Response Of Peripheral Blood Mononuclear Cells Research Articles

Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28-4-1BB Signaling in Bovine PBMCs.

Bovine viral diarrhea virus (BVDV), the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD), is an important pathogen of cattle and other wild animals throughout the world. BVDV infection typically leads to an impaired immune response in cattle. In the present study, we investigated the effect of Forsythoside A (FTA) on BVDV infection of bovine peripheral blood mononuclear cells (PBMCs). We found that Forsythoside A could not only promote proliferation of PBMCs and T cells activation but also inhibit the replication of BVDV as well as apoptosis induced by BVDV. FTA treatment could counteract the BVDV-induced overproduction of IFN-γ to maintain the immune homeostasis in bovine PBMCs. At same time, FTA can enhance the secretion of IL-2. What’s more, BVDV promotes the expression of CD28, 4-1BB and TRAF-2, which can be modulated by FTA. Our data suggest that FTA protects PBMCs from BVDV infection possibly via TRAF2-dependent CD28–4-1BB signaling, which may activate PBMCs in response to BVDV infection. Therefore, this aids in the development of an effective adjuvant for vaccines against BVDV and other specific FTA-based therapies for preventing BVDV infection.

Holi colours contain PM10 and can induce pro-inflammatory responses.

BackgroundAt Holi festivals, originally celebrated in India but more recently all over the world, people throw coloured powder (Holi powder, Holi colour, Gulal powder) at each other. Adverse health effects, i.e. skin and ocular irritations as well as respiratory problems may be the consequences. The aim of this study was to uncover some of the underlying mechanisms.MethodsWe analysed four different Holi colours regarding particle size using an Electric field cell counting system. In addition, we incubated native human cells with different Holi colours and determined their potential to induce a pro-inflammatory response by quantifying the resulting cytokine production by means of ELISA (Enzyme Linked Immunosorbent Assay) and the resulting leukocyte oxidative burst by flow cytometric analysis. Moreover, we performed the XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and Propidium iodide cytotoxicity tests and we measured the endotoxin content of the Holi colour samples by means of the Limulus Amebocyte Lysate test (LAL test).ResultsWe show here that all tested Holi colours consist to more than 40 % of particles with an aerodynamic diameter smaller than 10 μm, so called PM10 particles (PM, particulate matter). Two of the analysed Holi powders contained even more than 75 % of PM10 particles.Furthermore we demonstrate in cell culture experiments that Holi colours can induce the production of the pro-inflammatory cytokines TNF-α (Tumor necrosis factor-α), IL-6 (Interleukine-6) and IL-1β (Interleukine-1β). Three out of the four analysed colours induced a significantly higher cytokine response in human PBMCs (Peripheral Blood Mononuclear Cells) and whole blood than corn starch, which is often used as carrier substance for Holi colours. Moreover we show that corn starch and two Holi colours contain endotoxin and that certain Holi colours display concentration dependent cytotoxic effects in higher concentration. Furthermore we reveal that in principle Holi colours and corn starch are able to generate an oxidative burst in human granulocytes and monocytes. In Holi colour 1 we detected a fungal contamination.ConclusionsSome of the observed unwanted health effects of Holi colours might be explained by the high content of PM10 particles in conjunction with the possible induction of a pro-inflammatory response and an oxidative leukocyte burst.

Abstract 2210: Multiple antigens stimulating cellular therapy increases immune responses in patients with hepatocellular carcinoma

Abstract Background & Aims: Hepatocellular carcinoma (HCC) is one of the most common tumors in China, and frequently occurs in the patients with chronic hepatitis B virus (HBV) infection. To investigate whether adoptive cell therapy induces tumor specific immune responses in HCC patients and further brings clinical benefits, Multiple Antigens Stimulating Cellular Therapy (MASCT) was applied in addition to conventional therapies. Methods: Activated T cells that were prepared from the autologous peripheral blood mononuclear cells (PBMCs). Cells were stimulated and amplified with dendritic cells (DCs) pulsed by multiple tumor antigen peptides, including tumor associated antigen peptides and HBV antigen peptides. All patients received infusions of 5-10×107cells/kg of body weight of the resulting T cells every 1-3 months. The immune responses in the patients' PBMCs were examined before and after the treatment. Results: The resulting amplified cells were almost exclusively CD3+ (95% ± 1%), with a major percentage being CD3+CD8+ cells (70% ± 5%) and a minor proportion being CD3+CD4+(19% ± 3%) and CD3+CD56+ (14% ± 1%) cells, while there were insignificant amounts of CD4+CD25+Foxp3+ regulatory T cells (0.4% ± 0.1%). These T cells have restored immune functions and HLA-restricted specific cytotoxicity against HCC cells. After repeating treatment of MASCT, the frequency of regulatory T cells in the patients’ PBMCs was significantly decreased, and the proliferation and function of tumor antigen specific T cells were enhanced. The specific immune responses against each kind of tumor antigens were also detected in patients. And these immune responses were gradually increased during the treatment of MASCT. Moreover, in retrospective study, the disease control rate (DCR) in one year was found to be raised to 80% in stage B HCC patients receiving MASCT combined with conventional therapies. Conclusions: Our study has demonstrated that MASCT is a well tolerate immunotherapy in patients with HCC. Specific T cell responses against tumor antigens can be strongly induced in vivo. Our study also indicated that repeated MASCT treatment improves both the immunologic function and disease control of patients with HCC. Citation Format: Yanyan Han, Jing Huang, Ran Tao, Yabing Guo, Li Liu, Dongyun Wu, Jin Li, Xiangjun Zhou, Jinlin Hou. Multiple antigens stimulating cellular therapy increases immune responses in patients with hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2210.

Human eosinophils modulate peripheral blood mononuclear cell response to Schistosoma mansoni adult worm antigen invitro.

SummaryHigh numbers of eosinophils are observed in parasitic infections and allergic diseases, where they are proposed to be terminally differentiated effector cells that play beneficial role in host defence, or cause harmful inflammatory response. Eosinophils have been associated with killing of schistosomulae in vitro, but there is growing evidence that eosinophils can play additional immuno‐regulatory role. Here, we report results of a study that examines peripheral blood mononuclear cell (PBMC) cytokine responses to Schistosoma mansoni adult worm antigen (SWA) when stimulated alone or enriched with autologous eosinophils. Production of the Th‐2 type cytokines interleukin (IL)‐4, IL‐5 and IL‐13 was lower (P = 0·017, 0·018 and <0·001, respectively) in PBMC + eosinophil cultures than in PBMC‐only cultures stimulated with SWA. Substantial levels of IL‐13, IL‐10, interferon gamma and tumour necrosis factor alpha were recorded in cultures of eosinophils, but none of these cytokines showed significant association with the observed eosinophil‐induced drop in cytokine responses of PBMC. Transwell experiments suggested that the observed effect is due to soluble mediators that downmodulate production of Th‐2 type cytokines. This study shows that eosinophils may down‐modulate schistosome‐specific Th‐2 type cytokine responses in S. mansoni‐infected individuals. The mechanism of this immune modulation remains to be elucidated.

Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells

Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke.

Toll-like Receptor Expression and Signaling in Peripheral Blood Mononuclear Cells Correlate With Clinical Outcomes in Acute Hepatitis C Virus Infection.

Mechanisms by which spontaneous clearance of acute hepatitis C occurs are unclear. A critical role for the innate immune system and IFNL4 polymorphisms has been proposed. This study investigates whether Toll-like receptor (TLR) expression and signaling during acute hepatitis C correlates with clinical outcomes. Participants identified from the Australian Trial in Acute Hepatitis C and the Networks study were followed longitudinally from the time of diagnosis of acute hepatitis C. Peripheral blood mononuclear cells (PBMCs) and plasma were collected at and 2 time points after diagnosis. At each time point, TLR2, TLR4, and CD86 expression on peripheral blood monocytes, natural killer (NK) cells, and NK T cells was measured, as well as the response of PBMCs to stimulation with TLR ligands. Cytokine and chemokine levels were measured in stimulated PBMCs and plasma. We identified 20 participants with acute hepatitis C (10 with hepatitis C virus [HCV] monoinfection and 10 with HCV and human immunodeficiency virus coinfection). Eleven participants (55%) spontaneously cleared HCV. Acute hepatitis C and spontaneous clearance was associated with lower TLR4 expression on monocytes (P = .009) and NK cells (P = .029). Acute hepatitis C and spontaneous clearance was also associated with a reduced interferon γ response to TLR4 (P = .038) and TLR7/8 stimulation (P = .035), a reduced interleukin 6 response to TLR7/8 stimulation (P = .037), and reduced IFN-γ-inducible protein 10 (IP-10) response to TLR2 stimulation (P = .042). Lower plasma IP-10 levels were associated with spontaneous clearance (P = .001). These findings implicate TLR4 signaling as playing a critical role in the outcome of acute hepatitis C.

Allogeneic chondrogenically differentiated human mesenchymal stromal cells do not induce immunogenic responses from T lymphocytes in vitro

Background aimsIn regenerative medicine, the use of allogeneic cells could enable the development of “off the shelf” therapies for patients with critical size bone defects, reducing limitations observed with the use of autologous cells, such as cost and time to treat the patient. The idea of the use of allogeneic bone marrow mesenchymal stromal cells (BMSCs) has been of interest in tissue engineering studies. However, little is known about the properties of these cells upon differentiation. Chondrogenically differentiated BMSCs have already been shown to form endochondral bone in immunodeficient and immunocompetent animals. The success of this bone formation is dependent on the host's endogenous cells. This study investigates the interactions between allogeneic chondrogenically differentiated human bone marrow mesenchymal stromal cell (hBMSC) pellets and T lymphocytes in vitro. MethodsNon-chondrogenic (−transforming growth factor (TGF)β3) and chondrogenic hBMSC (+TGFβ3) pellets were directly co-cultured with unstimulated and CD3/CD28–stimulated peripheral blood mononuclear cells (PBMCs) for 7 days. hBMSC pellets from the co-culture were either fixed for histological analysis or quantitative real time polymerase chain reaction (qRT-PCR). PBMCs were harvested for flow cytometry. ResultsFlow cytometic analysis revealed that chondrogenically differentiated hBMSC pellets did not alter the number or proliferation of CD4+, CD8+ T cells or FoxP3+ T regulatory cells (CD4+CD25+CD127−). Chondrogenic hBMSC pellets did not induce immunogenic responses in unstimulated PBMCs. Infiltrating CD3 T cells were found in the matrix of hBMSC pellets. Furthermore, qRT-PCR demonstrated low levels of T-cell activation genes (CD25, CD69, PRF1 and GZMB) in addition to low gene expression levels of the pro-inflammatory gene tumor necrosis factor alpha (TNFα) in chondrogenically differentiated hBMSC pellets cultured with unstimulated PBMCs in comparison with non-chondrogenic hBMSC pellets. ConclusionsCollectively the results of this study demonstrate that allogeneic chondrogenically differentiated hBMSC pellets are non-immunogenic and do not induce the activation of destructive T-cell responses in vitro.

Human immune response to salivary proteins of wild-caught Phlebotomus papatasi.

Phlebotomine sand flies are the known vectors of Leishmania parasites. New approaches in vaccination against Leishmania have investigated the possibility of integrating Phlebotomus papatasi salivary proteins to enhance the immune response and protect against the transmission of the infection. The aim of the present study was to screen human immune responses to wild sand fly saliva and evaluate immunogenic salivary proteins. Blood samples were collected from donors in control and sand fly infested areas. Antibodies specific for sand fly antigens in donor plasma were probed using immunoblotting. In addition, recall proliferation capability of peripheral blood mononuclear cells (PBMC) was tested after sand fly salivary homogenates stimulation. The significant immunogenic salivary proteins (SPs) identified by immunoblotting were SP28, SP32, and SP36. A specific proliferative response of PBMC after stimulation with sand fly salivary homogenates was evident in donors that have antibody responses against sand fly salivary proteins. Individuals with antibody recognition to a higher number of salivary proteins (i.e., 3 or more SP bands) showed lower PBMC proliferative responses after in vitro stimulation with salivary gland homogenates (SGH) only in the sand fly infested, leishmaniasis free area. Interestingly, the presence of a humoral immune response to many SP antigens inversely correlates with a strong cell-mediated immune response (CMI). It was also noticed that some other heavily expressed antigens, in sand fly salivary homogenate, lack or have weak humoral immune reactivity in exposed individuals. Therefore, considering these antigens alone as CMI activators, without including the immunodominant humoral immune response proteins, needs future investigation.

Decreased Cell Wall Galactosaminogalactan in Aspergillus nidulans Mediates Dysregulated Inflammation in the Chronic Granulomatous Disease Host.

Invasive aspergillosis is a major threat to patients suffering from impaired neutrophil function, with Aspergillus fumigatus being the most common species causing this life-threatening condition. Patients with chronic granulomatous disease (CGD) not only develop infections with A. fumigatus, but also exhibit a unique susceptibility to infection with the normally nonpathogenic species Aspergillus nidulans. In this study, we compared the inflammatory cytokine response of peripheral blood mononuclear cells (PBMCs) from healthy and CGD patients to these two fungal species. CGD patients displayed evidence for a chronic hyperinflammatory state as indicated by elevated plasma IL-1β and TNF-α levels. PBMCs isolated from CGD patients secreted higher levels of IL-1β and TNF-α in response to A. nidulans as compared with A. fumigatus. The presence or absence of melanin in the cell wall of A. nidulans did not alter the cytokine release by healthy or CGD PBMCs. In contrast, A. fumigatus mutants lacking melanin stimulated higher levels of proinflammatory cytokine release from healthy, but not CGD PBMCs. Purified cell wall polysaccharides of A. nidulans induced a much higher level of IL-1β secretion by CGD PBMCs than did cell wall polysaccharides isolated from A. fumigatus. Using modified A. nidulans strains overexpressing galactosaminogalactan, we were able to show that the increased secretion of inflammatory cytokines by CGD PBMCs in response to A. nidulans are a consequence of low levels of cell wall-associated galactosaminogalactan in this species.

697. Phenotypic and Functional Characterisation of Human Anti-AAV CD8+ T Cells Using MHC Class I Tetramer-Associated Magnetic Enrichment

Introduction: Recombinant Adeno-Associated Vectors (AAV) represent nowadays the most largely used platform for in vivo gene therapy. Unfortunately, despite promising results in preclinical and clinical studies, pre-existing immunity against the viral capsid remains one of the major hurdles to the safety and efficiency of AAV-based gene transfer in Humans. Particularly, the impact of pre-existing anti-AAV CD8+ T lymphocytes on gene transfer remains poorly defined. This gap in knowledge can, in part, be attributed to the scarcity of AAV-specific CD8+ T cells among the peripheral blood mononuclear cells (PBMCs), and to the lack of relevant animal models. Experimental approach: In order to set up a more sensitive and comprehensive method to detect and characterise AAV-specific CD8+ T cells among PBMCs, we used Tetramer-Associated Magnetic Enrichment (TAME) to analyse the frequency and phenotype of AAV-specific CD8+ T cells by flow cytometry. To this end, we previously generated AAV-loaded tetramers using the UV-mediated peptide exchange technology. Results: We were able to detect AAV8 and AAV2 capsid-specific CD8+ T cells among PBMCs in all healthy donors tested (n>40), without any amplification, at frequencies ranging from 1.10−6 to 1.10−4 CD8+ T cells. Phenotypic assessment of the detected cells revealed a small proportion of memory CD45RO+ cells, a population that expectedly could have emerged after primary infection with wild-type AAV. Though AAV-specific CD8+ T cells were detectable by TAME in all donors tested, only few of them (11/42) had a positive response when cellular responses in PBMCs were assessed by anti-AAV IFN-γ ELISpot assays. Interestingly, we registered higher ex vivo frequencies and more frequent positive ELISpot responses in HLA-B7 donors then in HLA-A2's. Evaluation of anti-AAV antibody and neutralizing factor titres in the sera of the same donors revealed no correlation between humoral and cellular responses, as has been previously described. To further the functional assessment of the detected cells, we sorted AAV8 capsid-specific CD8+ T cells after TAME and expanded human primary T cell lines. Purity of the cell lines was checked using tetramer staining. We succeeded in generating several functional AAV-specific CD8+ T cell lines that upregulated CD107 and secreted IFN-γ, TNF-α, Granzyme B and Perforin when faced with AAV8-loaded target cells. Interestingly, some cell lines seemed to display a lack of reactivity that could not be attributed to a generally compromised functionality, suggesting that a fine tuning of AAV-specific CD8+ T cells’ activation might come into play, and is still under investigation. Conclusions and Perspectives: The dissimilarities observed ex vivo between humoral and cellular responses, as well as the different activation patterns registered in vitro highlight the need to gather multiple insights on capsid immunogenicity to better understand the onset of pre-existing anti-AAV immunity on recombinant AAV-based gene transfer and its impact on clinical outcome.

Air Pollution Particulate Matter Exposure Modulates Human Peripheral Blood Mononuclear Cell Host Immunity to Mycobacterium tuberculosis (M.tb)

Abstract Epidemiological studies indicate that indoor air pollution exposure increases susceptibility to M.tb infection and Tuberculosis (TB) development. Recently, we demonstrated that diesel exhaust particles and urban air pollution particulate matter (PM) impair human peripheral blood mononuclear cell (PBMC) and respiratory epithelial cell (A549) innate immune responses to M.tb via downregulation of TLR-dependent cytokines (IFN-γ, IL-1β IL-6, TNF-α), and antimicrobial peptides, respectively. Here we examined effects of real-world-derived air pollution PM from Iztapalapa, a municipality with high prevalence of air pollution and TB in Mexico City on innate and adaptive human immune responses to M.tb. Cytotoxic and inflammatory PBMC responses were examined upon exposure to PM2.5 (&amp;lt;2.5μm diameter) and PM10 (&amp;lt;10μm) from rainy, cold-dry and warm-dry weather seasons. PBMC from IGRA+ (immunodiagnostic evidence of M.tb exposure) and IGRA- donors were exposed for 20 hours to PM at 0.1, 1 and 10 mg/mL followed by additional 20-hour M.tb exposure. A PM dose-dependent inhibition of IFN-γ production was observed in M.tb-infected PBMC from IGRA+ but not IGRA- donors. Furthermore, the size range of PM, but not their seasonal source, affected the M.tb-induced IFN-γ production. No PM dose-dependent inhibition of IL-1β production was observed in M.tb-infected PBMC from either IGRA+ or IGRA− donors. PM10 from cold-dry and warm-dry seasons at high concentration caused cytotoxicity. This data suggests that urban ambient air pollution PM exposure (1) modulates M.tb-induced adaptive immune responses (suppression of IFN-γ but not IL-1β production in IGRA+ donors) and (2) potentially alters M.tb infection outcomes. Funding NIEHS 5R01ES020382-05

Truncated Escherichia coli thioredoxin induces proliferation of human blood mononuclear cells and production of reactive oxygen species as well as proinflammatory cytokines.

Thioredoxin (Trx) is a redox protein characterized by a Trx fold. A naturally occurring truncated human Trx, Trx 80, which lacks the C-terminal strand-helix of the Trx fold, stimulates proliferation of peripheral blood mononuclear cells (PBMCs). It has not been clear how Trx80 gains this function. This study investigates whether a peptide with substantial sequence difference from Trx80, but retaining an abridged Trx fold can elicit PBMC proliferation. We genetically truncated a carboxy-terminal β-α motif of Escherichia coli Trx to produce a peptide, Trx83, which shares low sequence identity with human Trx80. Addition of reduced-form Trx83 to resting human PBMCs promoted cell proliferation, while oxidized-form Trx83 lacked the function. By contrast, oxidized-form Trx80 exhibited a high activity in promoting PBMC proliferation, indicating the importance of sequence context of an abridged thioredoxin in influencing PBMC proliferation. Trx83 increases cellular reactive oxygen species and proinflammatory cytokines TNF-α and IL-1β, suggesting that Trx83 modulates inflammatory pathways. This notion is supported by the observation that cystine or cysteine abolishes the Trx83 induced PBMC proliferation. The PBMC stimulatory activity of Trx83 may have potential for pharmacological developments. Elicitation of primary proliferative responses of PBMCs by a protein is generally difficult. We show that Escherichia coli Trx83 with a truncated Trx fold induces PBMC proliferation, but only in the disulfide-reduced form. In contrast, oxidized-form human Trx80 is a potent stimulator. These results demonstrate that the sequence context of an abridged Trx fold is influential in inducing PBMC proliferation. The stimulatory effect of Trx83 is associated with an increase of inflammatory response. The possibility of eliciting PBMC proliferation and switching this activity on/off by redox control provides a perspective for developing Trx83 as a PBMC stimulatory agent. Copyright © 2016 John Wiley & Sons, Ltd.

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells.

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-β and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations.

Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients

Chlamydia pneumoniae is a cause of respiratory infection in adults and children. There is evidence for an association between atypical bacterial respiratory pathogens and the pathogenesis of asthma. We compared T helper (Th) responses in C. pneumoniae – infected peripheral blood mononuclear cells (PBMC) in patients with or without asthma. PBMC (1×106/mL) from asthmatic patients (N=11) and non-asthmatic controls (N=12) were infected or mock-infected for 1h +/− C. pneumoniae TW-183 at a multiplicity of infection (MOI)=1 and MOI=0.1, or cultured for 24h +/− Lactobacillus rhamnosus GG (LGG). Interleukin (IL)-4, IL-10, IL-12, Interferon (IFN)-gamma and total IgE levels were measured in supernatants (ELISA). C. pneumoniae infection led to an increase (>50%) of IgE levels in PBMC from asthmatics, compared with mock-infected on day 10; IgE wasn’t detected in non-asthmatics. C. pneumoniae – infected PBMC from asthmatics increased levels of IL-4 and IFN-gamma after 24h, compared with PBMC alone; levels of IL-10 and IL-12 were low. When uninfected-PBMC from asthmatics were LGG-stimulated, after 24h, IL-4 was undetectable, but IL-10, IL-12, and IFN-gamma increased, compared with PBMC alone. Thus, C. pneumoniae infection has the ability to induce allergic responses in PBMC of asthmatics, as evidenced by production of Th2 responses and IgE.

Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study.

Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses.Trial Registration: ClinicalTrials.gov NCT01269723

Baculovirus expression of the N-terminus of porcine heat shock protein Gp96 improves the immunogenicity of recombinant PCV2 capsid protein

Porcine circovirus type 2 (PCV2) causes significant economic losses to the swine industry worldwide. Heat shock proteins (Hsps) can be used as modulators to enhance both innate and adaptive immune responses. In the present study, recombinant baculoviruses expressing the PCV2Cap protein and the N-terminal 22–370 amino acids of porcine Gp96 (Gp96N), Hsp90, and Hsp70 (rBac-cap/Gp96N, rBac-cap/Hsp90 and rBac-cap/Hsp70, respectively) were constructed and the immune responses were examined in mice and piglets. The mouse experiments showed that rBac-cap/Gp96N increased the titers of specific anti-PCV2 neutralizing antibodies, proliferative responses of peripheral blood mononuclear cells (PBMCs) and IFN-γ levels compared to rBac-cap/Hsp90, rBac-cap/Hsp70, or rBac-cap. The pig experiments showed that the levels of anti-PCV2 antibody, proliferative responses of PBMCs, and IFN-γ in the rBac-cap/Gp96N groups were increased compared to those in rBac-cap group. There were no clear clinical signs of infection following PCV2 challenge in pigs inoculated with recombinant rBac-cap/Gp96N and rBac-cap, and the relative daily weight gains were higher than those in the challenge control (CC) group. The pathological lesions, extent of viremia, and viral loads of the vaccinated groups were milder than those in the CC group. Meanwhile, the extent of viremia and viral load present in the rBac-cap/Gp96N group were significantly lower than those in the rBac-cap group. These results indicated that porcine Gp96N effectively increased the humoral and cell-mediated immune responses of PCV2Cap. Gp96N presents an attractive adjuvant or immunotargeting strategy to enhance the protective efficacy of PCV2 subunit vaccines in swine.

Alcohol abuse and smoking alter inflammatory mediator production by pulmonary and systemic immune cells.

Alcohol use disorders (AUDs) and tobacco smoking are associated with an increased predisposition for community-acquired pneumonia and the acute respiratory distress syndrome. Mechanisms are incompletely established but may include alterations in response to pathogens by immune cells, including alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs). We sought to determine the relationship of AUDs and smoking to expression of IFNγ, IL-1β, IL-6, and TNFα by AMs and PBMCs from human subjects after stimulation with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). AMs and PBMCs from healthy subjects with AUDs and controls, matched on smoking, were cultured with LPS (1 μg/ml) or LTA (5 μg/ml) in the presence and absence of the antioxidant precursor N-acetylcysteine (10 mM). Cytokines were measured in cell culture supernatants. Expression of IFNγ, IL-1β, IL-6, and TNFα in AMs and PBMCs was significantly increased in response to stimulation with LPS and LTA. AUDs were associated with augmented production of proinflammatory cytokines, particularly IFNγ and IL-1β, by AMs and PBMCs in response to LPS. Smoking diminished the impact of AUDs on AM cytokine expression. Expression of basal AM and PBMC Toll-like receptors-2 and -4 was not clearly related to differences in cytokine expression; however, addition of N-acetylcysteine with LPS or LTA led to diminished AM and PBMC cytokine secretion, especially among current smokers. Our findings suggest that AM and PBMC immune cell responses to LPS and LTA are influenced by AUDs and smoking through mechanisms that may include alterations in cellular oxidative stress.

Dynamic changes in the proteome of human peripheral blood mononuclear cells with low dose ionizing radiation

Humans are continually exposed to ionizing radiation from natural as well as anthropogenic sources. Though biological effects of high dose radiation exposures have been well accepted, studies on low-to-moderate dose exposures (in the range of 50–500mGy) have been strongly debated even as researchers continue to search for elusive ‘radiation signatures’ in humans. Proteins are considered as dynamic functional players that drive cellular responses. However, there is little proteomic information available in context of human exposure to ionizing radiation. In this study, we determined differential expressed proteins in G0 peripheral blood mononuclear cells (PBMCs) from healthy individuals 1h and 4h after ‘ex vivo’ exposure with two radiation doses (300mGy and 1Gy). Twenty-three proteins were found to be significantly altered in irradiated cells when compared to sham irradiated cells with fold change ±1.5-fold (p≤0.05), with only three proteins showing ≥2.5-fold change, either with dose or with time. Mass spectrometry analyses identified redox sensor protein, chloride intracellular channel protein 1 (CLIC-1), the antioxidant protein, peroxiredoxin-6 and the pro-survival molecular chaperone 78KDa glucose regulated protein (GRP78) among the 23 modulated proteins. The mean coefficient of variation (CV) for the twenty-three radiation responsive protein spots was found to be 33.7% for 300mGy and 48.3% for 1Gy. We thus, conclude that the radiation proteomic response of G0 human PBMCs, which are in the resting stage of the cell cycle, involves moderate upregulation of protective mechanisms, with low inter-individual variability. This study will help further our understanding of cellular effects of low dose acute radiation in humans and contribute toward differential biomarker discovery.

Heparanase mRNA expression in peripheral blood mononuclear cell and heparanase activity monitor response to anticancer treatment and predict survival in pancreatic cancer

A 61 year old woman with active luminal Crohn's disease was successfully treated with infliximab induction therapy followed by 5 infusions every 8 weeks. However, symptoms returned in the weeks preceding the 7th and 8th infusions. The 9th infusion was therefore given only 4 weeks after the 8th infusion, but an acute severe anaphylactoid reaction occurred immediately after start of the infusion. Anti-infliximab IgG antibody concentration was high (100 U/ml) prior to the 8th infusion and up to 1 year after infliximab discontinuation (81 U/ml). Anti-infliximab IgE antibodies were not found, and the anti-infliximab antibodies did not cross react with adalimumab. One week after the anaphylactoid reaction to infliximab, adalimumab therapy was initiated. Twelve days after the first adalimumab administration (80 mg), a delayed hypersensitivity reaction occurred. This was likely caused by rapidly generated anti-adalimumab IgG antibodies (45 U/ml), as these antibodies appeared to be specific for adalimumab in that infliximab failed to compete with adalimumab/anti-adalimumab antibody binding ex vivo. In conclusion, immunogenicity to infliximab and adalimumab may be associated with both acute anaphylactoid reactions and delayed hypersensitivity reactions. Reactions may be precipitated by newly induced specific anti-drug antibodies rather than by cross-reactivity of previously generated antibodies.

Assessing the Effect of Orally Administered &lt;I&gt;Echinacea purpurea&lt;/I&gt; Extracts vs. Placebo on Pro-Inflammatory Cytokine Responses &lt;I&gt;Ex Vivo&lt;/I&gt;

Objective: To determine if aqueous, polysaccharide-containing Echinacea purpurea extracts taken orally increase pro-inflammatory cytokine responses ex vivo. Design: In two separate studies, the levels of TNF-alpha (TNF), interleukins 2 and 6 (IL-2 and IL-6) and interferon gamma (IFN-γ) secreted by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) from healthy adults randomized to take one of three E. purpurea formulations or placebo orally for 10 consecutive days were measured. Blood was obtained from participants at baseline and on days 2, 3, 7, and 10 while on study medication. PBMC were isolated and stimulated with PHA for 24 h, and supernatants collected for measurement of pro-inflammatory cytokine levels.Outcome Measures: Primary outcomes were peak concentrations of PHA-induced TNF, IL-2, IL-6, and IFN-γ from PBMC isolates collected while on study medication. Cytokine responses of PBMC from participants randomized to one of the Echinacea formulations were compared with those of placebo recipients by regression analysis.Results: Cytokine levels were obtained from mitogen-activated PBMC from 86 participants, collected while on study medication. No significant differences in the peak levels of PBMC-secreted TNF, IL-2, IL-6 and IFN-γ were observed between PBMC from those taking active Echinacea preparation vs. a placebo. After adjusting for age, a trend toward increased IL-6 secreted by PHA-stimulated PBMC isolated on day 3 of oral administration was observed for the group taking one of the E. purpurea formulations compared with placebo (P=0.064).Conclusions: Oral administration of E. purpurea did not significantly enhance peak pro-inflammatory cytokine responses in mitogen-stimulated PBMC.