Alpha Production Research Articles

Epicatechin suppresses the expression of C-C motif chemokine ligand 19 and ameliorates periodontitis

Periodontitis is a prevalent chronic inflammatory disease requiring the development of drug-based therapies. Epicatechin is a major polyphenol in cocoa extract and possesses numerous pharmacological properties. This study aimed to investigate the suppressive effect of epicatechin on C-C motif chemokine ligand 19 (CCL19)-mediated periodontitis using a mouse model. CCL19 expression was high in mouse gingiva with ligature-induced periodontitis. CCL19 expression was increased by lipopolysaccharide stimulation in murine gingival fibroblasts ESK-1 cells, and CCL19 induced the production of tumor necrosis factor alpha and interleukin-1 beta in mouse macrophage-like RAW264.7 cells. Importantly, lipopolysaccharide-enhanced CCL19 expression in ESK-1 cells was significantly suppressed by pretreatment with epicatechin. In addition, epicatechin administration significantly reduced alveolar bone resorption, and expression of Ccl19 and proinflammatory cytokines in the inflamed gingiva in high-fat diet-fed mice. These results demonstrate that epicatechin protects against periodontitis by reducing CCL19 levels, which may be a promising countermeasure for periodontitis in clinical practice.

Phthalocyanine derivative attenuates TNF-α production in macrophage culture and prevents alveolar bone loss in experimental periodontitis.

This study investigated the activity and mechanism of action of the iron tetracarboxyphthalocyanine (FeTcPc) on tumor necrosis factor alpha (TNF-α) production and its impact on experimental periodontitis. RAW 264.7 macrophages were treated with FeTcPc, activated with lipopolysaccharide (LPS) at 10 ng/mL, and the TNF-α levels were measured, as well as the nuclear factor kappa B (NF-κB) activation. Subsequently, a mouth gel containing 1% FeTcPc was topically administered to the gingival tissue of mice with periodontitis-induced ligatures. Bone loss and the gene expression of Tnfα, p65 (NF-κB), and receptor-activating nuclear factor kappa B ligand (Rankl) were quantified in gingival tissue. Finally, the systemic toxicity of FeTcPc was estimated in Galleria mellonella larvae. In an activated RAW 264.7 macrophage culture, 100 μM FeTcPc reduced TNF-α release and NF-κB activation. Regarding experimental periodontitis, topical application of mouth gel containing 1% FeTcPc blocked alveolar bone loss. Additionally, 1% FeTcPc reduced the expression of Tnfα, p65 (NF-κB), and Rankl in gingival tissue. Finally, administration FeTcPc at doses ranging from 1 to 1000 mg/kg did not cause acute systemic toxicity in G. mellonella. Overall, we demonstrated the potential of mouth gel containing FeTcPc as a therapeutic strategy for managing osteolytic inflammatory disorders, such as periodontitis.

Diaporthe foeniculina and D. eres, in addition to D. ampelina, may cause Phomopsis cane and leaf spot disease in grapevine.

Phomopsis cane and leaf spot (PCLS) disease, affecting grapevines (Vitis vinifera and Vitis spp.), has been historically associated with Diaporthe ampelina. Typical disease symptoms, comprising bleaching and black pycnidia, have also been associated with other Diaporthe spp. In this study, we conducted a molecular identification of the Diaporthe isolates isolated from grapevine canes from different geographic areas of southern Europe showing PCLS symptoms. Then, we investigated their morphological characteristics (including mycelium growth and production of pycnidia and alpha and beta conidia) in response to temperature. Finally, we artificially inoculated grapevine shoots and leaves with a subset of these isolates. Based on our results, PCLS etiology should be reconsidered. Though D. ampelina was the most crucial causal agent of PCLS, D. eres and D. foeniculina were also pathogenic when inoculated on green shoots and leaves of grapevines. However, D. rudis was not pathogenic. Compared to D. ampelina, D. eres and D. foeniculina produced both pycnidia and alpha conidia at lower temperatures. Thus, the range of environmental conditions favorable for PCLS development needs to be widened. Our findings warrant further validation by future studies aimed at ascertaining whether the differences in temperature requirements among species are also valid for conidia-mediated infection since it could have substantial practical implications in PCLS management.

The angiogenic role of the alpha 9-nicotinic acetylcholine receptor in triple-negative breast cancers.

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors α9 (α9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of α9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of α9-nAChRs in breast cancers, we generated a novel anti-α9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (α9 BsAb) for dissecting the molecular mechanism on α9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of α9-nAChR in nicotine-induced neovascularization of tumors. It revealed α9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in Matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of α9-nAChR in nicotine-mediated angiogenesis, the α9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2α), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting α9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting α9-nAChRs was designed and used to silence α9-nAChR expression and then evaluated the angiogenic role of α9-nAChRs. The results showed α9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, α9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for treatments of the α9 positive cancers.

Concurrent Ascaris infection modulates host immunity resulting in impaired control of Salmonella infection in pigs.

Ascaris is one of the most widespread helminth infections, leading to chronic morbidity in humans and considerable economic losses in pig farming. In addition, pigs are an important reservoir for the zoonotic salmonellosis, where pigs can serve as asymptomatic carriers. Here, we investigated the impact of an ongoing Ascaris infection on the immune response to Salmonella in pigs. We observed higher bacterial burdens in experimentally coinfected pigs compared to pigs infected with Salmonella alone. The impaired control of Salmonella in the coinfected pigs was associated with repressed interferon gamma responses in the small intestine and with the alternative activation of gut macrophages evident in elevated CD206 expression. Ascaris single and coinfection were associated with a rise of CD4-CD8α+FoxP3+ Treg in the lymph nodes draining the small intestine and liver. In addition, macrophages from coinfected pigs showed enhanced susceptibility to Salmonella infection in vitro and the Salmonella-induced monocytosis and tumor necrosis factor alpha production by myeloid cells was repressed in pigs coinfected with Ascaris. Hence, our data indicate that acute Ascaris infection modulates different immune effector functions with important consequences for the control of tissue-invasive coinfecting pathogens.IMPORTANCEIn experimentally infected pigs, we show that an ongoing infection with the parasitic worm Ascaris suum modulates host immunity, and coinfected pigs have higher Salmonella burdens compared to pigs infected with Salmonella alone. Both infections are widespread in pig production and the prevalence of Salmonella is high in endemic regions of human Ascariasis, indicating that this is a clinically meaningful coinfection. We observed the type 2/regulatory immune response to be induced during an Ascaris infection correlates with increased susceptibility of pigs to the concurrent bacterial infection.

The role of MNK1-mTORC1 pathway in modulating macrophage responses to Vibrio vulnificus infection.

Mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) plays a role in promoting the production of tumor necrosis factor alpha and interleukin-6 in macrophages during Vibrio vulnificus (Vv) infection. Inhibition or knockout of MNK1 in J774A.1 cells resulted in reduced cytokine production without affecting their transcription levels. MNK1 also impairs phagocytosis, bacterial clearance, and phagosome acidification in Vv-infected cells through the MNK1-mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. The findings highlight the importance of the MNK1-mTORC1 pathway in modulating macrophage responses to Vv infection.

Exploring In Vitro Immunomodulatory Properties of Moss Atrichum undulatum Extracts.

Bryophytes are rich sources of diverse secondary metabolites with a wide range of biological activities, including anti-inflammatory, antitumor and antimicrobial effects. The aim of this study was to investigate the chemical composition of extracts from two different genotypes (Serbian and Hungarian) of the axenic moss Atrichum undulatum and evaluate the immunomodulatory potential of the prepared extracts in vitro. Both genotypes of moss samples were cultivated in vitro and subsequently extracted in a Soxhlet apparatus with methanol or ethyl acetate. The highest concentration of total phenolic compounds was found in the methanolic extract of the Serbian genotype (54.25 mg GAE/g extract), while the ethyl acetate extract of the Hungarian genotype showed the highest concentration of phenolic acids (163.20 mg CAE/extract), flavonoids (35.57 mg QE/extract), and flavonols (2.25 mg QE/extract). The extracts showed anti-neuroinflammatory properties by reducing the production of reactive oxygen species, nitric oxide, and tumor necrosis factor alpha by lipopolysaccharide-stimulated microglial cells. Moreover, they mitigated the cytotoxic effects of the pro-inflammatory mediators produced by activated microglia on neurons. The data obtained suggest that extracts from A. undulatum moss have promising anti-neuroinflammatory and neuroprotective properties, making them interesting candidates for further research to combat neuroinflammation.

Investigating the impact of Mahā Mantra chanting on anxiety and depression : An EEG Rhythm Analysis Approach

Hare kṛṣṇa hare kṛṣṇa kṛṣṇa kṛṣṇa hare hare hare rāma hare rāma rāma rāma hare hare is the Mahā Mantra (MM). The Mantra is believed to be the sacred mantra from the ancient times. In the current research, an attempt has been made to study the brain dynamics of oscillatory changes after MM chanting. The mantra chanting produces a state of mind that has a positive effect on the individual’s brain. The electroencephalogram (EEG) signals are asynchronous in nature and record the brain's present state. In this study, forty individuals were assigned randomly into two (pre &post) groups,(n=20) with equal ratio of male and female and having mean age 25.5 yr. EEG signal was recorded in eyes closed condition both prior as well as after the MM chanting. The rhythms (Delta, theta, alpha, beta, and gamma) were extracted from the recorded EEG signals. To match the behavioural changes in the nature of the EEG, the power of the rhythm was computed and compared. According to the study, chanting MM leads to increase in alpha relative power in the central and parietal regions of the brain, which indicates that the brain has been refreshed and relieved. The study showed generous production of alpha, theta and delta waves which has been found in the study to be associated with the secretion of hormones or neurotransmitters causing dilation of blood vessels. As per literature it has been found that increase in relative alpha band power is associated with the secretion of hormones or neurotransmitters causing dilation of blood vessels which is further associated with the improvement in the mental well-being and feeling relaxed and releived, thus leading to decrease in anxiety and depression. The current research study proves the impact of the MM chanting leads to mental alleviation and mental well-being to a great extent.

Regulation of interferon alpha production by MAGUK-family protein CASK in H5N1-infected Macrophage

Regulation of interferon alpha production by MAGUK-family protein CASK in H5N1-infected Macrophage

Effects of Anti-Fibrotic Drugs on Transcriptome of Peripheral Blood Mononuclear Cells in Idiopathic Pulmonary Fibrosis.

Two anti-fibrotic drugs, pirfenidone (PFD) and nintedanib (NTD), are currently used to treat idiopathic pulmonary fibrosis (IPF). Peripheral blood mononuclear cells (PBMCs) are immunocompetent cells that could orchestrate cell-cell interactions associated with IPF pathogenesis. We employed RNA sequencing to examine the transcriptome signature in the bulk PBMCs of patients with IPF and the effects of anti-fibrotic drugs on these signatures. Differentially expressed genes (DEGs) between "patients with IPF and healthy controls" and "before and after anti-fibrotic treatment" were analyzed. Enrichment analysis suggested that fatty acid elongation interferes with TGF-β/Smad signaling and the production of oxidative stress since treatment with NTD upregulates the fatty acid elongation enzymes ELOVL6. Treatment with PFD downregulates COL1A1, which produces wound-healing collagens because activated monocyte-derived macrophages participate in the production of collagen, type I, and alpha 1 during tissue damage. Plasminogen activator inhibitor-1 (PAI-1) regulates wound healing by inhibiting plasmin-mediated matrix metalloproteinase activation, and the inhibition of PAI-1 activity attenuates lung fibrosis. DEG analysis suggested that both the PFD and NTD upregulate SERPINE1, which regulates PAI-1 activity. This study embraces a novel approach by using RNA sequencing to examine PBMCs in IPF, potentially revealing systemic biomarkers or pathways that could be targeted for therapy.

Blood Indices of the Patients with β-Thalassemia Minor Compared to the Patients with β-Thalassemia Minor-Alpha-Thalassemia

Objective: Thalassemia, as one of the most common genetic diseases, is a group of hereditary hemoglobin disorders due to a slight disturbance in the production of alpha and beta globin chains in the structure of hemoglobin occurs. There are still no clear criteria for differentiating thalassemia types based on hematological findings. In the current study, we aimed to evaluate the low-grade beta-thalassemia (β-thalassemia) indices in comparison with β-thalassemia minor with alpha-thalassemia (α-thalassemia).
 Methods and materials: In this descriptive-analytic study, 120 patients were enrolled, including 80 patients with minor β-thalassemia and 40 patients with minor β-thalassemia with α-thalassemia. Of all patients, 5cc blood samples were taken. The red blood cell parameters including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and hemoglobin were determined. The level of MCV> 80 and A2> 3.5 β-thalassemia minor and MCV <80 and A2 <3.5 were considered as elevated thalassemia or iron deficiency anemia.
 Results: The results showed that the mean of hemoglobin, HCT, MCH, MCHC, and MCV in the β-thalassemia group was significantly lower than that of the β-thalassemia with α-thalassemia group (P <0.0001). On the other hand, the level of these indices in the control group was significantly higher than in the two groups of patients (P<0.0001). The results showed that the percentage of hemoglobin A2 in the β-alpha-thalassemia group was 4.5 ± 0.91, significantly higher than the β-thalassemia group. The rate of hemoglobin and MCV was significantly lower in the β-thalassemia group compared to the silent and trait β+αthalassemia group. Also, the rate of hematocrit was significantly lower in the β-thalassemia group compared to the trait, although had no significant difference with the silence group.
 Conclusion: Based on our findings, despite the difference between some hematocrit indices in the patients with β-thalassemia and β-αthalassemia, these indices cannot be used as differential indices.

DNA corona on nanoparticles leads to an enhanced immunostimulatory effect with implications for autoimmune diseases

Autoimmune and inflammatory diseases are highly complex, limiting treatment and the development of new therapies. Recent work has shown that cell-free DNA bound to biological microparticles is linked to systemic lupus erythematosus, a prototypic autoimmune disease. However, the heterogeneity and technical challenges associated with the study of biological particles have hindered a mechanistic understanding of their role. Our goal was to develop a well-controlled DNA-particle model system to understand how DNA-particle complexes affect cells. We first characterized the adsorption of DNA on the surface of polystyrene nanoparticles (200 nm and 2 µm) using transmission electron microscopy, dynamic light scattering, and colorimetric DNA concentration assays. We found that DNA adsorbed on the surface of nanoparticles was resistant to degradation by DNase 1. Macrophage cells incubated with the DNA-nanoparticle complexes had increased production of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We probed two intracellular DNA sensing pathways, toll-like receptor 9 (TLR9) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), to determine how cells sense the DNA-nanoparticle complexes. We found that the cGAS-STING pathway is the primary route for the interaction between DNA-nanoparticles and macrophages. These studies provide a molecular and cellular-level understanding of DNA-nanoparticle-macrophage interactions. In addition, this work provides the mechanistic information necessary for future in vivo experiments to elucidate the role of DNA-particle interactions in autoimmune diseases, providing a unique experimental framework to develop novel therapeutic approaches.

12 Impact of progesterone signaling on tumor necrosis factor-alpha synthesis by luteal macrophages

Abstract Understanding the mechanism in which luteolysis occurs is an important step in improving the reproduction of livestock in the industry. The corpus luteum (CL) is a transient endocrine organ found on the ovary responsible for progesterone secretion. It is well known that prostaglandin F2-alpha (PGF) is responsible for luteal regression, however, the process by which luteal cells (LC) become sensitive to the luteolytic effects of PGF remain unclear. Therefore, the objective of this study was to determine if suppression of progesterone (P4) synthesis or blocking P4 signaling activates tumor necrosis factor (TNF)-alpha production by the luteal macrophages. Ovaries were collected from an abattoir and transported to the laboratory on ice. Ovary status (i.e. approximate days post ovulation), was determined by phenotypic description of ovarian structures. Only ovaries (n = 5) containing midcycle (d 12/13 post ovulation) CL were used in these experiments. Luteal tissue was isolated and dissociated to collect LC and and luteal macrophages (MAC). Isolated cells were incubated independently at 37°C at 5% CO2 for 24 h in 24-well plates. Luteal cells and MACs were then harvested and placed in the following conditions: 1) LC + MAC + DMSO (control), 2) LC + MAC + trilostane (TRIL 1.0mg/mL), 3) LC + MAC + TRIL 1.5 mg/mL, 4) MACS only, 5) MACS +LPS. Supernatant was harvested at 24 h whereas both cells and supernatant were harvested after 48 h, in vitro. Supernatant was immediately frozen for subsequent P4 analysis, via RIA. Data results were analyzed using a MIXED procedure of SAS and evaluated the effects of cell-type, treatment, and culture time. Statistical significance was determined at P < 0.05 and a tendency at 0.05 < P < 0.10. Overall, TRIL treatment reduced supernatant P4 concentration (8.3 and 6.8 ng/mL to 1.0 and 1.5 mg/mL TRIL, respectively) compared with controls (22.8 ng/mL; P = 0.0012); however there was not a difference when comparing the TRIL treated cultures. Progesterone concentrations tended to decrease over time (P = 0.0661). At 24 h in vitro, P4 concentrations were suppressed in 1.0 mg/mL TRIL and 1.5 mg/mL TRIL treated wells (5.3 and 4.2 ng/mL, respectively), compared with controls (16.4 ng/mL; P = 0.0014). Whereas P4 concentrations were reduced in the 1.5 mg/mL TRIL treated wells (1.6 ng/mL) compared with controls (9.95 ng/mL; P = 0.0419) and tended to differ in the1.0 mg/mL TRIL treated wells (2.4 ng/mL; P = 0.0634), at 48 h in vitro. Progesterone concentrations were undetectable in wells containing MAC only. Collectively, TRIL treatment effectively reduced P4 synthesis in LC cultures. Therefore, the greater concentration of TRIL is needed for P4 suppression in longer term culture systems. Additional analyses will be conducted to determine the effect of P4 suppression on macrophage function.

Chitosan and Sodium Hyaluronate Hydrogels Supplemented with Bioglass for Bone Tissue Engineering.

The aim of the study was to produce biocomposites based on chitosan and sodium hyaluronate hydrogels supplemented with bioglasses obtained under different conditions (temperature, time) and to perform an in vitro evaluation of their cytocompatibility using both indirect and direct methods. Furthermore, the release of ions from the composites and the microstructure of the biocomposites before and after incubation in simulated body fluid were assessed. Tests on extracts from bioglasses and hydrogel biocomposites were performed on A549 epithelial cells, while MG63 osteoblast-like cells were tested in direct contact with the developed biomaterials. The immune response induced by the biomaterials was also evaluated. The experiments were carried out on both unstimulated and lipopolysaccharide (LPS) endotoxin-stimulated human peripheral blood cells in the presence of extracts of the biocomposites and their components. Extracts of the materials produced do not exhibit toxic effects on A549 cells, and do not increase the production of proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin (IL-6) by blood cells in vitro. In direct contact with MG63 osteoblast-like cells, biocomposites containing the reference bioglass and those containing SrO are more cytocompatible than biocomposites with ZnO-doped bioglass. Using two testing approaches, the effects both of the potentially toxic agents released and of the surface of the tested materials on the cell condition were assessed. The results pave the way for the development of highly porous hydrogel-bioglass composite scaffolds for bone tissue engineering.

Influence of alpha-glutamil-tryptophan on the background and induced activity of factors of adaptive immunity for prevention

Background. During the ongoing COVID-19 pandemic and in the season of rising incidence of other respiratory infections, it is relevant to use preventive measures of non-specific prophylaxis. Synthetic peptides are widely considered as a tool. The representative of this group is the synthetic analogue of thymus regulatory peptides Thymogen, which has been used in Russia for more than 20 years in the treatment of acute and chronic infection diseases.The aim of the study. To evaluate the effect of Thymogen, a dosed nasal spray, on induced parameters of the immune system during prophylactic use in healthy volunteers.Materials and methods. Twenty healthy volunteers received Thymogen nasal dosed spray (JSC “Cytomed”, Russia) at a dose of 25 μg twice a day for 10 days. A comparative assessment of immunological parameters was carried out in dynamics: before the start of therapy, on days 6 and 11 of taking the drug and 14 days after the end of the course. Clinical observation was carried out from day 1 to day 11, registration of adverse events – the entire period of the study for 24 days. The first day was considered the day the drug was started.Results. In the course of the work, according to the data of immunological examination, a statistically significant increase in the virus-induced production of interferon alpha (INF-α) by a culture of peripheral blood cells was revealed. The growth rate was recorded on day 11 of taking Thymogen and persisted for 14 days after the end of the course. Significant differences in the dynamics of bactericidal and phagocytic activity of neutrophils, serum α- and γ-interferon were not obtained.Conclusion. The use of Thymogen spray at a dose of 25 μg for 10 days was safe and contributed to a significant induction of interferon-alpha in response to exposure to a viral pathogen, which allows us to recommend the drug for prophylactic use during the period of rising incidence of acute respiratory diseases.

Feeding spray-dried plasma to broilers early in life improved their intestinal development, immunity and performance irrespective of mycotoxins in feed.

Fungi that produce mycotoxins can grow on certain food products, such as grains and feed, and can cause a variety of health issues if consumed by animals, including chickens. The use of spray-dried plasma (SDP) is one strategy for combating the health problems caused by mycotoxins. In the present study, Ross 308 chickens (n = 960) were divided into four treatment groups. T1 group was given a control diet (corn-soybean meal), T2 group was given a control diet +2% SDP, T3 group was given a control diet +2% SDP + mixture mycotoxins and T4 group was givena control diet + mycotoxin mixture. The presence of SDP resulted in weight gain and decreased feed efficiency, whereas mycotoxins resulted in weight loss and increased feed efficiency. SDP increased the thymus' relative weight. The presence of mycotoxins increased the heterophile/lymphocyte ratio. The presence of mycotoxins reduced the production of IL-2 and macrophage inflammatory protein-3 Alpha (MIP-3a), whereas the presence of SDP increased the production of macrophage colony-stimulating Factor (M-CSF). SDP resulted in higher IgA concentrations in the intestinal and tracheal washes than mycotoxin. Finally, adding SDP to broiler diets boosts weight gain, feed efficiency, and immune system development. Our results provide information supporting that SDP is a promising tool for improving poultry immunity and performance.

Antibody-dependent enhancement and neutralization against CVB4 investigated in vitro and in silico through an agent-based model.

The infection with coxsackievirus B4 (CVB4) can be enhanced in vitro by antibodies directed against the viral capsid protein VP4. In peripheral blood mononuclear cells, antibody-dependent enhancement (ADE) of CVB4 infection leads to the production of interferon alpha (IFN-α). To investigate ADE of CVB4-induced production of IFN-α, an agent-based model was constructed with enhancing and neutralizing antibodies. The model recapitulates viral neutralization and ADE in silico. The enhancing and neutralizing activities of serum samples were evaluated in vitro to confront the model predictions with experimental results. Increasing the incubation time of CVB4 with serum samples improves virus neutralization in silico as well as in vitro. It also results in ADE at lower antibody numbers in silico, which is confirmed in vitro with IFN-α production at lower serum concentrations. Furthermore, incubation of CVB4 with serum at a low temperature does not induce IFN-α production in vitro. Thus, taken together our results suggest that enhancing antibodies bind cryptic epitopes, more accessible with longer incubation time and at higher temperature due to changes in capsid conformation, consistent with previous results indicating that enhancing antibodies are anti-VP4 antibodies.

Human CD4+CD25+ T cells expressing a chimeric antigen receptor against aberrant superoxide dismutase 1 trigger antigen-specific immunomodulation

Background aimsAmyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs’ immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate. MethodsA CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days. ResultsThe CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39. ConclusionsTaken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.

Bioactivity-Guided Fraction from Viscera of Abalone, Haliotis discus hannai Suppresses Cellular Basophils Activation and Anaphylaxis in Mice.

Basophils and mast cells are specialized effector cells in allergic reactions. Haliotis discus hannai (abalone), is valuable seafood. Abalone male viscera, which has a brownish color and has not been previously reported to show anti-allergic activities, was extracted with acetone. Six different acetone/hexane fractions (0, 10, 20, 30, 40, and 100%) were obtained using a silica column via β-hexosaminidase release inhibitory activity-guided selection in phorbol myristate acetate and a calcium ionophore, A23187 (PMACI)-induced human basophils, KU812F cells. The 40% acetone/hexane fraction (A40) exhibited the strongest inhibition of PMACI-induced-β-hexosaminidase release. This fraction dose-dependently inhibited reactive oxygen species (ROS) production and calcium mobilization without cytotoxicity. Western blot analysis revealed that A40 down-regulated PMACI-induced MAPK (ERK 1/2, p-38, and JNK) phosphorylation, and the NF-κB translocation from the cytosol to membrane. Moreover, A40 inhibited PMACI-induced interleukin (IL)-1β, IL-6, and IL-8 production. Anti-allergic activities of A40 were confirmed based on inhibitory effects on IL-4 and tumor necrosis factor alpha (TNF-α) production in compound (com) 48/80-induced rat basophilic leukemia (RBL)-2H3 cells. A40 inhibited β-hexosaminidase release and cytokine production such as IL-4 and TNF-α produced by com 48/80-stimulated RBL-2H3 cells. Furthermore, it's fraction attenuated the IgE/DNP-induced passive cutaneous anaphylaxis (PCA) reaction in the ears of BALB/c mice. Our results suggest that abalone contains the active fraction, A40 is a potent therapeutic and functional material to treat allergic diseases.

Essential oil and its nanoemulsion of Eucalyptus cladocalyx: chemical characterization, antioxidant, anti-inflammatory and anticancer activities

ABSTRACT Formulating a nanoemulsion (NE) of essential oil (EO) could enhance its efficiency while requiring lower concentrations. Eucalyptus cladocalyx F. Muell EO was rich in monoterpenes hydrocarbons. NE was prepared and the effect of surfactant (Tween 20, 40 and 80) and shearing time were investigated. The results showed that the best NE was formed using Tween 80 after 25 min of emulsification. Small droplet size (40 nm), low polydispersity index PDI (0.49), and stable zeta potential highlighted the excellent NE stability which was tested under storage conditions for 4 months. The results showed that the antioxidant and anticancer activities of NE were enhanced compared to free EO. Furthermore, NE and EO exhibited high anti-inflammatory effects by inhibiting nitric oxide (NO), Interleukin 6 (IL-6), and tumor necrosis factors alpha (TNF-α) production in liposaccharides (LPS)-induced RAW264.7 cells. In conclusion, a stable Eucalyptus cladocalyx-NE was produced, with improved biological activities.