Proliferation Of Peripheral Blood Lymphocytes Research Articles

Apoptosis and proliferation ofperipheral blood T-cells as alternative processes in pathogenesis of diabetes mellitus type 1

Apoptosis, along with proliferation, is a form of lymphocyte response to activating stimuli. In the early stages of cell differentiation, the apoptotic response prevails and it results to the formation of tolerance to inductor antigen. Mature lymphocytes proliferate in response to stimulation and it means the initial stage in the development of the immune response. Since in this case apoptosis and proliferation acts as alternative processes, their ratio can serve as a measure of the effectiveness of the cellular response to activating signals. The resistance of autoreactive T-cells to apoptosis is the main key point in the development of type 1 diabetes mellitus (T1DM). Autoreactive T-cells migrates from the bloodstream to the islet tissue of the pancreas and take an active part in b cells destruction. The resistance of autoreactive effector T-cells to apoptosis may suggest their high proliferative potential. Therefore, the comparative evaluation of apoptosis and proliferation of peripheral blood lymphocytes can give a more complete picture of their functional state and thus will help to reveal the causes of ineffective peripheral blood T-ceiis apoptosis in patients with T1DM and will help to understand more deeply the pathogenesis of the disease. in this article, the features of proliferative response of peripheral blood T-cells in patients with T1DM and in individuals with high risk of developing T1DM have been studied. Apoptosis of T-cell subpopulations has been investigated. The correlation between the apoptotic markers and the intensity of spontaneous and activation- induced in vitro T-cells proliferation of was revealed. it was determined, that autoreactive peripheral blood T-cells were resistant to apoptosis and demonstrated the increased proliferative potential in patients with T1DM and in individuals with high risk of developing T1DM.

Immunoprophylactic evaluation of recombinant gametocyte 22 antigen of Eimeria tenella in broiler chickens

Gametocyte proteins are being explored as potential vaccine candidates against Eimeria sp. in chicken since they are the components of the resilient oocyst wall. The aim of this study was to investigate the immunoprophylactic efficacy of recombinant Eimeria tenella gametocyte antigen 22 (EtGam22) in chickens against homologous oocyst challenge. Broiler chicks were subcutaneously immunized individually with 100μg of recombinant EtGam22 adjuvanted with Montanide ISA 71 VG at 7days of age and boosted 2weeks later. The immunized chickens were challenged individually with 1 × 104 sporulated oocysts of E. tenella 1week post-booster immunization. The anti-EtGam22 IgY and serum cytokine response was measured post-immunization. The results showed that the anti-EtGam22 IgY antibody, serum IFN-γ, IL-2, TGF-β, and IL-4 levels in chickens vaccinated with recombinant protein were significantly increased post-immunization as compared to unimmunized challenged controls (P < 0.05). The peripheral blood lymphocyte proliferation activity was also found significantly higher in EtGam22-immunized group on day 28, i.e., pre-challenge (P < 0.05). Upon homologous oocyst challenge, chickens immunized with rEtGam22 exhibited a significant drop in the total oocyst output per bird (246.78 ± 36.9 × 106, 45.23% reduction) and a significantly higher weight gain (497.7 ± 19.2g) as compared to unimmunized challenged controls. Taken together, these data indicate that EtGam22 is a potent immunogen for use as a subunit vaccine against cecal coccidiosis in chickens as it induces a diverse and robust immune response involving multiple cytokines and strong antibody titers.

Effects of dietary Enterococcus faecium NCIMB 11181 supplementation on growth performance and cellular and humoral immune responses in broiler chickens

This study evaluated the effects of dietary Enterococcus faecium NCIMB 11181 on growth performance and immune response in broiler chickens. A total of 360 1-day-old Arbor Acres male birds were randomly assigned to 4 treatments that administered different dosages of E. faecium (0, 5 × 107, 1 × 108, and 2 × 108 CFU E. faecium/kg diet). The results revealed that average daily gain (ADG) changed quadratically, while feed conversion rate (FCR) increased linearly from day 22 to 35 and day 1 to 35 (P < 0.05). Supplementation of E. faecium at 5 × 107CFU/kg diet resulted in increased ADG (P < 0.05) compared with the other groups. Birds fed with 2 × 108 CFU/kg E. faecium exhibited increased peripheral blood lymphocyte proliferation in response to concanavalin A (Con A) (P < 0.05) at day 35 and enhanced skin responses following phytohemagglutinin (PHA) injection (P < 0.05) at 12h. Serum lysozyme activity at day 21 increased linearly with dietary E. faecium concentration (P < 0.05), the highest activity was observed in the 1 × 108 and the 2 × 108 CFU E. faecium groups (P < 0.01). Serum levels of proinflammatory cytokines IL-1β, IL-2, IL-6, IFN-γ, and anti-inflammatory IL-4, IL-10 changed linearly or quadratically both at the initial and final phases (P < 0.05). In addition, BSA antibody titers were significantly increased following both primary and secondary inoculation when birds were fed with 1 × 108 or 2 × 108 CFU/kg E. faecium (P < 0.05). In comparison with other groups, birds received 5 × 107 CFU E. faecium exhibited the highest levels of serum IgG (P < 0.05) at day 35. Together, our results revealed that broiler diet supplemented with 5 × 107 CFU/kg E. faecium NCIMB 11181 was appropriate in relation to growth performance under normal conditions. Upon administration with higher dosages of E. faecium NCIMB 11181, obvious immune-stimulatory effects were observed following both cell-mediated and humoral immunity.

PD-1 blockade inhibits lymphocyte apoptosis and restores proliferation and anti-viral immune functions of lymphocyte after CP and NCP BVDV infection in vitro

Bovine viral diarrhea virus (BVDV) is an important virus that can cause extensive economic losses in both dairy and beef industry worldwide. Acute infection with BVDV results in peripheral blood lymphopenia, apoptosis and immunosuppression. Up-regulated programmed death-1 (PD-1) expression induces functional exhaustion of lymphocytes, inhibition of proliferation and apoptosis of lymphocytes during acute and chronic viral infections, such as HIV and HCV. However, there are no reports showing the role of PD-1 in peripheral blood lymphopenia, apoptosis and immunosuppression after acute BVDV infection. Accordingly, we measured the mRNA and protein expression of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) infected with BVDV, and analyzed the effects of PD-1 blockade on immune-associated function and activity in peripheral blood lymphocytes (PBLs). The results showed that both cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection stimulated the mRNA and protein expression of PD-1 and PD-L1 significantly. The upregulation of PD-1/PD-L1 was accompanied by the decreased PBLs proliferation and increased apoptosis. Additionally, PD-1 blockade restored proliferation, inhibited apoptosis, increased IFN-γ production and decreased BVDV load. Remarkably, the PD-1/PD-L1 interaction has a more substantial effect on the immunoregulation of inhibiting proliferation induced by CP BVDV infection. Our findings confirm that PD-1 plays a vital role in peripheral blood lymphopenia and apoptosis caused by acute BVDV infection, and provide new insights into exploring the immunopathological mechanisms of BVDV or other members of the Flaviviridae family, and a potential therapeutic strategy to control BVDV infection.

Analysis of Sister Chromatid Exchanges and Proliferation of Human Peripheral Blood Lymphocytes Exposed to Epoxiconazole

Abstract The potential genotoxic/cytotoxic effect of epoxiconazole was evaluated by means of sister chromatid exchanges (SCE) following the 24 and 48 h in vitro exposure of human peripheral blood lymphocytes to epoxiconazole at concentrations of: 5, 10, 25, 50 and 100 μg. ml–1. Dimethyl sulphoxide (DMSO), used as an epoxiconazole solvent, was used as a negative control and mitomycine (MMC) as a positive control. After the 24-hour exposure, we failed to observe a significant increase in SCE frequencies in comparison with the negative control, however, the concentrations of 10—100 μg.ml–1 caused a significant decrease in the proliferation index (PI; P &lt; 0.001). Also, the 48-hour exposure produced no significant alterations in the SCE frequencies in comparison with the control. At epoxiconazole concentrations ranging from 10 to 50 μg.ml–1 we recorded a moderate to strong, dose-dependent inhibition of PI (P &lt; 0.05; P &lt; 0.01; P &lt; 0.001), while at the highest dose (100 μg.ml–1) the reduction in PI compared to the control was less pronounced (P &lt; 0.05). The reduction in PI at the concentration range of 10—100 μg.ml–1 depended on the number of cells in the M1, M2 and M3 phases of the cell cycle per total number of 100 evaluated metaphases. Our results indicated a significant cytotoxic or cytostatic effect on human peripheral blood lymphocytes.

Immune response and protective efficacy of Eimeria tenella recombinant refractile body protein, EtSO7, in chickens

Refractile body protein, SO7, is a highly immunogenic protein which is essentially involved in the early development of Eimeria species infecting the domestic chicken. In the present study, the immune response and protective efficacy of recombinant Eimeria tenella SO7 (rEtSO7) protein was assessed in broiler chickens following homologous oocyst challenge. Broiler chicks were subcutaneously immunized with rEtSO7 antigen adjuvanted with Montanide ISA 71 VG on 7 and 21 days of age and protective efficacy of vaccination was evaluated in terms of body weight gain, lesion score and reduction in oocyst output. The peripheral blood lymphocyte proliferation, serum IgY response, and levels of interferon gamma (IFN-γ), interleukin 2 (IL-2), interleukin 4 (IL-4), tumor growth factor beta (TGF-β) and nitric oxide (NO) were assessed. The results revealed significant reduction (p < 0.05) in the oocyst output and increased weight gain in immunized birds as compared to unimmunized birds. Significantly increased levels of serum IgY, IFN-γ and proliferation of lymphocytes were evident in rEtSO7 immunized chickens. The results demonstrated that the recombinant protein could effectively elicit the cellular and humoral immune responses in immunized chickens, and provided significant protection against caecal coccidiosis in chickens.

Intraocular DHODH-inhibitor PP-001 suppresses relapsing experimental uveitis and cytokine production of human lymphocytes, but not of RPE cells

BackgroundUveitis is a potentially blinding inflammatory disease of the inner eye with a high unmet need for new therapeutic interventions. Here, we wanted to investigate the suppressive effect of the intraocular application of the small molecule dihydroorotate dehydrogenase (DHODH)-inhibitor PP-001 on experimental relapsing rat uveitis and furthermore determine its effect on proliferation and cytokine secretion of human peripheral blood lymphocytes (PBL) and human retinal pigment epithelial (RPE) cells in vitro.MethodsSpontaneously relapsing uveitis was induced in rats by immunization with interphotoreceptor retinoid-binding protein (IRBP) peptide R14. PP-001 was injected intravitreally after resolution of the primary disease to investigate further relapses. Proliferation and metabolic activity of phytohemagglutinin (PHA)-stimulated human peripheral lymphocytes with and without PP-001 and cytokine secretion were determined by XTT assay and bioplex bead assay. The RPE cell line ARPE-19 as well as primary human RPE cells treated with PP-001 or anti-vascular endothelial growth factor (VEGF) antibody bevacizumab were also investigated for metabolic activity and cytokine/chemokine secretion.ResultsInjection of PP-001 into rat eyes reduced the number of relapses by 70%, from 20 relapses (57% of the rats affected) in the control group to 6 relapses (33% of the rats) in the treatment group. In human PBL cultures, PP-001 reduced the proliferation in a dose-dependent manner. The secretion of several cytokines such as IL-17, IFN-γ, and VEGF was suppressed by PP-001, as previously observed with rat T cells in the experimental autoimmune uveitis (EAU) model. In contrast, human RPE cells were not affected by PP-001, while the anti-VEGF antibody bevacizumab severely impaired the secretion of various cytokines including VEGF.ConclusionsFor the first time, intravitreal injection of PP-001 demonstrated an effective, but transient reduction of relapses in the rat EAU model. In vitro PP-001 suppressed proliferation and cytokine/chemokine secretion of human lymphocytes, while neither human RPE cell line ARPE-19 nor primary RPE cells were affected.

Dairy Heifers Naturally Exposed to Fasciola hepatica Develop a Type 2 Immune Response and Concomitant Suppression of Leukocyte Proliferation.

ABSTRACTFasciola hepatica is a parasitic trematode of global importance in livestock. Control strategies reliant on anthelmintics are unsustainable due to the emergence of drug resistance. Vaccines are under development, but efficacies are variable. Evidence from experimental infection suggests that vaccine efficacy may be affected by parasite-induced immunomodulation. Little is known about the immune response to F. hepatica following natural exposure. Hence, we analyzed the immune responses over time in calves naturally exposed to F. hepatica infection. Cohorts of replacement dairy heifer calves (n = 42) with no prior exposure to F. hepatica, on three commercial dairy farms, were sampled over the course of a grazing season. Exposure was determined through an F. hepatica-specific serum antibody enzyme-linked immunosorbent assay (ELISA) and fluke egg counts. Concurrent changes in peripheral blood leukocyte subpopulations, lymphocyte proliferation, and cytokine responses were measured. Relationships between fluke infection and immune responses were analyzed by using multivariable linear mixed-effect models. All calves from one farm showed evidence of exposure, while cohorts from the remaining two farms remained negative over the grazing season. A type 2 immune response was associated with exposure, with increased interleukin-4 (IL-4) production, IL-5 transcription, and eosinophilia. Suppression of parasite-specific peripheral blood mononuclear cell (PBMC) proliferation was evident, while decreased mitogen-stimulated gamma interferon (IFN-γ) production suggested immunomodulation, which was not restricted to parasite-specific responses. Our findings show that the global immune response is modulated toward a nonproliferative type 2 state following natural challenge with F. hepatica. This has implications in terms of the timing of the administration of vaccination programs and for host susceptibility to coinfecting pathogens.

Construction of Vibrio mimicus ghosts as a novel inactivated vaccine candidate and its protective efficacy against ascites disease in grass carps (Ctenopharyngodon idella)

Vibrio mimicus (V. mimicus) is an important bacterial pathogen causing high mortality rates for fish and shellfish species. Prevention of vibriosis still remains a challenge, and an efficacious and safe vaccine that confers protection against V. mimicus needs to be developed. Here, with the purpose of generating V. mimicus bacterial ghosts (VmBGs), the bacteriophage PhiX174 lysis gene E was cloned into the shuttle plasmid pBV220 for construction of the recombinant temperature-sensitive lysis plasmid pBV220-lysisE. The pBV220-lysisE was then electroporated into V. mimicus strain 04–14, and the resultant transformants were used for production of VmBGs by inducing lysis gene E expression. The VmBGs were characterized by observing their morphology and structure on electron microscopy, and then the safety and vaccine potential of VmBGs were further evaluated in grass carps (Ctenopharyngodon idella), The results showed that the efficiency of VmBGs induction was about 99.91%, and no living bacteria survived after lyophilization. The obvious transmembrane lysis pores with loss of cytoplasmic materials were observed in VmBGs by electron microscopy. Fish vaccinated with VmBGs via intraperitoneal route showed significantly up-regulated expression of several immune-related genes (G-type lysozyme, IL-1β, MHC-I and MHC-IIβ) in the spleens, and higher serum agglutinating antibody level, serum bactericidal activity as well as peripheral blood lymphocytes proliferation responses compared to formalin-killed cells of V. mimicus (FKC) vaccinated fish and PBS control fish. More importantly, strong protection with relative survival rate of 76.67% was recorded in the VmBGs-vaccinated fish after challenge with V. mimicus strain 04–14, whereas the FKC vaccine and PBS control showed 40% and 0% survival rate, respectively. Overall, these findings suggest that the newly constructed VmBGs may be used as a promising vaccine candidate for the control of V. mimicus infection in fish.

Proliferation of Peripheral Blood Lymphocytes and Mesenchymal Stromal Cells Derived from Wharton's Jelly in Mixed and Membrane-Separated Cultures.

We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

LEI0258 microsatellite variability and its association with humoral and cell mediated immune responses in broiler chickens

Major histocompatibility complex (MHC) has a profound influence on disease resistance or susceptibility, productivity and important economic traits in chicken. Association of the MHC with a wide range of immune responses makes it a valuable predictive factor for the disease pathogenesis and outcome. The tandem repeat LEI0258 is a genetic marker which is located within the B locus of chicken MHC and strongly associated with serologically defined haplotypes. LEI0258 microsatellite marker was applied to investigate the MHC polymorphism in Ross 308 broiler chicken (N=104). Association of LEI0258 alleles with humoral and cell mediated immune responses to Newcastle disease (ND), Infectious bursal disease (IBD) and Avian influenza (AI) vaccines were also examined. LEI0258 polymorphism was determined by PCR-based fragment analysis, and association of LEI0258 alleles with immune responses were evaluated using multivariate regression analysis and GLM procedures. A total of seven alleles ranging from 195 to 448bp were found, including two novel alleles (263 and 362bp) that were unique in Ross 308 broiler population. Association study revealed a significant influence of MHC alleles on humoral and cellular immune responses in Ross population (P<0.05). Alleles 385 and 448bp were associated with increased peripheral blood lymphocyte proliferation response. Alleles 300, 362 and 448bp had a positive effect on immune responses to Infectious bursal disease vaccine, and allele 263bp was significantly correlated with elevated antibody titer against Newcastle disease vaccine. Results obtained from this study confirmed the important role of MHC as a candidate gene marker for immune responses that could be used in genetic improvement of disease-resistant traits and resource conservation in broiler population.

Evaluation of Peripheral Blood Lymphocytes Proliferation in Botteng Village, Mamuju Inhabitants Using Binucleate Index

Botteng Village in Mamuju, West Sulawesi was known for the high natural background radiation exposure. Botteng Village inhabitants exposed to high natural radiation in their daily life. Radiation exposure can inhibit the mitosis mechanism at various phases. Our previous study revealed that mitotic and nuclear division indexes in Botteng Village inhabitants were lower compared to control samples. To validate our previous study results here we evaluate the binucleate index in peripheral blood lymphocytes of Botteng Village inhabitants. Blood samples were collected from thirteen healthy adult subjects in Botteng Village and thirteen healthy adult subjects in normal background radiation area. Binucleate index was calculated as the proportion of binucleated cell (BNC) in 500 cells for each sample. Our study showed that the BI in Botteng Village was higher compared to control group (23.58 ± 9.60 vs 23.47 ± 6.24). Statistical analysis revealed that the different was not significant (p=0.973). It is possible that the small sample numbers used in this study were not adequate to represent the BI value in Botteng Village inhabitants. This study also showed that there was insignificant difference of BI in respect to gender and age for all samples. Further study using larger sample number should be conducted to ensure the possibility of BI to evaluate the effect of chronic low radiation dose exposure on lymphocytes proliferation.

Human Amniotic Mesenchymal Stem Cells Exhibit Similar Immunosuppressive Ability with Bone Marrow Mesenchymal Stem Cells and Possess a Higher Proliferation Activity and Clearer Stem Cell Properties in vitro

Objectives: Human bone marrow mesenchymal stem cells (hBMSCs) have been used for the prevention and treatment of acute graft-versus-host disease (aGVHD) in post-hematopoietic stem cell transplant patients. In this study, we compared the biological characteristics and immunosuppressive activity of human amniotic mesenchymal stem cells (hAMSCs) and hBMSCs to provide experimental evidence for the potential use of hAMSCs for treating aGVHD, thus solving the problem of insufficient hBMSCs sources. Methods: HAMSCs were isolated by enzymatic digestion. hBMSCs were isolated using Ficoll-Hypaque density gradients. The biological characteristics of both stem cell types were compared by morphological analysis, analysis of cell growth, cell cycle profiling, immunophenotyping, and immunofluorescence assays. An in vitro co-culture of MSCs and peripheral blood mononuclear cells (PBMCs) was performed and lymphocyte proliferation measured using the Cell Counting Kit-8(CCK-8) assay. IFN-γ production was determined in the co-culture supernatant using enzyme linked immunosorbent assay (ELISA). Results: Both hAMSCs and hBMSCs had fibroblast-like morphology. hAMSCs could be maintained for at least 15 culture passages, but hBMSCs started to show signs of aging and remarkably reduced proliferation at 6-7 passages. There was no significant difference in the proportion of cells in G2/M phase between hAMSCs and hBMSCs (P>0.05). Immunophenotyping revealed positive expression of CD105, CD90, and CD73 and negative expression of CD34, CD45, CD11b, CD19, and HLA-DR on the surface of both hAMSCs and hBMSCs. hAMSCs were positive for Oct-3/4, but hBMSCs were not. Both hAMSCs and hBMSCs expressed vimentin. PHA-stimulated PBMCs proliferation was inhibited by hAMSCs and hBMSCs. This inhibition was stronger, as the proportion of MSCs increased. There were not significant differences between the inhibitory effects of the two MSCs types on PBMCs proliferation (P>0.05). Interferon-γ (IFN-γ) production was lower when PBMCs were co-cultured with either hAMSCs or hBMSCs than when they were cultured alone (P 0.05). Conclusion: The results of this study demonstrated that hAMSCs have higher proliferation activity and clearer stem cell properties than hBMSCs. Both hAMSCs and hBMSCs were able to suppress the proliferation of allogeneic peripheral blood lymphocytes and reduce IFN-γ secretion stimulated by PHA in vitro.

Effect of Forsythia suspensa extract and chito-oligosaccharide alone or in combination on performance, intestinal barrier function, antioxidant capacity and immune characteristics of weaned piglets.

We investigated the effects of Forsythia suspensa extract (FSE) and chito-oligosaccharide (COS), alone or together, on performance and health status of weaned piglets. The treatments included a basal diet and three diets with 160 mg/kg COS, 100 mg/kg FSE, or 100 mg/kg FSE and 160 mg/kg COS. Supplementation with COS or FSE alone improved (P < 0.01) average daily gain and feed conversion ratio compared with the basal diet in the first 2 weeks. On day 14, COS or FSE supplementation separately produced stronger (P < 0.01) serum total antioxidant capacity and glutathione peroxidase activities and lower serum endotoxin (P < 0.05) and malondialdehyde (P < 0.01) concentrations, generated higher (P < 0.01) serum complement 4 concentration, peripheral blood lymphocyte proliferation and serum-specific ovalbumin antibody level than the basal diet. No differences in oxidative injury and immunity indices were detected on day 28. The combined FSE and COS produced similar results compared with FSE or COS when given alone. These data indicate FSE or COS can increase performance by modulating intestinal permeability, antioxidant status and immune function in younger pigs. There appears to be similar advantage in feeding the additives in combination over those obtained from feeding them separately.

Immunotoxicological Evaluation of Genetically Modified Rice Expressing Cry1Ab/Ac Protein (TT51-1) by a 6-Month Feeding Study on Cynomolgus Monkeys.

The present study was performed to evaluate the food safety of TT51-1, a new type of genetically modified rice that expresses the Cry1Ab/Ac protein (Bt toxin) and is highly resistant to most lepidopteran pests. Sixteen male and 16 female cynomolgus monkeys were randomly divided into four groups: conventional rice (non-genetically modified rice, non-GM rice), positive control, 17.5% genetically modified rice (GM rice) and 70% GM rice. Monkeys in the non-GM rice, positive control, and GM rice groups were fed on diets containing 70% non-GM rice, 17.5% GM rice or 70% GM rice, respectively, for 182 days, whereas animals in the positive group were intravenously injected with cyclophosphamide every other day for a total of four injections before the last treatment. Six months of treatment did not yield abnormal observations. Specifically, the following parameters did not significantly differ between the non-GM rice group and GM rice groups: body weight, food consumption, electrocardiogram, hematology, immuno-phenotyping of lymphocytes in the peripheral blood, mitogen-induced peripheral blood lymphocyte proliferation, splenocyte proliferation, KLH-T cell-dependent antibody response, organ weights and ratios, and histological appearance (p>0.05). Animals from the GM rice group differed from animals in the non-GM rice group (p<0.05) in several parameters: specifically, their body temperatures and serum alanine aminotransferase (ALT) levels were higher, whereas their levels of serum K+, Cl- and cytokines (IL-2, IL-4 and IL-5) were lower. Because dose- or time-dependent changes were not observed in this study and animals appeared histologically normal, the aforementioned differences were not considered to be adverse or related to the treatment with GM rice. In conclusion, a 6-month feeding study of TT51-1 did not show adverse immunotoxicological effects on cynomolgus monkeys.

Evaluation of immunogenicity and protective efficacy of recombinant ptfA of avian Pasteurella multocida.

Avian Pasteurella multocida is the causative agent of fowl cholera, a disease much affecting the poultry industry. In order to study the efficacy of the recombinant subunit vaccine constructed with ptfA gene of avian P. multocida, the ptfA gene fragment amplified by PCR from avian P. multocida was cloned into the prokaryotic expression vector pET32a and the recombinant plasmid pET32a-ptfA was obtained. The pET32a-ptfA was expressed in Escherichiacoli BL21(DE3) and the target protein rPtfA was purified. The purified protein was then mixed with Freund's adjuvant and the recombinant subunit vaccine was obtained. Three groups of chickens labeled as rPtfA, attenuated live vaccine and PBS were vaccinated with the recombinant subunit vaccine, attenuated live vaccine and PBS, respectively. Serum antibodies, peripheral blood lymphocyte proliferation (PBLP) and interferon-γ (IFN-γ) level secreted by peripheral blood lymphocyte were tested. The immunized chickens were finally challenged with virulent avian P. multocida and the protection rate was counted. Indirect ELISA showed the levels of antibodies in rPtfA and attenuated vaccine groups were most significantly higher than the other groups (P<0.01), and the former was slightly lower than the latter. Peripheral blood lymphocyte proliferation experiments and IFN-γ experiments indicated that SI value and the levels of IFN-γ induced by ConA in the two vaccine groups were significantly higher than those of the PBS groups (P<0.01), and that the attenuated vaccine group was higher than the rPtfA group. The protection rates of rPtfA and attenuated live vaccines were 45% and 75%, respectively. The results indicated that the PtfA recombinant subunit vaccine was capable of improving the immunity level and inducing a protective effect for the vaccinated chickens, but it was barely satisfactory.

Diethyldithiophosphate (DEDTP) Role in Proportion of Macrophages and Natural Killer Cells in Lymph Vessel and 4T1 Murine Breast Tumor Model

The diethyldithiophosphate (DEDTP) is a metabolite produced by the degradation of organophosphorus pesticides (POFs), and a dialkylphosphate (DAP) which is also chemically synthesized with a widespread commercial use and so the world population is chronically exposed to this compound. It is suggested that organophosphorus compounds produce deleterious effects on the immune system of the exposed population, since it has been demonstrated that DAPs affect peripheral blood lymphocytes activation and proliferation of T lymphocytes, furthermore, in vivo assays using C57 mice, the exposure to DEDTP enlarges skin tumors of BMK (Baby Mouse Kidney) cells, these results suggest that DEDTP exposure in vivo may modify the immune response and enhance the growth of tumor cells. Therefore, our objective is to determine the effect of DEDTP in the proportion of macrophages and natural killer cells (NKs) in lymph nodes and 4T1 breast cancer tumors in mice. To this end, we used Balb/c female mice of 6–8 weeks old, exposed for 8 days to 0.01 g/kg i.p. of DEDTP and inoculated with 1×105 4T1 cells. Tumors and lymph nodes were excised and measured. The percentage of total (F4/80+) and M2 (CD206+) macrophages and natural killer (NK) cells (CD355+) were assessed by flow cytometry. Exposure to DEDTP for 8 days increased the proportion of M2 macrophages. After 15 days of exposure to DEDTP, NK cells proportion was reduced but increased after 20 days. In mice challenged with 4T1 cells during DEDTP exposure, the number of total macrophages increased while the proportion of CD355+ cells in the lymph node proximal to the tumor decreased after 5 days; 10 days after, the number of M2 macrophages increased. Finally, after 20 days the number of M2 macrophages and NK cells in the proximal lymph node increased. In tumors, the number of infiltrating NK cells and total macrophages were reduced, followed by a reduction of M2 macrophages. Subsequently, after 15 days we observed an increase of infiltrating NK cells and M2 macrophages; after 20 days of tumor, M2 macrophages were increased. None of these changes had a significant impact on the establishment and development of tumors. With these results we conclude that exposure to DEDTP, in the absence of a particular antigen challenge, exerts immunomodulatory effects on individuals, apparently by inducing alternative activation of macrophages, however, this effect had no significant effect in the growth of 4T1 tumor cells. This could probably be because the more aggressive behavior of 4T1 tumor cells than BMK cells (previously tested on C57 mice) and the pronounced Th2 immune response profile of Balb/c mice (contrary to C57 animals). These results suggest that the effect of DEDTP on the immune system is not decisive against an aggressive tumor challenge, but may have relevance against a bacterial or parasitic infectious challenge.Support or Funding InformationCONACyT grant 153468

Effect of nutrient starvation on proliferation and cytokine secretion of peripheral blood lymphocytes.

Proliferating cancer cells are exposed to nutrient deprivation. Numerous previous studies have demonstrated how nutrient deprivation affects cancer cells; however, immune cells exposed to the identical conditions have not been completely examined. Furthermore, T-helper 2 lymphocyte predominance in certain neoplastic diseases has been reported; however, the mechanism remains unclear. The present study aimed to confirm whether nutrient deprivation affected proliferation and cytokine secretion of peripheral blood lymphocytes (PBLs). The proliferation of PBLs from healthy donors, cultured in a medium containing various glucose levels, was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. The expression levels of interleukin (IL)-4 and interferon (IFN)-γ among CD4(+) T cells, cultured with or without glucose and activated with phorbol 12-myristate 13-acetate and ionomycin, were examined using an intracellular cytokine staining method. The proliferation of PBLs cultured in a medium containing <100 mg/dl glucose of the standard blood sugar (BS) level was significantly reduced compared with the proliferation observed in a medium containing a standard BS level or higher. PBLs cultured in a glucose-free medium contained a significantly higher percentage of IL-4-positive and a lower percentage of IFN-γ-positive CD4(+) T cells compared with those cultured in a high-glucose medium. Nutrient deprivation suppressed the proliferation of PBLs, fostered the secretion of IL-4 and reduced secretion of IFN-γ. It is therefore possible that glucose-deficient microenvironments in local cancer tissues cause a partial immunodeficiency, which is advantageous to cancer growth.

The lympro® test: A fit for purpose validation of a flow cytometric assay to assess lymphocyte proliferation in peripheral blood lymphocytes in Alzheimer’s disease

The lympro® test: A fit for purpose validation of a flow cytometric assay to assess lymphocyte proliferation in peripheral blood lymphocytes in Alzheimer’s disease

A truncated C-terminal fragment of Mycobacterium tuberculosis HSP70 enhances cell-mediated immune response and longevity of the total IgG to influenza A virus M2e protein in mice

As the importance of virus-specific IgG2a and strong induction of Th1 type immune response for virus clearance was reported, conventional influenza vaccines induce a highly humoral immune response and fail to induce cytotoxic T-lymphocyte (CTL) immunity. Hence, in agreement with heat shock protein 70 (HSP70) acting as Th1 cytokine-like adjuvant, an Escherichia coli-expressed r4M2e.HSP70c fusion protein comprising C-terminus of Mycobacterium tuberculosis HSP70 genetically fused to four tandem repeats of influenza A virus M2e was constructed. Then, the case-control study was carried out to evaluate the humoral and cellular responses elicited against M2e in Balb/C mice by intramuscular immunization with r4M2e.HSP70c alone.Our results showed that r4M2e.HSP70c rather than control groups, r4M2e, r4M2e+Alum, or rHSP70c, significantly elevated both longevity and serum level of the total M2e-specific IgG antibody, induced a Th1 skewed humoral and cellular immune responses, increased the level of IFN-γ in BALF, and promoted the proliferation of peripheral blood lymphocytes. Furthermore, a virus challenge experiment revealed that mice vaccinated with r4M2e.HSP70c limited the severity of influenza A disease by 100% survival rate, less sever body weight loss and delaying the onset of morbidity in mice for 2days rather than other control groups. Here, we used r4M2e.HSP70c to stimulate M2e-specific antibody and cellular immune responses in Balb/C mice. The mHSP70c in the fusion form induced a long lasting Th1 skewed humoral and cellular immune responses against its associated protein. It seems anti-M2e antibodies limit viral replication and ameliorate influenza infection that allows the immune system to induce sterilizing HA-antibody against whole virion that leads to full protection against virulent influenza infection.