PHA-stimulated Cells Research Articles

ВЛИЯНИЕ ФАКТОРОВ РАДИАЦИОННОЙ И НЕРАДИАЦИОННОЙ ПРИРОДЫ НА КОНЦЕНТРАЦИЮ ТBX21 В ЛИЗАТАХ МОНОНУКЛЕАРОВ, СТИМУЛИРОВАННЫХ МИТОГЕНОМ

Purpose: To evaluate the effect of radiation and non-radiation factors on the intracellular concentration of the ТBX21 transcription factor in peripheral blood PHA-stimulated mononuclear cells of chronically exposed residents of the Techa Riverside settlements in the long-term period after the start of exposure. Material and methods: The main group consisted of 30 people aged 67–80 years with the mean dose to the red bone marrow 867±136 mGy, to thymus and peripheral lymphoid organs 125±20 mGy. The comparison group included 10 unexposed people aged 63–82 years. The main and the comparison groups had similar sex and ethnic composition. Peripheral blood mononuclear cells were PHA-stimulated for 24 hours. Cellular lysates were normalized by total protein concentration prior to being used for ELISA test. Results: After 24 hours of incubation the median and interquartile range of the intracellular TBX21 concentration in mononuclear cells was 34.2 (6.6–86.0) pg/ml after the mitogen stimulation, and 0 (0–24.9) pg/ml – without mitogen stimulation (р=0.001). In the comparison group these values made up 24.8 (0.2–47.6) and 13.0 (0–19.2) pg/ml, respectively Conclusion: The intracellular TBX21 concentration after 24-hour mitogen stimulation did not differ statistically significantly in chronically exposed and unexposed people, as well as in people from different dose groups. Statistically significant increase in TBX21 concentration in the lysates of mononuclear cells that were PHA-stimulated for 24 hours relative to non-mitogen stimulated cells was noted in chronically exposed people with medium and high doses to the red bone marrow. No correlation was revealed between the TBX21 concentration and dose characteristics, sex, and ethnicity of the studied individuals. The results are preliminary.

The Effect of Progestins on Cytokine Production in the Peripheral Blood Mononuclear Cells of Menopausal Women and Their Luminol-Dependent Chemiluminescence.

Steroid hormones are the key regulators of inflammatory and autoimmune processes. The role of steroid hormones is mostly inhibitory in these processes. The expression of IL-6, TNFα, and IL-1β, as markers of inflammation, and TGFβ, as a marker of fibrosis, could be useful tools to predict the response of an individual's immune system to the different progestins suitable for the treatment of menopausal inflammatory disorders, including endometriosis. In this study, the progestins P4 and MPA, as well as the novel progestin gestobutanoyl (GB), which possess potent anti-inflammatory properties towards endometriosis, were studied at a fixed concentration of 10 µM. Their influence on the production of the above cytokines in PHA-stimulated peripheral blood mononuclear cells (PBMCs) during 24 h incubation was evaluated by ELISA. It was found that synthetic progestins stimulated the production of IL-1β, IL-6, and TNFα and inhibited TGFβ production, while P4 inhibited IL-6 (33% inhibition) and did not influence TGFβ production. In the MTT-viability test, P4 also decreased PHA-stimulated PBMC viability by 28% during 24 h incubation, but MPA and GB did not have any inhibitory or stimulatory effects. The luminol-dependent chemiluminescence (LDC) assay revealed the anti-inflammatory and antioxidant properties of all the tested progestins, as well as some other steroid hormones and their antagonists: cortisol, dexamethasone, testosterone, estradiol, cyproterone, and tamoxifen. Of these, tamoxifen showed the most pronounced effect on the oxidation capacity of PBMC but not on that of dexamethasone, as was expected. Collectively, these data demonstrate that PBMCs from menopausal women respond differently to P4 and synthetic progestins, most likely due to distinct actions via various steroid receptors. It is not only the progestin affinity to nuclear progesterone receptors (PR), androgen receptors, glucocorticoid receptors, or estrogen receptors that is important for the immune response, but also the membrane PR or other nongenomic structures in immune cells.

β-ADRENERGIC RECEPTOR BLOCKERS AS A REGULATOR OF T CELL VIABILITY (IN THE MODEL SYSTEM OF THE JURKAT CELLS)

Our research aimed to establish the effectiveness of various β-adrenergic receptor blockers in the regulation of T cell proliferation in the model system of the Jurkat cells.Material and Methods. The research was conducted on the human leukemic mature T cells (Jurkat cells) (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany)) activated by phytohemagglutinin (PHA). Viability and proliferative activity of intact and PHA-stimulated Jurkat cells under the influence of Nebilet, Egilok, Betalok Zok, and Propranolol were studied with the MTT test. Results. Study results show that β-adrenergic receptors blockers selectively influence the activity of mitochondrial dehydrogenases, and therefore viability in both intact and mitogen-stimulated Jurkat cells. In particular, Nebilet, Betalok Zok, and Propranolol induced a statistically significant decrease of intact Jurkat cells viability by 63%, 20%, and 32%, respectively; Egilok and Betalok Zok (25mg) didn’t statistically significantly affect the PHA- activated Jurkat cells viability. Nebilet, Betalok Zok (50 mg), and Propranolol decreased the viability of the mitogen (PHA) activated Jurkat cells (for PHA dose 20μg/ml) and didn’t affect at a PHA dose of 50μg/ml.

Влияние рекомбинантного интерферона α-2b на цитокинпродуцирующую активность клеток иммунной системы у детей с COVID-19

The clinical efficacy of recombinant IFN-α2b in therapy for COVID-19 in children is the basis for studying the parameters of the cytokine production activity of immune system cells and will allow to optimize antiviral therapy regimens. Objective. To study the effect of recombinant IFN-α2b on serum IFN-α and IFN-γ concentrations and their synthesis by immune system cells in children with COVID-19. Materials and methods. Peripheral blood samples from 100 patients aged 1 to 17 years (1–7 years – 50 people, 8–17 years – 50 people) with a moderate course of COVID-19 were examined. Patients in the study group received recombinant IFN-α2b as Вклюpart of complex therapy. Patients in the comparison group received antiviral therapy with Arbidol®. Multiplex analysis was used to determine serum IFN-α and IFN-γ concentrations and the level of their synthesis by immune system cells. The enzyme-linked immunosorbent assay (ELISA) was used to determine serum concentrations of antibodies to IFN-α. Results. Combination therapy with recombinant IFN-α2b in children of both age groups led to an increase in serum IFN-α concentrations compared to baseline values prior to treatment, in children in the control group and patients with COVID-19 after treatment with Arbidol. There were no significant changes in serum IFN-α and IFN-γ concentrations and their synthesis in intact and PHA-stimulated cells in children of both age groups during treatment with Arbidol. Serum concentrations of antibodies to IFN-α during treatment with recombinant IFN-α2b did not depend on the age of children and remained within the reference range. Conclusion. A significant increase in serum IFN-α concentrations and restoration of its synthesis level induced by PHA to reference values indicate that the use of recombinant interferon medications with their antiviral and immunomodulatory effects should become an integral part of COVID-19 therapy. Key words: children, COVID-19, recombinant interferon IFN-α2b, serum IFN-α concentrations, IFN-α synthesis levels

PI3K, mTOR and GSK3 modulate cytokines’ production in peripheral leukocyte in temporal lobe epilepsy

IntroductionEpilepsy is a common pathological condition that predisposes individuals to seizures, as well as cognitive and emotional dysfunctions. Different studies have demonstrated that inflammation contributes to the pathophysiology of epilepsy. Indeed, seizures change the peripheral inflammatory pattern, which, in turn, could contribute to seizures. However, the cause of the altered production of peripheral inflammatory mediators is not known. The PI3K/mTOR/GSK3β pathway is important for different physiological and pharmacological phenomena. Therefore, in the present study, we tested the hypothesis that the PI3K/mTOR/GSK3β pathway is deregulated in immune cells from patients with epilepsy and contributes to the abnormal production of inflammatory mediators. MethodsPatients with temporal lobe epilepsy presenting hippocampal sclerosis and controls aged between 18 and 65 years-old were selected for this study. Peripheral blood was collected for the isolation of peripheral mononuclear blood cells (PBMC). Cells were pre-incubated with different PI3K, mTOR and GSK-3 inhibitors for 30 min and further stimulated with phytohaemaglutinin (PHA) or vehicle for 24 h. The supernatant was used to evaluate the production of IL-1β, IL-6, IL-10, TNF e IL-12p70. ResultsNon-selective inhibition of PI3K, as well as inhibition of PI3Kγ and GSK-3, reduced the levels of TNF and IL-10 in PHA-stimulated cells from TLE individuals. This stimulus increased the production of IL-12p70 only in cells from TLE individuals, while the inhibition of PI3K and mTOR enhanced the production of this cytokine. On the other hand, inhibition of GSK3 reduced the PHA-induced production of IL-12p70. ConclusionsHerein we demonstrated that the production of cytokines by immune cells from patients with TLE differs from non-epileptic patients. This differential regulation may be associated with the altered activity and responsiveness of intracellular molecules, such as PI3K, mTOR and GSK-3, which, in turn, might contribute to the inflammatory state that exists in epilepsy and its pathogenesis.

Cost-Effective Trap qPCR Approach to Evaluate Telomerase Activity: an Important Tool for Aging, Cancer, and Chronic Disease Research

OBJECTIVES:Telomeres are a terminal “DNA cap” that prevent chromosomal fusion and degradation. However, aging is inherent to life, and so is the loss of terminal sequences. Telomerase is a specialized reverse transcriptase encoded by self-splicing introns that counteract chromosome erosion. Telomerase activity is observed during early embryonic development, but after the blastocyst stage, the expression of telomerase reduces. The consequences of either insufficient or unrestrained telomerase activity underscore the importance of ongoing studies aimed at elucidating the regulation of telomerase activity in humans. In the present study, we aimed to standardize a simplified telomerase repeat-amplification protocol (TRAP) assay to detect telomerase activity in unstimulated and PHA-stimulated mononuclear cells.METHODS and RESULTS:Our optimized qPCR-based can efficiently evaluate telomerase activity. Quantification of protein and DNA between unstimulated and PHA-stimulated peripheral blood mononuclear cells revealed cellular activation and cell-cycle entry. The assay also showed that relative telomerase activity is significantly different between these two conditions, supporting the applicability of the assay. Furthermore, our findings corroborated that telomerase activity decreases with age.CONCLUSIONS:Telomeres and telomerase are implicated in aging and development of chronic diseases and cancer; however, difficulty in accessing commercial kits to investigate these aspects is a critical constraint in health surveillance studies. Our optimized assay was successfully used to differentiate telomerase activity between unstimulated and stimulated cells, clearly showing the reactivation of telomerase upon cell activation. This assay is affordable, reproducible, and can be executed in resource-limited settings.

Neonatal gut colonization by Bifidobacterium is associated with higher childhood cytokine responses

ABSTRACT The gut microbiota is a major stimulus for the immune system, and late acquisition of bacteria and/or reduced complexity of the gut flora may delay adaptive immune maturation. However, it is unknown how the gut bacterial colonization pattern in human infants is related to T cell activation during early childhood. We followed 65 Swedish children in the FARMFLORA cohort, from birth up to 3 years of age. In fecal samples collected at several time points during the first year of life, the gut colonization pattern was investigated with the use of both 16S rRNA next generation sequencing (NGS) and culture-based techniques. This was related to production of IL-13, IL-5, IL-6, TNF, IL-1β and IFN-γ by PHA-stimulated fresh mononuclear cells and to proportions of CD4+ T cells that expressed CD45RO at 36 months of age. Both NGS and culture-based techniques showed that colonization by Bifidobacterium at 1 week of age associated with higher production of IL-5, IL-6, IL-13, TNF and IL-1β at 36 months of age. By contrast, gut colonization by Enterococcus, Staphylococcus aureus or Clostridium in early infancy related inversely to induced IL-13, IL-5 and TNF at 3 years of age. Infants with elder siblings produced more cytokines and had a larger fraction of CD45RO+ T cells compared to single children. However, controlling for these factors did not abolish the effect of colonization by Bifidobacterium on immune maturation. Thus, gut colonization in early infancy affects T cell maturation and Bifidobacterium may be especially prone to induce infantile immune maturation.

Cellulose nanocrystals induce a dose-dependent effect on cytotoxicity and proliferative activity of human peripheral blood mononuclear cells

<p>Introduction. Cellulose nanocrystals (CNCs) as attractive natural materials, have numerous applications in the biomedical field. Their unique biomechanical characteristics, surface chemistry, low cost and sustainable nature make them an engaging alternative to conventional materials and potentiate their use as progressive material. Therefore, it is critical to evaluate the cytocompatibility and immunomodulatory properties of nCNCs, which have not been completely explored. The objective of this study was to examine a dose-dependent effect of native (n)CNCs on cytotoxicity and proliferative activity of human peripheral blood mononuclear cells (PBMNCs) in vitro. Methods. PBMNCs, obtained from the healthy blood donors, were cultivated with nCNCs. Cell viability was analyzed by flow cytometry assay, while proliferative activity was determined by MTT, [3H]-thymidine uptake assay and detection of IL-2 production. Results. The cytotoxicity results suggested that no concentration of nCNCs (50-400 mg/ml) affected necrosis of PBMNCs, whereas apoptosis was induced by the highest concentration of nCNCs compared to control (p<0.05). Unexpectedly, the highest concentration of nCNCs increased the metabolic activity of PHA-stimulated cells compared to control (p<0.05). In contrast to these findings, lower concentrations of nCNCs (50 mg/ml and 100 mg/ml) stimulated proliferation of PBMNCs (p<0.05 and p<0.001). It was followed by increased production of IL-2 (100 mg/ml) (p<0.001). Conclusion. The results suggest that non-cytotoxic concentrations of nCNCs modulate the proliferative activity of human PBMNCs, a phenomenon which has not been published up to now and which is relevant for further studies.</p>

Selective resistance profiles emerging in patient-derived clinical isolates with cabotegravir, bictegravir, dolutegravir, and elvitegravir

BackgroundIntegrase strand transfer inhibitors (INSTIs) are recommended for first-line HIV therapy based on their relatively high genetic barrier to resistance. Although raltegravir (RAL) and elvitegravir (EVG) resistance profiles are well-characterized, resistance patterns for dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) remain largely unknown. Here, in vitro drug selections compared the development of resistance to DTG, BIC, CAB, EVG and RAL using clinical isolates from treatment-naïve primary HIV infection (PHI) cohort participants (n = 12), and pNL4.3 recombinant strains encoding patient-derived Integrase with (n = 5) and without (n = 5) the E157Q substitution.ResultsPatient-derived viral isolates were serially passaged in PHA-stimulated cord blood mononuclear cells in the presence of escalating concentrations of INSTIs over the course of 36–46 weeks. Drug resistance arose more rapidly in primary clinical isolates with EVG (12/12), followed by CAB (8/12), DTG (8/12) and BIC (6/12). For pNL4.3 recombinant strains encoding patient-derived integrase, the comparative genetic barrier to resistance was RAL > EVG > CAB > DTG and BIC. The E157Q substitution in integrase delayed the advent of resistance to INSTIs. With EVG, T66I/A, E92G/V/Q, T97A or R263K (n = 16, 3, 2 and 1, respectively) arose by weeks 8–16, followed by 1–4 accessory mutations, conferring high-level resistance (> 100-fold) by week 36. With DTG and BIC, solitary R263K (n = 27), S153F/Y (n = 7) H51Y (n = 2), Q146 R (n = 3) or S147G (n = 1) mutations conferred low-level (< 3-fold) resistance at weeks 36–46. Similarly, most CAB selections (n = 18) resulted in R263K, S153Y, S147G, H51Y, or Q146L solitary mutations. However, three CAB selections resulted in Q148R/K followed by secondary mutations conferring high-level cross-resistance to all INSTIs. EVG-resistant viruses (T66I/R263K, T66I/E157Q/R263K, and S153A/R263K) retained residual susceptibility when switched to DTG, BIC or CAB, losing T66I by week 27. Two EVG-resistant variants developed resistance to DTG, BIC and CAB through the additional acquisition of E138A/Q148R and S230N, respectively. One EVG-resistant variant (T66I) acquired L74M/G140S/S147G, L74M/E138K/S147G and H51Y with DTG CAB and BIC, respectively.ConclusionsSecond generation INSTIs show a higher genetic barrier to resistance than EVG and RAL. The potency of CAB was lower than BIC and DTG. The development of Q148R/K with CAB can result in high-level cross-resistance to all INSTIs.

Effect of different loads of treadmill exercise on Th1/Th2 cytokine balance in rat splenocytes

The effect of moderate and overtraining exercise on Th1/Th2 balance was evaluated in rat splenocytes. Male Wistar rats were divided into sedentary control (C), moderately trained (MT; V=20m/min, 30min/day, 8 weeks), overtrained (OT; V=25m/min, 60min/day, 11 weeks) and recovered after overtraining (OR) (OT plus 2 weeks recovery) groups. At the end of study, cell viability, proliferation, interleukin 4 (IL-4) and interferon-γ (IFN-γ) secretion were evaluated in non-stimulated, phytohemagglutinin (PHA) and concavaline A (Con A)-stimulated splenocytes. Cell viability increased in MT and OR groups compared to control. Cell proliferation was higher in OR group than other groups. IL-4 concentration in PHA-stimulated cells from MT and OT groups, and IL-4 concentration in Con A-stimulated cells from OR and OT groups, were higher than the control group, but not for IFN-γ. In non-stimulated cells, IFN-γ/ IL-4 ratio was higher than MT and OT groups. In PHA and Con A-stimulated cells, IFN-γ/ IL-4 ratio was lower in exercise groups than control. We previously showed that moderate exercise increases Th1 cytokines in serum, but in splenocytes, Th2 or Th1 response may increase depending on the type of mitogen stimulation. Two-week recovery restored Th1/Th2 balance, only in non-stimulated splenocytes of overtrained animals.

Features of Cytokine Profile in Patients with Progressive TB-Induced Pulmonary Fibrosis Characterized by Various Intensities of Pulmonary Destructive Changes

Features of Cytokine Profile in Patients with Progressive TB-Induced Pulmonary Fibrosis Characterized by Various Intensities of Pulmonary Destructive Changes Production of pro- and anti-inflammatory cytokines (IFN-γ, IL-2, TNF-α, IL-8, IL-4, IL-10) in patients with pulmonary TB infection with various spread and severity of the process were investigated in in vitro PPD- and PHA-stimulated peripheral blood mononuclear cells (PBMCs). It was demonstrated that each type of MDR-TB lung fibrosis is determined by specific traits of a multilevel cytokine network functioning according to activity of cytokine-producing cells.

Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro.

Human adipose tissue-stromal derived cells (ASCs) are considered a perspective tool for regenerative medicine. Depending on the application mode ASC/allogeneic immune cell interaction can occur in the systemic circulation under plenty high concentrations of O2 and in target tissues at lower O2 levels. Here we examined the effects of allogeneic PHA-stimulated peripheral blood mononuclear cells (PBMCs) on ASCs under ambient (20%) oxygen and “physiological” hypoxia (5% O2). As revealed with microarray analysis ASCs under 20% O2 were more affected by activated PBMCs, which was manifested in differential expression of more than 300 genes, whereas under 5% O2 only 140 genes were changed. Altered gene pattern was only partly overlapped at different O2 conditions. Under O2 ASCs retained their proliferative and differentiative capacities, mesenchymal phenotype, and intracellular organelle' state. ASCs were proinflammatory activated on transcription level that was confirmed by their ability to suppress activation and proliferation of mitogen-stimulated PBMCs. ASC/PBMCs interaction resulted in anti-inflammatory shift of paracrine mediators in conditioning medium with significant increase of immunosuppressive LIF level. Our data indicated that under both ambient and tissue-related O2 ASCs possessed immunosuppressive potential and maintained functional activity. Under “physiological” hypoxia ASCs were less susceptible to “priming” by allogeneic mitogen-activated PBMCs.

Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

BackgroundCystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis.MethodsMorphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs).ResultsDespite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs.ConclusionsOur results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment.

Non-genomic rapid responses via progesterone in human peripheral T cells are not indirectly mimicked by sphingosine 1-phosphate

Progesterone is an endogenous immunomodulator that suppresses T cell activation during pregnancy. Progesterone has been shown to induce rapid responses that cause intracellular calcium ([Ca2+]i) elevation and acidification followed by inhibition of phytohemagglutinin (PHA)-stimulated proliferation. These rapid responses involve T cell plasma membrane sites, but the mechanisms remain unclear. Three new membrane progesterone receptors (mPRα/mPRβ/mPRγ) have been identified as expressed in T cells. These proteins have been identified as G-protein-coupled receptors. Recently, mPRs have been classified as progestin and adipoQ receptors (PAQRs). Furthermore, they have been suggested to be alkaline ceramidases, possibly involved in mediating sphingolipid signaling. Alkaline ceramidases are capable of converting ceramide to sphingosine, which might then be further phosphorylated sphingosine via sphingosine kinase to sphingosine 1-phosphate (S1P). This pathway could result in progesterone acting indirectly via S1P on membrane sphingosine 1-phosphate receptors (S1PRs) in T cells to induce rapid responses. Therefore, our aim was to investigate whether progesterone rapid responses occur indirectly in T cells via S1P. We found that S1P induces [Ca2+]i elevation however there was no change in intracellular pH. This is different from the situation with progesterone: S1P alone does not suppress PHA-stimulated cell proliferation and does not act synergistically with progesterone on the inhibition of PHA-induced cell proliferation. In contrast, S1P at 1μM is able to antagonize the proliferation inhibitory effect of progesterone. Thus the rapid responses that are induced by progesterone in human peripheral T cells probably do not involve indirect signaling via S1P and S1PRs.

Predicted Peptides from Non-Structural Proteins of Porcine Reproductive and Respiratory Syndrome Virus Are Able to Induce IFN-γ and IL-10

This work describes peptides from non-structural proteins (nsp) of porcine reproductive and respiratory syndrome virus (PRRSV) predicted as potential T cell epitopes by bioinfornatics and tested for their ability to induce IFN-γ and IL-10 responses. Pigs immunized with either genotype 1 or genotype 2 PRRSV attenuated vaccines (n=5/group) and unvaccinated pigs (n = 4) were used to test the peptides. Swine leukocyte antigen haplotype of each pig was also determined. Pigs were initially screened for IFN-γ responses (ELISPOT) and three peptides were identified; two of them in non-conserved segments of nsp2 and nsp5 and the other in a conserved region of nsp5 peptide. Then, peptides were screened for IL-10 inducing properties. Six peptides were found to induce IL-10 release in PBMC and some of them were also able to inhibit IFN-γ responses on PHA-stimulated cells. Interestingly, the IFN-γ low responder pigs against PRRSV were mostly homozygous for their SLA haplotypes. In conclusion, these results indicate that nsp of PRRSV contain T-cell epitopes inducing IFN-γ responses as well as IL-10 inducing segments with inhibitory capabilities.

Production of cytokines by mononuclear cells of hypertrophic adenoids in children with otitis media with effusion

Hypertrophic adenoids with otitis media with effusion is a common infectious disease and present a serious otological problem in children. Cytokines, potent inflammatory mediators, play important role in the initiation of immunological response in otitis media. Adenoids excised due to hypertrophy with or without chronic otitis media with effusion were used to isolate mononuclear cells. Secretion of cytokines by non-stimulated and PHA-stimulated cells was determined by specific ELISAs. We found a significant increase in the production of IL-5 and TNF-α secreted by adenoidal cells of children with otitis media with effusion compared to group with hypertrophic adenoids. No differences were found in the secretion of IL-8, IL-6, and IL-10 between these two groups of patients. Our results suggest a difference between the immunological responses in the course of hypertrophic adenoids with otitis media as compared to hypertrophic adenoids.

Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

BackgroundThe role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/ reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process.ResultsWe have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets - primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells.ConclusionsOur findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.

Programmed Death 1 Receptor ChangesEx Vivoin HIV-Infected Adults following Initiation of Highly Active Antiretroviral Therapy

This study investigates the short-term effects of highly active antiretroviral therapy (HAART) on programmed death 1 receptor (PD-1) expression and lymphocyte function. We compared lymphocytes from human immunodeficiency virus (HIV)-infected adults prior to the initiation of HAART with lymphocytes from the same subjects following 2 months of treatment. Short-term HAART resulted in a moderate increase in the expression of PD-1 on both CD4(+) and CD8(+) T cells; yet, there was still a significant reduction in viral load and recovery of CD4(+) T cells. After 2 months of HAART, lymphocytes from the subjects had a reduction in lymphoproliferative responses to phytohemagglutinin (PHA) and an increased response to the Candida recall antigen and the HIV antigen p24 compared to pretreatment lymphocytes. PHA-stimulated peripheral blood mononuclear cells (PBMCs) from samples obtained 2 months after HAART produced higher levels of Th-1 cytokines (gamma interferon [IFN-γ] and tumor necrosis factor alpha[TNF-α]) than the levels observed for samples taken before treatment was initiated. There were no significant changes in the proinflammatory cytokine interleukin-2 (IL-2) or Th-2 cytokines (IL-4, IL-5, and IL-10) in the corresponding samples. Ex vivo PD-1 blockade significantly augmented PHA-induced lymphoproliferation as well as the levels of Th-1 cytokines and to a lesser extent the levels of Th-2 cytokines in PBMC cultures. The ability to downregulate PD-1 expression may be important in enhancing immune recovery in HIV infection.

FasL Expression in Activated T Lymphocytes Involves HuR-mediated Stabilization

A prolonged activation of the immune system is one of the main causes of hyperproliferation of lymphocytes leading to defects in immune tolerance and autoimmune diseases. Fas ligand (FasL), a member of the TNF superfamily, plays a crucial role in controlling this excessive lymphoproliferation by inducing apoptosis in T cells leading to their rapid elimination. Here, we establish that posttranscriptional regulation is part of the molecular mechanisms that modulate FasL expression, and we show that in activated T cells FasL mRNA is stable. Our sequence analysis indicates that the FasL 3'-untranslated region (UTR) contains two AU-rich elements (AREs) that are similar in sequence and structure to those present in the 3'-UTR of TNFα mRNA. Through these AREs, the FasL mRNA forms a complex with the RNA-binding protein HuR both in vitro and ex vivo. Knocking down HuR in HEK 293 cells prevented the phorbol 12-myristate 13-acetate-induced expression of a GFP reporter construct fused to the FasL 3'-UTR. Collectively, our data demonstrate that the posttranscriptional regulation of FasL mRNA by HuR represents a novel mechanism that could play a key role in the maintenance and proper functioning of the immune system.

Defective T-cell Proliferation and IL-2 Production in a Subgroup of Patients with Coronary Artery Disease.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by innate and adaptive immune responses to a variety of microbial and self-antigens. Given the crucial role of adaptive immunity in the pathogenesis of atherosclerosis, this study was performed to investigate the proliferative response of peripheral blood mononuclear cells (PBMC) and interleukin (IL)-2 production in patients with coronary artery disease (CAD). In this study, 25 patients with chronic stable CAD and 25 healthy individuals were investigated. The PBMCs were separated and stimulated with phytohaemagglutinin (PHA). MTT assay was performed to measure cell viability and proliferation. IL-2 concentrations in cell culture supernatants were determined by Enzyme-Linked Immunosorbent Assay. PHA-stimulated cells revealed a significantly increased optical density (OD) in both groups of patients (p=0.004) and controls (p<0.001). However, the patient group showed a significantly lower Stimulation index (SI) (p=0.001). Upon in vitro stimulation with PHA, IL-2 levels were significantly increased in both groups of patients and controls (p<0.001). However, IL-2 concentrations were significantly lower in the patient group (p=0.018). Six patients showed defective IL-2 production, whereas similar finding was not observed in the normal control subjects (p=0.022). PBMCs from patients with coronary artery disease showed defective PHA-induced mitogenesis and IL-2 production. Considering the autoimmune nature of atherosclerosis, decreased IL-2 production may potentially enhance the atherogenic process, leading to spontaneous activation of autoreactive T lymphocytes.