Mitogen-induced Proliferation Research Articles

In vitro analysis on the adhesion potential of Lactiplantibacillus plantarum from infant faeces and its gastrointestinal localization, growth promotion, and immunomodulation in Wistar rats: a preliminary study.

Lactic acid bacteria, found in heterogenous niches, are known for their health-endorsing properties and are in demand as prospective probiotics. Hence, the scientific community around the globe is in continuous search for novel and new potential strains with extensive applicability and minimum risk. In this context, the present study evaluated the efficiency of Lactiplantibacillus plantarum (P2F2) of human origin, a highly autoaggregating and coaggregating (with pathogens) strain, for its colonization, growth promotion, and immunomodulation. Results indicated moderate hydrophobicity on adhesion to xylene and n-hexadecane and weak electron-donating properties with chloroform. The biofilm of P2F2 formed on polystyrene was strong and highly correlated to exopolysaccharide production. The autoaggregation was moderately correlated with hydrophobicity and biofilm production. It was noted that the P2F2 strain modulated the gut microbiota and increased intestinal villi length in Wistar rats. The lipid and glucose profiles remained intact. P2F2 treatment increased the activity of reactive oxygen species-generating cells in the peritoneal cavity, besides augmenting the mitogen-induced splenocyte proliferation and maintained the immunoglobulins at the normal level. Results from this study conclusively suggest that the strain P2F2 adheres to the intestine and modulates the gut ecosystem besides enhancing cell-mediated immunity without altering the serological parameters tested.

Oral administration of tilapia hydrolysate peptides ameliorates the immune-related side effects of cyclophosphamide in BALB/c mice

Bioactive peptide derived from fish is a novel and promising immunomodulating agent. However, the use of tilapia peptides as adjuvant therapy to ameliorate chemotherapy-induced immunosuppression in vivo has not been previously studied. In this study, tilapia hydrolysate peptides (THP) with a low molecular weight distribution (<3 kDa) were obtained from tilapia processing byproducts using Flavourzyme hydrolysis. These peptides showed no cytotoxicity to RAW264.7 macrophage cells and splenocytes. THP promoted the proliferation of RAW264.7 macrophage cells under basal conditions and enhanced mitogen-induced splenocyte proliferation. When treated with LPS-induced RAW264.7 macrophage cells, THP exhibited anti-inflammatory properties by inhibiting iNOS expression and NO production. To evaluate the in vivo immunomodulatory activity of THP, BALB/c mice were assigned to four groups (n = 7) and received either water (Control and CY group) or THP at doses of 400 mg/kg (Low-THP) and 800 mg/kg (High-THP) body weight through oral gavage for total 12 days. All groups, except the control, were injected with 100 mg/kg BW of cyclophosphamide (CY) on day 7. The high dose of THP positively influenced the recovery of peripheral white blood cell counts. Both doses of THP improved the proliferation ability of splenic T cells and immune organ indices. Additionally, the results of pro-inflammatory gene expression and histological examination suggested that both doses of THP alleviated CY-induced spleen damage. Consequently, oral administration of THP effectively ameliorated the immune-related side effects of CY, including reductions of peripheral white blood cell count, inhibition of splenic lymphocytes proliferation, atrophy of the spleen and thymus, and spleen damage.

Study of the immunotoxic properties of pegylated hyaluronidase

The development of safe drugs occupies a special place in the pharmaceutical industry. One of the main tasks of its preclinical phase is to evaluate possible toxic effects of the developed drug on the body and on various systems, including the immune system. The aim of our work was to study immunotoxic properties of pegylated hyaluronidase (PEG- HYAL). Material and methods. Mice F1(CBA/C57Bl/6) were divided into subgroups which were intragastric and intraperitoneally administered with PEG-HYAL in different dosages (50, 500, 1250, 2500 and 5000 U/kg). The number of antibody-producing cells in the spleen, the mass and cellularity of central and peripheral immune organs, phagocytic activity of peritoneal macrophages and neutrophils, delayed hypersensitivity reaction (DHR), level of hemagglutinin to sheep erythrocytes, spontaneous and mitogen-induced splenocyte proliferation were determined. Results. PEG-HYAL did not induce DHR, did not suppress phagocyte activity of peritoneal macrophages, at a dose of 50 ED/kg did not significantly affect the hemagglutinin content to erythrocytes, but at a dose of 500 ED/kg did statistically significantly reduce the titer of specific antibodies. When experimental animals were exposed to PEG-HYAL at doses of 50 and 500 U/kg, spontaneous and mitogen-induced proliferation of splenocytes decreased. Conclusions. The PEG-HYAL trial produced results that can be used to substantiate the administration of the PEG-HYAL-based medication.

Effects of 2-acetyl-4-tetrahydroxybutylimidazole exposure during gestation and lactation on maternity and offspring immune function in Balb/c mice

2-Acetyl-4-tetrahydroxybutylimidazole (THI), a by-product of Class Ⅲ caramel color, is generally recognized to cause lymphopenia in mammals. However, it remains unknown whether THI exposure during gestation and lactation causes damage to the immune system of offspring. In this study, pregnant Balb/c mice were gavaged with 0, 0.5, 2.5 and 12.5 mg/kg THI from gestation day (GD) 6 to postanal day (PND) 21, after which we treated another batch of dams from GD6 to PND21 and the offspring for 3 weeks after weaning with 0, 2, 10, 50 mg/L THI in drinking water respectively, and investigated the immunological anomalies of dams and offspring. The results showed that lymphopenia was observed in dams but not in weaning pups on PND21, which were exposed to THI during gestation and lactation. 2 mg/L THI and 2.5 mg/kg THI began to cause a remarkable reduction of the numbers of white blood cells and lymphocytes in dams. Besides both the cellular and the humoral immune response was not affected in weaning pups, which were measured by plaque-forming cell (PFC) assay and delayed-type hypersensitivity (DTH) assay respectively. Furthermore, THI could be detected in the plasma of dams with a dose-dependent manner, but not in that of both female and male weaning pups. In both male and female offspring being treated with 10 and 50 mg/L THI for another 3 weeks after weaning, lymphocytopenia was observed and T lymphocytes including CD4+ and CD8+ cells were significantly reduced in their spleens except lymph nodes. 10 and 50 mg/L THI treatment increased CD4+ and CD8+ single positive cells in thymus of female and male weaning mice. Mitogen-induced proliferation ability of T cells in the spleen and lymph nodes was impaired in female weaning mice exposed 50 mg/L THI, while male weaning mice treated with 10 and 50 mg/L THI showed impairment in the spleen but not lymph nodes. Based on the results in this study, no observed adverse effect level (NOAEL) for 3-week THI treatment in weaning mice was considered to be 2 mg/L (0.30 mg/kg bw for female mice and 0.34 mg/kg bw for male mice). And NOAEL for THI treatment in dams might be set to 0.5 mg/kg bw/day. Collectively from the perspective of NOAEL, offspring are not more sensitive than dams or adult mice.

Human Regulatory Macrophages Derived from THP-1 Cells Using Arginylglycylaspartic Acid and Vitamin D3.

Regulatory macrophages (Mregs) are unique in that they have anti-inflammatory and immunosuppressive properties. Thus, treating inflammatory diseases using Mregs is an area of active research. Human Mregs are usually generated by culturing peripheral blood monocytes stimulated using a macrophage colony-stimulating factor with interferon (IFN)-γ. Herein, we generated Mregs with an elongated cell morphology from THP-1 cells that were stimulated with phorbol 12-myristate 13-acetate and cultured with both arginylglycylaspartic acid and vitamin D3. These Mregs regulated macrophage function, and respectively downregulated and upregulated the expression of pro-inflammatory and immunosuppressive mediators. They also expressed Mregs-specific markers, such as dehydrogenase/reductase 9, even when exposed to such inflammatory stimulants as IFN-γ, lipopolysaccharide, purified xenogeneic antigen, and xenogeneic cells. The Mregs also exerted anti-inflammatory and anticoagulatory actions in response to xenogeneic cells, as well as exerting immunosuppressive effects on mitogen-induced Jurkat T-cell proliferation. Our method of generating functional Mregs in vitro without cytokines is simple and cost-effective.

Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation

BackgroundDiacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I).MethodsWe assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed.ResultsDGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition.ConclusionsOur study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.

Bioactive compounds in Layacha brown rice (Oryza sativa L.) improve immune responses in mice via activation of transcription factor Nrf2

Dysregulated or weak immunity is caused by ageing, chemo-radiotherapy, COVID-19, infections, steroids, pollutants and toxins. Bioactive foods are required for boosting immunity. In the present study, eight rice (Oryza sativa L.) varieties were selected from the repository of 23,250 Indian germplasm accessions. The immunomodulatory effects of these rice varieties were assessed in vitro and in vivo. Layacha-rice-methanolic-extract (Larimex) significantly enhanced innate (>20%) and adaptive (>10%) immune responses evinced from higher bacterial phagocytosis by macrophages, increased mitogen-induced T-cell proliferation (11%) and scavenged reactive oxygen species (ROS) (70–80%). Larimex activated transcription factor Nrf2 and its downstream genes NQO1, HO1 and Txnrd1 in immune cells. Larimex significantly improved immune responses (>20%) only in cells from wild type but not Nrf2 knock-out mice indicating its causal role in boosting immunity. Untargeted metabolomics of Layacha rice showed preponderance of metabolic pathways and bioactive compounds, which activate Nrf2 in mammalian cells. Layacha rice can be a suitable food for boosting immunity.

Immunoregulatory actions of calf thymus extract (TFX®) in vitro in relation to its effect on expression of mitogen activated protein kinases.

The in vitro immunotropic actions of a calf thymus extract - thymus factorX(TFX®) preparation were investigated. The preparation did not lower the viability of the A549 epithelial cell line and mouse bone marrow cells in the investigated concentration range. TFX® exhibited a co-stimulatory action of concanavalin A (Con A)-induced mouse thymocyte proliferation and partially restored the mitogen-induced proliferation capability of mouse thymocytes exposed to hydrocortisone (HC). The preparation also inhibited Herpes virus-1 (HSV-1) replication in A549 cells when preincubated with the virus and when added to the infected cells. In addition, it weakly inhibited lipopolysaccharide (LPS)-induced TNF α, IL-1β and IL-6 by the THP-1 monocyte cell line. The determination of mitogen activated protein kinase (MAPK) expression in Jurkat T cells revealed strong increases in ERK-2 kinase and p38α subunits. In WEHI 231 immature B cells, TFX® elevated p38α, and had a particularly strong elevating effect on p38γ. In HL-60 myeloblastic cells, the expression of p38α, β and γ was not detectable, almost blocked for p38δ and JNK, but accompanied by an increase in ERK-1. In turn, the effects of TFX® in J744E macrophages resulted in a strong increase in p38γ expression, moderate elevations of ERK and a drop in p38δ. Significant increases in MAPK expression were also found in cells from the lymphoid organs. In the bone marrow cell population, p38α, β and γ, in thymocytes p38α, γ and δ, and in splenocytes p38β and γ, subunit expression was elevated. We conclude that the changes in MAPK expression may be attributed to cell maturation and differentiation, and explain the beneficial therapeutic effects of TFX®.

Malabaricone C, a constituent of spice Myristica malabarica, exhibits anti-inflammatory effects via modulation of cellular redox

The present study primarily focuses on the efficacy of Malabaricone C (Mal C) as an anti-inflammatory agent. Mal C inhibited mitogen-induced T-cell proliferation and cytokine secretion. Mal C significantly reduced cellular thiols in lymphocytes. N-acetyl cysteine (NAC) restored cellular thiol levels and abrogated Mal C-mediated inhibition of T-cell proliferation and cytokine secretion. Physical interaction between Mal C and NAC was evinced from HPLC and spectral analysis. Mal C treatment significantly inhibited concanavalin A-induced phosphorylation of ERK/JNK and DNA binding of NF-κB. Administration of Mal C to mice suppressed T-cell proliferation and effector functions ex vivo. Mal C treatment did not alter the homeostatic proliferation of T-cells in vivo but completely abrogated acute graft-versus-host disease (GvHD)-associated morbidity and mortality. Our studies indicate probable use of Mal C for prophylaxis and treatment of immunological disorders caused due to hyper-activation of T-cells.

Investigating anti-inflammatory and immunomodulatory properties of brivaracetam and lacosamide in experimental autoimmune encephalomyelitis (EAE)

PurposeInflammation plays a role in drug-resistant epilepsy (DRE). We have previously reported an increased proportion of CD4 T cells displaying a pro-inflammatory profile in the peripheral blood of adults with DRE. Specific anti-epileptic drugs (AEDs) exhibit immunomodulatory properties that could increase the risk of infections but also contribute to their beneficial impact on DRE and other neurological diseases. The impact of novel generation AEDs on the profile of immune cells and on neuroinflammatory processes remains unclear. MethodsWe compared the influence of brivaracetam and lacosamide on the activation of human and murine peripheral immune cells in vitro and in vivo in active experimental autoimmune encephalomyelitis (EAE), a common mouse model of central nervous system inflammation. ResultsWe found that brivaracetam and lacosamide at 2.5 μg/ml did not impair the survival and activation of human immune cells, but a higher dose of 25 μg/ml decreased mitogen-induced proliferation of CD8 T cells in vitro. Exposure to high doses of brivaracetam, and to a lesser extent lacosamide, reduced the proportion of CD25+ and CD107a+ CD8+ human T cells in vitro, and the frequency of CNS-infiltrating CD8+ T cells at EAE onset and CD11b+ myeloid cells at peak in vivo. Prophylactic administration of brivaracetam or lacosamide did not delay EAE onset but significantly improved the clinical course in the chronic phase of EAE compared to control. ConclusionNovel generation AEDs do not impair the response to immunization with MOG peptide but improve the course of EAE, possibly through a reduction of neuroaxonal damage.

The 22nd Chromatography Component of the Fasciola gigantica Excretory-Secretory Products Decreased the Proliferation of Peripheral Blood Mononuclear Cells from Buffalo.

The 22nd chromatography component (F22) of the Fasciola gigantica excretory-secretory products (FgESP) shows better diagnostic value than the FgESP, and diagnostic methods based on F22 have also been established. Thus, exploring its immunomodulatory function and potential as a molecular vaccine candidate is attractive. In the present study, the effect of F22 on the mitogen-induced proliferation of buffalo peripheral blood mononuclear cells (PBMCs) in the innate immune response was preliminarily studied using the FgESP as a control. PBMCs were incubated with concanavalin A (ConA) and phytohemagglutinin (PHA) at optimal (1 µg/well) or suboptimal (0.25 µg/well) doses coupled with FgESP and F22 at different doses (1-16 µg/well). Cell proliferation was then assessed by microenzyme reaction colorimetry (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay). In addition, the components of F22 were also explored by mass spectrometry and then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to infer their functions. The results indicated that FgESP decreased the proliferation of PBMCs stimulated with ConA and PHA at specific doses, whereas F22 significantly decreased the proliferation of PBMCs stimulated with ConA and PHA at both optimal and suboptimal doses (p < 0.05). Two hundred and sixteen proteins were identified in F22, and these included 86 proteins that could be assigned to more than one pathway and some with robust immunomodulatory ability. Further studies should be performed to investigate the immunomodulatory function of F22 in the adaptive immune response, and the components of F22 can be further studied as potential vaccine candidate molecules.

Divergent Roles of Ephrin-B2/EphB4 Guidance System in Pulmonary Hypertension.

Smooth muscle cell (SMC) expansion is one key morphological hallmark of pathologically altered vasculature and a characteristic feature of pulmonary vascular remodeling in pulmonary hypertension. Normal embryonal vessel maturation requires successful coverage of endothelial tubes with SMC, which is dependent on ephrin-B2 and EphB4 ligand-receptor guidance system. In this study, we investigated the potential role of ephrin-B2 and EphB4 on neomuscularization in adult pulmonary vascular disease. Ephrin-B2 and EphB4 expression is preserved in smooth muscle and endothelial cells of remodeled pulmonary arteries. Chronic hypoxia-induced pulmonary hypertension was not ameliorated in mice with SMC-specific conditional ephrin-B2 knockout. In mice with global inducible ephrin-B2 knockout, pulmonary vascular remodeling and right ventricular hypertrophy upon chronic hypoxia exposure were significantly diminished compared to hypoxic controls, while right ventricular systolic pressure was unaffected. In contrast, EphB4 receptor kinase activity inhibition reduced right ventricular systolic pressure in hypoxia-induced pulmonary hypertension without affecting pulmonary vascular remodeling. Genetic deletion of ephrin-B2 in murine pulmonary artery SMC, and pharmacological inhibition of EphB4 in human pulmonary artery smooth muscle cells, blunted mitogen-induced cell proliferation. Loss of EphB4 signaling additionally reduced RhoA expression and weakened the interaction between human pulmonary artery smooth muscle cells and endothelial cells in a three-dimensional coculture model. In sum, pulmonary vascular remodeling was dependent on ephrin-B2-induced Eph receptor (erythropoietin-producing hepatocellular carcinoma receptor) forward signaling in SMC, while EphB4 receptor activity was necessary for RhoA expression in SMC, interaction with endothelial cells and vasoconstrictive components of pulmonary hypertension.

Comparison of mitogen-induced proliferation in child and adult healthy groups by flow cytometry revealed similarities.

The proliferation of antigen-specific lymphocyte clones, the initial step in acquired immunity, is vital for effector functions. Proliferation tests both in immunology research and diagnosis are gaining attendance gradually, while the use of adult healthy individuals as controls of pediatric patients is a question. This study aimed to investigate and compare mitogen-stimulated proliferation responses of total lymphocytes and T- and B-lymphocyte subsets in adult and children healthy donors. Nineteen children and 20 adult healthy donors were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) purified from peripheral blood samples of the donors, by Ficoll gradient centrifugation, were stained with CFSE and were cultured in a 37℃ CO2 incubator for 120h with the absence or existence of polyclonal activators: PHA and CD-Mix. After cell culture, PBMCs were stained with monoclonal antibodies against CD4 and CD19, and proliferation percentages of CD4+ T and CD19+ B cells, together with total lymphocytes were determined by flow cytometry. This study revealed similarities between children and adult age groups, concerning mitogenic stimulation of the lymphocytes. The only difference was a significantly high proliferation of pediatric CD4+ T cells in response to PHA. CD4+ T cell responses against PHA were inversely correlated with altering age. When pediatric individuals were distributed into age groups of 0-2years, 3-5years, and 6-18years, PHA responses of CD4+ cells were found to be diminished with advancing age. These findings propose the possibility of enrollment of adult healthy individuals as controls for pediatric patients.

Early-life maternal deprivation affects the mother-offspring relationship in domestic pigs, as well as the neuroendocrine development and coping behavior of piglets.

Early-life adversity may have programming effects on the psychological and physiological development of offspring. Domestic pigs (Sus scrofa) are an excellent model species for studying these effects because of their many physiological similarities to humans. Piglets from 10 sows were subjected to daily 2-h maternal deprivation on postnatal days (PND) 2–15 alone (DA) or in a group of littermates (DG). Control piglets (C) from 10 sows stayed with their mothers. Mother-offspring interaction, milk oxytocin, and cortisol were analyzed. An open-field/novel-object (OF/NO) test was performed with piglets on PNDs 16 and 40. Plasma cortisol and immune parameters were determined on PND 5 and 16. Two piglets from each group and sow were sacrificed on PND 20 and stress-related gene expression in the limbic system and prefrontal cortex (PFC), as well as splenic lymphocyte proliferative abilities, were examined. The milk cortisol of sows increased during the first separation of mother and offspring on the second day of lactation, whereas milk oxytocin did not change. The increase in cortisol by the OF/NO test on PND 16 was greater in C piglets than in DA and DG ones. DA piglets showed less agitated behavior than DG and C piglets in the OF/NO test at PND 16, but appeared more fearful. On PND 40, DA piglets showed more arousal than DG and C piglets in the OF/NO test. Neither plasma IgA nor N/L ratios in blood nor mitogen-induced proliferation of spleen lymphocytes were affected by deprivation. We found a higher mRNA expression of CRHR1 in the hypothalamus and a higher expression of MR in the hippocampus in DA piglets than in DG ones. The expression of GR, MR, and CRHR1 genes in the PFC was reduced by maternal deprivation, however, the expression of arginine vasopressin and oxytocin receptors was not affected. Repeated maternal deprivation induces sustained effects on stress reactivity and behavior of domestic piglets. Some of these effects were buffered by the presence of littermates. In addition, we found sex-specific differences in behavior and gene expression.

Baicalin protected mice against radiation-induced lethality: A mechanistic study employing in silico and wet lab techniques

Baicalin is a main active ingredient of the dried root of Scutellaria and has been extensively employed in Traditional Chinese Medicine for the treatment of asthma, fever, and psoriasis. Based on the reports of antioxidant, anti-inflammatory, anti-infection, and anti-tumor activities of baicalin, we have explored its radioprotective efficacy using in vitro and in vivo experimental model systems. In the present study, we have investigated the radioprotective, immunomodulatory, and anti-inflammatory properties of baicalin using wet lab and in silico approaches. It was observed that pre-treatment of murine splenic lymphocytes with baicalin protected cells against radiation-induced cell death possibly by decreasing the cellular reactive oxygen species levels. Prophylactic oral administration of baicalin offered significant increase in endogenous spleen colony counts and an enhancement in the survival of mice. We have also observed that baicalin suppressed mitogen-induced splenic lymphocyte proliferation and IL-2 production. It also inhibited the production of nitric oxide in RAW 264.7 cells in response to elicitation of lipopolysaccharide. Further, in silico study was performed to evaluate the possible mechanism of radioprotection and immunomodulation by selecting different pro-inflammatory mediators such as COX2, Lck, NIK, and IKK-β which have a significant role in radioprotection, lymphocyte activation, and inflammation. Our molecular docking and molecular dynamics study show that baicalin has a significant predicted binding affinity with COX2, Lck, NIK, and IKK-β. These in silico results can explain the experimentally observed radioprotective, immunosuppressive, and anti-inflammatory properties of baicalin. Thus, radioprotection offered by baicalin may be because of its antioxidant, anti-inflammatory, and immunomodulatory properties.

Photoperiodic regulation of the splenocyte immune responses in the fresh water snake, Natrixpiscator

Photoperiod and melatonin are important regulators of immunity. We hypothesized that these two factors play an important role in the regulation of immune responses in the Natrix piscator. Animals were kept in either short or long days and splenocyte immune responses were studied. Respiratory burst activity of splenocytes was assessed through reduction of nitrobluetetrazolium salt while production of nitric oxide was assessed indirectly by nitrite assay. Density gradient centrifugation was used to isolate splenic lymphocytes which were utilized to study proliferation with and without mitogens. Super oxide production by splenocytes was reduced significantly in the cultures obtained from animals kept either in short or long days. Nitrite release was decreased when animals were subjected to long days. The photoperiodic alterations acted differentially on proliferations of the splenic lymphocytes. Spontaneous and mitogen-induced proliferation of splenic lymphocytes were enhanced in cultures obtained from snakes maintained in short days when compared with cultures from snakes obtained either from long day or natural day length conditions. In vitro melatonin significantly enhanced the splenic lymphocyte proliferation of the cultures obtained from animals kept in long days when compared with splenic lymphocyte proliferations of the cultures obtained from long day animals or the animals kept in natural day length conditions. We found evidence which suggest that photoperiod may influence seasonal energy budgets and induce adjustments which optimize energy allocation for costly physiological processes such as immune function. In seasonally breeding animals such as Natrix piscator, the pineal hormone melatonin assists in the suppression of reproduction and elevation of immunity, which are the crucial adaptation for perpetuation of species.

Excessive Accumulation of Intracellular Ca2+ After Acute Exercise Potentiated Impairment of T-cell Function.

Ca2+ is an important intracellular second messenger known to regulate several cellular functions. This research aimed to investigate the mechanisms of exercise-induced immunosuppression by measuring intracellular calcium levels, Ca2+-regulating gene expression, and agonist-evoked proliferation of murine splenic T lymphocytes. Mice were randomly assigned to the control, sedentary group (C), and three experimental groups, which performed a single bout of intensive and exhaustive treadmill exercise. Murine splenic lymphocytes were separated by density-gradient centrifugation immediately (E0), 3h (E3), and 24h after exercise (E24). Fura-2/AM was used to monitor cytoplasmic free Ca2+ concentration in living cells. The combined method of carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry was used for the detection of T cell proliferation. The transcriptional level of Ca2+-regulating genes was quantified by using qPCR. Both basal intracellular Ca2+ levels and agonist (ConA, OKT3, or thapsigargin)-induced Ca2+ transients were significantly elevated at E3 group (p<0.05 vs. control). However, mitogen-induced cell proliferation was significantly decreased at E3 group (p<0.05 vs. control). In parallel, the transcriptional level of plasma membrane Ca2+-ATPases (PMCA), sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), TRPC1, and P2X7 was significantly downregulated, and the transcriptional level of IP3R2 and RyR2 was significantly upregulated in E3 (p<0.01 vs. control). In summary, this study demonstrated that acute exercise affected intracellular calcium homeostasis, most likely by enhancing transmembrane Ca2+ influx into cells and by reducing expression of Ca2+-ATPases such as PMCA and SERCA. However, altered Ca2+ signals were not transduced into an enhanced T cell proliferation suggesting other pathways to be responsible for the transient exercise-associated immunosuppression.

Dysregulated retinoic acid signaling in airway smooth muscle cells in asthma.

Vitamin A deficiency has been shown to exacerbate allergic asthma. Previous studies have postulated that retinoic acid (RA), an active metabolite of vitamin A and high-affinity ligand for RA receptor (RAR), is reduced in airway inflammatory condition and contributes to multiple features of asthma including airway hyperresponsiveness and excessive accumulation of airway smooth muscle (ASM) cells. In this study, we directly quantified RA and examined the molecular basis for reduced RA levels and RA-mediated signaling in lungs and ASM cells obtained from asthmatic donors and in lungs from allergen-challenged mice. Levels of RA and retinol were significantly lower in lung tissues from asthmatic donors and house dust mite (HDM)-challenged mice compared to non-asthmatic human lungs and PBS-challenged mice, respectively. Quantification of mRNA and protein expression revealed dysregulation in the first step of RA biosynthesis consistent with reduced RA including decreased protein expression of retinol dehydrogenase (RDH)-10 and increased protein expression of RDH11 and dehydrogenase/reductase (DHRS)-4 in asthmatic lung. Proteomic profiling of non-asthmatic and asthmatic lungs also showed significant changes in the protein expression of AP-1 targets consistent with increased AP-1 activity. Further, basal RA levels and RA biosynthetic capabilities were decreased in asthmatic human ASM cells. Treatment of human ASM cells with all-trans RA (ATRA) or the RARγ-specific agonist (CD1530) resulted in the inhibition of mitogen-induced cell proliferation and AP-1-dependent transcription. These data suggest that RA metabolism is decreased in asthmatic lung and that enhancing RAR signaling using ATRA or RARγ agonists may mitigate airway remodeling associated with asthma.

Differential Leukocyte MicroRNA Responses Following Pan T Cell, Allorecognition and Allosecretome-Based Therapeutic Activation

Effective immunomodulation of T-cell responses is critical in treating both autoimmune diseases and cancer. Our previous studies have demonstrated that secretomes derived from control or methoxypolyethylene glycol mixed lymphocyte alloactivation assays exerted potent immunomodulatory activity that was mediated by microRNAs (miRNA). The immunomodulatory effects of biomanufactured miRNA-based allo-secretome therapeutics (SYN, TA1, IA1 and IA2) were compared to Pan T-cell activators (PHA and anti-CD3/CD28) and lymphocyte alloactivation. The differential effects of these activation strategies on resting peripheral blood mononuclear cells (PBMC) were assessed via T-cell proliferation, subset analysis and miRNA expression profiles. Mitogen-induced PBMC proliferation (> 85%) significantly exceeded that arising from either allostimulation (~ 30%) or the pro-inflammatory IA1 secretome product (~ 12%). Consequent to stimulation, the ratio of CD4 to CD8 cells of the resting PBMC (CD4:CD8; 1.7 ± 0.1) decreased in the Pan T cell, allrecognition and IA1 activated cells (averages of 1.1 ± 0.2; 1.2 ± 0.1 and 1.0 ± 0.1). These changes arose consequent to the expansion of both CD4+CD8+ and CD4–CD8– populations as well as the shrinkage of the CD4 subset and the expansion of the CD8 T cells. Importantly, these activation strategies induced vastly different miRNA expression profiles which were associated with significant differences in cellular differentiation and biological function. These findings support the concept that the “differential patterns of miRNA expression” regulate the biologic immune response in a “lock and key” manner. The biomanufacturing of miRNA-enriched secretome biotherapeutics may be a successful therapeutic approach for the systemic treatment of autoimmune diseases (TA1) and cancer (IA1).

Modulation of Donor Graft By the Steroidal Lactone Withaferin A Alleviates Post-Transplant Acute GVHD Via Nrf-2 Independent T-Reg Induction

Acute Graft Versus Host Disease (aGVHD), resulting from an immune mediated attack of donor cells against recipient's healthy tissues remains the bugbear of allogeneic Bone Marrow Transplantation (allo-BMT). Considering the heterogeneous nature of aGVHD, associated high incidence of morbidity and mortality, severe side effects and limited success of current immunosuppressive prophylactic regimens, we were encouraged to develop a novel, safe and easy-to-use intervention for prophylaxis of aGVHD.Our group, for the first time ever, employed an approach of donor graft modulation with Withaferin A (WA) prior to allogeneic transplantation to decrease the allo-reactivity of donor lymphocytes for mitigating post-transplant aGVHD. WA is a steroidal lactone isolated from the medicinal plant Withania somnifera . Our previous studies have shown the immune-suppressive efficacy of WA on T lymphocytes in vitro {Gambhir et al Toxicol Appl Pharmacol 2015; 289: 297}. Extending those findings, we demonstrate the anti-GVHD potential of WA in a murine model of aGVHD.Transient (2h) treatment of lymphocytes of C57Bl/6 mice with 1µM WA inhibited mitogen induced activation, proliferation and cytokine secretion (Figure 1A) in vitro without inducing significant cell death (≤10% at 1µM WA). In order to evaluate graft modulatory potential of WA for alleviating aGVHD in vivo, we validated and characterized a MHC mismatched and High Dose Myeloablative Chemo-Conditioning (HDMCC) based C57BL/6-to-BALB/c aGVHD model earlier reported by Sadeghi et al {Bone Marrow Transplant. 2008;42:807}, except we increased the splenocyte dose to 42*106 per recipient to intensify aGVHD manifestation. In this model, we observed early mortality with a median survival of 9 days (Figure 1B), clinically and histologically relevant characteristics of aGVHD accompanied by cytokine storm in recipients of HDMCC + allo-graft (n=33) compared to HDMCC alone (n=35).Infusion of donor graft incubated with 1µM WA in vitro for 2 h prior to transplantation abrogated the post-transplant cytokine storm. On day +3, levels of IFN-g, IL-1b, IL-2, IL-6, TNF-a and IL-17 were suppressed by 3.21, 3.18, 1.57, 3.80, 1.70 and 4.32 folds respectively in WA group compared to Vehicle Control (VC) group. Conversely levels of the anti-inflammatory cytokine, IL-10, increased by 1.86 fold in WA group as compared to VC group. The levels of IFN-g, IL-1b and IL-2 continued to be low on day +7 in WA group compared to VC group by 4.53, 1.39 and 1.93 folds respectively.Further,survival was significantly improved in recipients of allo-graft modulated with WA compared to VC [Hazard Ratio = 11.86 (7.164 to 50.06)] (Figure 1C). GVHD associated tissue damage was also markedly reduced in the WA group as compared to VC group (Figure 1D) . At the same time reconstitution of lymphoid, myeloid and erythroid lineages was also achieved in peripheral blood with complete and stable donor chimerism observed even at day +69. Clinical scoring of aGVHD using minor modification of Hill's method {Blood 1997; 90:3204} showed that mice from VC group had a median score of 11 (10-12) on a scale of 15, with moderate to severe clinical GVHD features on day +15. In contrast, mice from WA group, scored 1 (1-2) with mild clinical GVHD features (Figure 1E).In order to elucidate the cellular mechanism of action of WA, we studied its effect on T cell differentiation. We found that WA induced T-regulatory (Treg) differentiation in CD4+ T helper cells in vitro . WA is known to activate the transcription factor Nuclear Respiratory Factor-2 (Nrf-2) in a Keap1-independent and Pten/Pi3k/Akt-dependent mechanism. Using Nrf-2 knockout mice, we found that Treg induction by WA was independent of Nrf-2 (Figure 1F) .In conclusion, we are the first group to show pre-clinical evidence for therapeutic efficacy of WA by in vitro modulation of donor graft prior to transplantation. WA suppressed two causal events in GVHD pathophysiology namely, ‘immune-priming’ and ‘release of myriad cytokines’. In addition, WA also induced Treg differentiation in donor T cells resulting in improved overall survival and reduction in aGVHD associated morbidity without compromising the reconstitution of host immune system. We are currently investigating the GVL effect of WA modulated graft in order to successfully transfer this novel approach to the clinics.This work was supported by the Department of Biotechnology (6242 P61/RGCB/PMD/DBT/ARMR2015). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.