contain both excitatory and inhibitory inputs, and therefore allow for investigation of excitatory/inhibitory signaling in an easily accessible model system. While previous studies have focused on postsynaptic GABAergic inhibitory mechanisms, this study investigated the hypothesis that GABAergic signaling also has presynaptic inhibitory function. This hypothesis was tested by loading synaptogreen C4 dye (also called FM1-43) into presynaptic vesicles in the presence of GABA at Lumbricus terrestris longitudinal muscle synapses. GABA treatment significantly reduced the fluorescence intensity observed at these synapses, suggesting that GABAergic signaling does indeed have a presynaptic inhibitory mechanism.

Effects of Cinobufagin on the Proliferation, Migration, and Invasion of H1299 Lung Cancer Cells.

Cinobufagin (CB), with its steroidal nucleus structure, is one of the major, biologically active components of Chan Su. Recent studies have shown that CB exerts inhibitory effects against numerous cancer cells. However, the effects of CB regarding the metastasis of non-small cell lung cancer (NSCLC) and the involved mechanisms need to be further studied. The purpose of the present study aimed to report the inhibitory function of CB against proliferation and metastasis of H1299 cells. CB inhibited proliferation of H1299 lung cancer cells with an IC50 value of 0.035±0.008 μM according to the results of MTT assays. Antiproliferative activity was also observed in colony forming cell assays. In addition, 5-ethynyl-2'-deoxyuridine (EdU) retention assays revealed that CB significantly inhibited the rate of DNA synthesis in H1299 cells. Moreover, results of the scratch wound healing assays and transwell migration assays displayed that CB exhibited significant inhibition against migration and invasion of H1299 cells. Furthermore, CB could concentration-dependently reduce the expression of integrin α2, β-catenin, FAK, Src, c-Myc, and STAT3 in H1299 cells. These western blotting results indicated that CB might target integrin α2, β-catenin, FAK and Src to suppress invasion and migration of NSCLC, which was consistent with the network pharmacology analysis results. Collectively, findings of the current study suggest that CB possesses promising activity against NSCLC growth and metastasis.

Rumination and schizotypal personality traits

<p>Although ru&shy;mination and schizotypal traits can be considered transdiagnostic phenomena and can occur within non-clinical population as well, a relatively small number of research has been carried out on the topic involving both patient and non clinical participants. The aim of this study is to examine the rela&shy;tionship between schizotypal traits and rumination using a transdiagnostic approach, involving participants living with psychotic disorders and sine morbo individuals.</p>. <p>We recruited participants living with psychotic disorders (paranoid schizophrenia, hebephrenia, schizoaffective disorder, etc.) (n = 30) and controls who had not been diagnosed with any mental illnesses (n = 67). The connection between rumination and schizotypal traits was examined by self-report questionnaire method in a cross-sectional arrangement. The Oxford-Liverpool Inventory was used to measure schizotypal traits, and the Ruminative Thought Style Questionnaire was used to determine the level of rumination.&nbsp;</p>. <p>Schizotypal symptoms (&beta; = 0.575; p &lt; 0.001), especially cognitive disorganization (&beta; = 0.459; p &lt; 0.001) and unusual experiences (&beta; = 0.221; p = 0.029) significantly explained the degree of rumination.&nbsp;</p>. <p>Our results support the hypothesis that the association between rumination and schizotypic traits is due to decreased cognitive inhibitory functions.</p>.

ZDHHC16 restrains osteogenic differentiation of bone marrow mesenchymal stem cells by inhibiting phosphorylation of CREB

AimsThe osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) plays a critical role in fracture healing. Osteogenic differentiation is regulated by a variety of post-translational modifications, but the function of protein palmitoylation in osteogenesis remains largely unknown. MethodsOsteogenic differentiation induction of hBMSCs was used in this study. RT‒qPCR and immunoblotting assays (WB) were used to test marker genes of osteogenic induction. Alkaline phosphatase (ALP) activity, ALP staining and Alizarin red staining were performed to evaluate osteogenesis of hBMSCs. Signal finder pathway reporter array, co-immunoprecipitation and WB were applied to elucidate the molecular mechanism. A mouse fracture model was used to verify the in vivo function of the ZDHHC inhibitor. Key findingsWe revealed that palmitic acid inhibited Runx2 mRNA expression in hBMSCs and identified ZDHHC16 as a potential target palmitoyl acyltransferase. In addition, ZDHHC16 decreased during osteogenic induction. Next, we confirmed the inhibitory function of ZDHHC16 by its knockdown or overexpression during osteogenesis of hBMSCs. Moreover, we illustrated that ZDHHC16 inhibited the phosphorylation of CREB, thus inhibiting osteogenesis of hBMSCs by enhancing the palmitoylation of CREB. With a mouse femur fracture model, we found that 2-BP, a general inhibitor of ZDHHCs, promoted fracture healing in vivo. Thus, we clarified the inhibitory function of ZDHHC16 during osteogenic differentiation. SignificanceCollectively, these findings highlight the inhibitory function of ZDHHC16 in osteogenesis as a potential therapy method for fracture healing.

WITHDRAWN: Interleukin-35 dampens T helper 22 phenotype shift in CD4+CD25+CD127dim/- regulatory T cells in primary biliary cholangitis

The phenotype shift in regulatory T cells (Tregs) contributes to immunopathogenesis of autoimmune diseases. The current study was aimed to investigate the regulatory function of interleukin-35 (IL-35) to T helper 22 (Th22) cell phenotype shift in Tregs in primary biliary cholangitis (PBC). Fifty-five PBC patients and twenty-four controls were enrolled. CD4+CD25+CD127dim/- Tregs and Th22 cells were investigated by flow cytometry. Forkhead box P3 (FoxP3) and aryl hydrocarbon receptor (AhR) mRNA levels were assessed by real-time polymerase chain reaction. Plasma IL-10 and IL-22 levels were measured by ELISA. Purified Tregs were stimulated with exogenous IL-35, and were co-cultured with autologous CD4+CD25- T cells. Cellular proliferation and cytokine production was measured. Purified Tregs were also cultured into Th22 condition in the presence or absence of exogenous IL-35, and Th22 phenotype were assessed. PBC patients had lower levels of Treg percentage, FoxP3 mRNA, and plasma IL-10, while had higher levels of Th22 proportion, AhR mRNA, and plasma IL-22. Tregs from PBC patients showed reduced immunosuppressive activity, which presented as increased cellular proliferation, interferon-γ production and decreased IL-35/IL-10 secretion in co-culture system. Tregs shifted into Th22 phenotype in PBC patients with elevated CCR4, CCR6, and CCR10 expression as well as increased IL-22 production. IL-35 not only enhanced inhibitory function of Tregs but also suppressed phenotype shift of Tregs into Th22 phenotype in PBC patients. This process was accompanied by elevation of IL-10 and transforming growth factor-β1 secretion by Tregs from PBC patients. The present data suggested that reduced IL-35 might be insufficient to maintain Tregs function and phenotype shift from Tregs into Th22 phenotype in PBC patients.

Therapeutic effects of KRM-II-81, positive allosteric modulator for α2/3 subunit containing GABAA receptors, in a mouse model of Dravet syndrome.

Introduction: Dravet syndrome (DS) is an intractable epilepsy syndrome concomitant with neurodevelopmental disorder that begins in infancy. DS is dominantly caused by mutations in the SCN1A gene, which encodes the α subunit of a voltage-gated Na channel. Pre-synaptic inhibitory dysfunction is regarded as the pathophysiological mechanism, but an effective strategy for ameliorating seizures and behavioral problems is still under development. Here, we evaluated the effects of KRM-II-81, a newly developed positive allosteric modulator for α 2/3 subunit containing GABAA receptors (α2/3-GABAAR) in a mice model of DS both in vivo and at the neuronal level. Methods: We used knock-in mice carrying a heterozygous, clinically relevant SCN1A mutation (background strain: C57BL/6J) as a model of the DS (Scn1a WT/A1783V mice), knock-in mouse strain carrying a heterozygous, clinically relevant SCN1A mutation (A1783V). Seizure threshold and locomotor activity was evaluated by using the hyperthermia-induced seizure paradigm and open filed test, respectively. Anxiety-like behavior was assessed by avoidance of the center region in locomotor activity. We estimated a sedative effect by the total distance traveled in locomotor activity and grip strength. Inhibitory post synaptic currents (IPSCs) were recorded from a hippocampal CA1 pyramidal neuron in an acutely prepared brain slice. Results: KRM-II-81 significantly increased the seizure threshold of Scn1a WT/A1783V mice in a dose-dependent manner. A low dose of KRM-II-81 specifically improved anxiety-like behavior of Scn1a WT/A1783V mice. A sedative effect was induced by relatively high dose of KRM-II-81 in Scn1a WT/A1783V mice, the dose of which was not sedative for WT mice. KRM-II-81 potentiated IPSCs by increasing its decay time kinetics. This effect was more prominent in Scn1a WT/A1783V mice. Discussion: Higher activation of α2/3-GABAAR by KRM-II-81 suggests a compensatory modification of post synaptic inhibitory function against presynaptic inhibitory dysfunction in Scn1a WT/A1783V. The increased sensitivity for KRM-II-81 may be relevant to the distinct dose-dependent effect in each paradigm of Scn1a WT/A1783V mice. Conclusion: Selective activation for α2/3-GABAAR by KRM-II-81 could be potential therapeutic strategy for treating seizures and behavioral problems in DS.

Effects of aqueous ginger extract on smooth muscle contraction in bovine cecum: in vitro study.

Intestinal hypomotility cause health risks and economic losses and is considered as an important digestive disorder that efforts to find prokinetic drugs can solve this major problem. This study investigated the effects of Zingiber officinale aqueous extract (ZOAE) on caecal smooth muscle contractions in healthy cows. To perform in vitro tests, cecum strips connected to the organ bath. Ginger aqueous extract caused concentration-dependent contraction in caecal smooth muscle with an effective threshold concentration of 6.00 mg L-1. The strongest contraction was caused at a concentration of 100 mg L-1 with an average contraction of 141%. To evaluate the possible mechanisms underlying the contractile effect on cecum strips, atropine, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and verapamil completely inhibited aqueous extract induced smooth muscle contractions, while addition of hexamethonium had no effect on the contraction process. The lack of reduction of contractions caused by the extract in the presence of hexamethonium indicates that presence of acetylcholine-like constituents independent of nicotinic receptors. The inhibitory properties of atropine and 4-DAMP indicate that at least part of the prokinetic effect of the extract is due to stimulating the muscarinic receptors, especially M3 receptors. Also, verapamil inhibitory function proves that the extract acting by L-type calcium channels. The results suggest that the ZOAE has a potential prokinetic effect which may provide a pharmacological base to its medicinal or prophylactic use in caecal motility disorders.

Cytokine profiles in healthy animals and in cows with purulent-necrotic limb processes

One of the typical and mandatory reactions of the body to trauma or infectious agents for damage to any tissues and organs is the acute phase reaction, which is an induced increase in the synthesis followed by an increase in the blood and tissues of a number of proteins with immunological, bactericidal, antioxidant and inhibitory functions. The purpose of the study is to determine the levels of cytokines in clinically healthy cows, pigs and dogs, as well as in cows with purulent-necrotic processes of the limbs. The levels of pro-inflammatory cytokines and anti-inflammatory IL-10 were determined in blood serum of cows, pigs and dogs. Cows with purulent-necrotic lesions of the limbs (n=26) were divided into: 1st group (n=8) – acute form of purulent-necrotic lesions of the distal limbs; 2nd group (n=8) – generalized lesions; group 3 (n=10) – recurrent foci in the area of the fingers. The content of TNF-α, IL-1ß and IL-10 interleukins in blood serum was determined by the immunoenzymatic method according to the standard protocol. It was established that the blood levels of pro-inflammatory TNF-α and IL-1ß in clinically healthy cows are significantly lower than anti-inflammatory IL-10, for the ratio of IL-10:TNF-α – 3.3:1, and IL- 10: IL- 1ß - 9.5:1, therefore, for cattle under physiological norms, an inherent anti-inflammatory cytokine profile. In clinically healthy pigs, the anti-inflammatory cytokine profile turned out to be the most pronounced, as the cytokine indices in them were significantly higher: IL-10:TNF-α – 19.4:1; IL-10:IL-1β – 13.9:1. In the blood of clinically healthy dogs, the cytokine indices between IL-10:IL-1ß are extremely low - 1.5:1, TNF-α : IL-1ß - 0.2:1, even lower, and between IL-10:TNF-α - 8.8:1, and therefore the anti-inflammatory cytokine profile is much lower according to the totality of cytokine indices. In cows with an acute form of necrobacteriosis, compared with clinically healthy animals, the level of TNF-α in the blood is 5.6 times higher (Р&lt;0.001), and IL-1ß is 3.4 times higher (Р&lt;0.001), due to their increase index by 1.7 times, up to 4.9:1. Under such conditions, the level of IL-10 increases only 1.8 times (р&lt;0.05). Animals with the generalized form are characterized by a critical increase in the blood level of TNF-α by 16.8 times and IL-1ß by 17.8 times (p &lt;0.001), while the level of IL-10 remains unchanged compared to the acute form. Cytokine index IL-10:TNF-α acquires a critical value - 0.4:1, and IL-10 to IL-1ß - 1:1. In animals with recurrent lesions, low levels of IL-1ß, especially IL-10, are characteristic. However, the concentration of TNF-α remains quite high and exceeds the normal value by 12.6 times (p&lt;0.001). Therefore, various clinical forms of necrobacterial lesions of the limbs in cows have a compensatory or non-compensatory nature of cytokinemia, with an imbalance of the functionality of acute-phase proteins due to the insufficient capacity of the inhibitory potential of sick cows. Key words: cows, pigs, dogs, cytokines, serum, clinically healthy animals, inflammation.

High-Level rAAV Vector Production by rAdV-Mediated Amplification of Small Amounts of Input Vector.

The successful application of recombinant adeno-associated virus (rAAV) vectors for long-term transgene expression in clinical studies requires scalable production methods with genetically stable components. Due to their simple production scheme and the high viral titers achievable, first generation recombinant adenoviruses (rAdV) have long been taken into consideration as suitable tools for simultaneously providing both the helper functions and the AAV rep and cap genes for rAAV packaging. So far, however, such rAdV-rep/cap vectors have been difficult to generate and often turned out to be genetically unstable. Through ablation of cis and trans inhibitory function in the AAV-2 genome we have succeeded in establishing separate and stable rAdVs for high-level AAV serotype 2 Rep and Cap expression. These allowed rAAV-2 production at high burst sizes by simple coinfection protocols after providing the AAV-ITR flanked transgene vector genome either as rAAV-2 particles at low input concentrations or in form of an additional rAdV. With characteristics such as the ease of producing the required components, the straightforward adaption to other transgenes and the possible extension to further serotypes or capsid variants, especially the rAdV-mediated rAAV amplification system presents a very promising candidate for up-scaling to clinical grade vector preparations.