Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis

Toll‐like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT‐PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti‐TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre‐incubation of PBMCs from healthy donors with anti‐TLR2 led to reduced expression of IFN‐γ and IL‐12p70, but not of IL‐4 and IL‐10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.     

Apolipoprotein A-I as a candidate serum marker for the response to lithium treatment in bipolar disorder

The molecular basis of bipolar disorder (BD) is still unknown as is the mechanism through which lithium, the therapy of choice, exerts its effects in treatment of BD. So far, no biomarkers exist to facilitate diagnosis of BD or treatment evaluation. To investigate whether BD and its treatment with lithium leaves a characteristic signature in the serum proteome, we used SELDI‐TOF MS to analyze individual serum samples from BD patients treated with lithium (BD‐plus‐Li, n=15) or other drugs (BD‐minus‐Li, n=10) and from healthy controls (n=15). Interestingly, features of 28 kDa (one peak) and 14 kDa (three peaks) showed a decreased level in the BD‐minus‐Li group and a level restored to that of the control group in the BD‐plus‐Li group. To reveal the identity of these features, we subjected pooled serum samples from both BD groups to the 2‐D DIGE technology and identified 28 kDa apolipoprotein A‐I (apo A‐I) and three 14 kDa fragments thereof as upregulated in the BD‐plus‐Li group. Immunoturbidimetry, a routine clinical assay, verified the characteristic apo A‐I signature in individual serum samples. In conclusion, we propose apo A‐I as a candidate marker that can visualize response to lithium treatment at the serum protein level.     

Interleukin-28A promotes IFN-γ production by peripheral blood mononuclear cells from patients with Behçet’s disease

Behçet’s disease (BD) is an autoimmune disease of unknown etiology. Interleukin-28A (IL-28A) promotes immune responses and may participate in the pathogenesis of autoimmune diseases. To examine the role of IL-28A in the pathogenesis of BD, we measured the expression of IFN-γ and IL-17 by IL-28A-stimulated peripheral blood mononuclear cells (PBMCs) from 19 patients with BD and 16 healthy individuals. We found that IFN-γ and IL-17 were undetectable in the sera from BD patients and control subjects. The mRNA expression and protein production of IFN-γ by IL-28A-stimulated PBMCs from BD patients were significantly increased compared to healthy individuals. No significant difference was observed in the mRNA expression and protein production of IL-17 by IL-28A-stimulated PBMCs between BD patients and normal individuals.     

Transcellular distribution heterogeneity of Annexin A5 represents a protective response to lupus-related thrombophilia: a pilot Proteomics-based study

Lupus-related vascular events are becoming a formidable obstacle to the improvement of long-term prognosis of systemic lupus erythematosus (SLE) and the existent findings lack for systematization. Proteomics is a strategic approach but its applications in this regard are rare and primarily involve proteome acquisition or biomarker screening, rather than functional identification. To provide further insight, we investigated the proteomic diversity of peripheral blood mononuclear cells (PBMCs) in SLE and the possible role of the identified Annexin A5 (AnxA5) in pathogenesis. The study involved 214 SLE and 183 healthy women. The two-dimensional electrophoresis gel images showed 649 ± 25 and 676 ± 19 protein spots from the PBMCs of the patients and controls, respectively. From these protein spots, 30 differentially expressed proteins were chosen, and 16 of these proteins were identified by mass spectrometer. Western blotting confirmed the over-expressed candidate, AnxA5, from the PBMCs of the patients (SLE:control=1.607:1, P=0.0004), but ELISAs indicated decreased levels of sera AnxA5 in the patients compared to healthy donors (SLE vs. control=26.8 ± 3.0 vs. 49.0 ± 3.3 ng/mL, P<0.0001). A positive correlation was demonstrated between the manifestation of thrombosis and AnxA5 (Mann-Whitney Z=-2.084, P=0.037), not anti-AnxA5, while searching for correlations between clinical parameters and the two molecular levels of patient sera. The coagulation assays using plasma from SLE patients revealed that elevated AnxA5 could shorten prothrombin time, activated partial thromboplastin time and prolonged thrombin time (P<0.001). Our data demonstrated the proteomic differences in the PBMCs between SLE patients and healthy persons. Moreover, the heterogeneous transcellular distribution, increased intracellular concentrations and decreased serum levels of AnxA5 represent a protective response to lupus-related thrombophilia; AnxA5 mostly participate in the common coagulation pathway in the thrombogenesis of SLE.     

Increased Expression of IL-22 Is Associated with Disease Activity in Behcet’s Disease

Objective

Interleukin (IL)-22 has been reported to be involved in the development of autoimmune diseases. This study aimed to analyze the expression and potential role of IL-22 in the pathogenesis of Behcet’s disease (BD).

Methods

The levels of IL-22 in patient sera or supernatants of cultured peripheral blood mononuclear cells (PBMCs) and CD4⁺T cells were detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to evaluate the frequency of IL-22–producing CD4⁺ T cells. IL-22 mRNA from erythema nodosum skin lesions was examined using real time quantitative RT-PCR.

Results

BD patients with active uveitis showed a significantly higher expression of IL-22 in the supernatants of stimulated PBMCs and CD4⁺T cells compared with BD patients without active uveitis and normal controls. An increased frequency of IL-22-producing CD4⁺T cells was also found in BD patients with active uveitis. IL-22 mRNA expression was elevated in erythema nodosum skin lesions. In BD patients, a high IL-22 level in the supernatant of stimulated PBMCs correlated with the presence of retinal vasculitis and erythema nodosum.

Conclusions

IL-22 was associated with disease activity in BD and correlated with the presence of small vessel inflammation, suggesting that it may be involved in its pathogenesis.     

MALDI-TOF/TOF-MS reveals elevated serum haptoglobin and amyloid A in Behcet's disease

Behcet's disease (BD) is a multisystemic autoimmune disease with unclear etiology and pathogenesis. To screen aberrant serum proteins in BD, serum samples were obtained from eight male BD patients with active uveitis and eight male healthy volunteers with informed consent. The serum samples from active BD patients and normal controls were pooled. Highly abundant serum proteins (albumin and IgG) were depleted from these two samples using an affinity capture based kit. The obtained samples were subjected to two-dimensional gel electrophoresis (2-DE). Protein spots were visualized with the "blue silver" staining. Differently expressed proteins were subsequently identified by matrix-assisted laser desorption /ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Western blot and enzyme-linked immunosorbent assay (ELISA) were performed using the serum samples from 18 patients with active BD, 6 patients with inactive BD, 22 patients with Vogt-Koyanagi-Harada (VKH) syndrome, and 20 healthy volunteers to validate the results of 2-DE and MS. Proteomic profiles of the pooled samples were compared, and approximately 800 protein spots were observed in each of the gels. Expression levels of four of the protein spots in active BD were significantly higher than those in the normal controls. Mass spectrometric protein identification revealed that the four protein spots corresponded to two proteins: haptoglobin (Hp) and serum amyloid A (SAA). Western blot and ELISA showed that Hp was only overexpressed in active BD but not in inactive BD, VKH syndrome, or healthy controls. An obvious band of SAA was detected in 72.2% of the serum samples from BD patients, whereas a vague band of this protein was found in 10.0% of the tested normal samples and 9.1% of VKH samples. Our results revealed a significantly increased expression of Hp and SAA in serum of active BD patients. These two proteins may be involved in the development of BD.     

Macrophage migration inhibitory factor in zinc-allergic systemic contact dermatitis

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose expression has been found to be critical to the generation of the antigen-specific immune response. Recent studies suggested that MIF plays a role in the initiation and maintenance of allergic disease. The aim of this study was to investigate whether MIF is involved in the pathogenesis of zinc-allergic systemic contact dermatitis. A 49-year-old Japanese woman developed facial edema, blepharedema and pruritic edematous erythema with papules over the entire body. Based of the results of a metal patch test, drug lymphocyte stimulating test and drug challenge test, diagnosis of zinc-allergic systemic contact dermatitis was made. Serum MIF and TNF-alpha levels of the patient, 20 healthy controls and other 6 patients who showed positive reaction to metal patch test were measured by an ELISA. Moreover we examined MIF production of peripheral blood mononuclear cells (PBMCs) from our patient, 3 healthy controls and other 2 patients who showed positive reaction to metal patch test at various metal concentrations. The patient's serum showed high MIF and TNF-alpha levels compared to healthy controls and other metal allergy patients. Furthermore, zinc stimulation of patient's PBMC showed higher MIF and TNF-alpha secretion compared with healthy subjects. The MIF content of 2 patients with other metal allergy was not significantly increased after metal stimulation. Our data suggest that zinc in the peripheral blood of zinc-allergic patients induce PBMCs to produce increased MIF levels, which could lead to systemic contact dermatitis.     

Analysis of peripheral blood lymphocyte phenotypes and Th1/Th2 cytokines profile in the systemic immune responses of Helicobacter pylori infected individuals

H. pylori elicits specific humoral and cellular immune responses in the mucosal immune system. However, the type and extent of T lymphocyte response in the systemic immune system is not clear for H. pylori positive patients. In this study, peripheral blood T lymphocyte phenotypes and serum Th1/Th2 based cytokines of 32 H. pylori positive patients were analyzed and compared to those of healthy controls. While αβ TCR⁺ lymphocytes and their phenotype analysis were not significantly different to those of healthy controls, the percentage of pan γδ TCR⁺ lymphocytes was up to 2.4 times greater in the H. pylori positive group then in healthy controls. Furthermore, significant increases in IL‐10 concentrations in serum samples of H. pylori patients indicated that their immune systems had switched toward a Th2 type immune response. The correlation between phenotype and type of T cell response in the peripheral blood during H. pylori infection is discussed.     

Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up

Recent findings indicate that microglia in Alzheimer’s disease (AD) is senescent whereas peripheral blood mononuclear cells (PBMCs) could infiltrate the brain to phagocyte amyloid deposits. However, the molecular mechanisms involved in the amyloid peptide clearance remain unknown. Autophagy is a physiological degradation of proteins and organelles and can be controlled by pro-inflammatory cytokines. The purpose of this study was to evaluate the impact of inflammation on autophagy in PBMCs from AD patients at baseline, 12 and 24 months of follow-up. Furthermore, PBMCs from healthy patients were also included and treated with 20 μM amyloid peptide 1–42 to mimic AD environment. For each patient, PBMCs were stimulated with the mitogenic factor, phytohaemagglutin (PHA), and treated with either 1 μM C16 as an anti-inflammatory drug or its vehicle. Autophagic markers (Beclin-1, p62/sequestosome 1 and microtubule-associated protein-light chain 3: LC3) were quantified by western blot and cytokines (Interleukin (IL)-1β, Tumor necrosis Factor (TNF)-α and IL-6) by Luminex X-MAP^® technology. Beclin-1 and TNF-α levels were inversely correlated in AD PBMCs at 12 months post-inclusion. In addition, Beclin-1 and p62 increased in the low inflammatory environment induced by C16. Only LC3-I levels were inversely correlated with cognitive decline at baseline. For the first time, this study describes longitudinal changes in autophagic markers in PBMCs of AD patients under an inflammatory environment. Inflammation would induce autophagy in the PBMCs of AD patients while an anti-inflammatory environment could inhibit their autophagic response. However, this positive response could be altered in a highly aggressive environment.     

系统性红斑狼疮患者外周血单个核细胞Toll样受体9的表达及血清白介素-10水平的研究

目的:研究Toll样受体9(TLR-9)在系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)上的表达水平及SLE患者血清白介素-10水平,探讨发病机制。方法:从23例活动期、19例缓解期SLE患者和20例正常对照组中分离PBMCs,利用反转录-聚合酶链反应(RT-PCR)法检测PBMCs中TLR9 mRNA的表达水平,利用酶联免疫吸附试验法检测其血清白介素-10水平。结果:活动期SLE患者PBMCs的TLR-9mRNA表达高于缓解组(P<0.01)及正常对照(P<0.01),缓解期和正常对照组相比,差异无统计学意义(P>0.05)。SLE活动期患者血清IL-10水平显著高于缓解期患者(P<0.01),并均高于正常对照组(P<0.01)。结论:活动期SLE患者PBMC的TLR9 mRNA的表达水平增高;并且活动期及缓解期SLE患者血清IL-10水平升高可能与TLR9 mRNA表达的上调相关。     

Decreased interleukin 27 expression is associated with active uveitis in Beh?et’s disease

Instruction

Interleukin 27 (IL-27) is an important regulator of the proinflammatory T-cell response. In this study, we investigated its role in the pathogenesis of Behçet’s disease (BD).

Methods

IL-27 mRNA in peripheral blood mononuclear cells (PBMCs) was examined by performing RT-PCRs. Cytokine levels in sera or supernatants of PBMCs, naïve CD4⁺ T cells, dendritic cells (DCs) and DC/T cells were determined by enzyme-linked immunosorbent assay. We used RNA interference in naïve CD4⁺ T cells to study the role of interferon regulatory factor 8 (IRF8) in the inhibitory effect of IL-27 on Th17 cell differentiation. Flow cytometry was used to evaluate the frequency of IL-17- and interferon γ–producing T cells.

Results

The expression of IL-27p28 mRNA by PBMCs and IL-27 in the sera and supernatants of cultured PBMCs were markedly decreased in patients with active BD. A higher frequency of IL-17-producing CD4⁺ T (Th17) cells and increased IL-17 production under Th17 polarizing conditions were observed in patients with active BD. IL-27 significantly inhibited Th17 cell differentiation. Downregulation of IRF8 by RNA interference abrogated the suppressive effect of IL-27 on Th17 differentiation. IL-27 inhibited the production of IL-1β, IL-6 and IL-23, but promoted IL-10 production, by DCs. IL-27-treated DCs inhibited both the Th1 and Th17 cell responses.

Conclusions

The results of the present study suggest that a decreased IL-27 expression is associated with disease activity in BD patients. Low IL-27 expression may result in a higher Th1 and Th17 cell response and thereby promote the autoinflammatory reaction observed in BD. Manipulation of IL-27 may offer a new treatment modality for this disease.     

Effect of Vitamin D on Peripheral Blood Mononuclear Cells from Patients with Psoriasis Vulgaris and Psoriatic Arthritis

BackgroundPsoriasis, a chronic skin disease with or without joint inflammation, has increased circulating proinflammatory cytokine levels. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis and osteoclast activity, as well as regulation of immune response. We aimed to study osteoclast differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris and psoriatic arthritis, in response to 1,25(OH)₂D₃.MethodsSerum levels of bone turnover markers were measured by ELISA in patients with psoriasis vulgaris and psoriatic arthritis, and healthy controls. PBMCs were isolated and cultured with or without RANKL/M-CSF and 1,25(OH)₂D₃. Osteoclast differentiation and cytokine secretion were assessed.ResultsPsoriatic arthritis patients had lower osteocalcin, as well as higher C-telopeptide of type I collagen and cathepsin K serum levels compared with psoriasis vulgaris patients and controls. RANKL/M-CSF-stimulated PBMCs from psoriatic arthritis patients produced higher proinflammatory cytokine levels and had a differential secretion profile in response to 1,25(OH)₂D₃, compared with psoriasis vulgaris and control PBMCs.ConclusionsOur data confirmed altered bone turnover in psoriatic arthritis patients, and demonstrated increased osteoclastogenic potential and proinflammatory cytokine secretion capacity of these PBMCs compared with psoriasis vulgaris and controls. 1,25(OH)₂D₃ abrogated these effects.     

The Elevated Secreted Immunoglobulin D Enhanced the Activation of Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis

Immunoglobulin D (IgD) is a surface immunoglobulin that is expressed as either membrane IgD (mIgD) or secreted IgD (sIgD). Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) are still unclear. In this study, we compared the expression of sIgD, mIgD and IgD receptor (IgDR) in RA patients and healthy controls, and investigated the effect of sIgD on the function of PBMCs. We found that the levels of sIgD, mIgD and IgDR were significantly higher in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with soluble receptor activator of nuclear factor-κB ligand (sRANKL), rheumatoid factor (RF) and C-reactive protein (CRP) in RA patients. Strikingly, IgD could enhance the proliferation of PBMCs and induce IL-1α, IL-1β, TNF-α, IL-6 and IL-10 production from PBMCs. Moreover, the percentage of activated T cell subsets (CD4⁺CD69⁺, CD4⁺CD154⁺) and activated B cell subsets (CD19⁺CD23⁺, CD19⁺CD21⁺, CD19⁺IgD⁺ and CD19^-CD138⁺) were increased by IgD. The percentage of unactivated T cell subset (CD4⁺CD62L⁺) and immature B cell subset (CD19⁺IgM⁺IgD^-) were decreased by IgD in PBMCs. Furthermore, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Our results demonstrate that IgD enhanced the activation of PBMCs, which may contribute to RA pathogenesis. Therefore, IgD could be a potential novel immunotherapeutic target for the management of RA.     

Elevation of human tumor necrosis factor-like weak inducer of apoptosis in peripheral blood mononuclear cells is correlated with disease activity and lupus nephritis in patients with systemic lupus erythematosus

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a recently identified proinflammatory cytokine of the TNF superfamily. Studies have indicated that TWEAK plays an important role in renal, vascular injury and immune disease. The aim of this study was to explore the expression of the TWEAK in peripheral blood mononuclear cells (PBMCs) and analyze the correlation between TWEAK and disease activity and renal damage of SLE. The expression of TWEAK in PBMCs was determined by RT-PCR and western blot. SLE disease activity was evaluated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) 2000 score. Next were analyzed the correlations of TWEAK mRNA and protein to serum IL-10, MCP-1 and some laboratory parameters of SLE disease activity. Subjects comprised 48 patients with SLE including 25 patients with renal damage and 23 without, 20 patients with rheumatoid arthrithis (RA) and 15 healthy controls. The results showed that TWEAK expressions in PBMCs from SLE patients were significantly higher than that in RA patients or healthy controls, especially higher in those patients with renal disease. Elevated production of TWEAK is correlated positively and significantly with SLEDAI, proteinuria, serum anti-dsDNA, IL-10 and MCP-1, but inversely associated with serum complements. Our results suggested that TWEAK in PBMCs is positively related to SLE disease activity and might be involved in the pathogenesis of SLE.     

Dysregulated Serum IL-23 and SIRT1 Activity in Peripheral Blood Mononuclear Cells of Patients with Rheumatoid Arthritis

Sirtuin 1 (Sirt1) is a class III histone deacetylase (HDAC) that modulates gene expression and is involved in the regulation of proinflammatory cytokines. Interleukin-23 (IL-23) is produced by activated macrophages and dendritic cells and could fuel the progression of rheumatoid arthritis (RA). The goal of our study was to evaluate serum IL-23 levels and both Sirt1 activity and expression in peripheral blood mononuclear cells (PBMCs) in patients with RA compared to healthy controls (HC) and to determine the relationship between Sirt1 activity/expression and IL-23 levels. We assessed apoptosis in PBMCs of RA patients and its association with Sirt1 expression and serum IL-23. Serum IL-23 levels were increased in RA patients in comparison with controls. We found a positive correlation between the levels of serum IL-23 and serum IL-6 in RA patients. Decreased cytoplasmic Sirt1 activity was observed in RA patients with severe disease compared to HC. The expression of Sirt1 protein was significantly decreased in PBMCs of RA patients compared to HC using western blotting. Serum IL-23 levels correlated positively with the cytoplasmic Sirt1 activity in RA patients. Apoptosis rate of PBMCs isolated from RA patients was increased compared to HC and correlated negatively with the expression of Sirt1 protein and serum IL-23 levels. Levels of serum IL-23 and Sirt1 activity and expression were disturbed in RA parallel to increased PBMC apoptosis. Our findings might provide the rationale for the development of new therapeutic approaches in RA.     

Circulating intercellular adhesion molecules in blood and bronchoalveolar lavage in Behçet's disease

The aim of this study was to evaluate circulating intercellular adhesion molecule-1 (cICAM-1) in serum and in bronchoalveolar lavage (BAL), as a marker for the inflammatory process in patients with active Behçet''s disease (BD). Circulating ICAM-1 was tested by an enzyme linked immuno-sorbent assay in serum and in BAL of patients with BD. These values were compared to those of patients with tuberculosis and to healthy controls. Increased levels of circulating ICAM-1 were found in serum from patients with active BD compared to healthy controls (p < 0.01). Similar levels of serum cICAM-1 were found in BD and tuberculosis. Additionally, both BD and tuberculosis patients exhibited high levels of cICAM-1 in BAL fluid, suggesting that this increase may be a result of the immune system activation in inflammatory sites. Circulating ICAM-1 seemed to have a good discriminative power in identifying active BD, being elevated in all active stages (p < 0.01) compared to remission BD stage. No differences were found in active BD patients depending upon the clinical manifestations. These results suggest that cICAM-1 may be involved in leucocyte adhesion and migration into the vessel wall of the lung. Circulating forms are derived from molecules expressed on the surface of activated cells, as a result of an inflammatory process.     

Impaired release of antimicrobial peptides into nasal fluid of hyper-IgE and CVID patients

Background

Patients with primary immunodeficiency (PID) often suffer from frequent respiratory tract infections. Despite standard treatment with IgG-substitution and antibiotics many patients do not improve significantly. Therefore, we hypothesized that additional immune deficits may be present among these patients.

Objective

To investigate if PID patients exhibit impaired production of antimicrobial peptides (AMPs) in nasal fluid and a possible link between AMP-expression and Th17-cells.

Methods

Nasal fluid, nasopharyngeal swabs and peripheral blood mononuclear cells (PBMCs) were collected from patients and healthy controls. AMP levels were measured in nasal fluid by Western blotting. Nasal swabs were cultured for bacteria. PBMCs were stimulated with antigen and the supernatants were assessed for IL-17A release by ELISA.

Results

In healthy controls and most patients, AMP levels in nasal fluid were increased in response to pathogenic bacteria. However, this increase was absent in patients with common variable immunodeficiency (CVID) and Hyper-IgE syndrome (HIES), despite the presence of pathogenic bacteria. Furthermore, stimulation of PBMCs revealed that both HIES and CVID patients exhibited an impaired production of IL-17A.

Conclusion

CVID and HIES patients appear to have a dysregulated AMP response to pathogenic bacteria in the upper respiratory tract, which could be linked to an aberrant Th17 cell response.