Th1-type Immune Response Research Articles

Role of dietary protein and exercise on biomarkers of immune activation in older patients during hospitalization.

The aim of this study was to examine the effect of short-term protein supplementation (aiming to consume 1.2g protein/kg body weight per day) combined with moderate resistance training on 3days of the week on tryptophan-kynurenine metabolism in 40 older patients with hip fracture. Secondary outcomes for physical recovery were hand-grip strength and chair-rise score. Older patients with hip fracture exhibited higher degrees of immune activation, detected by increased neopterin and kynurenine to tryptophan levels compared with reference values for healthy elderly with no significant differences between those who received the exercise-protein intervention compared to the control. Increasing dietary protein intake during hospitalization did not alleviate the Th1-type immune response in the elderly patient. On the other hand, muscularity per se may affect immune activation responses following injury, as improvements in maximum hand-grip strength with the intervention were related to decreases in neopterin levels.

462: Immunomodulatory activity of Vitamin D

Vitamin D is involved in several immune-regulatory pathways, having functional overlaps with progesterone. The link between the immunological actions of the two molecules might lie in the regulation of the vitamin D receptor in the T cells by progesterone. Progesterone induces a Th2 type immune response, suppressing cell-mediated anti-foetal response during pregnancy. Vitamin D exerts similar effects, by controlling naive T-cell maturation into Th1 - Th2 type, and Th17 cells. Several of the immunological effects of progesterone are manifested via its downstream mediator, called PIBF. The percentage of PIBF-positive lymphocytes is significantly reduced in women prone to pregnancy loss, preterm labour and threatened miscarriage. The aim of this study was to investigate, whether PIBF might be a link between the immunomodulatory effects of progesterone and vitamin D. PHA-activated peripheral lymphocytes, obtained from healthy women during the follicular and luteal phases of the cycle, were treated with different concentrations of progesterone or vitamin D for 24h. Another set of lymphocytes were subjected to a standard concentration of progesterone and increasing concentrations of vitamin D. PIBF production was detected by immunohistochemistry and Western blot. Vitamin D induces PIBF production in activated human peripheral lymphocytes in vitro. Lymphocytes taken during the luteal phase responded better to both progesterone and vitamin D than those taken during the follicular phase, suggesting that vitamin D acts more efficiently in progesterone-primed lymphocytes. In line with this, peripheral lymphocytes, treated with increasing concentrations of vitamin D together with a standard concentration of progesterone, produced significantly more PIBF than those treated with similar concentrations of vitamin D in the absence of progesterone. These findings indirectly confirm that progesterone induces VDR in the lymphocytes. For the first time, it is suggested that vitamin D, by inducing PIBF, cooperates with progesterone in regulating the immune response during pregnancy.

Heat-killed Mycobacterium paragordonae therapy exerts an anti-cancer immune response via enhanced immune cell mediated oncolytic activity in xenograft mice model.

A therapeutic strategy capable of skewing toward a Th1-type immune response is crucial for cancer treatment. Recently, we reported Mycobacterium paragordonae (Mpg) as a potential live vaccine for mycobacterium infections. In this study, we explored the immunotherapeutic potential of heat-killed Mpg (HK-Mpg) in a mouse tumor xenograft model and elucidated its underlying antitumor mechanisms. MC38 cells derived from murine colon adenocarcinoma were implanted by subcutaneously injecting mice. The anticancer effects of HK-Mpg therapy were compared with HK-M. bovis BCG, an effective adjuvant for cancer immunotherapy. HK-Mpg treatment enhanced tumor reduction and mouse survival. Furthermore, HK-Mpg treatment synergistically enhanced the anticancer therapeutic effect of cisplatin. In addition, HK-Mpg enhanced inflammatory cytokine production and recruitment of immune cell into tumor-infiltrating sites and splenocytes in vaccinated mice. Our mechanistic study demonstrates that HK-Mpg therapy elicits a strong antitumor immune response in mice, mainly through natural killer cell-mediated oncolytic activity via the activation of dendritic cells (DCs) and by enhancing inflammatory cytokines production such as IL-12 from DC. Hence, HK-Mpg can be a potential immunotherapy adjuvant, enhancing the effect of cancer chemotherapy.

Eosinophil Activation by Toll-Like Receptor 4 Ligands Regulates Macrophage Polarization.

Eosinophils are terminally differentiated granulocytes that have long been considered as destructive cells associated with Th2 type immune responses such as allergic inflammation and helminth infections. Recently, eosinophils have been actively studied as multifunctional leukocytes regulating an array of physiological responses through interaction with other immune cells. In this study, we examined the expression and function of Toll-like receptors (TLRs) in eosinophilic EoL-1 cells and demonstrated the expression of a number of immune mediators in activated EoL-1 cells and their interaction with the macrophage cell line THP-1 upon TLR4 ligand stimulation. EoL-1 cells differentiated with butyrate increased expression of TLR3, TLR4, and TLR7 at mRNA and protein level with flow cytometry analysis. Mature eosinophils derived from human cord blood CD34+ cells were subjected to RNA-sequencing, and showed the expression of a panel of TLR transcripts and TLR4 was the most highly expressed TLR. Among the cognate ligands of TLR3, TLR4, and TLR7, lipopolysaccharide (LPS) or palmitic acid significantly increased mRNA expression of immune mediators in differentiated EoL-1 cells. Notably, Western blot analysis of palmitic acid-treated differentiated EoL-1 cells showed significantly up-regulated expression of Th2 type cytokines and transcription factors driving eosinophil differentiation. To evaluate functional significance of TLR4 ligand-stimulated eosinophils, we added conditioned media (CM) from EoL-1 cells to differentiated THP-1 cells and assessed the expression of M1 macrophage or M2 macrophage-related markers. M1 and M2 macrophage markers were significantly upregulated by CM from LPS and palmitic acid stimulated EoL-1 cells, respectively. In addition, the adipose tissue of obese mice, where eosinophils are decreased due to obesity-induced inflammation, showed significantly decreased frequency of M2 macrophages, despite an increase in the total macrophage numbers. Based on these collective data, we proposed that eosinophils regulate both inflammatory and anti-inflammatory polarization of macrophages through functional changes induced by different TLR4 ligands.

Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK-MAPK and NF-κB Signal Pathways through TLR4.

The potD gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system. In this study, we found PotD in Haemophilus (Glaesserella) parasuis could actively stimulate both humoral immune and cellular immune responses and elevate lymphocyte proliferation, thus eliciting a Th1-type immune response in a murine immunity and infection model. Stimulation of Raw 264.7 macrophages with PotD validated that Toll-like receptor 4, rather than 2, participated in the positive transcription and expression of pro-inflammatory cytokines IL–1β, IL–6, and TNF–α using qPCR and ELISA. Blocking signal-regulated JNK–MAPK and RelA(p65) pathways significantly decreased PotD-induced pro-inflammatory cytokine production. Overall, we conclude that vaccination of PotD could induce both humoral and cellular immune responses and provide immunoprotection against H. parasuis challenge. The data also suggest that Glaesserella PotD is a novel pro-inflammatory mediator and induces TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through JNK–MAPK and RelA(p65) pathways.

FEATURES OF SYSTEM IMMUNITY IN WOMEN WITH ENDOMETRIOSIS AND GENITAL INFECTION

External genital endometriosis is an inflammatory, estrogen-dependent disease that develops predominantly in women of reproductive age and is characterized by the presence of pain syndrome and infertility. Today, endometriosis is one of the most common gynecological diseases in women of reproductive age, however, the etiology and pathogenesis of it are not completely clear. Violations of systemic immunity are most important in the pathogenesis of endometriosis. The literature data on the features of the immune response in endometriosis in combination with genital infection are few and contradictory. Purpose – to study the features of systemic immunity in women with external genital endometriosis and pathogens of genital infection.A total of 159 women with external genital endometriosis were examined. The main lymphocyte subpopulations, the functional activity of neutrophils and peripheral blood monocytes, and the content of cytokines in the blood serum were studied. A study of systemic immunity was performed in women with 1-2 and 3-4 stages of endometriosis, as well as depending on the presence of pathogens of genital infection. The presence of Chlamydia trachomatis, Ureaplasma spp., Mycoplasma genitalium, HSV1, 2/CMV, HPV in the endometrium, peritoneal fluid, and endometrioid heterotopies was determined. Statistical processing was performed using the IBM SPSS Statistics Version 22.2 statistical analysis software package.According to the results of the study, it was found that women with endometriosis of stages 1-2 show signs of systemic inflammation with a predominance of the Th2 type of immune response and inhibition of cellular immunity. A particular feature of HPV was an increase in T-NK lymphocytes, a decrease in IL-2 and neutrophil functional activity. The presence of Ureaplasma spp./Mycoplasma genitalium was characterized by a decrease in cellular immunity and an increase in T-NK cells. Only with HPV and Ureaplasma spp./Mycoplasma genitalium decreased synthesis of IL-2, IL-6. With 3-4 stages, the most significant changes in immunity were found in groups of women with genital infection. When HPV – a high level of IgA, increased IgM, IL-8. With Ureaplasma spp./Mycoplasma genitalium – inhibition of cellular immunity, high levels of IgA, reduction of neutrophil phagocytic activity.Thus, in women with endometriosis in the presence of pathogens of genital infection revealed features that may contribute to the development and progression of the disease.

The Essential Role Played by B Cells in Supporting Protective Immunity Against Trichuris muris Infection Is by Controlling the Th1/Th2 Balance in the Mesenteric Lymph Nodes and Depends on Host Genetic Background.

How B cells contribute to protective immunity against parasitic nematodes remains unclear, with their importance as accessory cells underexplored. In this study, anti-CD20 monoclonal antibody (α-CD20 mAb)-mediated depletion of B cells from C57BL/6 mice revealed an important role for B cells in supporting Th2 immune responses and thus expulsion of Trichuris muris (T. muris). C57BL/6 mice normally mount mixed Th1/Th2 immune responses to T. muris and expel the parasite by the third week post infection. However, B cell-depleted C57BL/6 had significantly reduced Th2-type cytokines post infection and failed to expel the parasite. IFN-γ production in the MLN of C57BL/6 mice receiving α-CD20 mAb treatment was not affected, collectively resulting in an overall change in Th1/Th2 balance in favor of Th1. Further, the expression of IFN-γ and IFN-γ-induced genes at the effector site, the gut, was significantly increased in the absence of B cells. Interestingly, and in complete contrast, BALB/c mice, which mount strongly polarized Th2 immune responses, rather than mixed Th1/Th2 immune responses, were still able to expel T. muris in the absence of B cells. We thus hypothesized that the B cell plays a critical role in enabling strong Th2 responses in the context of mixed Th1/Th2 settings, with the role becoming redundant in highly Th2 polarized environments. In support of this, neutralization of IFN-γ in B cell depleted C57BL/6 restored resistance against T. muris infection. Thus, our data suggest an important role of B cells in supporting Th2-type immune responses in mixed IFN-γ-rich Th1/Th2 settings.

Parenteral-Oral Immunization with Plant-Derived HBcAg as a Potential Therapeutic Vaccine against Chronic Hepatitis B.

Chronic hepatitis B (CHB) is the cause of severe liver damage, cirrhosis, and hepatocellular carcinoma for over 240 million people worldwide. Nowadays, several types of treatment are being investigated, including immunotherapy using hepatitis B core antigen (HBcAg) assembled into highly immunogenic capsid-like particles (CLPs). Immunogenicity of plant-produced and purified HBcAg, administered parenterally or intranasally, was previously reported. In this study, a novel parenteral–oral vaccination scheme is proposed using plant-derived HBcAg preparations. The antigen for injection was obtained via transient expression in Nicotiana benthamiana. HBcAg-producing transgenic lettuce was lyophilized and used as an orally delivered booster. The intracellular location of plant-produced HBcAg CLPs implies additional protection in the digestive tract during oral immunization. BALB/c mice were intramuscularly primed with 10 µg of the purified antigen and orally boosted twice with 5 or 200 ng of HBcAg. A long-lasting and significant systemic response after boosting with 200 ng HBcAg was induced, with anti-HBc titer of 25,000. Concomitantly, an insignificant mucosal response was observed, with an S-IgA titer of only 500. The profile of IgG isotypes indicates a predominant Th1 type of immune response, supplemented by Th2, after injection–oral vaccination. The results demonstrate that a low dose of parenteral–oral immunization with plant-derived HBcAg can elicit a specific and efficient response. This study presents a potential new pathway of CHB treatment.

The Effect of Size, Dose, and Administration Route on Zein Nanoparticle Immunogenicity in BALB/c Mice.

BackgroundZein-based carriers are a promising delivery system for biomedical applications. However, few studies involve systematic investigation on their in vivo biocompatibility and immunogenicity.PurposeThe objective of this study was to identify the immunogenicity, type of immune response, biocompatibility and systemic recall immune response of zein nanoparticles administrated via different routes in mice.Animals and methodsFemale Balb/c mice were selected as the animal model in this paper. The effect of particle size, dose and inoculation routes on immunogenicity were systematically explored. The mice were challenged at week 50 via intramuscular and subcutaneous routes to investigate the systemic recall immune responses of zein nanoparticles. Hematoxylin and eosin staining was performed to investigate the biocompatibility of zein nanoparticles at injection sites.ResultsThe administration of zein particles by parenteral routes led to a long-term systemic immune response. Particle size did not affect zein-specific IgG antibody titers. IgG antibody titers and inflammatory cell infiltration at the injection sites resulting from intramuscular zein particle injection were significantly higher than those from subcutaneous injection of the same dose. For intramuscular inoculation, dose-dependent IgG antibody titers were observed after the third inoculation, while no significant difference was found via the subcutaneous route. For both routes, IgG titer showed a time-dependent decrease at all dose levels from week 5 onward, and finally plateaued at week 28. The IgG subtype assay showed a predominant Th2-type immune response for both administration routes. Challenge with zein nanoparticles at week 50 led to a significant increase in specific IgG titer at all dose levels, indicating systemic recall immune responses. Interestingly, IgG antibody levels in the subcutaneous groups showed a delayed decrease compared to those of the intramuscular injection groups at all dose levels.ConclusionThis study indicated that immunogenicity may be one of the key challenges of using zein nanoparticles as carriers via parenteral administration. Further investigation is needed to illustrate zein immunogenicity in other forms, and the possible effect of systemic recall immune response on in vivo pharmacokinetic characteristics.

Peptide-based vaccine successfully induces protective immunity against canine visceral leishmaniasis

Dogs are the main reservoir of zoonotic visceral leishmaniasis. Vaccination is a promising approach to help control leishmaniasis and to interrupt transmission of the Leishmania parasite. The promastigote surface antigen (PSA) is a highly immunogenic component of Leishmania excretory/secretory products. A vaccine based on three peptides derived from the carboxy-terminal part of Leishmania amazonensis PSA and conserved among Leishmania species, formulated with QA-21 as adjuvant, was tested on naive Beagle dogs in a preclinical trial. Four months after the full course of vaccination, dogs were experimentally infected with Leishmania infantum promastigotes. Immunization of dogs with peptide-based vaccine conferred immunity against experimental infection with L. infantum. Evidence for macrophage nitric oxide production and anti-leishmanial activity associated with IFN-γ production by lymphocytes was only found in the vaccinated group. An increase in specific IgG2 antibodies was also measured in vaccinated dogs from 2 months after immunization. Additionally, after challenge with L. infantum, the parasite burden was significantly lower in vaccinated dogs than in the control group. These data strongly suggest that this peptide-based vaccine candidate generated cross-protection against zoonotic leishmaniasis by inducing a Th1-type immune response associated with production of specific IgG2 antibodies. This preclinical trial including a peptide-based vaccine against leishmaniasis clearly demonstrates effective protection in a natural host. This approach deserves further investigation to enhance the immunogenicity of the peptides and to consider the possible engineering of a vaccine targeting several Leishmania species.

Extraction, isolation, immunoregulatory activity, and characterization of Alpiniae oxyphyllae fructus polysaccharides

For further applications of Alpiniae oxyphyllae fructus in modern clinical medicine, Alpiniae oxyphyllae fructus polysaccharide (AOFP) was studied in the present work. The extraction conditions of AOFP were optimized by the response surface method with a Box-Behnken design. The maximum extraction rate of AOFP was 3.18%. An anion-exchange DEAE-52 cellulose column and a Sephadex G-100 gel column were used to isolate the AOFP, and three polysaccharides (AOFP1, AOFP2, AOFP3) were obtained. All three polysaccharides possessed immunoregulatory activity, but the effects of AOFP1 were greater than the other two polysaccharides. AOFP1 significantly stimulated Th1- and Th2-type immune responses and specific immune responses. Meanwhile, the characterization of AOFP1 was studied. AOFP1 was composed of arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid at a molar ratio of 16.46:12.7:4.9:17.11:4.35:6.52:6 with an average molecular weight of 43.4 kDa. These results suggest that AOFP1 can be developed as a natural immunomodulatory drug.

A new multi-epitope peptide vaccine induces immune responses and protection against Leishmania infantum in BALB/c mice.

Visceral leishmaniasis (VL) is a tropical and subtropical disease which is endemic in more than eighty countries aroundthe world. Leishmania infantum is one of the main causative agents of VL disease. Currently, there is no approved-to-market vaccine for VL therapy. In this study, we evaluated cellular and humoral immune responses induced by our newly designed multi-epitope vaccine in BALB/c mice. Four antigenic proteins, including histone H1, sterol 24-c-methyltransferase (SMT), Leishmania-specific hypothetical protein (LiHy), and Leishmania-specific antigenic protein (LSAP) were chosen for the prediction of potential immunodominant epitopes. Moreover, to enhance vaccine immunogenicity, two toll-like receptors 4(TLR4) agonists, resuscitation-promoting factors of Mycobacterium tuberculosis (RpfE and RpfB), were employed as the built-in adjuvants. Immunization with the designed multi-epitope vaccine elicited a robust Th1-type immune response, compared to other groups, as shown by increased levels of IL-2, IFN-γ, TNF-α, and IgG2a. Furthermore, a significant decrease was observed in Th-2-type-related cytokines such as IL-4 in immunized mice. The designed construct also induced a significant reduction in parasite load (p < 0.0001), conferring protection against L. infantum challenge. This study could be promising in gaining insight towards the potential of peptide epitope-based vaccines as effective protective approaches against Leishmania species.

Effects of regulatory T cells on glyceraldehyde-3-phosphate dehydrogenase vaccine efficacy against Schistosoma japonicum

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a candidate subunit vaccine that induces protective immunity and elicits partial resistance to Schistosoma japonicum upon mouse and livestock vaccination. This study aimed to evaluate the effect of regulatory T cells (Tregs), which were defined as CD4+CD25+Foxp3+ cells, on the efficacy of a GAPDH vaccine against S. japonicum. BALB/c female mice were randomly divided into five groups as follows: normal, infected control, anti-CD25 monoclonal antibody (anti-CD25 mAb), GAPDH group, and co-treated with anti-CD25 mAb and GAPDH group. The worm reduction and liver egg reduction rates in the GAPDH group were 32.46% and 35.43%, respectively, which increased to 60.09% and 58.78%, respectively, after anti-CD25 mAb administration. Compared with those in the infected control group, the percentage of Tregs in the spleen decreased significantly when GAPDH and anti-CD25 mAb were used either alone or in combination. Furthermore, secretions associated with the Th1 response increased in splenocytes of the anti-CD25 mAb group, whereas the Th1 and Th2 responses increased in splenocytes of the GAPDH and co-treated groups. Compared to that in the infected control group, granuloma diameter in the GAPDH and co-treated groups increased slightly, but there were no significant differences among the groups. Our results indicate that the protective effect of the GAPDH vaccines can be improved by decreasing Tregs and enhancing the Th1- and Th2-type immune responses. Therefore, anti-CD25 mAb and GAPDH might exert synergistic effects to clear parasites by decreasing the frequency of Tregs and increasing the Th1- and Th2-type immune responses.

The protective effects against cyclophosphamide (CTX)-induced immunosuppression of three glucomannans

The protective effects against cyclophosphamide (CTX)-induced immunosuppression of three glucomannans from Dendrobium officinale (DOP), Aloe (ASP), and Konjac (KGM) respectively are investigated from the aspects of cellular and humoral immunity. Results showed that glucomannans enhanced the secretion of T helper type 1 (Th1) cytokines and the proportion of CD4 T lymphocytes, promoted the subpopulation of T lymphocytes by up-regulating T-bet and GATA-3 mRNA expressions. Meanwhile, glucomannans increased the secretion of immunoglobulins, stimulated the differentiation of plasmocytes by up-regulating IRF4, Blimp-1 and XBP-1 mRNA expressions. In addition, DOP had better effects on promoting T lymphocytes function and immunoglobulins production, KGM showed better effects in increasing cytokines production, while ASP had better effects in stimulating Th1 type immune response. These results suggested that all three glucomannans from different sources exhibited immunoregulatory function, however, each expressed their bioactivities in different mechanisms.

Associations Between Glutathione-S-Transferase Genotypes and Bronchial Hyperreactivity Patients With Di-isocyanate Induced Asthma. A Follow-Up Study.

Introduction: Di-isocyanates TDI (toluene di-isocyanate), MDI (diphenylmethane di-isocyanate), and HDI (hexamethylene di-isocyanate) are the most common chemicals causing occupational asthma. Di-isocyanate inhalation has been reported to induce oxidative stress via reactive oxygen and nitrogen species leading to tissue injury. Glutathione transferases (GSTs) and N-acetyltransferases (NATs) are detoxifying enzymes whose general function is to inactivate electrophilic substances. The most important genes regulating these enzymes, i.e., GSTM1, GSTP1, GSTT1, NAT1, and NAT2 have polymorphic variants resulting in enhanced or lowered enzyme activities. Since inability to detoxify harmful oxidants can lead to inflammatory processes involving activation of bronchoconstrictive mechanisms, we studied whether the altered GST and NAT genotypes were associated with bronchial hyperreactivity (BHR) in patients with di-isocyanate exposure related occupational asthma, irrespective of cessation of di-isocyanate exposure, and adequacy of asthma treatment.Methods: Polymerase chain reaction (PCR) based methods were used to analyze nine common polymorphisms in GSTM1, GSTM3, GSTP1, GSTT1, NAT1, and NAT2 genes in 108 patients with diagnosed occupational di-isocyanate-induced asthma. The genotype data were compared with spirometric lung function and BHR status at diagnosis and in the follow-up examination on average 11 years (range 1–22 years) after the asthma diagnosis. Serum IgE and IL13 levels were also assessed in the follow-up phase.Results: An association between BHR and GSTP1 slow activity (Val105/Val105) genotype was demonstrated in the subjects at the follow-up phase but not at the diagnosis phase. Moreover, the patients with the GSTP1 slow activity genotype exhibited characteristics of Th-2 type immune response more often compared to those with the unaltered GSTP1 gene. Interestingly, all 10 patients with the GSTP1 slow activity genotype had both the GSTM3 slow activity genotype and the unaltered GSTT1 gene.Discussion: The results suggest associations of the low activity variants of the GSTP1 gene with BHR. The fact that these associations came up only at the follow-up phase when the subjects were not any more exposed to di-isocyanates, and used asthma medication, suggest that medication and environmental factors influence the presentation of these associations. However, due to the exploratory character of the study and relatively small study size, the findings remain to be confirmed in future studies with larger sample sizes.

Association of the CCR5Δ32 Mutant Genotype with Sickle Cell Disease in Egyptian Patients

Sickle cell disease is considered the most common single base mutation in the world, with >250,000 new patients being discovered each year. It consists of a wide spectrum of clinical presentations and complications. The CCR5Δ32 is the mutant genotype of C–C chemokine receptor 5 (CCR5). It is widely distributed due to several micro organisms that target macrophages in different populations. Theoretically, CCR5Δ32 confers an advantage to sickle cell disease patients. The chronic inflammatory response is the main pathogenesis in sickle cell disease, thus, the presence of the null CCR5Δ32 mutant genotype prevents the Th1-type immune response caused by the CCR5 chemokine receptor. This study aimed to define the true incidence of the CCR5Δ32 mutant genotype and to correlate its presence with the clinical and/or the radiological findings in sickle cell disease patients. We proposed decreased morbidity and prolonged survival of sickle cell disease patients carrying the CCR5Δ32 genotype. The study showed relatively the same prevalence (5.1%) of the CCR5Δ32 mutant genotype found in 500 sickle cell disease patients when compared to 1000 healthy controls (5.0%) with the same ethnic background. Despite the near prevalence of the incidence to controls, we suggest that CCR5Δ32 is relatively beneficial to sickle cell disease patients as polymorphic patients showed uncomplicated clinical presentation in contrast to other patients without the CCR5Δ32.

Enhancement of autophagy as a strategy for development of new DNA vaccine candidates against Japanese encephalitis

For decades, an on-going concerted effort has been made to develop a universal DNA vaccine to combat the looming threat of a potential outbreak of the emerging Japanese encephalitis virus (JEV) infection. However, effective strategies are urgently required to counter poor immunogenicity and insufficient long-term protection. Recent reports have confirmed the critical role of autophagy in antigen presentation, long-term immune memory and immune responses against JEV. In this study, JEV prM and E protein with strong immunogenicity were fused with microtubule-associated protein 1 light chain 3 (LC3) encoding gene to construct an autophagy-mediated pJME-LC3 DNA vaccine. Researches indicated significant increase of autophagosomes or LC3 Ⅱ expression in pJME-LC3 transfected cells. Furthermore, prME-LC3 fused protein was observed co-localized with GFP-LC3 to autophagosomes, which means it was successfully targeted to autophagosomes. After immunizing with pJME-LC3, mice were detected highest proportion of CD3+CD8+ T lymphocytes, CD8+ effector memory T cells (TEMs) and JEV specific cytotoxic T lymphocyte (CTL) activity to eliminate JEV. pJME-LC3 also enhanced IgG2a antibody in serum and cytokines IFN-γ, IL-12 produced by splenocytes, thus skew toward Th1 type immune response by activating the JAK2/STAT1 signaling pathway and upregulating expression of transcription factor T-bet. Notably, mice immunized with pJME-LC3 showed highest survival rate and long-lasting neutralizing antibody when challenged with virulent JEV, which were consistent with augment in percentage of CD4+ central memory T cells (TCMs). In brief, our studies suggested that autophagy can be used as a optimization strategy to enhance JEV specific immune response and long-term immune memory. Our attempt will contribute towards future efforts to develop an efficacious JEV vaccine.

Dual immune functions of IL-33 in inflammatory bowel disease.

Interleukin-33 (IL-33) has emerged as a critical regulator in a variety of diseases, including inflammatory bowel disease (IBD). IL-33 can be produced by various tissues and cells, and typically induces Th2-type immune responses via binding to the receptor ST2. In addition, accumulated data have shown that IL-33 also plays a modulatory role in the function of regulatory T cells (Tregs), B cells, and innate immune cells such as macrophages and innate lymphoid cells (ILCs). IBD, including Crohn's disease and ulcerative colitis, are characterized by aberrant immunological responses leading to intestinal tissue injury and destruction. Although IL-33 expression is increased in IBD patients and correlates with the patients' disease activity index, mechanistic studies to date have demonstrated both pathogenic and protective roles in animal models of experimental colitis. In this review, we will summarize the roles and mechanisms of IL-33 in IBD, which is essential to understand the pathogenesis of IBD and determine potential therapies.

Abstract CT044: AST-008, a TLR9 agonist spherical nucleic acid, activated NK cells, T cells, and cytokines in healthy subjects in a Phase I clinical trial

Abstract Background Exicure develops spherical nucleic acid (SNA) constructs, which are 3-dimensional arrangements of oligonucleotides where the nucleic acids are densely packed and radially oriented around a nanoparticle. SNAs have properties that are distinct from the “linear” nucleic acids (i.e., nucleic acids not arranged in the SNA format), which include, most importantly, increased cellular uptake compared to linear nucleic acids. AST-008 is an SNA configuration of a toll-like receptor 9 (TLR9) agonist oligonucleotide, designed to trigger anti-tumor immune responses in patients with cancer. AST-008 is intended to be administered intratumorally in combination with checkpoint inhibitors for the treatment of solid tumors. AST-008 has potent antitumor activity as a monotherapy and synergizes with anti-PD-1 antibody therapy in several preclinical tumor models. Materials and Methods AST-008 has been evaluated in a Phase 1 study (Protocol number: AST-008-101). The safety, tolerability, pharmacokinetics and pharmacodynamics of single ascending doses of AST-008 were studied in healthy volunteers. Four dose levels of AST-008 were evaluated in four cohorts. Results The results indicated that AST-008 was safe and well tolerated after single subcutaneous injections. No serious adverse events or dose limiting toxicity were reported. The most common adverse events observed were flu-like symptoms, injection site reactions, and reversible, short-lived leukopenia and neutropenia. AST-008 induced an innate immune response after administration to healthy volunteers. Cytokine and chemokine analysis indicated that a Th1-type immune response was elicited. IL-12 (p40), IL-1RA, IL-6, IP-10, and MCP-1 were consistently induced across all doses. AST-008 elicited 9.5 fold and 3.5 fold increases in the fraction of activated T cells and natural killer cells, respectively, compared to baseline at the highest dose tested. These results suggest that the cytokines and immune cell activation elicited by AST-008 will initiate and propagate the cancer immunity cycle in the presence or absence of checkpoint inhibitors. Pharmacokinetic analysis revealed that the concentration of AST-008 in plasma peaked 2 hours after dosing and was dose proportional. Conclusions AST-008 elicited no serious adverse events or dose limiting toxicity at the doses tested. AST-008 is a potent innate immune activator and exhibits pharmacodynamic properties that are expected to result in anti-tumor effects in patients with cancer. A Phase 1b/2 study of AST-008 in combination with pembrolizumab in cancer patients is ongoing. Citation Format: Weston L. Daniel, Ulrike Lorch, Simon Coates, Alice S. Bexon, Scott Mix. AST-008, a TLR9 agonist spherical nucleic acid, activated NK cells, T cells, and cytokines in healthy subjects in a Phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT044.

Abstract 3217: Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA

Abstract Recent studies have highlighted the critical role of CD103+/CD141+ dendritic cells (DCs) in antitumor immunity. CDX-301 is a soluble, recombinant human FLT3 ligand (Flt3L). Flt3L is a hematopoietic cytokine which stimulates the proliferation and differentiation of various blood cell progenitors, including CD103+/CD141+ DCs. CDX-301 is in clinical development for multiple cancers and may hold significant opportunity for synergistic development in combination with other immunotherapies, in particular toll-like receptor 9 (TLR9) agonists that are known to promote maturation and activation of DCs. AST-008 is a TLR9 agonist oligonucleotide in a spherical nucleic acid (SNA) format and is in clinical development for multiple cancers in combination with pembrolizumab. SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. AST-008 induces potent TH1-type immune responses in vitro, in mice and non-human primates, and has shown potent antitumor activity as a monotherapy and enhanced checkpoint inhibitor activity in several murine tumor models. AST-008 increases tumor-infiltrating lymphocytes, interferon-inducible gene expression, and activation and expansion of CD8+ T cells with reduced T-regulatory cells in the tumor microenvironment. We examined the effects of the combination of CDX-301 and a murine version of AST-008, muAST-008, in mice on DCs and on antitumor efficacy in a tumor model. Mice were implanted with MC38 murine colon adenocarcinoma tumor cells (day 0) and animals were treated with either CDX-301 (5 μg, days 2-8 i.p.) only, muAST-008 only (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30 p.t.), or the combination of CDX-301 (5 μg, days 2-8) with muAST-008 (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30). Treatment with CDX-301 and muAST-008 showed an additive effect in retarding tumor growth and prolonging survival. To help understand the mechanisms involved in the antitumor effects, animals were treated with a similar course of CDX-301 and muAST-008 and spleens were harvested for flow cytometry evaluation 2 days after the last CDX-301 dose. For these studies, we also included a second DC activating agent, an agonist anti-CD40 mAb FGK45.5 (50 μg i.p. together with the last dose of CDX-301) among the combinations. We observed a significant increase in the percentage of CD103+CD8+ cDCs by the addition of muAST-008 to CDX-301 treatment. In addition, muAST-008 led to the up-regulation of activation markers on dendritic cells, which was markedly enhanced when combined with CD40 activation. These data demonstrate that muAST-008 leads to systemic activation of CDX-301 expanded DCs, leading to more potent anti-tumor immunity and support the potential of combining CDX-301 and AST-008 in augmenting the immunotherapy of cancers. Citation Format: Lawrence J. Thomas, Li-Zhen He, Lauren E. Gergel, Eric M. Forsberg, Elizabeth Q. Do, James M. Boyer, April R. Baronas, Mallary Rocheleau, Kathleen M. Borrelli, Anna Wasiuk, Jeffrey Weidlick, Henry C. Marsh, Bart R. Anderson, SubbaRao Nallagatla, Richard Kang, Ekambar R. Kandimalla, Tibor Keler. Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3217.