Efficacy of abatacept treatment for focal segmental glomerulosclerosis and minimal change disease: A systematic review of case reports, case series, and observational studies
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INTRODUCTION: The efficacy of abatacept has been demonstrated mainly in case reports, case series, and observational studies with small sample size. With current evidence, it is premature to conclude that abatacept is an effective treatment for nephrotic syndrome. MATERIALS AND METHODS: We searched MEDLINE, SCOPUS, and Cochrane Library until December 2019 for studies including patients with focal segmental glomerulosclerosis (FSGS) or minimal change disease (MCD) treated with abatacept. Proteinuria recovery and remission were outcomes of interest. Presence of urinary CD80 level of B7-1 staining on kidney biopsies was also reported. RESULTS: A total of 11 studies (n = 32) were included in the systematic review. 60% of patients were male. The median age was 27.5 years (range 5.2 - 72 years). Approximately 90.6% had FSGS, while 9.4% had MCD. With median follow-up of 12 months (IQR 6.38), only 15 patients (46.9%) showed response in proteinuria reduction, and 12 patients (43.8%) achieved remission with abatacept. Serious adverse events were reported in 12.5%. Additionally, we observed that patients with positive B7-1 staining on kidney biopsies had higher odds of achieving remission with abatacept (likelihood ratio 18.25; p < 0.001). We found no significant correlation between elevated CD80 levels and remissions. CONCLUSION: The efficacy of abatacept therapy for FSGS or MCD was only 43.8%. Serious adverse effects are common. However, our study suggested that abatacept should be considered only in patients with positive B7-1 staining on kidney biopsy because these patients tend to respond to treatment.

Efficacy of different hemodialysis methods on dendritic cell marker CD40 and CD80 and platelet activation marker CD62P and P10 in patients with chronic renal failure.

BACKGROUND: Chronic renal failure (CRF) has become a major public health concern, which increases the risk of stroke and systemic thromboembolism. Therefore, therapeutic strategies are in urgent requirement. This study was conducted for investigating efficacy of hemodialysis (HD), hemodiafiltration (HDF), and hemoperfusion (HP) in patients with CRF and the correlation with the presence of complications following HD therapy. METHODS: The therapeutic effect, living quality, biochemical indicators, and dry weight were detected before and after the treatment regimens. Flow cytometry was conducted to detect expressions of dendritic cell markers (CD40 and CD80) and platelet activation markers (CD62P and P10), and the relationship between their expression and therapeutic effect as well as the association of these expressions with complications was analyzed. RESULTS: After HD therapy, patients presented with decreased serum creatinine, serum phosphorus, triglyceride, parathyroid hormone, and ß2 -MG expression; increased hemoglobin, plasma albumin expressions, and dry weight; and enhanced therapeutic effect and living quality. CD62P and P10 expressions decreased, while CD40 and CD80 expressions increased following HD therapy. The therapeutic effect improved in patients with low expressions of CD40 and CD80 and high expressions of CD62P and P10 following HP treatment and complications were lower after treatment of HDF and HP. CONCLUSION: The aforementioned results indicated that CRF patients treated with HP exhibited higher expression of CD40 and CD80 and lower expression of CD62P and P10, suggesting that HP is conferred to have better efficacy than HDF and HD. Therefore, HP may be a promising clinical regimen for treatment of CRF patients.

Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen-Driven Local Proliferation.

CD4(+)Foxp3(+) regulatory T cells (Tregs) are key immune suppressors that regulate immunity in diverse tissues. The tissue and/or inflammatory signals that influence the magnitude of the Treg response remain unclear. To define signals that promote Treg accumulation, we developed a simple system of skin inflammation using defined Ags and adjuvants that induce distinct cytokine milieus: OVA protein in CFA, aluminum salts (Alum), and Schistosoma mansoni eggs (Sm Egg). Polyclonal and Ag-specific Treg accumulation in the skin differed significantly between adjuvants. CFA and Alum led to robust Treg accumulation, with >50% of all skin CD4(+) T cells being Foxp3(+) In contrast, Tregs accumulated poorly in the Sm Egg-inflamed skin. Surprisingly, we found no evidence of inflammation-specific changes to the Treg gene program between adjuvant-inflamed skin types, suggesting a lack of selective recruitment or adaptation to the inflammatory milieu. Instead, Treg accumulation patterns were linked to differences in CD80/CD86 expression by APC and the regulation of CD25 expression, specifically in the inflamed skin. Inflammatory cues alone, without cognate Ag, differentially supported CD25 upregulation (CFA and Alum > Sm Egg). Only in inflammatory milieus that upregulated CD25 did the provision of Ag enhance local Treg proliferation. Reduced IL-33 in the Sm Egg-inflamed environment was shown to contribute to the failure to upregulate CD25. Thus, the magnitude of the Treg response in inflamed tissues is controlled at two interdependent levels: inflammatory signals that support the upregulation of the important Treg survival factor CD25 and Ag signals that drive local expansion.

Differential gene expression in Mycobacterium bovis challenged monocyte-derived macrophages of cattle.

A functional genomics approach was used to examine the immune response for transcriptional profiling of PBMC M. bovis infected cattle and healthy control cattle to stimulation with bovine tuberculin (purified protein derivative PPD-b). Total cellular RNA was extracted from non-challenged control and M. bovis challenged MDM for all animals at intervals of 6 h post-challenge, in response to in-vitro challenge with M. bovis (multiplicity of infection 2:1) and prepared for global gene expression analysis using the Agilent Bovine (V2) Gene Expression Microarray, 8 × 60 K. The pattern of expression of these genes in PPD bovine stimulated PBMC provides the first description of an M. bovis specific signature of infection that may provide insights into the molecular basis of the host response to infection. Analysis of these mapped reads showed 2450 genes (1291 up regulated and 1158 down regulated) 462 putative natural antisense transcripts (354 up-regulated and 108 down regulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). The results provided enrichment for genes involved top ten up regulated and down regulated panel of genes, including transcription factors proliferation of T and B lymphocytes. The highest differentially-expressed genes were associated to immune and inflammatory responses, immunity, differentiation, cell growth, apoptosis, cellular trafficking and regulation of lipolysis and thermogenesis. Microarray results were confirmed in infected cattle by RT qPCR to identify potential biomarkers TLR2, CD80, NFKB1, IL8, CXCL6 and ADORA3 of bovine tuberculosis.

B7-1 Is Not Induced in Podocytes of Human and Experimental Diabetic Nephropathy.

The incidence of progressive kidney disease associated with diabetes continues to rise worldwide. Current standard therapy with angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers achieves only partial renoprotection, increasing the need for novel therapeutic approaches. Previous studies described B7-1 induction in podocytes of patients with proteinuria, including those with FSGS and type 2 diabetic nephropathy (DN). These findings sparked great excitement in the renal community, implying that abatacept, a costimulatory inhibitor that targets B7-1, could be a novel therapy for diabetic renal disease. Given previous concerns over the value of B7-1 immunostaining and the efficacy of abatacept in patients with recurrent FSGS after renal transplantation, we investigated B7-1 expression in human and experimental DN before embarking on clinical studies of the use of B7-1 targeting strategies to treat proteinuria in DN. Immunohistochemical analysis of kidney specimens using different antibodies revealed that B7-1 is not induced in podocytes of patients with DN, independent of disease stage, or BTBR ob/obmice, a model of type 2 diabetes. These results do not support the use of abatacept as a therapeutic strategy for targeting podocyte B7-1 for the prevention or treatment of DN.

Any value of podocyte B7-1 as a biomarker in human MCD and FSGS?

Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are the most common causes of nephrotic syndrome in children and in young adults. Relapsing MCD carries the risk of severe complications and prolonged immunosuppression, whereas FSGS remains largely untreatable and urgently needs more effective treatments. Recently, induction of B7-1 (CD80), an immune-related protein expressed by antigen-presenting cells, was observed in podocytes of MCD and FSGS patients, suggesting that B7-1 plays a role in the pathogenesis of these diseases, and hence that abatacept, a B7-1 inhibitor, could be a possible treatment. Since previous studies raised serious concerns regarding the reliability of immunohistochemical assays for B7-1 detection and the efficacy of B7-1 inhibitory treatment, we investigated B7-1 podocyte expression in MCD and FSGS patients. Using different primary antibodies and immunohistochemical assays, no significant upregulation of podocyte B7-1 was detected in patients' biopsies compared with controls. To further confirm our findings, we analyzed mice with adriamycin-induced nephropathy, a model of human FSGS, and mice injected with LPS as additional control. Podocyte B7-1 was not observed in mice injected with adriamycin or LPS either. In conclusion, since B7-1 is not induced in podocyte of MCD and FSGS patients, the antiproteinuric action of abatacept, if confirmed, may not be the result of an effect on podocyte B7-1.

Early activation of teleost B cells in response to rhabdovirus infection.

UNLABELLED: To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM(+)) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM(+) cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM(+) cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM(+) cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM(+) cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM(+) cell proliferation, a consistent effect on IgM(+) cell survival was detected. IMPORTANCE: Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have demonstrated that VHSV infection provoked immediate transcriptional effects on B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-κB and increased IgM(+) cell survival. Interestingly, VHSV activated B lymphocytes toward an antigen-presenting profile, suggesting an important role of IgM(+) cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells.

Follicular B cells in thyroids of mice with spontaneous autoimmune thyroiditis contribute to disease pathogenesis and are targets of anti-CD20 antibody therapy.

B cells are required for development of spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice where they function as important APCs for activation of CD4(+) T cells. Depletion of B cells using anti-CD20 effectively inhibits SAT development. The goals of this study were to characterize the B cells that migrate to thyroids in SAT, and to determine whether anti-CD20 effectively targets those B cells in mice with established SAT. The results showed that most thyroid-infiltrating B cells in mice with SAT are follicular (FO) B cells. Expression of CD80, CD86, and CD40 was significantly increased on FO, but not marginal zone, splenic B cells after SAT development. Thyroid-infiltrating and peripheral blood B cells had lower expresion of CD20 and CD24 compared with splenic and lymph node FO B cells. Despite reduced CD20 expression, anti-CD20 depleted most B cells in thyroids of mice with established SAT within 3 d. B cell depletion in thyroids of mice given anti-CD20 was more complete and longer lasting than in spleen and lymph nodes and was comparable to that in blood. Circulation of B cells was required for effective and rapid removal of B cells in thyroids because preventing lymphocyte egress by administration of FTY720 abrogated the effects of anti-CD20 on thyroid B cells. Therefore, the FO subset of B cells preferentially contributes to SAT development and persistence, and anti-CD20 targeting of FO B cells effectively eliminates B cells in the target organ even though thyroid B cells have decreased CD20 expression.

Impact of cardiac surgery on the expression of CD40, CD80, CD86 and HLA-DR on B cells and monocytes.

OBJECTIVE: We measured and compared changes in the percentage of cells expressing CD80, CD86, CD40, HLA-DR and the expression of these molecules on B cells and monocytes of patients who underwent either on-pump, mini on-pump or off-pump cardiac surgery. METHODS: Blood samples from patients who underwent either on-pump, mini on-pump or off-pump cardiac surgery were collected before surgery, instantly after surgery and on the 1(st), 3(rd) and 7(th) days after surgery. Surface expression of CD80, CD86, CD40 and HLA-DR molecules was determined by flow cytometry. RESULTS: Our results show that all three surgical techniques altered the expression of these molecules, as well as the percentage relative number of specific cell populations. We identified statistically significant differences when comparing different surgical techniques. On-pump surgery revealed a more pronounced impact on the phenotype of immune system cells than the other techniques. Therefore, it is likely that the function of immune cells is changed the most by on-pump surgery. We found a lower decrease in the number of CD80(+) monocytes and a lower drop in the CD40 expression on monocytes in off-pump patients in comparison with on-pump patients. CONCLUSION: All the types of cardiac surgical techniques, off-pump, on-pump and modified mini-invasive on-pump, are associated with changes in CD80, CD86, CD40 and HLA-DR expression. We found several significant differences in the expression of the selected molecules when we compared all three groups of patients.

Enhanced suppressive capacity of tumor-infiltrating myeloid-derived suppressor cells compared with their peripheral counterparts.

Although the main site of action for myeloid-derived suppressor cells (MDSCs) is most likely the tumor microenvironment, so far the study of these cells has been largely restricted to spleen-derived MDSCs. In this study, we compared the suppressive capacity of splenic and tumor-derived MDSCs in different subcutaneous mouse tumor models. We investigated which suppressive mechanisms were involved. Finally, we investigated whether MDSCs and regulatory T cells (Treg ) cooperate in the suppression of T-cell responses. In all models, splenic granulocytic MDSCs (grMDSC) strongly suppress CD4(+) T-cell proliferation while the suppressive effect on CD8(+) T cells is less pronounced. Splenic monocytic MDSCs (moMDSC) have a lower suppressive capacity, compared to grMDSC, on both CD4(+) and CD8(+) T-cell proliferation. Both grMDSC and moMDSC isolated from the tumor have a much stronger suppressive activity compared to MDSCs isolated from the spleen of tumor-bearing mice, associated with a higher NO2 (-) production by the tumor-derived moMDSC and arginase activity for both subsets. The expression of CD80 is also elevated on tumor-derived grMDSC compared with their peripheral counterparts. Direct contact with tumor cells is required for the upregulation of CD80 and CD80(+) MDSCs are more suppressive than CD80(-) MDSCs. Coculture of Treg and MDSCs leads to a stronger suppression of CD8(+) T-cell proliferation compared to the suppression observed by Treg or MDSCs alone. Thus, we showed that tumor-infiltrating MDSCs possess a stronger suppressive capacity than their peripheral counterparts and that various suppressive mechanisms account for this difference.

The Effect of Akkermansia muciniphila and Its Outer Membrane Vesicles on MicroRNAs Expression of Inflammatory and Anti-inflammatory Pathways in Human Dendritic Cells.

Probiotics play a crucial role in immunomodulation by regulating dendritic cell (DC) maturation and inducing tolerogenic DCs. Akkermansia muciniphila affects inflammatory response by elevating inhibitory cytokines. We aimed to evaluate whether Akkermansia muciniphila and its outer membrane vesicles (OMVs) affect microRNA-155, microRNA-146a, microRNA-34a, and let-7i expression of inflammatory and anti-inflammatory pathways. Peripheral blood mononuclear cells (PBMCs) were isolated from the healthy volunteers. To produce DCs, monocytes were cultivated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were allocated into six subgroups: DC + Lipopolysaccharide (LPS), DC + dexamethasone, DC + A. muciniphila (MOI 100, 50), DC + OMVs (50 µg/ml), and DC + PBS. The surface expression of human leukocyte antigen-antigen D related (HLA-DR), CD86, CD80, CD83, CD11c, and CD14 was examined using flow cytometry, and the expression of microRNAs was assessed using qRT-PCR, and the levels of IL-12 and IL-10 were measured using ELISA. A. muciniphila (MOIs 50, 100) could significantly decrease IL-12 levels relative to the LPS group. The IL-10 levels were decreased in the DC + LPS group than the DC + dexamethasone group. Treatment with A. muciniphila (MOI 100) and OMVs could elevate the concentrations of IL-10. DC treatment with LPS led to a significant increment in the expression of microRNA-155, microRNA-34a, and microRNA-146a. The expression of these microRNAs was reversed by A. muciniphilia and its OMVs treatment. Let-7i increased in treatment groups compared to the DC + LPS group. A. muciniphilia (MOI 50) had a substantial effect on the expression of HLA-DR, CD80, and CD83 on DCs. Therefore, DCs treatment with A. muciniphila led to induce tolerogenic DCs and the production of anti-inflammatory IL-10.

Increased expression of programmed death (PD)-1 and its ligand PD-L1 correlates with impaired cell-mediated immunity in high-risk human papillomavirus-related cervical intraepithelial neoplasia.

Impaired local cellular immunity contributes to the pathogenesis of persistent high-risk human papillomavirus (HR-HPV) infection and related cervical intraepithelial neoplasia (CIN), but the underlying molecular mechanisms remain unclear. Recently, the programmed death 1/programmed death 1 ligand (PD-1/PD-L1; CD279/CD274) pathway was demonstrated to play a critical role in attenuating T-cell responses and promoting T-cell tolerance during chronic viral infections. In this study, we examined the expression of PD-1 and PD-L1 on cervical T cells and dendritic cells (DCs), respectively, from 40 women who were HR-HPV-negative (-) or HR-HPV-positive (+) with CIN grades 0, I and II-III. We also measured interferon-γ, interleukin-12 (IL-12) and IL-10 in cervical exudates. The most common HPV type was HPV 16, followed by HPV 18, 33, 51 and 58. PD-1 and PD-L1 expression on cervical T cells and DCs, respectively, was associated with HR-HPV positivity and increased in parallel with increasing CIN grade. The opposite pattern was observed for CD80 and CD86 expression on DCs, which decreased in HR-HPV+ patients in parallel with increasing CIN grade. Similarly, reduced levels of the T helper type 1 cytokines interferon-γ and IL-12 and increased levels of the T helper type 2 cytokine IL-10 in cervical exudates correlated with HR-HPV positivity and CIN grade. Our results suggest that up-regulation of the inhibitory PD-1/PD-L1 pathway may negatively regulate cervical cell-mediated immunity to HPV and contribute to the progression of HR-HPV-related CIN. These results may aid in the development of PD-1/PD-L1 pathway-based strategies for immunotherapy of HR-HPV-related CIN.

Site-specific dynamics of CD11b+ and CD103+ dendritic cell accumulations following ozone exposure.

Pulmonary dendritic cells (DCs) are among the first responders to inhaled environmental stimuli such as ozone (O(3)), which has been shown to activate these cells. O(3) reacts with epithelial lining fluid (ELF) components in an anatomically site-specific manner dictated by O(3) concentration, airway flow patterns, and ELF substrate concentration. Accordingly, the anatomical distribution of ELF reaction products and airway injury are hypothesized to produce selective DC maturation differentially within the airways. To investigate how O(3) affects regional airway DC populations, we utilized a model of O(3)-induced pulmonary inflammation, wherein C57BL/6 mice were exposed to 0.8 ppm O(3) 8 h/day for 1, 3, and 5 days. This model induced mild inflammation and no remarkable epithelial injury. Tracheal, but not more distant airway sites, and mediastinal lymph node (MLN) DC numbers were increased significantly after the third exposure day. The largest increase in each tissue was of the CD103(+) DC phenotype. After 3 days of exposure, fewer DCs expressed CD80, CD40, and CCR7, and, at this same time point, total MLN T cell numbers increased. Together, these data demonstrate that O(3) exposure induced site-specific and phenotype changes in the pulmonary and regional lymph node DC populations. Possibly contributing to ozone-mediated asthma perturbation, the phenotypic changes to DCs within pulmonary regions may alter responses to antigenic stimuli. Decreased costimulatory molecule expression within the MLN suggests induction of tolerance mechanisms; increased tracheal DC number may raise the potential for allergic sensitization and asthmatic exacerbation, thus overcoming O(3)-induced decrements in costimulatory molecule expression.

Characterization of effector memory CD8+ T cells in the synovial fluid of rheumatoid arthritis.

Little is known about the cellular characteristics of CD8(+) T cells in rheumatoid arthritis (RA). We addressed this by investigating whether the frequency of the CD8(+) T cell subsets and their phenotypic characteristics are altered in the peripheral blood and synovial fluid (SF) from patients with RA. In this study, CD8(+) T cells, mainly CD45RA(-) effector memory (EM) CD8(+) T cells, were increased significantly in the SF, but not in the peripheral blood from RA patients, compared with healthy controls. The synovial EM CD8(+) T cells were activated phenotypes with high levels of CD80, CD86, and PD-1, and had a proliferating signature in vivo upon Ki-67 staining, whereas the Fas-positive cells were prone to apoptosis. In addition, EM CD8(+) T cells in the SF were less cytotoxic, as they expressed less perforin and granzyme B. In particular, the proportions of synovial fluid mononuclear cells that were CCR4(+)CD8(+) T cells and IL-4-producing CD8(+) T cells (i.e., Tc2 cells) were significantly higher than those in peripheral blood mononuclear cells of patients with RA and healthy controls. In addition, the number of IL-10-producing CD8(+) suppressor T (Ts) cells increased significantly in the SF of RA patients. Especially, CD8(+) T cells were inversely correlated with disease activity. These findings strongly suggest that EM CD8(+) T cells in the SF are increased, likely because of inflammation, and they may be involved in modulating inflammation, thereby affecting the development and progression of RA.

Functional reconstitution of defective myeloid dendritic cells in chronic hepatitis C infection on successful antiviral treatment.

OBJECTIVE: Poor cellular trafficking and suboptimal T-cell responses in liver, the hall marks of chronic hepatitis C virus (CHC) infection, might be attributed to defective antigen presentation. Controversy exists regarding role of myeloid dendritic cells (DCs) in CHC and response to antiviral treatment. This study examines functional status of DCs before and after completion of treatment with the aim to find any modulatory effect. DESIGN: Frequency and functions of monocyte-derived DCs (mo-DCs) were evaluated in CHC (n = 25), before the start of therapy (CHC(0) ). These patients were then put on treatment with peg-interferon-α plus ribavirin for 24 or 48 weeks, and the mo-DC functions were evaluated after 6 months of completion of treatment (CHC(6) ) again, using multicolour flow cytometry, endocytosis assay, cytokine assay and mixed lymphocyte reaction. RESULTS: Pre-treatment frequency of mo-DCs in CHC(0) was lower than that in healthy controls, which became close to normal in patients who achieved virological response (SVR+, n = 20) but not in non-responders (SVR-, n = 5). Pre-treatment levels of CD83, CD80 and CD86 on mo-DC in SVR(0) +, but not SVR(0) -, got upregulated after lipopolysaccharide stimulation supporting the hypothesis that DCs play deciding role in response to therapy. Post-treatment allostimulatory and phagocytosing capacity of mo-DCs in SVR+ patients indicated regain in functional capacity in these patients but not in SVR- patients. CONCLUSIONS: Our results indicate that DCs in CHC patients exhibiting mature and functional phenotype prior to therapy achieve sustained virological response suggesting that functional modulation of defective DCs is directly associated with successful response to therapy.

Areca nut extracts suppress the differentiation and functionality of human monocyte-derived dendritic cells.

BACKGROUND AND OBJECTIVE: Areca quid chewing, a major risk factor contributing to the occurrence of oral cancer and precancer, has been reported to be associated with the severity and high prevalence of periodontal diseases in areca quid chewers. As dendritic cells are critically involved in the regulation of innate and adaptive immunity in oral mucosa, the objective of the present study was to investigate the effect of areca nut extracts (ANE) on the differentiation and reactivity of dendritic cells derived from monocytes. MATERIAL AND METHODS: Human peripheral blood monocytes were cultured in the presence of granulocyte-monocyte colony-stimulating factor and interleukin-4 for 7 d to generate dendritic cells. To examine the effect of ANE on the generation of dendritic cells, the monocytes were exposed to ANE throughout the 7 d culture period. In addition, the effect of ANE on the maturation of monocyte-derived dendritic cells induced by lipopolysaccharide (LPS) was examined. RESULTS: Monocytes cultured in granulocyte-monocyte colony-stimulating factor and interleukin-4 exhibited a typical phenotype of dendritic cells, as evidenced by the heightened expression of human leukocyte antigen (HLA)-DR, CD11c and the co-stimulatory molecules CD40, CD80 and CD86. Exposure of the monocytes to ANE did not influence the expression of HLA-DR and CD11c, but markedly attenuated the proportion of CD40-positive cells and the mean fluorescence intensity of CD86. The expression of co-stimulatory molecules in LPS-activated dendritic cells was not affected, whereas the mRNA expression of interleukin-12 induced by LPS was markedly suppressed by ANE treatment in a concentration-dependent manner. CONCLUSION: These results suggest that ANE exposure interfered with the differentiation of dendritic cells from monocytes. Moreover, the functionality of mature monocyte-derived dendritic cells was attenuated in the presence of ANE.

Antigen-presenting cell marker expression and phagocytotic activity in periodontal ligament cells.

BACKGROUND: Periodontal ligament (PDL) cells are the main cellular constituents of the periodontium, maintain the integrity of the connective tissue, and impact pathology in periodontitis. The aim of this study was to analyze whether PDL cells recognize foreign particles and participate in the immune response to periodontal pathogens. METHODS: Expression of surface proteins characteristic of antigen-presenting cells (APCs) (major histocompatibility complex [MHC] class II, CD40, CD80, CD86) was analyzed in PDL cells after challenge with the cytokines interleukin (IL)-1ß, IL-17A, and interferon-gamma (IFN-γ) or with heat-killed Aggregatibacter actinomycetemcomitans using real-time PCR and flow cytometry. Confocal laser scanning microscopy, transmitted light microscopy, flow cytometry, and time-lapse microscopy were applied to analyze their phagocytotic capacity of collagen (carboxylate-modified microspheres), non-periodontal (Escherichia coli) and periodontal (Aggregatibacter actinomycetemcomitans) pathogens. Furthermore, it was examined whether cytokine activation of PDL cells affects the phagocytosis of collagen or bacteria. RESULTS: PDL cells upregulated MHC class II after cytokine stimulation on transcriptional level, whereas co-stimulatory molecules characteristic of professional APCs were not induced. Analyses on protein level revealed that MHC class II was not constitutively expressed in all PDL cell lines used. PDL cells phagocytosed both collagen and bacteria via acidic vesicles, suggesting the formation of phagosomes. Phagocytosis could be partially inhibited by inhibitors of phagocytosis, i.e., dynasore and wortmannin. Pre-incubation with cytokines did not further enhance the phagocytosis rate of collagen or bacteria. CONCLUSIONS: These results suggest that PDL cells do not only represent bystanders in periodontal infections, but display non-professional APC characteristics, suggesting possible participation in immune reactions of the oral cavity.

Effect of platelet-rich plasma on growth and antigenic profile of human osteoblasts and its clinical impact.

PURPOSE: In recent years, there has been widespread clinical use of platelet-rich plasma (PRP) to facilitate the regeneration of different tissues. However, few data are available on the effect of PRP on parameters other than cell growth. The aim of the present study was to evaluate the effect of PRP on the cell cycle, antigenic profile, and proliferation of primary cultured human osteoblasts. MATERIALS AND METHODS: The cells in the present study were derived from human bone sections obtained from healthy volunteers during third molar surgery. PRP was prepared from human venous blood and used to culture the cell line obtained from the same patient. Flow cytometry was used to study the cell cycle, antigenic profile, and proliferation. RESULTS: The treatment of osteoblasts with PRP modified the expression of CD54, CD80, CD86, and HLA-DR antigens. PRP treatment increased cell proliferation in the short term, but the cell proliferation capacity diminished in the long term, perhaps owing to cell exhaustion. No change in the cell cycle profile was observed in the PRP-cultured cells. CONCLUSIONS: These results suggest that PRP treatment accelerates bone neoformation with no cell cycle changes that might carry a risk of malignant transformation.

Miltefosine effectively modulates the cytokine milieu in Indian post kala-azar dermal leishmaniasis.

BACKGROUND: The increasing incidence of unresponsiveness to antimonials in leishmaniasis prompted the use of newer drugs such as miltefosine. Miltefosine influences macrophage effector functions, but its effect on patients with post kala-azar dermal leishmaniasis (PKDL) has not been evaluated. METHODOLOGY: The immunomodulatory activity of miltefosine was evaluated in patients with PKDL by studying the expression of activation markers (CD14 and CD16) and costimulatory molecules (CD80 and CD86) on circulating monocytes, levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 6, interleukin 1ß, and interleukin 8) and anti-inflammatory cytokines (interleukin 10, transforming growth factor ß, interleukin 4, and interleukin 13) in serum and peripheral blood mononuclear cell culture supernatants, and serum nitrite and arginase activity. RESULTS: Miltefosine on circulating monocytes upregulated expression of CD16 and CD86 and reduced that of CD14. Miltefosine also induced a significant increase in circulating levels of pro-inflammatory cytokines with a concomitant decrease in anti-inflammatory cytokines. Its macrophage activating potential was evidenced by its ability to decrease serum arginase activity and increase serum nitrite. CONCLUSIONS: Miltefosine increased the proportion of monocytes that have a pro-inflammatory phenotype, which was accompanied by an enhanced secretion of pro-inflammatory cytokines and increased levels of serum nitrite. The decrease in anti-inflammatory cytokine levels and serum arginase activity collectively indicated that miltefosine triggered a robust T-helper 1 response that facilitated parasite elimination.

Prostatectomy restores the maturation competence of blood dendritic cell precursors and reverses the abnormal expansion of regulatory T lymphocytes.

OBJECTIVE: To verify the presence of deviated dendritic cell (DC) precursors and of suppressor lymphocytes (Treg) in tumor bearing prostate cancer (PCa) patients and to monitor the corrective effect of tumor ablation. METHODS: Monocytes isolated from the blood of patients before and 1 month after prostatectomy were allowed to reach complete maturation (mDC) ex vivo in a clinical grade two-step process. T-regulatory cells were identified in the lymphocyte cell fraction by the CD4(+)CD25(high)FoxP3(+)/CD4(+)CD25(high)CD127(low/-) phenotype. RESULTS: Despite loss of the monocytes marker CD14, cytokine-matured DCs of tumor bearing patients expressed lower levels of the costimulatory molecule CD80 and of the maturation markers CD83 and CCR7 compared to mDC of normal subjects (NS, P = 0.001, 0.001, and 0.008, respectively). Prostatectomy restored CD80, CD83, and CCR7 expression to values not different from those of NS (P = 0.15, 0.60, and 0.71) and significantly higher than those of the pre-surgery state (CD83, P = 0.0003 and CCR7, P = 0.002). The frequency of Tregs, identified as either CD4 + CD25(high)FoxP3(+) or CD4(+)CD25(high)CD127(low/-), was significantly higher in pre-surgery patients than in NS (P = 0.0001 and 0.0003) and significant recovery of the CD4(+)CD25(high)CD127(low/-) (P = 0.0005) was observed after surgery. CONCLUSIONS: The presence of defective DC precursors and suppressor lymphocytes in the tumor-bearing, but not tumor-free stage, positions the latter as the ideal setting for clinical success of PCa vaccine therapy.