Induced Lymphocyte Proliferation Research Articles

Fresh Human Umbilical Cord Arteries as a Potential Source for Small-Diameter Vascular Grafts.

The demand for small-diameter vascular grafts has been globally increased but still lacks optimal solutions in this category. This study evaluated the feasibility of utilizing human pretreated fresh and nondecellularized umbilical cord arteries (hUCAs) as vascular grafts without needing any immunosuppression process. A mixed lymphocyte reaction assay revealed that hUCAs did not induce lymphocyte proliferation or cytokine production. To assess the in vivo inflammatory response, hUCAs were buried in fatty tissue under the skin of the abdominal wall in the left and right iliac fossas of rats. The average sizes of the implanted hUCAs remained consistent at 30 days post implantation. To evaluate xenogeneic transplantation, hUCAs were grafted to the abdominal aorta below the kidney of Wister rats. Remarkably, all rats exhibited positive revascularization and perfusion, maintaining blood pressure values of around 110/70 mmHg. Doppler ultrasound consistently indicated good circulation, with the three separate echogenic layers corresponding to the three arterial wall layers throughout the assessment period. Grafted rats exhibited normal motor behavior, accompanied by positive responses to thermal and pain stimulation. Blood biochemical values and whole blood cell counts showed no significant differences between pre and post-transplantation. Histological analysis of the grafts revealed no calcification or thrombosis, and a mild chronic inflammatory response was presented. In conclusion, hUCAs maintained their structural and functional properties after transplantation in rats without immunosuppression. This highlights their potential as a source for allogeneic, readily accessible, small-diameter vascular grafts.

A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

A Vaccine-Pulsed Dendritic Cell-Based In Vitro Assay to Induce Lymphocyte Proliferation

Interaction between MSCS and blood mononuclear cells during <i>in vitro</i> co-cultivation in the presence of a three-dimensional artificial matrix mimicking regenerating bone tissue

Bone tissue repair and regeneration is a complex process involving many cells and controlled by multiple factors. Immune cells and cytokines play a crucial role in regulating the balance of bone formation and resorption. However, the immunomodulatory mechanism of bone regeneration is still unclear. Nevertheless, the reciprocal regulatory influence of immunocompetent cells and mesenchymal stem cells (MSCs) is well known. MSCs and immunocompetent cells secrete various cytokines, growth factors, and extracellular matrix molecules that play important roles in regulating hematopoiesis, angiogenesis, immune and inflammatory responses. Several studies confirm that different molecules expressed by MSCs may induce lymphocyte proliferation. Therefore, the study of the mutual influence of MSCs and blood mononuclear cells during in vitro co-cultivation, even in the presence of an artificial matrix mimicking regenerating bone tissue, is relevant and expedient. In this experimental series, the studies were performed at the interface between living and non-living substrate phases thus mimicking the “regenerating bone / hematopoietic microenvironment” system. A series of separated in time experiments was performed on a plastic surface (2D culture model) and in the presence of three-dimensional artificial matrices mimicking regenerating bone tissue (3D culture model).

Lactobacillus plantarum surface-displayed FomA (Fusobacterium nucleatum) protein generally stimulates protective immune responses in mice.

A significant correlation is observed between Fusobacterium nucleatum (F. nucleatum) and the evolution of inflammatory bowel disease (IBD). Particularly, FomA, a critical pathogenic element of F. nucleatum, inflicts substantial detriment to human intestinal health. Our research focused on the development of recombinant Lactobacillus plantarum that expresses FomA protein, demonstrating its potential in protecting mice from severe IBD induced by F. nucleatum. To commence, two recombinant strains, namely L. plantarum NC8-pSIP409-pgsA'-FomA and NC8-pSIP409-FnBPA-pgsA'-FomA, were successfully developed. Validation of the results was achieved through flow cytometry, ELISA, and MTT assays. It was observed that recombinant L. plantarum instigated mouse-specific humoral immunity and elicited mucosal and T cell-mediated immune responses. Significantly, it amplified the immune reaction of B cells and CD4+T cells, facilitated the secretion of cytokines such as IgA, IL4, and IL10, and induced lymphocyte proliferation in response to FomA protein stimulation. Finally, we discovered that administering recombinant L. plantarum could protect mice from severe IBD triggered by F. nucleatum, subsequently reducing pathological alterations and inflammatory responses. These empirical findings further the study of an innovative oral recombinant Lactobacillus vaccine.

Does Panax notoginseng (Sanqi) inhibit H22 tumor growth via improving the host’s cellular immunity?

Ginsenoside Rg3 has many functions, including anti-tumor activity, antioxidant activity, and immune system modulatory activity. Interestingly, There are two stereoisomers exist in Rg3, 20(S)-ginsenoside Rg3 [20(S)-Rg3] and 20(R)-ginsenoside Rg3 [20(R)-Rg3], respectively. These two isomers have different pharmacological effects due to their stereospecificity. In this study, we compared their effect of stimulating the ConA to induce lymphocyte proliferation and increase the cytokines IL-2 and IFN-γ levels in mice and improve the cellular immunity of the host. The study predicted that ginsenoside (Rg3) could increase Th1-type cytokines interleukin-2 and interferon-γ and stimulate ConA-induced lymphocyte proliferation in mice, thereby improving cellular immunity in H22 mice. The effects of the Rg3 R-form were significantly greater than Rg3 S-form.

Does Panax notoginseng (Sanqi) inhibit H22 tumor growth via improving the host’s cellular immunity?

Ginsenoside Rg3 has many functions, including anti-tumor activity, antioxidant activity, and immune system modulatory activity. Interestingly, There are two stereoisomers exist in Rg3, 20(S)-ginsenoside Rg3 [20(S)-Rg3] and 20(R)-ginsenoside Rg3 [20(R)-Rg3], respectively. These two isomers have different pharmacological effects due to their stereospecificity. In this study, we compared their effect of stimulating the ConA to induce lymphocyte proliferation and increase the cytokines IL-2 and IFN-γ levels in mice and improve the cellular immunity of the host. The study predicted that ginsenoside (Rg3) could increase Th1-type cytokines interleukin-2 and interferon-γ and stimulate ConA-induced lymphocyte proliferation in mice, thereby improving cellular immunity in H22 mice. The effects of the Rg3 R-form were significantly greater than Rg3 S-form.

PIL-12 delivered by polymer based nanovector for anti-tumor genetherapy

Finding more effective and safe non-viral vectors to transfer genes into cancer cells has become the key of immune gene therapy for cancer. Herein a triblock compound MPEG2000–PDLLA4000–MPEG2000 modified by cationic liposome DOTAP was used as a non-viral vector DOTAP/MPEG2000–PDLLA4000–MPEG2000 (DMPM) to effectively transfer interleukin (IL)-12 plasmid (pIL-12) into tumor tissue. IL-12 produced by transfected tumor cells successfully inducing lymphocyte proliferation and promoting interferon-γ (IFN-γ) secretion, which resulted in tumor cells death. The ability of DMPM to transfer pIL-12 and the immune effect induced by IL-12 in cells had been explored. The anti-tumor effect, mechanism and safety of pIL-12/DMPM in mice cancer model were investigated in this study. Our results showed that the pIL-12 transferred by DMPM was highly expressed both in CT26 cells and B16-F10 cells. IL-12 expressed in the culture supernatant of transfected tumor cells stimulated lymphocyte proliferation and promoted IFN-γ secretion. The experimental result confirmed that pIL-12/DMPM therapy significantly reduced tumor growth in mice model. We designed the nanocomposite DMPM to deliver pIL-12 for cancer treatment and explored its therapeutic efficacy and the underlying anti-tumor mechanism. Our study suggested pIL-12 loaded by DMPM complex would be an effective strategy for cancer treatment.

The Effect of Curcumin-Loaded Glucan Nanoparticles on Immune Cells: Size as a Critical Quality Attribute

Curcumin is known for its multiple health benefits, largely due to its antioxidant and anti-inflammatory properties. It has been extensively studied as a therapeutic agent, however, it does not have good clinical efficacy due to its poor water solubility and bioavailability. Despite accepting the encapsulation of this compound in polymeric particles as one of the most promising strategies to increase its therapeutic value, these nanoparticles have fallen short of expectations due to a lack of assessment of their possible adverse effects on the immune system. Therefore, in this work, we report on a new method to encapsulate curcumin into glucan nanoparticles and their effects on cells of the immune system were evaluated. Two different-sized curcumin-loaded glucan NPs (GluCur 100 and GluCur 380) were produced, each with an encapsulation efficiency close to 100%, and were characterized regarding their size distribution, surface properties, and morphology. The results revealed the greatest hemolytic effect and cytotoxicity for the smallest particles (100 nm) tested in human PBMCs and RAW 264.7 cells. Although GluCur 380 NPs showed a weaker ROS production, they were able to inhibit the production of NO by macrophages. Furthermore, we found that the coagulation time was not affected by both sized-particles as well as platelet function. Additionally, both nanoparticles induced lymphocyte proliferation and TNF-α secretion by Mo-DCs. In conclusion, this report emphasizes the importance of the immunotoxicity assessment and how this is dependent on the intrinsic properties of nanomaterials, hopefully contributing to increasing the safety of nanomedicines.

Biocompatibility of Dextran-Coated 30 nm and 80 nm Sized SPIONs towards Monocytes, Dendritic Cells and Lymphocytes

Dextran-coated superparamagnetic iron oxide nanoparticles (SPIONDex) of various sizes can be used as contrast agents in magnetic resonance imaging (MRI) of different tissues, e.g., liver or atherosclerotic plaques, after intravenous injection. In previous studies, the blood compatibility and the absence of immunogenicity of SPIONDex was demonstrated. The investigation of the interference of SPIONDex with stimulated immune cell activation is the aim of this study. For this purpose, sterile and endotoxin-free SPIONDex with different hydrodynamic sizes (30 and 80 nm) were investigated for their effect on monocytes, dendritic cells (DC) and lymphocytes in concentrations up to 200 µg/mL, which would be administered for use as an imaging agent. The cells were analyzed using flow cytometry and brightfield microscopy. We found that SPIONDex were hardly taken up by THP-1 monocytes and did not reduce cell viability. In the presence of SPIONDex, the phagocytosis of zymosan and E. coli by THP-1 was dose-dependently reduced. SPIONDex neither induced the maturation of DCs nor interfered with their stimulated maturation. The particles did not induce lymphocyte proliferation or interfere with lymphocyte proliferation after stimulation. Since SPIONDex rapidly distribute via the blood circulation in vivo, high concentrations were only reached locally at the injection site immediately after application and only for a very limited time. Thus, SPIONDex can be considered immune compatible in doses required for use as an MRI contrast agent.

Hypoparathyroidism-Retardation-Dysmorphism Syndrome due to a Variant in the Tubulin-Specific Chaperone E Gene as a Cause of Combined Immune Deficiency.

BackgroundHypoparathyroidism-retardation-dysmorphism (HRD) syndrome is a disease composed of hypoparathyroidism, growth retardation, severe developmental delay, and typical dysmorphic features caused by the tubulin-specific chaperone E gene variant. Many patients succumb in infancy to HRD due to overwhelming infections mainly caused by Pneumococcus spp. Knowledge related to the immune system in these patients is scarce.PurposeTo define the immune phenotype of a cohort of HRD patients including their cellular, humoral, and neutrophil functions.MethodsThe study included HRD patients followed at Soroka University Medical Center. Clinical and immunological data were obtained, including immunoglobulin concentrations, specific antibody titers, lymphocyte subpopulations, lymphocyte proliferation, and neutrophil functions.ResultsNine patients (5 females and 4 males) were enrolled, aged 6 months to 15 years. All received amoxicillin prophylaxis as part of a routine established previously. Three patients had bacteremia with Klebsiella, Shigella spp., and Candida. Three patients had confirmed coronavirus disease 19 (COVID-19), and two of them died from this infection. All patients had normal blood counts. Patients showed high total IgA and IgE levels, low anti-pneumococcal antibodies in spite of a routine vaccination schedule, and reduced frequency of naive B cells with increased frequency of CD21lowCD27- B cells. All patients had abnormal T-cell population distributions, including reduced terminally differentiated effector memory CD8, inverted CD4/CD8 ratios, and impaired phytohemagglutinin (PHA)-induced lymphocyte proliferation. Neutrophil superoxide production and chemotaxis were normal in all patients tested.ConclusionHRD is a combined immunodeficiency disease with syndromic features, manifesting in severe invasive bacterial and viral infections.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-022-01380-9.

Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum.

Ureaplasma diversum is a bacterial pathogen that infects cattle and can cause severe inflammation of the genital and reproductive systems. Lipid-associated membrane proteins (LAMPs), including GUDIV-103, are the main virulence factors in this bacterium. In this study, we heterologously expressed recombinant GUDIV-103 (rGUDIV-103) in Escherichia coli, purified it, and evaluated its immunological reactivity and immunomodulatory effects in bovine peripheral blood mononuclear cells (PBMCs). Samples from rabbits inoculated with purified rGUDIV-103 were analysed using indirect enzyme-linked immunosorbent assay and dot blotting to confirm polyclonal antibody production and assess kinetics, respectively. The expression of this lipoprotein in field isolates was confirmed via Western blotting with anti-rGUDIV-103 serum and hydrophobic or hydrophilic proteins from 42 U. diversum strains. Moreover, the antibodies produced against the U. diversum ATCC 49783 strain recognised rGUDIV-103. The mitogenic potential of rGUDIV-103 was evaluated using a lymphoproliferation assay in 5(6)-carboxyfluorescein diacetate succinimidyl ester–labelled bovine PBMCs, where it induced lymphocyte proliferation. Quantitative polymerase chain reaction analysis revealed that the expression of interleukin-1β, toll-like receptor (TLR)-α, TLR2, TLR4, inducible nitric oxide synthase, and caspase-3–encoding genes increased more in rGUDIV-103–treated PBMCs than in untreated cells (p < 0.05). Treating PBMCs with rGUDIV-103 increased nitric oxide and hydrogen peroxide levels. The antigenic and immunogenic properties of rGUDIV-103 suggested its suitability for immunobiological application.

Evaluation of Immunotropic Activity of Iridoid-Anthocyanin Extract of Honeysuckle Berries (Lonicera caerulea L.) in the Course of Experimental Trichinellosis in Mice.

Our experiment determined the immunotropic activity of a natural, iridoid-anthocyanin extract from honeysuckle berry (Lonicera caerulea L.) (LC). The extract was administered to mice infected with Trichinella spiralis, orally at a dose of 2 g/kg bw, six times at 24 h intervals (from day 3 prior to the infection to day 3 post-infection (dpi) with T. spiralis. At 5, 7, 14, and 21 dpi, samples of blood, spleen, and mesenteric lymph nodes (MLN) were collected, and isolated lymphocytes were analyzed by flow cytometry. The splenocyte proliferation was estimated with MTT testing, and the intensity of intestinal and muscle infection was also studied. LC stimulated the local immune system by inducing lymphocyte proliferation in the spleen 7 dpi and altered the percentage and absolute count of B (CD19+) and T (CD3+, CD8+) cells 7, 14, and 21 dpi in the peripheral blood. LC extract affected the dynamics of expulsion of adult Trichinella from the intestines and prolonged the intestinal phase of the infection but did not change the number of larvae in the muscles. These results suggest that Lonicera caerulea L. fruit extract modulates murine cellular immune response during intestinal phase of T. spiralis infection but shows no antiparasitic activity.

Mathematical Modeling of Proliferative Immune Response Initiated by Interactions Between Classical Antigen-Presenting Cells Under Joint Antagonistic IL-2 and IL-4 Signaling

During an adaptive immune response from pathogen invasion, multiple cytokines are produced by various immune cells interacting jointly at the cellular level to mediate several processes. For example, studies have shown that regulation of interleukin-4 (IL-4) correlates with interleukin-2 (IL-2) induced lymphocyte proliferation. This motivates the need to better understand and model the mechanisms driving the dynamic interplay of proliferation of lymphocytes with the complex interaction effects of cytokines during an immune response. To address this challenge, we adopt a hybrid computational approach comprising of continuous, discrete and stochastic non-linear model formulations to predict a system-level immune response as a function of multiple dependent signals and interacting agents including cytokines and targeted immune cells. We propose a hybrid ordinary differential equation-based (ODE) multicellular model system with a stochastic component of antigen microscopic states denoted as Multiscale Multicellular Quantitative Evaluator (MMQE) implemented using MATLAB. MMQE combines well-defined immune response network-based rules and ODE models to capture the complex dynamic interactions between the proliferation levels of different types of communicating lymphocyte agents mediated by joint regulation of IL-2 and IL-4 to predict the emergent global behavior of the system during an immune response. We model the activation of the immune system in terms of different activation protocols of helper T cells by the interplay of independent biological agents of classic antigen-presenting cells (APCs) and their joint activation which is confounded by the exposure time to external pathogens. MMQE quantifies the dynamics of lymphocyte proliferation during pathogen invasion as bivariate distributions of IL-2 and IL-4 concentration levels. Specifically, by varying activation agents such as dendritic cells (DC), B cells and their joint mechanism of activation, we quantify how lymphocyte activation and differentiation protocols boost the immune response against pathogen invasion mediated by a joint downregulation of IL-4 and upregulation of IL-2. We further compare our in-silico results to in-vivo and in-vitro experimental studies for validation. In general, MMQE combines intracellular and extracellular effects from multiple interacting systems into simpler dynamic behaviors for better interpretability. It can be used to aid engineering of anti-infection drugs or optimizing drug combination therapies against several diseases.

Physical, functional and biochemical features of Nanoskin® bacterial cellulose scaffold as a potential carrier for cell transference

We evaluated the feasibility of Nanoskin®, a bacterial cellulose (BC) nanofiber membrane, as a potential scaffold for cell transference therapy. Nanoskin® does not induce lymphocyte proliferation, but it does not kill lymphocytes. Human mesenchymal stem cells (MSCs) and lymphocytes produced significantly elevated amounts of interleukin (IL)-10 (p = 0.0005; p = 0.02) when in contact with Nanoskin®. In conclusion, the Nanoskin® can be a promising biomaterial for use as a cell carrier-scaffold in cell transference therapies.

Combination of conserved recombinant proteins (NP & 3M2e) formulated with Alum protected BALB/c mice against influenza A/PR8/H1N1 virus challenge.

Influenza is one of the most important agents of pandemic outbreak causing substantial morbidity and mortality. Vaccination strategies of influenza must be adapted annually due to constant antigenic changes in various strains. Therefore, the present study was conducted to evaluate protective immunity of the conserved influenza proteins. For this purpose, three tandem repeats of M2e (3M2e) and NP were separately expressed in E. coli and were purified using column chromatography. Female Balb/c mice were injected intradermally with a combination of the purified 3M2e and NP alone or formulated with Alum (AlOH3) adjuvant in three doses. The mice were challenged by intranasal administration of H1N1 (A/PR/8/34) 2weeks after the last vaccination. The results demonstrated that recombinant NP and M2e proteins are immunogenic and could efficiently elicit immune responses in mice compared to non-immunized mice. The combination of 3M2e and NP supplemented with Alum stimulated both NP and M2e-specific antibodies, which were higher than those stimulated by each single antigen plus Alum. In addition, the secretion of IFN-γ and IL-4 as well as the induction of lymphocyte proliferation in mice received the mixture of these proteins with Alum was considerably higher than other groups. Moreover, the highest survival rate (86%) with the least body weight change was observed in the mice immunized with 3M2e and NP supplemented with Alum followed by the mice received NP supplemented with Alum (71%). Accordingly, this regimen can be considered as an attractive candidate for global vaccination against influenza.

Novel Mutations in RASGRP1 Are Associated with Immune Dysregulation and Predispose to EBV-Induced Lymphoma

& I. Somekh and B. Marquardt contributed equally§ C. Klein and R. Somech contributed equallyBackgroundRAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency disorder in one patient (Salzer et al, Nat Immunol 2016). Clinically, the disease was characterized by recurrent episodes of pneumonia, bronchiectasis, severe failure to thrive, CD4 T-cell lymphopenia, elevated TCRγδ cell proportions as well as low-grade Epstein-Barr virus (EBV)-associated B cell lymphoma. Immunologically, RASGRP1 deficiency is associated with defective proliferation and activation of T and B cells, respectively. Here, we report 3 novel patients with RASGRP1 deficiency from 2 unrelated consanguineous kindred.MethodsOne patient of Turkish origin (P1) and 2 Palestinian 1st degree cousins (P2, P3) were evaluated. Genetic analysis using whole exome sequencing was conducted. Immunological and biochemical assays were performed on primary patient material and genetically engineered T-cell lines using CRISPR/Cas9.ResultsClinical findings for all three patients included failure to thrive, hepatosplenomegaly, lymphadenopathy, and recurrent pulmonary infections resulting in bronchiectasia. Two patients had overt auto-immunity (AIHA and autoimmune thrombocytopenia, respectively). One patient had increased anti-nuclear antibody titers. One patient (P1) developed EBV-associated diffuse large B cell lymphoma. He underwent autologous hematopoietic stem cell transplant due to relapse after combined rituximab and NHL-BFM-90 treatment protocol and is currently in remission at the age of 14 years. P2 had deceased at the age of 3 years due to disseminated intravascular coagulation. P3 developed diffuse large B cell lymphoma at the age of 3 years, treated with R-CHOP protocol and is currently in remission.Immune workup of P2 and P3 revealed low levels of T-cell receptor excision circles (TREC), an abnormal T-cell receptor (TCR) Vß repertoire and a markedly increased proportion of TCRγδ cells. Phytohemagglutinin (PHA)-induced lymphocyte proliferation and anti-CD3 mitogen proliferation responses were decreased. EBV-viral load in peripheral blood was elevated for both patients, as measured by PCR.Genetic analysis by whole exome sequencing identified an autosomal recessive homozygous mutations in RASGRP1. P1 had an inversion (c.649_650inv; p.Glu217Ser) and P2/P3 had a deleterious frameshift mutation (c.1111_1114del; p.Asp371Ilefs*7). The variants were confirmed by Sanger sequencing and segregated with the disease phenotype. To confirm the functional relevance of the RASGRP1 mutations, we engineered Jurkat cells via CRISPR/Cas9 editing. RASGRP1 -/- Jurkat cells showed decreased phosphorylation of ERK1/2 and decreased CD69 expression in comparison to wildtype cells. Jurkat cells with RASGRP1 knockin mutants are currently under analysis.ConclusionsRASGRP1 deficiency is a novel primary immunodeficiency disorder associated with immune dysregulation and susceptibility to EBV-induced B-cell malignancies. Early diagnosis of RASGRP1 deficiency is critical. Even though clinical remission of EBV-lymphoma may be reached using high dose chemotherapy regimens, long term cures may only be reached by allogeneic hematopoietic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

The effect of green tea extracts on lymphocyte proliferation: A study in mice inoculated with Listeria monocytogenes

Elimination of L. monocytogenes in spleen and liver were dependent on CD4 and CD8 T cells. Since previous studies had shown that green tea extract (GTE) stimulated the production of IL-12, IFN-g, and TNF-a, this recent study was aimed to prove that GTE could induce lymphocyte proliferation. Balb/c mice were randomly divided into 11 groups, each group consisted of 5 mice. Group I, the mice were not given green tea nor bacteria. Group II, the mice were not given tea but they were inoculated with L. monocytogenes. Group III to V, the mice were treated with green tea polyphenon-60 in different doses (1.5, 3, and 6 mg/day). Group VI to VIII, the mice were treated with EGCG (0.5, 1, and 2 mg/day). Group IX to XI, the mice were treated with EGC (0.15, 0.3, and 0.6 mg/day). The mice in group III to XI were treated with those GTE orally for 14 days. They were inoculated with 104 L. monocytogenes intra peritoneally on the 10th day. All the mice were sacrificed on day 15 to examine the number of lymphoblasts in spleens. The results revealed that the number of lymphoblasts increased in all groups of mice treated with green tea polyphenon-60, EGCG, and EGC. Green tea polyphenon-60 had the highest effect to increase the number of lymphoblasts in mice inoculated with L. monocytogenes. It was concluded that green tea extracts could induce lymphocyte proliferation.

Synergistic effect of GRA7 and profilin proteins in vaccination against chronic Toxoplasma gondii infection

Toxoplasmosis is a zoonotic disease with worldwide prevalence in humans and warm-blooded animal populations. In livestock Toxoplasma gondii is the causal agent of significant economic losses since it can cause abortions in goats and sheep. It is estimated that one third of the world population is infected. Although there are effective therapies for acute infection, these are sometimes poorly tolerated, teratogenic, and have a long administration time. Considering the deficiencies that exist related to the prevention and treatment of toxoplasmosis, the development of a safe and effective vaccine would be extremely valuable in fighting against this infection. In the present work, we characterize for the first time the adjuvant and immunogenic potential of a recombinant profilin protein (rTgPF), in a vaccine formulation alone or in combination with the well-known GRA7 antigen candidate in a murine toxoplasmosis model. Since TgPF acts as a ligand for TLR11 and 12 inducing innate immune responses that promote type 1 adaptive responses, we first study the capacity of the mix rGRA7 + rTgPF to initiate an immune response by evaluating dendritic cell activation. Both rTgPF and rGRA7 induces activation of mouse BMDCs more efficiently than the single proteins, evidenced by increased expression of CD80 and CD86 co-stimulatory proteins and secretion of IL-6, IL-10 and IL-12 cytokines after in vitro stimulation. The sum of the effects of rGRA7 and rTgPF on BMDCs maturation led us to assay them in a vaccination protocol. BALB/c mice vaccinated with this mix elicited a Th1-biased immunity via the induction of lymphocyte proliferation, activation of CD4+T cells and increased IFN-γ production that resulted in enhanced protection against chronic Toxoplama gondii infection. Profilin per se induce only cellular immunity but augments the effect of rGRA7 immune responses when used together, thus allowing us to postulate rTgPF as a potential adjuvant in a protein vaccine.

Photoperiodic manipulation modulates the innate and cell mediated immune functions in the fresh water snake, Natrix piscator

Objectives of the current work were to investigate the role of photoperiod and melatonin in the alteration of immune responses in a reptilian species. Animals were kept on a regimen of short or long days. Blood was obtained and leucocytes were isolated to study various innate immune responses. Lymphocytes were separated from blood by density gradient centrifugation and were used to study proliferation. Respiratory burst activity was measured through nitrobluetetrazolium reduction assay while nitric oxide production by leucocytes was assayed by nitrite assay. Lymphocytes were isolated and used to study proliferation with and without B and T cell mitogens. Photoperiodic manipulation acted differentially on leucocyte counts. Nitrite release was increased while superoxide production was decreased in cultures obtained from the snakes kept on the short day regimen. Significant enhancement of mitogen induced lymphocyte proliferation was observed in cultures from the animals kept in either long or short days compared to cultures from the animals kept in natural ambient day length. Use of in vitro melatonin showed that lymphocytes from the animals, kept in long days, were more reactive. Photoperiod induces changes in immune status which may permit adaptive functional responses in order to maintain seasonal energetic budgets of the animals. Physiological responses (like elevated immune status) are energetically expensive, therefore, animals have evolved a strategy to reduce immune functions at times when energy is invested in reproductive activities. Natrix piscator breeds from September to December and elevated pineal hormone in winter suppresses reproduction while immunity is stimulated.

Melatonin mediates monochromatic light–induced proliferation of T/B lymphocytes in the spleen via the membrane receptor or nuclear receptor

Our studies found that melatonin mediates the monochromatic light–induced lymphocyte proliferation in chickens. However, melatonin receptor subtypes contain membrane receptor (Mel1a/Mel1b/Mel1c) and nuclear receptor (Retinoic acid receptor–related orphan receptor [ROR] α/RORβ/RORγ) and are characteristic with cell specificity. This study compared receptor pathway of melatonin, which mediated the monochromatic light–induced T/B lymphocyte proliferations in chickens. Newly hatched chicks were randomly divided into white light, red light, green light (GL), and blue light groups. Green light promoted the membrane receptor expression in the spleen but decreased the nuclear receptor level compared with that of red light. These changes were accompanied by increase of T/B lymphocyte proliferation and plasma melatonin level under GL. Pinealectomy reversed aforementioned changes and resulted in no differences among the light-treated groups. Supplementation of exogenous melatonin enhanced GL-induced T/B lymphocyte proliferation in the spleen but was reversed by Mel1c antagonist prazosin and RORα agonist SR1078 and enhanced by RORα antagonist SR3335. However, Mel1b antagonist 4P-PDOT and RORγ antagonist GSK increased the stimulation effect of melatonin on GL-induced T lymphocyte proliferation but no effect on the B-lymphocyte proliferation. These results indicate that melatonin promotes the GL-induced T lymphocyte proliferation through Mel1b, Mel1c, and RORα/RORγ; however, the Mel1a, Mel1c, and RORα may be involved in the B lymphocyte proliferation.