Peripheral Blood Mononuclear Cell Production Research Articles

Effects of 2-Year Nutritional and Lifestyle Intervention on Oxidative and Inflammatory Statuses in Individuals of 55 Years of Age and over at High Cardiovascular Risk.

Obesity and overweight are disorders with high impact on the morbidity and mortality of chronic diseases, such as type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD). We aim to assess the effects of 2-year nutritional and lifestyle intervention on oxidative and inflammatory status in individuals of 55 years of age and over at high CVD risk. Participants (n = 100 individuals of 55 years of age and over living in the Balearic Islands, Spain) were randomized into control and intervention group. Anthropometric and haematological parameters, blood pressure and physical activity were measured before and after the intervention. Oxidative and inflammatory biomarkers in plasma, urine, peripheral blood mononuclear cells (PBMCs) and neutrophils were determined. A higher reduction in abdominal obesity, blood pressure and triglycerides levels was observed after a 2-year intervention. An improvement of oxidative stress and proinflammatory status was demonstrated with a significant reduction in myeloperoxidase, xanthine oxidase, malondialdehyde and monocyte chemoattractant protein-1 (MCP1) levels, and an increase in polyphenols in plasma was observed. A decrease in reactive oxygen species production in PBMCs and neutrophils levels after zymosan and lipopolysaccharide activation was found in the intervention group with respect to the control group. The intervention with hypocaloric Mediterranean Diet and customized physical activity improves oxidative stress and proinflammatory status and could contribute to decreasing the CVD risk.

Preclinical anti-tumor activity of small-molecule oral PD-L1 checkpoint inhibitors.

e14558 Background: The PD-1/PD-L1 checkpoint axis is a validated target in oncology, and immunotherapy with antibody approaches have proven efficacy across various tumor types. Oral small-molecule inhibitors of PD-L1 may offer advantages over antibody approaches by enabling tunable on-target engagement and the potential for better tissue penetration and improved efficacy. Here we report the preclinical in vitro activity of small-molecule inhibitors of PD-L1 possessing a novel mechanism of action, favorable pharmacokinetics, and demonstrated in vivo efficacy in a mouse colon adenocarcinoma model. Methods: In vitro activity was assessed in a Jurkat T cell NFAT reporter assay and PD-L1 reduction was confirmed in CHO-K1 cells expressing human PD-L1 (CHO-K1-hPD-L1) and in peripheral blood mononuclear cells (PBMCs) from healthy donors. T cell activation assays were conducted with PBMCs from healthy donors. Non-specific cytokine release was evaluated in human whole blood. Pharmacokinetic (PK) evaluations were conducted in rodents and non-human primates (NHP). In vivo efficacy was evaluated in an MC38 tumor humanized PD-L1 and PD-1 mouse model. Results: Small-molecule inhibitors of PD-L1 were able to disrupt PD-1:PD-L1 and PD-L1:CD80 interactions through the reduction of PD-L1 expression on the cell surface via a novel internalization mechanism, resulting in PD-L1 degradation (EC50s ranged from 1.9 – 24 nM in CHO-K1-hPD-L1 and primary human myeloid cells). Lead PD-L1 inhibitors mediated potent activation of T cells in a NFAT reporter assay (EC50s of 13 and 18 nM) as well as dose responsive elevations in IL-2 production in human PBMCs stimulated with staphylococcal enterotoxin B. Compound treatment did not elicit non-specific cytokine release in human whole blood, supportive of favorable immune safety. PK profiles showed low systemic clearance in rodents and NHP. In an MC38 tumor model, once daily oral administration of compounds at 3, 10, and 30 mg/kg resulted in dose responsive tumor reduction that was associated with T cell activation and infiltration into tumors. Conclusions: Oral small-molecule PD-L1 inhibitors possessing a novel mechanism of action and the ability to mediate T cell activation in primary human immune cell types were identified. These compounds display in vivo anti-tumor efficacy comparable to anti-PD-L1 antibody and possess favorable preclinical profiles for further development, with the potential for all-oral treatment regimens for oncology.

Inflammatory and Oxidative Stress Markers Related to Adherence to the Mediterranean Diet in Patients with Metabolic Syndrome.

Metabolic syndrome (MetS) is characterized by increased pro-oxidative stress and a pro-inflammatory state. Several studies emphasized the protective effect of the Mediterranean dietary pattern (MDP). To assess the oxidative and inflammatory state according to the adherence to MDP using biomarkers in patients with MetS. Antioxidant and pro-inflammatory biomarkers were determined in plasma, peripheral blood mononuclear cells (PBMCs), and neutrophils of adults (aged 55–75 years old; 60% women) with MetS living in Mallorca (Spain). Anthropometrics, dietary intake by a validated semi-quantitative 143-item food frequency questionnaire, and a Dietary Inflammatory Index were measured. Patients with low adherence to MDP showed higher levels of glycated haemoglobin A1c and triglycerides, and lower levels of HDL cholesterol. Plasma levels of interleukin-1β, IL-6, IL-15, tumour necrosis factor α, xanthine oxidase, and ghrelin, and activities of superoxide dismutase, and myeloperoxidase were higher in subjects with low adherence to the MDP. Reactive oxygen species production in PBMCs and neutrophils stimulated with lipopolysaccharide was higher in participants with low adherence to the MDP. Patients with MetS and higher adherence to the MDP showed less altered anthropometric parameters, blood biochemical profile, and better oxidative and inflammatory status.

Semi-Refined Carrageenan Attenuates Lipopolysaccharide-Induced Reactive Oxygen Species Production in Peripheral Blood Mononuclear Cells and Cell Membrane Alterations in Leukocytes

Aim: To assess the effects of semi-refined carrageenan (E407a) on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation in peripheral blood mononuclear cells (PBMCs) and LPS-mediated cell membrane alterations in leukocytes. Methods: Blood samples collected from 8 intact rats were incubated with E407a (10 mg/ml), E407a (50 mg/ml), LPS (1 µg/ml), E407a (10 mg/ml) + LPS (1 µg/ml), E407a (50 mg/ml) + LPS (1 µg/ml) and without those compounds (controls) for 2 h in RPMI 1640 medium enriched with 5% fetal bovine serum. ROS generation in PBMCs obtained from the incubated samples was estimated by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. The impact of E407a, LPS and their mixture on leukocyte cell membranes was evaluated spectrofluorimetrically using the fluorescent probe 2-(2¢-hydroxy-phenyl)-5-phenyl-1,3-oxazole. Results: Expectedly, incubation with LPS induced ROS generation in PBMCs and decreased the lipid order of cell membranes in leukocytes. E407a alone was found to alter neither ROS production in PBMCs, nor membrane lipid order in leukocytes. Semi-refined carrageenan partially reduced LPS-mediated ROS overproduction in PBMCs and cell membrane alterations in leukocytes. Conclusion: E407a attenuates LPS-induced alterations of redox homeostasis in rat PBMCs and LPS-mediated modifications of cell membrane lipid order in leukocytes.

Impaired mitochondrial ATP production, reduced mitochondrial spare respiratory capacity, and increased oxidative stress in PBMCs are associated with aging in adult EGAT population

Aging is associated with the degeneration of several tissues, and that degeneration has an impact on the structure and function of organs. Aging is the most potential risk factor for chronic diseases, particularly dementia. Mitochondrial function is one of the important processes to maintain the metabolism in cells and organs. Mitochondrial function in peripheral blood mononuclear cells (PBMCs) can be a biomarker for several chronic disease. However, the association between aging and mitochondrial function from PBMCs of healthy adult population has not been elusive. Thus, the present study aims to explore the association between aging and mitochondrial function in human PBMCs. The participants were recruited from Electricity Generating Authority of Thailand (EGAT). The demographic data were collected. The basic laboratory parameters were obtained by venipuncture. The PBMCs were also isolated from the venous blood of the participant and then underwent a mitochondrial function test by Seahorse assay. The mitochondrial oxidative stress and mass were assessed by MitoSox and MitoTracker fluorescent dyes and were quantified by flow cytometry. The data of 1,753 participants remained in the final analysis. The associations of age and mitochondrial respiratory parameters were assessed by linear regression analysis. The covariates including underlying diseases of participants and the basic laboratory parameters obtained in this study were used to evaluate the independent associations of age on mitochondrial functions. The general characteristics of the participants in this study were shown in Table 1. The increased age associated with the reduction in mitochondrial ATP production (beta coefficient [95% confident interval] = -0.59 [-0.76,-0.43]) and mitochondrial spare respiratory capacity (-3.30 [-3.65,-2.95]) in PBMCs. Furthermore, the higher age is associated with the higher mitochondrial oxidative stress/mass ratio (0.0045 [0.0040,0.0050]). After adjusted with underlying diseases or basic laboratory parameters, these associations were still in the same direction as univariate analysis. Aging was associated with mitochondrial dysfunction in PBMCs as indicated by the reduction in mitochondrial ATP production and mitochondrial spare respiratory capacity and elevation of mitochondrial oxidative stress/mass ratio. Further investigation on these findings might be useful to develop novel disease interventions or biomarkers for chronic diseases, particularly dementia.

Novel in vitro invariant natural killer T cell functional assays

BackgroundInvariant Natural Killer T (iNKT) cells are innate lymphocytes bridging the innate and adaptive immune systems and are critical first responders against cancer and infectious diseases. iNKT cell phenotype and functionality are studied using in vitro stimulation assays assessing cytokine response and proliferation capabilities. The most common stimulant is the glycolipid α-Galactosyl Ceramide (α-GalCer), which stimulates iNKT cells when presented by CD1d, an MHC class I-like molecule expressed by antigen-presenting cells (APC). Another stimulant used is α-GalCer-loaded DimerX, a CD1d-Ig fusion protein which stimulates iNKT cells in an APC-independent fashion. Here, we demonstrate use of the PBS-57-loaded CD1d-tetramer as an APC-independent stimulant, where PBS-57 is an α-GalCer analogue. MethodsUsing healthy fresh (n = 4) and frozen (n = 7) peripheral blood mononuclear cells (PBMCs), 10-h cytokine response (measuring IFN-γ production) and 10-day proliferation assays were performed assessing iNKT functionality using α-GalCer, CD1d-tetramer and DimerX stimulants. ResultsAll stimulants effectively induced IFN-γ production in both fresh and frozen PBMC. After the 10-h activation, CD1d-tetramer was significantly more effective than α-GalCer (p = 0.032) in inducing IFN-γ production in fresh PBMC and significantly more effective than both α-GalCer (p = 0.004) and DimerX (p = 0.021) in frozen PBMC. Similarly, all stimulants induced strong proliferation responses in all samples, although this was only significant in the frozen PBMC. No significant differences in proliferation were observed between stimulants. SignificanceThis study supports PBS-57-loaded CD1d-tetramer as an effective in vitro APC-independent iNKT cell stimulant, which is comparable to or even more effective than α-GalCer and DimerX. As CD1d is downregulated during infectious disease and cancer as evasion strategies, in vitro assays which are APC-independent can assist in providing objective insight to iNKT activation by not relying on CD1d expression by APCs. Overall, the novel CD1d-tetramer stimulation equips researchers with an expanded “toolkit” to successfully assess iNKT cell function.

Impact of Sodium Butyrate Treatment in LPS-Stimulated Peripheral Blood Mononuclear Cells of Poorly Controlled Type 2 DM.

Type 2 diabetes mellitus (T2DM) is associated with chronic low-grade inflammation, which is marked by the dysregulation of innate and adaptive immune responses. Therefore, reducing inflammation, possibly through an immunoregulatory agent, may play a role in T2DM treatment. Butyrate is the most potent short-chain fatty acid (SCFA), and it exerts anti-inflammatory properties by inhibiting histone deacetylase activity. As an immunoregulatory agent, sodium butyrate can inhibit nuclear factor kB (NF-kB) activation and reduce the production of pro-inflammatory cytokines in immune cells. The aim of the study was to measure the level of plasma butyrate in poorly controlled T2DM and normoglycemic participants and to compare the response of peripheral blood mononuclear cells (PBMCs) to sodium butyrate treatment between the groups by measuring production of the following cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γ, IL-13, and IL-10. The in vitro study examined the PBMCs of 15 participants with poorly controlled T2DM and 15 normoglycemic participants. PBMCs were cultured with the following stimulations for two days at a temperature of 37°C and 5% CO2: 100 ng/mL lipopolysaccharide (LPS), 1 mM sodium butyrate, or a combination of 100 ng/mL LPS and 1 mM sodium butyrate. Plasma butyrate was measured using gas chromatography-mass spectrometry, and cytokines from culture supernatant were analyzed using magnetic beads multiplex assay. Plasma butyrate levels in participants with poorly controlled T2DM did not significantly differ from those in normoglycemic participants (p = 0.105). Compared to treatment with an LPS-stimulated PBMC culture, treatment with 1 mM sodium butyrate reduced the levels of TNF-α (p < 0.039) and IFN-γ (p < 0.038) in normoglycemic participants. The same general trend was seen in PBMC from participants with poorly controlled T2DM, but higher variability appeared to preclude statistical significance. These data suggest that butyrate may modulate inflammatory cytokine production in human PBMCs, but more research is needed to determine if butyrate is anti-inflammatory in poorly controlled T2DM.

The Soluble Factor from Oral Cancer Cell Lines Inhibits Interferon-γ Production by OK-432 via the CD40/CD40 Ligand Pathway.

Simple SummaryOK-432 is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously reported that OK-432 treatment can induce the production of high levels of IFN-γ from peripheral blood mononuclear cells (PBMCs). Moreover, the IFN-γ production from PBMCs by OK-432 is impaired by conditioned media (CM) from oral cancer cells. To determine the inhibitory mechanism of IFN-γ production by CM, the genes involved in IFN-γ production was retrieved by cDNA microarray analysis. We found that CD40 played a key role in IFN-γ production via IL-12 production. Although the expression levels of CD40 were upregulated by OK-432 treatment in PBMCs, CM inhibited OK-432-induced CD40 expression. These findings suggest that uncertain soluble factor(s) in CM may suppress IFN-γ production via the CD40/CD40L–IL-12 axis in PBMCs.(1) Background: OK-432 is a penicillin-killed, lyophilized formulation of a low-toxicity strain (Su) of Streptococcus pyogenes (Group A). It is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously showed that (i) OK-432 treatment induces a high amount of IFN-γ production from peripheral blood mononuclear cells (PBMCs), and (ii) conditioned medium (CM) from oral cancer cells suppresses both the IFN-γ production and cytotoxic activity of PBMCs driven by OK-432. The aim of this study was to determine the inhibitory mechanism of OK-432-induced IFN-γ production from PBMCs by CM. (2) Methods: We performed cDNA microarray analysis, quantitative RT-PCR, and ELISA to reveal the inhibitory mechanism of CM. (3) Results: We found that CD40 plays a key role in IFN-γ production via IL-12 production. Although OK-432 treatment upregulated the expression levels of the IL-12p40, p35, and CD40 genes, CM from oral cancer cells downregulate these genes. The amount of IFN-γ production by OK-432 treatment was decreased by an anti-CD40 neutralizing antibody. (4) Conclusions: Our study suggests that uncertain soluble factor(s) produced from oral cancer cells may inhibit IFN-γ production from PBMCs via suppressing the CD40/CD40L–IL-12 axis.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans–induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Vitamin K2 Suppresses Proliferation and Inflammatory Cytokine Production in Mitogen-Activated Lymphocytes of Atopic Dermatitis Patients through the Inhibition of Mitogen-Activated Protein Kinases.

Vitamin K2 is suggested to have a suppressive effect on the peripheral blood mononuclear cells (PBMCs) of pediatric atopic dermatitis patients. We examined the molecular targets of vitamin K2 to suppress proliferation and cytokine production in T-cell mitogen-activated PBMCs of atopic dermatitis patients from the viewpoint of mitogen-activated protein kinase signaling molecules. The study population included 16 pediatric vitamin K2 patients and 21 healthy subjects. The effect of vitamin K2 on concanavalin A-activated PBMC proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell counting assays. T-helper (Th)1/Th2/Th17 cytokine profiles in plasma and PBMC-culture supernatants were analyzed by a cytometric beads array assay. Mitogen-activated protein kinase signaling molecules in concanavalin A-activated PBMCs were examined by enzyme-linked immunosorbent assay (ELISA) assays. At 10-100 µM, vitamin K2 significantly suppressed the proliferation of mitogen-activated PBMCs derived from atopic dermatitis patients and healthy subjects (p < 0.05). The interleukin (IL)-10 concentrations in plasma and the PBMC culture supernatants of atopic dermatitis patients were significantly higher than those of healthy subjects (p < 0.05). The IL-2 concentrations in the culture supernatants of atopic dermatitis PBMCs were significantly lower than those of healthy PBMCs (p < 0.05). Vitamin K2 significantly inhibited the IL-17A, IL-10, and tumor necrosis factor α (TNF-α) production (p < 0.05), and increased the IL-2 production (p < 0.01) in the culture supernatant of atopic dermatitis PBMCs. At 10-100 µM, vitamin K2 markedly decreased the of Mek1, extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and SAPK/c-Jun N-terminal kinase (JNK) expression in atopic dermatitis PBMCs (p < 0.05). Vitamin K2 is suggested to attenuate activated T-cell immunity in atopic dermatitis patients through the inhibition of mitogen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways.

The effect of horse placenta extract on human peripheral blood mononuclear cells

Introduction and Aim:Placental extracts due to the unique biological properties have been widely utilized in various fields of medicine. The objective of the study was to investigate the effect of horse placenta extract on human peripheral blood mononuclear cells (PBMCs). Materials and Methods:The placenta extract was obtainedfrom the mares. PBMCs were isolated from venous blood samples from healthy volunteers. To assess the toxic effect of the placenta extract (in final protein concentrations of 1500, 750, 375, 187.5, 93.75, 46.88 ?g/ml) an MTT test was employed. As a control, cultures of PBMCs untreated with an extract were used. The cytotoxicity of the extract was examined by using propidium iodide (PI). A CFSE test was utilized to evaluate T-cell proliferation. The levels of reactive oxygen species (ROS) were also evaluated. An enzyme-linked immune-sorbent assay was used to determine the level of interleukin secretion. Results:According to the results of MTT assay, the placenta extract cytotoxicity was observed at a concentration of 1500 ?g/ml and 750 ?g/ml. PI test showedthat the placenta extract solution had no toxic effect on PBMCs after 72 h (concentration range from 1500 to 46.9 ?g/ml). However, toxic effect was observed after 72 hours of exposure to extract at a concentration of 1500 to 3250 ?g/ml, where cytotoxicitywas 4.6 and 12.0%, respectively (p ? 0.05). The 2-hour incubation of cells with placenta extract at concentrations of 1500 ?g/ml and 375 ?g/ml led to a significant decrease in ROS level (p ? 0.05). In addition, T-cell proliferation was considerably reduced at an extract's concentration of 1500 ?g/ml and 375 ?g/ml compared to the control (p ? 0.05). The detected increase in the production of human IL-4 PBMC (24-hour incubation) was dependent on the concentration of the placenta extract. Conclusion:The results of the study indicate that the horse placenta extract does not possess cytotoxicity towards human PBMCs. Moreover, we observed the immune-modulating properties of placenta extract. Keywords:Horse placenta extract; cytotoxicity; immune modulation.

Downregulation of Inflammatory Cytokine Release from IL-1β and LPS-Stimulated PBMC Orchestrated by ST2825, a MyD88 Dimerisation Inhibitor.

The inflammatory process implicates homeostasis disruption and increased production of inflammatory mediators. Myeloid differentiation primary response 88 (MyD88) is an essential protein recruited after lipopolysaccharide (LPS) and interleukin (IL)-1β stimulation, a process that converges in nuclear factor kappa B (NF-κB) activation, as well as a transcription of several genes of both pro- and anti-inflammatory cytokines. The inhibition of MyD88 has shown efficacy by decrease inflammatory response, and has demonstrated potential application as a therapeutic target in chronic diseases. In this study, we investigate the effect of MyD88 dimerisation inhibitor ST2825 on cytokine production from rhIL-1β and LPS-stimulated peripheral blood mononuclear cells (PBMC) from healthy blood donors (HBD). ST2825 significantly downregulates the production of IFN-γ, IL-6, IL-12, IL-2, IL-15, IL-7, VEGF, IL-1Ra, IL-4, IL-5, IL-13 and IL-9 (p < 0.05) in LPS-stimulated PBMC. Moreover, ST2825 had a relatively low impact on IL-1β signalling pathway inhibition, showing that only a few specific cytokines, such as IFN-γ and IL-1Ra, are inhibited in rhIL-1β-stimulated PBMC (p < 0.01). In conclusion, MyD88 dimerisation inhibitor ST2825 showed high efficacy by inhibiting pro- and anti-inflammatory cytokine production in LPS-stimulated PBMC. Moreover, although rhIL-1β induced a sustained cytokine production (p < 0.05), ST2825 did not show a significant effect in the secretion of neither pro- nor anti-inflammatory cytokines in rhIL-1β-stimulated PBMC.

Neutrophil extracellular trap from Kawasaki disease alter the biologic responses of PBMC.

Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is an acute systemic vasculitis syndrome that mainly occurs in infants under 5 years of age. In the current manuscript, we were aiming to analyze the role of neutrophil extracellular traps (NETs) in the pathogenesis of KD, especially their interplay with peripheral blood mononuclear cells (PBMCs). Neutrophils were exposed to 20 nM phorbol myristate acetate (PMA), we found that neutrophils of KD patients were more likely to form NETs compared with healthy controls (HCs). Furthermore, PBMCs were cultured with NETs for 24 h, and we observed that NETs significantly increased the cell viability, suppressed cell apoptosis, and enhanced the pro-inflammatory cytokines production and NF-κB activation in PBMCs from KD patients. In addition, with the stimulation of NETs, the expression of vascular endothelial growth factor A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α) were increased, which were related with the pathological mechanism of KD. At last, we examined the activation of phosphoinositide 3 kinase (PI3K)/Akt signaling, and we found NETs treatment obviously enhanced the activation of PI3K and Akt. In conclusion, these findings suggested that the formation of NETs may alter the biologic responses of PBMC and affect the vascular injury in KD.

Immune response in cutaneous leishmaniasis patients with healing vs. non-healing lesions

The outcome of Leishmania infection mainly depends upon the Leishmania species which causes the disease and the generation of the type of host immune response, the healing process and protection in leishmaniasis depends upon induction of Th1 response. In this study, the Th1/Th2 cytokine profile in cutaneous leishmaniasis (CL) is evaluated. This study was carried out in leishmaniasis clinic of CRTSDL, TUMS, during March 2018 to March 2019. Peripheral blood mononuclear cells (PBMC) of volunteers with active healing and non-healing lesion (s) of cutaneous leishmaniasis (CL), volunteers with and without history of CL were cultured and stimulated with Soluble Leishmania antigen (SLA). The supernatants were collected and the levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method. The results showed a significantly higher levels of IFN-γ in volunteers with active CL healing form (p<0.005), history of CL (p<0.005) than healthy volunteers. A significantly (p<0.005) higher level of IFN-γ was seen in volunteers with active healing form of lesion than non-healing form. There was a significantly (p<0.005) higher level of IL-10 in volunteers with a history of non-healing form and active non-healing form of CL. There was no significant difference in IL-5 production in PBMC of different groups. IFN-γ production starts at early stage of cutaneous leishmaniasis and enhance during course of lesion healing, IFN-γ level is significantly higher in all patients compared to healthy volunteers, IFN-γ is significantly higher in patients with healing form than non-healing form of lesion.

In vitro Interaction of Pseudomonas aeruginosa Biofilms With Human Peripheral Blood Mononuclear Cells.

The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was <10% upon 24 h incubation of PBMC with P. aeruginosa biofilms. PBMC incubated for 24 h with preformed P. aeruginosa biofilms were significantly more activated compared to PBMC incubated alone. Interestingly, a marked activation of CD56+CD3− natural killer (NK) cells was observed that reached 60% of NK cells as an average of different donors. In the culture supernatants of PBMC co-cultured with P. aeruginosa biofilms, not only pro-inflammatory (IL-1β, IFN-γ, IL-6, and TNF-α) but also anti-inflammatory (IL-10) cytokines were significantly increased as compared to PBMC incubated alone. Furthermore, incubation of biofilms with PBMC, caused a statistically significant increase in the CFU number of P. aeruginosa, as compared to biofilms incubated without PBMC. In order to assess whether PBMC products could stimulate the growth of P. aeruginosa biofilms, we incubated preformed P. aeruginosa biofilms with or without supernatants obtained from the co-cultures of PBMC with biofilms. In the presence of the supernatants, the CFU count of biofilm-derived P. aeruginosa, was two to seven times higher than those of biofilms incubated without supernatants (P < 0.01). Overall, the results obtained shed light on the reciprocal interaction between human PBMC and P. aeruginosa biofilms. P. aeruginosa biofilms induced PBMC activation and cytokine secretion but, in turn, the presence of PBMC and/or PBMC-derived components enhanced the number of P. aeruginosa biofilm associated bacteria. This may indicate a successful bacterial defensive/persistence strategy against immune response.

Simultaneous analysis of saturated and unsaturated oxylipins in ‘ex vivo’ cultured peripheral blood mononuclear cells and neutrophils

Oxylipins are a family of saturated and unsaturated fatty acids peroxidation products with bioactive properties. We have developed an improved method for the measurement of ex vivo oxylipin production by peripheral blood mononuclear cells (PBMCs) and neutrophils. We aimed to develop an analytical method to determine the production rates of polyunsaturated fatty acids (PUFAs), PUFA-oxylipin, and saturated-oxylipins by stimulated PBMCs and neutrophils based on solid phase extraction and HPLC-MS/MS technology. A UHPLC system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer was used to identify and quantify oxylipin production. For each oxylipin and PUFA their differential response was calculated with respect to a deuterated internal standard factor (ISF). To calculate oxylipin and PUFAs in the culture samples, the individual ISF was used for each oxylipin and PUFA with respect to the deuterated internal standard. PBMCs and neutrophils showed a different pattern of oxylipin production and fatty acid secretion. Lipopolysaccharide (LPS) did not stimulate oxylipin production or fatty acids secretion in PBMCs, whereas phorbol myristate acetate (PMA) stimulation increased the production rate of 5-HETE, 15-HETE, 15-HEPE, 17-DoHE, PGE2, AA, and DHA. LPS stimulation decreased 16-hydroxyl-palmitatte (16−OHPAL) production and DHA secretion in neutrophils, while PMA stimulation increased the production rate of AA and its derivate oxylipins, 5-HETE, 15-HETE, and PGE2. In conclusion, we have developed a new method to determine oxylipins derived from both saturated and unsaturated fatty acids in culture cell media. This method has enough sensitivity, and accuracy, to determine oxylipin production and fatty acid secretion by PBMCs and neutrophils.

Cytokine profiles in peripheral blood mononuclear cells from patients with cystic echinococcosis

IntroductionCystic echinococcosis (CE) is a chronic zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus), which affects domestic and wild carnivores as the definitive host and ungulates as intermediate hosts. In intermediate hosts, both Th1 and Th2 cells are involved in the immune responses to an echinoccocal infection. This study aimed to investigate production of IL-4, IL-10, and IFN-γ cytokines in peripheral blood mononuclear cells (PBMCs) of CE patients before and after surgical treatment. MethodsTo evaluate cytokine production in response to E. granulosus antigens, we investigated IL-4, IL-10, and IFN-γ production in PBMCs of 20 CE patients in response to hydatid cyst fluid antigen (HCF-Ag) before and after surgical treatment using ELISA. ResultsThe mean IL-4 production from HCF-Ag stimulated PBMCs was significantly decreased (p < 0.05), while IFN-γ was significantly increased in HCF-Ag stimulated PBMCs in patients after surgery (p = 0.005).Furthermore, our results showed that there is no significant difference between IL-10 production in patients before and after treatment (p = 0.562). ConclusionsOur data Indicated production of IL-4 in cultured PBMCs of CE patients stimulated with HCF-Ag was decreased significantly. While, production of IFN-γ was increased significantly in responses to HCF Ag after surgery. We concluded that the evaluation of IL-4 and IFN-γ in HCF-Ag stimulated PBMCs of CE patients should be considered as a useful marker in the follow up of patients with cystic echinococcosis.

Immunomodulatory Mechanism of Acyclic Nucleoside Phosphates in Treatment of Hepatitis B Virus Infection

Current treatment with nucleos(t)ide analogs (NUCs) safely controls the replication of hepatitis B virus (HBV) and improves prognosis in patients with HBV. However, the inability to completely clear HBV is problematic, and novel therapies are desired. It has been believed that all NUCs have similar functions to inhibit HBV reverse transcriptase. However, our recent findings that only acyclic nucleoside phosphonates (ANPs; adefovir dipivoxil and tenofovir disoproxil fumarate) had an additional effect of inducing interferon (IFN)-λ3 in the gastrointestinal tract suggests that ANPs are not only distinct from nucleoside analogs (lamivudine and entecavir) in their structures but also in their functions. Because enteric lipopolysaccharide (LPS) can cross the intestine and affect peripheral blood mononuclear cells (PBMCs), we hypothesized that orally administered ANPs could have further additional effects to modulate LPS-mediated cytokine profile in PBMCs. This study showed that pretreatment of PBMCs, from either healthy volunteers or patients with HBV, with ANPs inhibited LPS-mediated interleukin (IL)-10 production, which reciprocally induced IL-12p70 and tumor necrosis factor-α production in a dose-dependent manner. Furthermore, the combination of IFN-α and ANPs synergistically enhanced LPS-mediated IL-12p70 production in PBMCs. Mechanistic analyses revealed that cellular metabolites of ANPs directly bound the Akt protein, inhibiting its translocation to the plasma membrane, thereby impairing Akt phosphorylation. Therefore, pretreatment of PBMCs with ANPs impairs LPS-mediated IL-10 production. Among NUCs, only ANPs have an additional pharmacological effect modulating LPS-mediated cytokine production, which is expected to produce favorable immune responses toward HBV elimination. This additional immunomodulation by ANPs in PBMCs, as well as IFN-λ3 induction in the gastrointestinal tract, provides insights into HBV treatment.

Combined in vitro IL-12 and IL-15 stimulation promotes cellular immune response in dogs with visceral leishmaniasis.

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.

Galectin Domain Containing Protein from Haemonchus contortus Modulates the Immune Functions of Goat PBMCs and Regulates CD4+ T-Helper Cells In Vitro.

Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleukin (IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction. The results demonstrated that rHc-GDC was successfully cloned, expressed in expression vector as well as in the gut surface of adult H. contortus worm and successful binding with PBMCs surface were observed. Immunoblotting results revealed that rHc-GDC is an important active protein of H. contortus excretory and secretory products. Moreover, the interaction of rHc-GDC with host cells increased the production of Th2, Th9 cells, IL4, IL-9, PBMC proliferation, nitric oxide, and cell migration. No effects of rHc-GDC were observed on PMBC apoptosis, production of Th1 cells, and secretions of IFN-γ and IL-10 cytokines. These findings indicate that recombinant GDC protein from H. contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro.