Stimulate Cytokine Production Research Articles

P.825 Twenty year follow-up study on antipsychotic treatment, physical morbidity, and mortality in a nationwide cohort of 62,250 patients with schizophrenia (FIN20)

Next to aluminum salts, squalene nanoemulsions comprise the most widely employed class of adjuvants in approved vaccines. Despite their importance, the mechanisms of action of squalene nanoemulsions are not completely understood, nor are the structure/function requirements of the oil composition.In this study, we build on previous work that compared the adjuvant properties of nanoemulsions made with different classes of oil structures to squalene nanoemulsion. Here, we introduce nanoemulsions made with polyprenols derived from species of the Pinaceae family as novel vaccine adjuvant compositions. In contrast with long-chain triglycerides that do not efficiently enhance an immune response, both polyprenols and squalene are comprised of multimeric isoprene units, which may represent an important structural property of oils in nanoemulsions with adjuvant properties.Oils derived from species of the Pinaceae family were formulated in nanoemulsions, with or without a synthetic Toll-like receptor 4 (TLR4) ligand, and characterized regarding physicochemical and biological activity properties in comparison to squalene nanoemulsions.Oils were extracted from species of the Pinaceae family and used to prepare oil-in-water nanoemulsions by microfluidization. Emulsion droplet diameter stability was characterized by dynamic light scattering. Nanoemulsions were evaluated for in vitro biological activity using human whole blood, and in vivo biological activity in mouse, pig, and ferret models when combined with pandemic influenza vaccine antigens.Nanoemulsions comprised of Pinaceae-derived polyprenol oils demonstrated long-term physical stability, stimulated cytokine production from human cells in vitro, and promoted antigen-specific immune responses in various animal models, particularly when formulated with the TLR4 ligand glucopyranosyl lipid adjuvant (GLA).Pinaceae-derived nanoemulsions are compatible with inclusion of a synthetic TLR4 ligand and promote antigen-specific immune responses to pandemic influenza antigens in mouse, pig, and ferret models.

Fish oil supplementation with various lipid emulsions suppresses in vitro cytokine release in home parenteral nutrition patients: a crossover study

Long-chain n-3 polyunsaturated fatty acids modulate immune cell functions. The primary objective of this study was to evaluate the impact of different lipid emulsions (LEs) with supplemented doses of fish oil (FO) on serum cytokine concentration and in vitro cytokine production in patients with intestinal failure on home parenteral nutrition (HPNPs). We hypothesized that FO supplementation would diminish lipopolysaccharide (LPS)-stimulated cytokine production. Twelve HPNPs receiving Smoflipid for at least 3 months were given FO (Omegaven) for a further 4 weeks. After this cycle, the patients were randomized to subsequently receive 1 cycle with Lipoplus and 1 cycle with ClinOleic for 6 weeks or vice versa plus 4 weeks of added Omegaven after each cycle in a crossover design. Comparison of the baseline LE regimens showed lower LPS-stimulated production of IL-1β in the HPNPs on Lipoplus than on the Smoflipid and ClinOleic regimens, as well as lower IL-8 compared to the Smoflipid regimen. Omegaven reduced IL-8 concentration in serum under the Lipoplus regimen and diminished LPS-stimulated production of IL-1β under the Smoflipid and ClinOleic. IL-6 and TNF-α production was depressed only in those on Smoflipid. Irrespective of the LE used, the HPNPs compared to the healthy controls showed higher IL-6, IL-8, and TNF-α concentrations in serum and LPS-stimulated production of IL-6 as well as lower n-6/n-3 long-chain polyunsaturated fatty acids in the erythrocyte phospholipids. LPS-stimulated production of IL-6 correlated negatively with the parenteral dose of eicosapentaenoic acid + docosahexaenoic acid. In conclusion, FO-supplemented parenteral nutrition suppresses in vitro cytokine production.

Pharmacologic studies of CDH17/CD3 bispecific antibodies to treat colorectal and pancreatic cancers.

116 Background: Immunotherapy has emerged as a new hope for cancer patients who do not respond to conventional therapies. In addition to the widely investigated immune checkpoint inhibitors such as PD-L1 antibody, bispecific T cell redirecting antibodies have also drawn much attention from pharmaceutical companies. The popularity of bispecific T cell engager has been increasing since the US FDA’s accelerated approval of blinatumomab for treatment of haematological malignancies. The clinical efficacy of such engagers in solid tumors have however remained to be demonstrated. Cadherin-17 (CDH17) is a promising immunotherapeutic target for gastrointestinal (GI) cancers. It is a highly tumor-specific antigen with restricted expression limited to the tight junction of intestine that is normally inaccessible to biologics. CDH17 overexpression correlates with tumor burden and poor prognosis. Methods: Fully humanized CDH17/CD3 bispecific antibodies were generated. T cell-mediated cytotoxicity was tested in vitro and in vivo. Safety was addressed in cynomolgus monkeys with intestinal CDH17 level equivalent to that of human. Results: One lead antibody, ARB202 exhibited high-affinity binding to CDH17 and CD3. In the presence of CDH17-positive pancreatic cancer cells, ARB202 stimulated in vitro IL-2 release in CD3/CD28-expanded PBMC. In the absence of tumor cells ARB202 stimulated cytokine production in PBMCs required over 600-fold greater concentrations. ARB202 specifically directed in vitro T cell killing of GI cancer cells expressing CDH17, but not of cells lacking CDH17 expression. In mouse xenograft models reconstituted with human immune cells, ARB202 demonstrated significant tumor growth inhibition with concomitant IL-2 response. No dose-dependent toxicities on cynomolgus monkeys were observed over a 3-log dose range. ARB202 displayed favourable in vivo pharmacokinetic profiles in mice and monkeys. ARB202 is a stable bispecific antibody retaining functional activity after incubation for a month at 37°C at pH6.0. Conclusions: ARB202 is a promising candidate for clinical trials. A proprietary cell line is generated for the pilot manufacturing of ARB202 for IND enabling studies.

Suppression of monosodium urate crystal-induced inflammation by inhibiting TGF-β-activated kinase 1-dependent signaling: role of the ubiquitin proteasome system.

Monosodium urate (MSU) crystals activate inflammatory pathways that overlap with interleukin-1β (IL-1β) signaling. However, the post-translational mechanisms involved and the role of signaling proteins in this activation are unknown. In the present study, we investigated the intracellular signaling mechanisms involved in MSU-induced activation of THP-1 macrophages and human nondiseased synovial fibroblasts (NLSFs) and the in vivo efficacy of an inhibitor of tumor growth factor-β (TGF-β)-activated kinase 1 (TAK1), 5Z-7-oxozeaenol, in MSU-induced paw inflammation in C57BL/6 mice. THP-1 macrophage activation with MSU crystals (25-200 µg/ml) resulted in the rapid and sustained phosphorylation of interleukin-1 receptor-activated kinase 1 (IRAK1 Thr209) and TAK1 (Thr184/187) and their association with the E3 ubiquitin ligase TRAF6. At the cellular level, MSU inhibited the deubiquitinases A20 and UCHL2 and increased 20s proteasomal activity, leading to a global decrease in K63-linked ubiquitination and increase in K48-linked ubiquitination in THP-1 macrophages. While MSU did not stimulate cytokine production in NLSFs, it significantly amplified IL-1β-induced IL-6, IL-8, and ENA-78/CXCL5 production. Docking studies and MD simulations followed by TAK1 in vitro kinase assays revealed that uric acid molecules are capable of arresting TAK1 in an active-state conformation, resulting in sustained TAK1 kinase activation. Importantly, MSU-induced proinflammatory cytokine production was completely inhibited by 5Z-7-oxozeaenol but not IRAK1/4 or TRAF6 inhibitors. Administration of 5Z-7-oxozeaenol (5 or 15 mg/kg; orally) significantly inhibited MSU-induced paw inflammation in C57BL/6 mice. Our study identifies a novel post-translational mechanism of TAK1 activation by MSU and suggests the therapeutic potential of TAK1 in regulating MSU-induced inflammation.

Domain 4 of pneumolysin from Streptococcus pneumoniae is a multifunctional domain contributing TLR4 activating and hemolytic activity

The pneumolysin (Ply) protein of Streptococcus pneumoniae is composed of four domains and possesses several different but related activities. In this study, recombinant Ply and two truncated forms, Ply domain 1-3 and Ply domain 4 (rPly4), were expressed and characterized regarding their participation in apoptosis, the stimulation of cytokine production, hemolytic activity and virulence. rPly4 activated murine bone marrow-derived dendritic cells in a Toll-like receptor (TLR) 4-dependent manner. The rPly4 alone was able to produce hemolytic activity at high concertation and penetrate the lipid bilayer. We further demonstrated that domain 4 of Ply involved in the virulence of the bacteria in mouse model. In the absence of apoptotic activity, the virulence level caused by rPly4 was similar to that of full length Ply. Our data suggested that domain 4 of Ply alone with TLR4 agonist and hemolytic activity may play roles in virulence of Streptococcus pneumoniae.

332 Young Scholar Presentation: regulation of immune signaling by extracellular vesicles

Abstract Exosomes and other extracellular vesicles traffic a variety of protein and nucleic acid cargo throughout the body. Immune cells especially utilize these vesicles for communication, and they are being studied in clinical settings for their potential to act as disease markers, but also to serve as drug delivery systems. In animal science, a large part of the research around exosomes has centered around their presence in milk, as well as their bioavailability and potential impact for the neonate and for humans; milk-derived exosomes are enriched with immune-related proteins and microRNA that may relate to maternal health, mammary function, or neonate development. We sequenced miRNA isolated from colostrum of dairy cows predicted to feature either moderate or high degree of immune suppression, but found minimal differences (2 differentially expressed miRNA of 343 miRNA analyzed) between the two groups. Immune suppression exhibited during early lactation may also be related to altered exosome-mediated communication between immune cells. We studied the ability of bovine exosomes to alter immune responses of primary bovine monocyte-derived macrophages and found that exosomes alone or in combination with LPS were able to stimulate cytokine production to a greater extent than LPS alone. The ability of exosomes to transport cytokine, bioactive lipid, and regulatory RNA cargo suggests potential involvement in transition cow health. Altered exosome content and function have been associated with uterine infection, mastitis, and metabolic dysfunction; future studies with exosome signaling may help clarify the complexities of transition cow immune function and point to strategies to support immunity.

A Brief Review about the Effect of Honey on Diabetic Foot Ulcer; from In Vitro Studies to Clinical Trials

Diabetes mellitus has many complications; one of the most important ones is diabetic foot ulcer (DFU). Due to the increasing prevalence of diabetes in the world and evidences of the role of honey in treatment of DFU, we decided to review the effect of honey on DFU. We searched the keywords of “honey”, “diabetic foot ulcer”, “treatment”, “diabetes mellitus”, “wound dressing” and “wound healing” in indexing systems of PubMed, Scopus and ISI Web of Science between 2007 to 1 Jun 2017, in English and Persian languages. Out of 120 articles published, 20 articles met the inclusion criteria, including one in vivo study, four in vitro studies and 15 human studies. The results showed the efficacy of honey as a wound healing agent in some types of wounds. Mechanisms of healing methods included hyper osmosis, stimulating cytokine production, producing transforming growth factor-β1 and etc. In some articles, honey was used in combination with other medications like Commiphora molmol, Nigella sativa and povidone iodine. Honey showed minor side effects, in some studies, like mild burning or minor sensation. Despite these complications, the treatment was not stopped and in some cases the disorder was resolved by changing the combination ingredients. Although, the reviewed studies showed healing effect for honey in DFU, more studies are needed to confirm the efficacy.

In vitro cytokine synthesis in unstimulated and mitogen-stimulated peripheral blood mononuclear cells from individuals with schizophrenia

Increasing evidence has shown that the immune system is involved in the schizophrenia development, with alterations in immune cell reactivity being one possible factor contributing to its pathogenesis. The purpose...

Vaccine adjuvant activity of emulsified oils from species of the Pinaceae family

BackgroundNext to aluminum salts, squalene nanoemulsions comprise the most widely employed class of adjuvants in approved vaccines. Despite their importance, the mechanisms of action of squalene nanoemulsions are not completely understood, nor are the structure/function requirements of the oil composition. PurposeIn this study, we build on previous work that compared the adjuvant properties of nanoemulsions made with different classes of oil structures to squalene nanoemulsion. Here, we introduce nanoemulsions made with polyprenols derived from species of the Pinaceae family as novel vaccine adjuvant compositions. In contrast with long-chain triglycerides that do not efficiently enhance an immune response, both polyprenols and squalene are comprised of multimeric isoprene units, which may represent an important structural property of oils in nanoemulsions with adjuvant properties. Study designOils derived from species of the Pinaceae family were formulated in nanoemulsions, with or without a synthetic Toll-like receptor 4 (TLR4) ligand, and characterized regarding physicochemical and biological activity properties in comparison to squalene nanoemulsions. MethodsOils were extracted from species of the Pinaceae family and used to prepare oil-in-water nanoemulsions by microfluidization. Emulsion droplet diameter stability was characterized by dynamic light scattering. Nanoemulsions were evaluated for in vitro biological activity using human whole blood, and in vivo biological activity in mouse, pig, and ferret models when combined with pandemic influenza vaccine antigens. ResultsNanoemulsions comprised of Pinaceae-derived polyprenol oils demonstrated long-term physical stability, stimulated cytokine production from human cells in vitro, and promoted antigen-specific immune responses in various animal models, particularly when formulated with the TLR4 ligand glucopyranosyl lipid adjuvant (GLA). ConclusionPinaceae-derived nanoemulsions are compatible with inclusion of a synthetic TLR4 ligand and promote antigen-specific immune responses to pandemic influenza antigens in mouse, pig, and ferret models.

Differential recognition of Candida tropicalis, Candida guilliermondii, Candida krusei, and Candida auris by human innate immune cells.

BackgroundThe deep-seated infections caused by the Candida genus are associated with a high mortality rate, and Candida albicans is the most frequent species associated with these diseases. The fungal wall is composed of macromolecules not synthesized by the host, and therefore is a source of ligands recognized by innate immune cells.MethodsWe performed a comparative study analyzing the cell wall composition and organization of Candida tropicalis, Candida guilliermondii, Candida krusei, and Candida auris, along with their ability to stimulate cytokine production and phagocytosis by human innate immune cells.ResultsWe found that the wall of these species had the basic components already described in C. albicans, with most of the chitin and b1,3-glucan located underneath the mannan layer. However, the walls of C. krusei and C. auris were rich in chitin and the former had a lower content of mannans. C. guilliermondii contained changes in the mannan and the b1,3-glucan levels. These species were differentially phagocytosed by human macrophages and stimulated cytokine production in a dectin-1-dependent pathway. C. krusei showed the most significant changes in the tested parameters, whereas C. auris behaved like C. albicans.ConclusionOur results suggest that the cell wall and innate immune recognition of C. tropicalis, C. guilliermondii, C. krusei, and Candida auris is different from that reported for C. albicans.

The role of senescence of bone marrow cells in acute kidney injury

BackgroundThe prevalence of acute kidney injury (AKI) in elderly patients has grown considerably. Age-associated changes in the immune system can be one of the critical factors determining AKI outcomes. This study aimed to investigate the role of senescence of bone marrow (BM)-derived cells in the development of AKI, focusing on the immune response.MethodsFemale 7-week-old C57BL/6 mice were irradiated and treated with BM cells from either 48-week-old or 8-week-old male mice. Ischemia-reperfusion injury (IRI) was induced, and their functional deterioration, histological tubular damage, and inflammatory responses were compared. For the in-vitro study, lipopolysaccharide (LPS)-stimulated cytokine production by BM cells from old and young mice were examined.ResultsAt 24 hours after IRI, there was no significant difference in the number of circulating immune cells between the mice transplanted with old or young BM cells. However, the mice with old BM cells showed less functional deterioration and histological tubular injury than those with young BM cells. Moreover, macrophage infiltration and renal cytokine interleukin (IL)-12 levels were lower in the mice with old BM cells at 24 hours post-IRI. Consistently, the in vitro study showed that LPS-induced production of cytokines interferon-γ, monocyte chemoattractant protein-1, and IL-10 was attenuated in cultured old BM cells, suggesting that age-related functional changes in these cells may lead to reduced inflammation in IRI.ConclusionImmunosenescence could affect the susceptibility and response to renal IRI. Further studies specifically addressing age-related alterations can help in the development of treatment strategies for elderly patients with AKI.

Prenatal immune challenge induces behavioral deficits, neuronal remodeling, and increases brain nitric oxide and zinc levels in the male rat offspring

Schizophrenia is a severe mental disorder with numerous etiological susceptibilities. Maternal infection is a key risk factor for schizophrenia. Prenatal lipopolysaccharide (LPS) infection stimulates cytokine production that affects brain development. In the present study, we aimed to investigate the effect of prenatal LPS injection at gestational day (GD) 14–16 on behavioral paradigms, and neuronal morphology in the prefrontal cortex (PFC), basolateral amygdala (BLA), nucleus accumbens (NAcc) and ventral hippocampus (VH) at two critical ages of development: pre-pubertal (postnatal day 35, PD35) and post-pubertal (PD60) age in male rats. We also evaluated the effects of LPS on nitric oxide (NO) and zinc (Zn) levels in seven brain areas (PFC, VH, amygdala, brainstem, striatum and dorsal hippocampus) at PD35 and PD60. LPS induced hyperlocomotion in a novel environment and reduced social contact as well as increased the levels of NO and Zn in the PFC, brainstem and amygdala as observed in other animal models of schizophrenia-related behavior. Furthermore, we found that LPS-treated rats presented post-pubertal neuronal hypertrophy in the PFC and BLA and decreased spine density in the NAcc. The neuronal morphology of neurons in the VH in LPS-treated rats remained unaltered. Interestingly, the anxiogenic-related behavior correlated with neuronal hypertrophy observed in the BLA. Our findings suggest that the behavioral and neural modifications observed in our model could be mediated by the long-lasting alterations in Zn and NO levels in the brain.

Psychopharmacology: neuroimmune signaling in psychiatric disease-developing vaccines against abused drugs using toll-like receptor agonists.

Since substance use disorders have few or no effective pharmacotherapies, researchers have developed vaccines as immune-therapies against nicotine, cocaine, methamphetamine, and opioids including fentanyl. We focus on enhancing antibody (AB) production through stimulation of toll-like receptor-5 (TLR5) during active vaccination. The stimulating adjuvant is Entolimod, a novel protein derivative of flagellin. We review the molecular and cellular mechanisms underlying Entolimod's actions on TLR5. Entolimod shows excellent efficacy for increasing AB levels to levels well beyond those produced by anti-addiction vaccines alone in animal models and humans. These ABs also significantly block the behavioral effects of the targeted drug of abuse. The TLR5 stimulation involves a wide range of immune cell types such as dendritic, antigen presenting, T and B cells. Entolimod binding to TLR5 initiates an intracellular signaling cascade that stimulates cytokine production of tumor necrosis factor and two interleukins (IL-6 and IL-12). While cytokine release can be catastrophic in cytokine storm, Entolimod produces a modulated release with few side effects even at doses 30 times greater than doses needed in these vaccine studies. Entolimod has markedly increased AB responses to all of our anti-addiction vaccines in rodent models, and in normal humans. Entolimod and TLR5 stimulation has broad application to vaccines and potentially to other psychiatric disorders like depression, which has critical inflammatory contributions that Entolimod could reduce.

Exercise Increases Mucosal-associated Invariant T Cell Cytokine Expression but Not Activation or Homing Markers.

To determine the MAIT cell response to submaximal exercise on activation and homing marker expression and stimulated cytokine production. Twenty healthy, young, recreationally active males cycled for 40 min at 86% of VT after an overnight fast. Peripheral blood mononuclear cells were isolated and labeled to identify specific MAIT cell populations using flow cytometry. Cytokine production after stimulation was also determined. Mucosal-associated invariant T cells were 2.9% of T cells and increased to 3.9% after exercise and with recovery whereas cell numbers significantly increased by 91.5% after exercise before returning to resting levels. Chemokine and activation marker absolute cell number significantly increased while expression levels remained constant but the high levels of CCR5 may help direct MAIT cells to sites of inflammation. After stimulation, TNFα expression significantly increased after exercise before returning to baseline with a similar trend for IFNγ. The MAIT cell numbers undergo a partial biphasic response after submaximal exercise and appear to be preferentially mobilized within T cells; however, the magnitude of the submaximal response was attenuated relative to maximal exercise. Stimulated MAIT cells increase TNFα expression, indicating greater responsiveness to pathogens after acute exercise.

Novel Therapeutic Approaches to Allosensitization and Antibody-mediated Rejection.

Modification of pathogenic antibodies for autoimmune diseases illuminated the biologic relevance of B cells, plasma cells, and pathogenic antibodies in autoimmunity. They have also rejuvenated interest in how B cells mediate multiple effector functions that include antibody production, antigen presentation to T cells, costimulation, and the production of immune stimulating and immune modulatory cytokines. Repurposing these drugs from autoimmunity and cancer immunotherapy has yielded important advancements in the care of antibody-mediated rejection patients and novel drug development aimed at HLA desensitization have recently emerged. We now stand on an important threshold that promises many advances in the care of our allosensitized patients. We hope that these initial advances will encourage basic scientist, clinical investigators, industry, National Institutes of Health, our academic societies, and the Food and Drug Administration to continue support of these important objectives. These advances clearly have implications for sensitized patients receiving solid organ transplants and antibody-mediated rejection treatment. Modification of alloimmunity and alloantibodies will also have relevance to xenotransplantation where the xenoantibodies present a formidable obstacle to advancement of this important therapy. Working together, we can advance transplant therapeutics where biologic agents are likely to play novel and important roles. Here, we discuss novel drugs emerging in this area.

RAGE Signaling in Skeletal Biology.

The receptor for advanced glycation end products (RAGE) and several of its ligands have been implicated in the onset and progression of pathologies associated with aging, chronic inflammation, and cellular stress. In particular, the role of RAGE and its ligands in bone tissue during both physiological and pathological conditions has been investigated. However, the extent to which RAGE signaling regulates bone homeostasis and disease onset remains unclear. Further, RAGE effects in the different bone cells and whether these effects are cell-type specific is unknown. The objective of the current review is to describe the literature over RAGE signaling in skeletal biology as well as discuss the clinical potential of RAGE as a diagnostic and/or therapeutic target in bone disease. The role of RAGE and its ligands during skeletal homeostasis, tissue repair, and disease onset/progression is beginning to be uncovered. For example, detrimental effects of the RAGE ligands, advanced glycation end products (AGEs), have been identified for osteoblast viability/activity, while others have observed that low level AGE exposure stimulates osteoblast autophagy, which subsequently promotes viability and function. Similar findings have been reported with HMGB1, another RAGE ligand, in which high levels of the ligand are associated with osteoblast/osteocyte apoptosis, whereas low level/short-term administration stimulates osteoblast differentiation/bone formation and promotes fracture healing. Additionally, elevated levels of several RAGE ligands (AGEs, HMGB1, S100 proteins) induce osteoblast/osteocyte apoptosis and stimulate cytokine production, which is associated with increased osteoclast differentiation/activity. Conversely, direct RAGE-ligand exposure in osteoclasts may have inhibitory effects. These observations support a conclusion that elevated bone resorption observed in conditions of high circulating ligands and RAGE expression are due to actions on osteoblasts/osteocytes rather than direct actions on osteoclasts, although additional work is required to substantiate the observations. Recent studies have demonstrated that RAGE and its ligands play an important physiological role in the regulation of skeletal development, homeostasis, and repair/regeneration. Conversely, elevated levels of RAGE and its ligands are clearly related with various diseases associated with increased bone loss and fragility. However, despite the recent advancements in the field, many questions regarding RAGE and its ligands in skeletal biology remain unanswered.

Immune Modulation in Pediatric Sepsis.

The initial host immune response to sepsis in children is characterized by a proinflammatory surge that can be associated with fever, capillary leak, and organ dysfunction. There is, however, a concurrent anti-inflammatory response that results in hyporesponsiveness of innate and adaptive immune cells. When severe, this response is termed immunoparalysis and is known to be associated with prolonged organ dysfunction, increased risk for nosocomial infection, and death in septic adults and children. Sepsis-induced immune suppression can be defined in the laboratory by reduced whole blood ex vivo - stimulated cytokine production capacities, reduced expression of human leukocyte antigen (HLA)-DR on circulating monocytes, and reduced absolute cell counts. While anti-inflammatory therapies have largely been unsuccessful at improving outcomes from adult and pediatric sepsis, the use of immunostimulatory therapies such as granulocyte macrophage colony-stimulating factor (GM-CSF) in patients with sepsis-induced immunoparalysis shows promise. A greater understanding of the risk factors for immunoparalysis along with the development and execution of immunophenotype-specific clinical trials of strategies to optimize innate and adaptive immune function are needed to further improve outcomes in septic children.

The Immunomodulatory Activity of Polysaccharides from the Medicinal Mushroom Amauroderma rude (Agaricomycetes) Is Mediated via the iNOS and PLA2-AA Pathways.

Our earlier work indicated that the polysaccharides of Amauroderma rude (AR) appear to have an effect on immunoregulation. However, the pathways are not clear. In this paper, we discuss the immunomodulatory mechanisms of A. rude to provide a scientific basis for its possible use as a food. Amauroderma rude increased the expression of iNOS and P38 in the Raw246.7 cell line. When the AR concentration reached 150 μg/mL, the expressions of iNOS and P38 increased 23.0% and 191.7%, respectively. When the AR concentrations were 50 μg/mL, the concentrations of cytokines IL-2, TNF-α, and IFN-γ were 33.65 pg/mL, 12.53 pg/mL and 42.56 pg/mL. When AR reached 200 μg/mL, the lgA and lgM levels were 0.73 μg/mL and 1.5 μg/mL. When AR reached 400 μg/mL, the lgG level reached 1.65 μg/mL by ELISA assay. When 4.8 mg AR were orally administered, IL-2, TNF-α, IFN-γ, PGE2, and LTB4 increased dramatically, to 0.17 pg/mL, 0.16 pg/mL, 0.15 pg/mL, 30.71 pg/mL, and 18.68 pg/mL, respectively. The concentrations of lgA, lgM, and lgG, AA, and PLA2 also increased significantly to 2.62 pg/mL, 2.14 pg/mL, 2.06 pg/mL, 5.23 μg/mL and 3.68 ng/mL, respectively. With 4.8 mg AR p.o., iNOS protein expression increased 16.8% and P38 increased 234.0%. These results indicate that A. rude polysaccharides stimulate cytokine production and activate the iNOS, PLA2-AA, and MAPK pathways during the immunomodulatory process.

Molecular profile of scpA and sdaB virulence genes in Streptococcus pyogenes isolated from pharyngitis

Group A Streptococci (GAS) or Streptococcus pyogenes is an important pathogen which causes a wide-ranging of diseases for human. This study was carried out in Ear Nose Throat (ENT) department in Al–Habboby Teaching Hospital, Thi-Qar province, south of Iraq during the period from October 2015 to April 2016. Two hundred and ten swabs were collected from patients infected with pharyngitis. 152 (72.3 %) showed positive growth with S. pyogenes. GAS isolates were subjected to detect two virulence genes (scpA and sdaB) by conventional PCR technique using specific primer pairs and DNA sequencing analysis. The sequencing of PCR products produced from bacterial DNA showed significant alignments identities (96-99%) to the S. pyogenes which are located in BLAST-NCBI Genbank. The six sequences of Streptococcus pyogenes scpA and sdaB genes determined in this study have been deposited in the GenBank under the accession numbers MF49318-MF497323. Phylogenetic analysis of S. pyogenes based upon the neighbour-joining of partial scpA and sdaB gene sequences showed that these sequences were derived from Streptococcal genes. In addition, S. pyogenes can produce several exotoxins that have the potential to damage the host tissues either directly or through the stimulation of cytokine production.

Gene Set Enrichment Analysis Suggests That Increased Rituximab-Mediated NK Cell Cytotoxicity after Vitamin D Substitution Is Driven By Upregulation of Interferon Alpha (IFN-a) Isoforms

Introduction: We recently showed that vitamin D deficiency leads to decreased overall survival of DLBCL-patients treated with rituximab-chemotherapy (Bittenbring et al, JCO, 2014). We hypothesized that rituximab-mediated NK cell-cytotoxicity is more effective at higher vitamin D levels. This was confirmed by vitamin D substitution of healthy volunteers, which increased their rituximab-mediated cytotoxicity in vitro against the Daudi lymphoma cell line. To unveil the molecular mechanisms behind this finding, resting NK cells before and after vitamin D supplementation were isolated from those volunteers and a whole transcriptome analysis was performed.Methods: We collected PBMCs from eight healthy volunteers with vitamin D deficiency before and after vitamin D substitution to > 30 ng/ml 25-OH vitamin D3. NK cells were isolated from PBMCs by magnetic depletion of all non-NK cells. Purity of the CD16+ cells was confirmed by flow cytometry. After isolating total RNA, we performed a microarray analysis using an Affymetrix Gene-Chip 2.0 ™. The signals were normalized using the LMA algorithm. For pathway analysis, gene set enrichment analysis (GSEA) was used. A two-step approach was chosen. Firstly, we separated 7.705 genes due to their involvement in the NK cell-mediated immune response according to the Gene Ontology database, irrespective of their differential expression. This dataset was used separately for specific analysis of the NK cell-cytotoxicity pathway to increase sensitivity. Secondly, the complete data set of 48.145 genes was used in an exploratory analysis in an attempt to screen for other dysregulated pathways involved in the immune response and vitamin D homeostasis. We used gene sets provided from the Molecular Signature Database. A significance level of < 0.05 for p and False Discovery Rate (FDR) was chosen. Real-time quantitative PCR was performed to confirm the results.Results: The NK cell-associated cytotoxicity pathway was found to be significantly upregulated after restoration of normal vitamin D levels in the specific analysis. The most significantly overexpressed genes in the gene set were five IFN-α subtypes (IFN-α2, IFN-α4, IFN-α6, IFN-α7, and IFN-α10) as well as IFN-κ. The exploratory analysis showed an upregulation of the response to type I interferon pathway and regulation of type I interferon mediated signaling pathway. The most upregulated genes in those pathways were again the IFN-α subtypes mentioned above. Other pathways involved in the immune response were found to be downregulated after vitamin D substitution, like interferon gamma response; cytokine production and chemotaxis. The common denominator of these pathways was the downregulation of three toll-like receptor genes (TLR-8, TLR-7, TLR-2).Conclusion: The increased expression of specific IFN-α subtypes could explain the increased rituximab-mediated NK cell-cytotoxicity after vitamin D substitution in deficient individuals. To the best of our knowledge, this is the first study to suggest a role for vitamin D in IFN-α regulation. TLRs are known to stimulate cytokine production in NK cells including IFN-α. It can be assumed, that the observed upregulation of IFN-α genes after vitamin D substitution leads to a negative feedback on positive regulators of cytokine production like TLR, causing their downregulation once vitamin D levels are restored. This implies a comprehensive role of vitamin D in IFN-α biosynthesis in human NK cells. DisclosuresStilgenbauer:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.