Exposure to the plasticizer, Di-(2-ethylhexyl) phthalate during juvenile period exacerbates autism-like behavior in adult BTBR T + tf/J mice due to DNA hypomethylation and enhanced inflammation in brain and systemic immune cells.

Epigenetic modifications are known to play a crucial role in the behavioral modifications through regulation of gene expression. Environmental factors are known to regulate genetic transcription through DNA methylation which is one of the mechanisms of epigenetic modification. Di-2-ethylhexyl phthalate (DEHP) is one of the most abundant phthalate plasticizers in day-to-day products. Prenatal/postnatal DEHP administration has been reported to cause inflammation as well as behavioral dysregulation, however it is not known if exposure to DEHP during juvenile stage affects peripheral/neuronal inflammation and autism-like symptoms in BTBR mice at adulthood. This study investigated effect of DEHP exposure during juvenile period on DNA methylation (global DNA methylation/DNMT1 expression) and inflammation (IL-17A, IL-6, MCP-1, TNF-α) in CD4 + T cells/CD11c + DCs and cortex, and autism-like symptoms (three-chambered sociability test, self-grooming and marble burying test) in asocial BTBR and social C57 mice at adulthood. Our data reveal that BTBR mice exposed to DEHP during juvenile period have hypomethylated DNA/DNMT1 expression in CD11c + DCs and cortex as compared to vehicle-exposed BTBR mice. It was associated with upregulated inflammation in periphery [plasma IL-6/IL-17A, CD11c + DCs (IL-6/MCP-1/TNF-α), and CD4+ T cells (IL-17A)] and cortex (IL-6, MCP-1, TNF-α), and aggravation in autism-like symptoms in DEHP-treated BTBR mice. These data propose that exposure of DEHP during juvenile period may affect autism-like behavior and inflammation in BTBR mice at adulthood through epigenetic regulation. Therefore, underlying genetic predisposition may play a crucial role in worsening of autistic symptoms in ASD subjects in adulthood if they are exposed to environmental pollutants such as DEHP during juvenile period.

Antinematode Activity of Abomasum Bacterial Culture Filtrates against Haemonchus contortus in Small Ruminants.

Haemonchosis is a parasitic disease of small ruminants that adversely affects livestock production. Haemonchus contortus is one of the most prevalent nematode parasites that infect the abomasum of small ruminants. This parasite reduces milk production, overall growth and sometimes causes the death of the infected animals. The evaluation of the biocontrol potential of some abomasum bacterial isolates against H. contortus is investigated in this study. Out of which, three isolates-Comamonas testosteroni, Comamonas jiangduensis, Pseudomonas weihenstephanesis-show significant effect against the nematode L3, adult, and egg hatch inhibition assays. Various concentrations of metabolites from these bacteria are prepared and applied in different treatments compared with control. In the case of adult mortality assay, 50% metabolites of C. testosteroni and P. weihenstephanesis show 46% adult mortality, whereas C. jiangduensis shows 40% mortality. It is observed that decreasing the concentration of bacterial metabolite, lowers nematode mortality. The minimum nematode mortality rate is recorded at the lowest filtrates concentration of all the bacterial isolates. The same trend is observed in egg hatch inhibition assay, where the higher concentration of bacterial culture filtrates shows 100% inhibition of H. contortus egg. It is concluded that the effect of bacterial culture filtrates against H. contortus is dose-dependent for their activity against nematode L3, adult, and inhibition of egg hatchment.

Green Synthesis, Characterization, Enzyme Inhibition, Antimicrobial Potential, and Cytotoxic Activity of Plant Mediated Silver Nanoparticle Using Ricinus communis Leaf and Root Extracts.

The need of non-toxic synthesis protocols for nanoparticles arises developing interest in biogenic approaches. The present project was focused on cost effective, environment congenial synthesis of Ag nanoparticles and their biological applications. Leaf and root extracts of Ricinus communis were used as a reducing and stabilizing agent in synthesis process. A Proposed mechanism in published literature suggested that Indole-3-acetic acid, l-valine, triethyl citrate, and quercetin-3-0-p-d-glucopyranoside phytoconstituents of Ricinus communis act as reducing and capping agents. The synthesized Ag NPs were characterized with a help X-ray diffractometer, Transmission electron microscopy, UV-Vis spectrophotometry and Fourier Transform Infrared Spectroscopy (FTIR). The XRD results inveterate the synthesis of pure nano size crystalline silver particles. The FTIR data revealed the possible functional groups of biomolecules involved in bio reduction and capping for efficient stabilization of silver nanoparticles. TEM analysis confirmed the almost spherical morphology of synthesized particles with mean size 29 and 38 nm for R-Ag-NPs (root) and L-Ag-NPs (leaf), respectively. The stability of synthesized nanoparticles was examined against heat and pH. It was observed that synthesized nanoparticles were stable up to 100 °C temperature and also showed stability in neutral, basic and slightly acidic medium (pH 05-06) for several months while below pH 5 were unstable. The synthesized silver nanoparticles had promising inhibition efficiency in multiple applications, including as bactericidal/fungicidal agents and Urease/Xanthine oxidase enzymes inhibitors. The cytotoxicity of synthesized nanoparticles shows that the concentration under 20 µg/mL were biologically compatible.

Dysregulation of Ki-67 Expression in T Cells of Children with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by behavioral abnormalities such as impairments in social function and deficits in communication. The etiology of autism is unknown in most cases, but many studies have pointed towards the immune system as a causative agent in autism. Specific studies implicated lymphocytes, natural killer (NK) cells, monocytes, cytokines, and specific transcription factors in the development of ASD. The protein Ki-67 is n expressed in the proliferating cells and is used as a tool in several disorders. Ki-67 plays a crucial role in many neurological diseases. However, Ki-67 role in ASD is not fully understood. In this study, we investigated the possible role of Ki-67 expression in autistic children. We compared Ki-67 production in CD3+, CD4+, CD8+, CXCR4+, CXCR7+, CD45R+, HLA-DR+, GATA3+, Helios+, and FOXP3+ peripheral blood mononuclear cells (PBMCs) in autistic children to typically developing (TD) controls using immunofluorescence staining. We also determined Ki-67 mRNA levels in PBMCs using RT-PCR. The results revealed that autistic children had significantly increased numbers of CD3+Ki-67+, CD4+Ki-67+, CD8+Ki-67+, CXCR4+Ki-67+, CXCR7+Ki-67+, CD45R+Ki-67+, HLA-DR+Ki-67+, CXCR4+GATA3+, GATA3+Ki-67+ cells and decreased Helios+Ki-67+ and FOXP3+Ki-67+ cells compared with TD controls. In addition, the autistic children showed upregulation of Ki-67 mRNA levels compared with TD controls. Further studies need to be carried out to assess the exact role of Ki-67 and its therapeutic potential in ASD.

Bruton's tyrosine kinase inhibition attenuates oxidative stress in systemic immune cells and renal compartment during sepsis-induced acute kidney injury in mice.

Sepsis is a life-threatening condition which affects multiple organs including the kidney. Sepsis-induced acute kidney injury (AKI) is a major health burden throughout the globe. Pathogenesis of sepsis-induced AKI is complex; however, it involves both innate and adaptive immune cells such as B cells, T cells, dendritic cells (DCs), macrophages, and neutrophils. Bruton's tyrosine kinase (BTK) is reportedly involved in inflammatory and oxidative signaling in different immune cells, however its contribution with respect to sepsis-induced AKI has not been delineated. This study attempted to investigate the role of BTK and its inhibition on oxidizing enzymes NADPH oxidase (NOX-2) and inducible nitric oxide synthase (iNOS) in DCs, neutrophils, and B cells during AKI. Our data reveal that BTK is activated in DCs, neutrophils, and B cells which causes an increase in AKI associated biochemical markers such as serum creatinine/blood urea nitrogen, renal myeloperoxidase activity, and histopathological disturbances in renal tubular structures. Activation of BTK causes upregulation of NOX-2/iNOS/nitrotyrosine in these immune cells and kidney. Treatment with BTK inhibitor, Ibrutinib causes attenuation in AKI associated dysfunction in biochemical parameters (serum creatinine/blood urea nitrogen, renal myeloperoxidase activity) and oxidative stress in immune cells and kidney (iNOS/NOX2/lipid peroxides/nitrotyrosine/protein carbonyls). In summary, the current investigation reveals a compelling role of BTK signaling in sepsis-induced AKI which is evident from amelioration of AKI associated renal dysfunction after its inhibition.

5-Aminoisoquinolinone, a PARP-1 Inhibitor, Ameliorates Immune Abnormalities through Upregulation of Anti-Inflammatory and Downregulation of Inflammatory Parameters in T Cells of BTBR Mouse Model of Autism.

Autism spectrum disorder (ASD) covers a range of neurodevelopmental disorders involving impairments in communication and repetitive and stereotyped patterns of behavior and reciprocal social interaction. 5-Aminoisoquinolinone (5-AIQ), a PARP-1 inhibitor, has neuroprotective and anti-inflammatory effects. We investigated the influence of 5-AIQ-treatment in BTBR T+ Itpr3tf/J (BTBR) mice as an autism model and used flow cytometry to assess the effect of 5-AIQ on FOXP3, Helios, GATA3, IL-9, IL-10 and IL-17A production by CXCR6+ and CD4+ T cells in the spleen. We also confirmed the effect of 5-AIQ treatment on expression of FOXP3, Helios, GATA3, IL-17A, IL-10, and IL-9 mRNA and protein expression levels in the brain tissue by quantitative PCR and western blotting. Our results demonstrated that 5-AIQ-treated BTBR mice had significantly increased numbers of CXCR6+FOXP3+, CXCR6+IL-10+, and CXCR6+Helios+ cells and decreased numbers of CD4+GATA3+, CD4+IL-9+, and CD4+IL-17A+ cells as compared with those in untreated BTBR mice. Our results further demonstrated that treatment with 5-AIQ in BTBR mice increased expression for FOXP3, IL-10, and Helios, and decreased expression for GATA3, IL-17A, and IL-9 mRNA. Our findings support the hypotheses that 5-AIQ has promising novel therapeutic effects on neuroimmune dysfunction in autism and is associated with modulation of Treg and Th17 cells.

Genome-Based Drug Target Identification in Human Pathogen Streptococcus gallolyticus.

Streptococcus gallolysticus (Sg) is an opportunistic Gram-positive, non-motile bacterium, which causes infective endocarditis, an inflammation of the inner lining of the heart. As Sg has acquired resistance with the available antibiotics, therefore, there is a dire need to find new therapeutic targets and potent drugs to prevent and treat this disease. In the current study, an in silico approach is utilized to link genomic data of Sg species with its proteome to identify putative therapeutic targets. A total of 1,138 core proteins have been identified using pan genomic approach. Further, using subtractive proteomic analysis, a set of 18 proteins, essential for bacteria and non-homologous to host (human), is identified. Out of these 18 proteins, 12 cytoplasmic proteins were selected as potential drug targets. These selected proteins were subjected to molecular docking against drug-like compounds retrieved from ZINC database. Furthermore, the top docked compounds with lower binding energy were identified. In this work, we have identified novel drug and vaccine targets against Sg, of which some have already been reported and validated in other species. Owing to the experimental validation, we believe our methodology and result are significant contribution for drug/vaccine target identification against Sg-caused infective endocarditis.

The α9α10 nicotinic acetylcholine receptors antagonist α-conotoxin RgIA reverses colitis signs in murine dextran sodium sulfate model.

Nicotinic acetylcholine receptors can regulate inflammation primarily through the vagus nerve via the cholinergic anti-inflammatory pathway. α9α10 nicotinic receptors (nAChRs) are a new promising target for chronic pain and inflammation. Recently, α9α10 selective α-conotoxin antagonists were shown to have antinociception effect in neuropathic and tonic inflammatory pain animal models. However, limited data available on the role of α9α10 nAChRs in experimental colitis. In this study, we report for the first time, the role of α9α10 nAChRs in the dextran sodium sulfate (DSS) experimental animal colitis model. We determined the effect of the α9α10 nAChRs antagonist, α-conotoxin RgIA (α-RgIA) in DSS-induced colitis model in adult male and female C57BL/6 J mice. DSS solution was freely given in the drinking water for seven consecutive days, and tap water was given on the 8th day. We then sacrificed mice on day 8 to examine the entire colon. Disease severity, colon tissue histology, and tumor necrosis factor-α (TNF-α) were evaluated. The lower doses (0.02 and 0.1 nmol/mouse, s.c.) of α-RgIA treatment in DSS-treated mice were inactive, whereas the higher dose (0.2 nmol/mouse, s.c.) reversed the disease activity index (DAI) score, loss of body weight, total histological damage score, as well as the colonic level of TNF-α compared to the DSS-control group. Moreover, the highest dose of α-RgIA (0.2 nmol/mouse, s.c.) significantly rescued the colon length shortening in DSS-treated mice compared to the DSS-control mice. The availability of α9*-selective conotoxins has opened new avenues in pharmacology research and potential targets in inflammatory disorders.

The Role of Nicotinic Receptors in the Attenuation of Autism-Related Behaviors in a Murine BTBR T + tf/J Autistic Model.

Nicotinic receptors are distributed throughout the central and peripheral nervous system. Postmortem studies have reported that some nicotinic receptor subtypes are altered in the brains of autistic people. Recent studies have demonstrated the importance of nicotinic acetylcholine receptors (nAChRs) in the autistic behavior of BTBR T + tf/J mouse model of autism. This study was undertaken to examine the behavioral effects of targeted nAChRs using pharmacological ligands, including nicotine and mecamylamine in BTBR T + tf/J and C57BL/6J mice in a panel of behavioral tests relating to autism. These behavioral tests included the three-chamber social interaction, self-grooming, marble burying, locomotor activity, and rotarod test. We examined the effect of various oral doses of nicotine (50, 100, and 400 mcg/mL; po) over a period of 2 weeks in BTBR T + tf/J mouse model. The results indicated that the chronic administration of nicotine modulated sociability and repetitive behavior in BTBR T + tf/J mice while no effects observed in C57BL/6J mice. Furthermore, the nonselective nAChR antagonist, mecamylamine, reversed nicotine effects on sociability and increased repetitive behaviors in BTBR T + tf/J mice. Overall, the findings indicate that the pharmacological modulation of nicotinic receptors is involved in modulating core behavioral phenotypes in the BTBR T + tf/J mouse model. LAY SUMMARY: The involvement of brain nicotinic neurotransmission system plays a crucial role in regulating autism-related behavioral features. In addition, the brain of the autistic-like mouse model has a low acetylcholine level. Here, we report that nicotine, at certain doses, improved sociability and reduced repetitive behaviors in a mouse model of autism, implicating the potential therapeutic values of a pharmacological intervention targeting nicotinic receptors for autism therapy. Autism Res 2020, 13: 1311-1334. © 2020 International Society for Autism Research, Wiley Periodicals, Inc.

CXC chemokine receptor 3 antagonist AMG487 shows potent anti-arthritic effects on collagen-induced arthritis by modifying B cell inflammatory profile.

Several studies have suggested that chemokine receptors are important mediators of inflammatory response in rheumatoid arthritis (RA). B cells are also known to play an important role in RA pathology. C-X-C chemokine receptor type 3 (CXCR3) is considered a potential therapeutic target in different inflammatory diseases; however, the mechanism remains unclear. Here, we evaluated the potentially protective effect of AMG487, a selective CXCR3 antagonist, in collagen-induced arthritis (CIA) mouse model. CIA mice were treated with AMG487 (5 mg/kg) every 48 h, from day 21 until day 41. We then investigated the effect of AMG487 on NF-κB p65-, NOS2-, MCP-1-, TNF-α-, IFN-γ, IL-4-, and IL-27-producing CD19+ B cells in the spleen through flow cytometry. We also evaluated the mRNA and protein expression levels of these molecules using RT-PCR and western blotting in the knee tissues. Our results revealed that AMG487-treated mice showed decreased NF-κB p65-, NOS2-, MCP-1-, and TNF-α-, and increased IL-4-, and IL-27-producing CD19+ B cells compared with the control mice. Additionally, AMG487 treatment significantly down regulated NF-κB p65, NOS2, TNF-α, and IFN-γ, and upregulated IL-4 and IL-27 mRNA and protein expression levels compared with the control. Thus, our study shows that AMG487 exerts its anti-arthritic effect by potently downregulating inflammatory B cell signaling. Based on our observations, we propose that AMG487 could serve as a potential novel therapeutic agent for inflammatory and autoimmune diseases, including RA.

Inhibition of tyrosine kinase signaling by tyrphostin AG126 downregulates the IL-21/IL-21R and JAK/STAT pathway in the BTBR mouse model of autism.

Autism spectrum disorder (ASD) comprises a broad range of neurodevelopmental disorders that are associated with deficits in social interaction and communication. The tyrosine kinase inhibitor tyrphostin AG126 represents a promising therapeutic agent for several neuroinflammatory disorders. There are currently no treatments available that can improve ASD and we previously showed that AG126 treatment exerts beneficial effects on BTBR T+ Itpr3tf/J (BTBR) mice, a model for autism that shows the core features of ASD; however, the immunological mechanisms and molecular targets associated with this effect were previously unclear. This study was undertaken to delineate the neuroprotective effect of AG126 on BTBR mice. Here, using this mouse model, we investigated the effects of AG126 administration on IL-21R, IL-21, IL-22, TNF-α, NOS2, STAT3, IL-27, and Foxp3 production by CD8+ T cells in the spleen by flow cytometry. We further explored the mRNA and protein expression of IL-21, IL-22, IL-1ß, TNF-α, NOS2, JAK1, STAT3, IL-27, and Foxp3 in brain tissue by RT-PCR, and western blotting. We found that BTBR mice treated with AG126 exhibited significant decreases in IL-21R-, IL-21-, IL-22-, TNF-α-, NOS2-, STAT3-producing, and increases in IL-27- and Foxp3-producing, CD8+ T cells. Our results further demonstrated that AG126 treatment effectively decreased IL-21, IL-22, IL-1ß, TNF-α, NOS2, JAK1, and STAT3, and increased IL-27 and Foxp3 mRNA and protein expression in brain tissues. Our findings suggest that AG126 elicits a neuroprotective response through downregulation of the IL-21/IL-21R and JAK/STAT pathway in BTBR mice, which could represent a promising novel therapeutic target for ASD treatment.

Therapeutic treatment with Ibrutinib attenuates imiquimod-induced psoriasis-like inflammation in mice through downregulation of oxidative and inflammatory mediators in neutrophils and dendritic cells.

Psoriasis is clinically characterized by well-demarcated silvery plaques which may appear on the extremities, scalp, and sacral area. The multidimensional interactions among innate immune cells [neutrophils and dendritic cells (DCs)], adaptive immune cells and skin resident cells result in characteristic features of psoriatic inflammation such as acanthosis, hyperkeratosis, and parakeratosis. Tec family kinases are involved in the pathogenesis of several inflammatory diseases. One of them is Bruton's tyrosine kinase (BTK) which is reported to carry out inflammatory and oxidative signaling in neutrophils and DCs. Effect of BTK inhibitor with regard to psoriatic inflammation has not been explored previously especially in a therapeutic setting. In the current investigation, effect of BTK inhibitor, Ibrutinib on oxidative/inflammatory signaling in dermal/splenic neutrophils [phosphorylated BTK (p-BTK), inducible nitric oxide synthase (iNOS), nitrotyrosine], CD11c + DCs (p-BTK, iNOS, nitrotyrosine, MCP-1, TNF-α) and enzymatic antioxidants [superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR)] in imiquimod (IMQ)-induced psoriatic inflammation was evaluated using therapeutic mode. Our results show that IMQ treatment led to induction of p-BTK expression along with concomitant increase in oxidative stress in neutrophils, and CD11c + DCs in skin/periphery. Therapeutic treatment with Ibrutinib caused attenuation of IMQ-induced oxidative stress in CD11c + DCs and neutrophils. Further there were dysregulations in antioxidants enzymes (SOD/GPx/GR) in the skin of IMQ-treated mice, which were corrected by Ibrutinib. In short, our study reveals that BTK signaling in neutrophils and CD11c + DCs upregulates oxidative stress which is concomitant with psoriatic inflammation in mice. Ibrutinib attenuates psoriasis inflammation through downregulation of oxidative stress in these innate immune cells.

The Stat3 inhibitor, S3I-201, downregulates lymphocyte activation markers, chemokine receptors, and inflammatory cytokines in the BTBR T+ Itpr3tf/J mouse model of autism.

Autism is a complex neurodevelopmental disorder with a high incidence rate. It is characterized by deficits in communication, a lack of social skills, cognitive inflexibility, and stereotypical behaviors. Autism has been gradually increasing in children over the past several years, without the existence of an effective treatment. BTBR T+ Itpr3tf/J (BTBR) mice serve as an accepted model to evaluate autistic-like behaviors as they display core behavioral symptoms displayed in autism. Previous findings showed that S3I-201, a selective Stat3 inhibitor, can be used to treat neuroinflammation disorders. Previously, we showed that S3I-201 treatment has therapeutic effects on autism-like behaviors, and Th1/Th17 and regulatory T cells in BTBR mice. The objective of the present study was to further explore the role of S3I-201 in BTBR mice, and this was performed by investigating the effects of S3I-201 treatment on lymphocyte activation markers (CD4+CD25+ and CD4+CD69+), chemokine receptors (CD4+CCR6+, CD4+CCR7+, CD4+CXCR4+, and CD4+CXCR5+), and proinflammatory cytokines (CD4+IL-6+ and CD4+TNF-α+) in the spleen cells of BTBR and C57BL/6 (C57) mice. The mRNA and protein expression levels of CD69, CCR6, CCR7, CXCR4, CXCR5, IL-1ß, IL-6, and TNF-α were examined in the brain tissues, and in BTBR mice, a significant decrease in CD25, CD69, CCR6, CCR7, CXCR4, CXCR5, IL-6, and TNF-α producing CD4+ T cells was observed. The present findings suggest that treatment with S3I-201 may be a therapeutic approach to improve immune abnormalities in a subgroup of autistic subjects.