Higher Expression Of HLA-DR Research Articles

Maturation profiles of peripheral blood dendritic cells in patients with endogenous uveitis

To determine whether or not maturation of dendritic cells (DCs) is associated with pathogenesis of endogenous uveitis, we analyzed expression of maturation markers, including CD80, CD86, and human leukocyte antigen (HLA)-DR, in peripheral blood (PB) DCs for comparison between healthy controls (HCs) and uveitis patients. A total of 21 patients and 16 HCs were included. Flow cytometric analysis was performed using PB DCs during the active phase of intraocular inflammation. CD86 and HLA-DR expression was higher in PB DCs from uveitis patients versus HCs, whereas that of CD80 was not significantly different. Levels of CD86 and HLA-DR expression tended to parallel those of inflammatory activity, and decreased after anti-inflammatory therapy. However, expression of CD86 and HLA-DR, even in remission, was not completely down-regulated to the low levels found in HCs. Our results indicate the maturation of DCs may play a role in the pathogenesis of endogenous uveitis. The relatively high expression of HLA-DR and co-stimulatory molecules even in the quiescence of inflammation suggests maturation of DCs may be associated with chronicity and recurrence of uveitis.

Differential effects of monophosphoryl lipid A and cytokine cocktail as maturation stimuli of immunogenic and tolerogenic dendritic cells for immunotherapy

Immunotherapy using monocyte-derived dendritic cells (MDDC) is increasingly being considered as alternative therapeutic approach in cancer, infectious diseases and also in autoimmunity when patients are not responsive to conventional treatments. In general, generation of MDDC from monocytes is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the culture to obtain mature DCs suitable for therapy. For DC maturation, different combinations of pro-inflammatory mediators and Toll-like receptor ligands have been tested, obtaining DCs that differ in their properties and the type of immune response they promote. Therefore, it is necessary to find an optimal cytokine environment for DC maturation to obtain a cellular product suitable for DC-based immunotherapeutic protocols. In this study, we have evaluated in vitro the effects of different maturation stimuli on the viability, phenotype, cytokine profile, stability and functionality of immunogenic and tolerogenic (1α,25-dihydroxyvitamin D(3)-treated) MDDC. Maturation was induced using the clinical grade TLR4-agonist: monophosphoryl lipid A (LA), compared to the traditional cytokine cocktail (CC; clinical grade TNF-α, IL-1β, PGE2) and a combination of both. Our results showed the combination of CC+LA rendered a potent immunogenic DC population that induced the production of IFN-γ and IL-17 in allogeneic co-cultures, suggesting a Th17 polarization. Moreover, these immunogenic DCs showed a high surface expression of CD83, CD86, HLA-DR and secretion of IL-12p70. When aiming to induce tolerance, using LA to generate mature TolDC did not represent a clear advantage, and the stability and the suppressive capability exhibited by CC-matured TolDC may represent the best option. Altogether, these findings demonstrate the relevance of an appropriate maturation stimulus to rationally modulate the therapeutic potential of DCs in immunotherapy.

Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis

In this study, we investigated the hypothesis that regulatory T cells (T(reg)) are involved in the immunomodulatory effects of statins on rheumatoid arthritis (RA) patients. The 12-week study cohort consisted of 55 RA patients and 42 control subjects allocated to either a group treated with atorvastatin (AT) (20 mg/day) or a non-AT group. T(reg) numbers, suppressive function, serum inflammatory markers, and disease activity were evaluated before and after the therapy. Furthermore, the effects of AT on the frequency and suppressive function of T(reg) were determined in vitro. Our data revealed that the suppressive function of T(reg) from RA patients significantly decreased compared with that of control subjects. AT significantly reduced erythrosedimentation, C-reactive protein, and disease activity. Concomitantly, T(reg) numbers and suppressive functions were significantly improved by AT. Consistent with the in vivo experiments, AT promoted the generation of T(reg) from primary T cells and enhanced preexisting T(reg) function in vitro. Moreover, we showed that PI3K-Akt-mTOR and ERK signal pathways were involved in the induction of T(reg) by AT. In conclusion, AT significantly increased T(reg) numbers and restored their suppressive function in the RA patients, and this may be relevant in the modulation of uncontrolled inflammation in this disorder.

Chronic differentiation of human mucosal IgA-secreting cells during B cell depletion therapy with rituximab. (161.3)

Abstract During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. We previously demonstrated that at steady-state, most human peripheral blood antibody-secreting cells produce IgA and express mucosal homing receptors, and suggested their generation in mucosal immune responses. In the current study, we analyzed the blood of patients who received B cell depletion therapy with rituximab (anti-CD20) for treatment of rheumatoid arthritis before and at 2-9months after rituximab infusion by flow cytometry. While CD20+ B cells were reduced to <0.02% of their intial numbers before therapy, CD19+/CD27hi/CD20- antibody-secreting cells remained detectable in the blood at 26-119% of their initial numbers. These circulating antibody-secreting cells expressed IgA that bound to bacterial antigens, beta7 integrin and CCR10 before and during B cell depletion, suggesting their mucosal origin. High expression of HLA-DR and Ki-67 and in vitro migration towards CCL28 and CXCL12 qualified these cells as recently activated plasmablasts, reflecting the survival of functional B cells during rituximab treatment. Consistently, IgA+ plasmablasts and plasma cells were identified in the lamina propria during B cell depletion. Our data implicate that a population of mucosal B cells is resistant to rituximab and self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.

Potential confusion of contaminating CD16+ myeloid DCs with anergic CD16+ NK cells in chimpanzees

Precise identification of NK-cell populations in humans and nonhuman primates has been confounded by imprecise phenotypic definitions. A common definition used in nonhuman primates, including chimpanzees, is CD3(-) CD8α(+) CD16(+) , and this is the dominant NK-cell phenotype in peripheral blood. However, recent data suggest that in chimpanzees a rare CD8α(-) CD16(+) population also exists. Herein, we present evidence validating the existence of this rare subset in chimpanzee peripheral blood, but also demonstrating that gating on CD3(-) CD8α(-) CD16(+) cells can inadvertently include a large number of CD16(+) myeloid DCs (mDCs). We confirmed the inclusion of mDCs in CD3(-) CD8α(-) CD16(+) gated cells by demonstrating high expression of CD11c, BDCA-1 and HLA-DR, and by the lack of expression of NKp46 and intracellular perforin. We also functionally validated the CD8α(-) NK-cell and mDC populations by mutually exclusive responsiveness to a classical NK-cell stimulus, MHC class I-deficient cells, and a prototypic mDC stimulus, poly I:C, respectively. Overall, these data demonstrate common problems with gating of NK cells that can lead to erroneous conclusions and highlight a critical need for consensus protocols for NK-cell phenotyping.

Contribution of Thomsen-Friedenreich antigens to bladder cancer malignancy: Characterization of cell line models

A particular type of glycans has been associated with cancer, the O-linked glycosidic Thomsen-Friedenreich (TF) antigens -T and Tn, and the sialylated forms sT and sTn. The expression of TF antigens in bladder cancer (BC) has also been reported, although the correlation with prognosis is controversial. Furthermore, the molecular basis underlying their expression and the role played by the enzymes sialyltransferases (STs), responsible for the expression of sialylated forms, are unknown [1]. For a better understanding, we chose to study the cell line models: MCR and HT1376, exhibiting a low cell surface expression of the sTn and sT respectively, that were transduced with “ST6GalNac.I” or “ST3Gal.I” STs cDNA. The resulted transduced cells MCRST6GalNac1 and HT1376ST3Gal1 where purified by immune-magnetic sorting for the sTn, and selected clones for the expression of sT. Our preliminary results show differences concerning to cell adhesion ability and immunogenicity. Namely, dendritic cells (DCs) and MCRST6GalNac1 in co-cultures, results in an increased expression of TGF-b and IL-10, and decreased of TNF-a and IL-6. After adhesion, MCR negative control (MCRNC) induces higher maturation on DCs (higher expression of HLA-DR, CD80, CD83 and CD86). Concerning to phagocytosis, it was seen that HT1376ST3Gal1 are apparently more resistant to phagocytosis by macrophages, in respect to negative control (HT1376NC), and MCRST6GalNac1 less resistant to phagocytosis by DCs, in respect to MCRNC. Author details CEDOC, Departamento de Imunologia, Faculdade de Ciencias Medicas, Universidade Nova de Lisboa, Lisboa, Portugal. Departimento di Patologia Sperimentale, Universita di Bologna, Bologna, Italia.

Peritoneal macrophage priming in cirrhosis is related to ERK phosphorylation and IL‐6 secretion

Bacterial infections are common complications arising in patients with cirrhosis and ascites. Translocation of bacterial DNA is a dynamic process that is associated with an increased inflammatory response and a poor prognosis in this setting. The aim of this study was to study whether peritoneal macrophages remain in a chronic primed status to allow a rapid response to subsequent events of bacterial translocation. Peritoneal monocyte-derived macrophages were isolated from 25 patients with cirrhosis and non-infected ascites and compared with donor's blood monocytes. Activation cell-surface markers were screened using flow-cytometry, and the phosphorylation state of ERK 1/2, p38 MAP Kinase, PKB/Akt and transcription factors c-Jun and p65 NFκB were evaluated using Western blot. Synthesis of tumour necrosis factor alpha, interleukin 6 (IL-6) and interleukin-10 (IL-10) at baseline and in response to bacterial stimuli was evaluated using ELISA. A high expression of CD54, CD86 and HLA-DR at baseline was displayed by peritoneal macrophages. Increased phosphorylated levels of ERK1/2, protein kinase B (PKB) and c-Jun, together with IL-6 production, were observed in peritoneal macrophages at baseline compared with donors' blood monocytes. A positive correlation was established between basal IL-6 levels and extracellular signal-regulated kinase (ERK) phosphorylation in peritoneal macrophages from patients with cirrhosis (r=0·9; P=0·005). Addition of lipopolysaccharide induced higher phosphorylation levels of all studied signalling intermediates than synthetic-oligodeoxydinucleotides, but similar end-stage p65 NFκB. A sustained immune response is present in ascitic fluid of cirrhotic patients, even in the temporal absence of bacterial antigens. This would facilitate a fast response, probably controlled by IL-6, against repeated bacterial-DNA translocation or in liver chronic inflammation.

Emergence of Dendritic Cells in the Myocardium after Acute Myocar­dial Infarction - Implications for Inflammatory Myocardial Damage

Dendritic cells (DC) are crucial for T cell mediated immune responses. Recently, we observed a significant decrease in circulating myeloid DC precursors in patients with acute myocardial infarction (AMI). The aim of the present study was to investigate whether myeloid DC are present in infarcted myocardium. Myocardial specimens of 10 patients with AMI and 7 accident victims (controls) were collected after autopsy. In immunostainings the presence of DC (CD209+, fascin+), T cells (CD3+), macrophages (CD68+), and HLA-DR expression was analyzed. Significantly higher numbers of CD209+-DC (97 vs. 44 cells/0.25 mm2, p=0.03), fascin+-DC (54 vs. 8 cells/0.25 mm2, p=0.02), T cells (27 vs. 6 cells/0.25 mm2, p=0.02), and macrophages (44 vs. 6 cells/0.25 mm2, p=0.01) associated with high HLA-DR expression were detected in infarcted myocardium. Frequent colocalizations of DC and T cells were observed. In occluded coronary arteries numerous DC, T cells, macrophages and high HLA-DR expression were found. We show that DC are present in infarcted myocardium after AMI. High HLA-DR expression and the colocalization with T cells suggest that they might trigger an immune response leading to further myocardial damage.

T-Cell Dysfunction in HIV-1–Infected Patients With Impaired Recovery of CD4 Cells Despite Suppression of Viral Replication

CD4 T-cell recovery is impeded in some HIVinfected patients despite successful combination antiretroviral therapy (cART) with suppressed HIV RNA. We hypothesized that T-cell dysfunction would be increased in these patients. In the Danish HIV Cohort Study, we identified HIV-1-infected patients initiating cART with a CD4 cell count <100 cells per microliter, followed by HIV RNA <50 copies per milliliter for 3 years. Patients with a CD4 count <200 cells per microliter after 3 years were identified as cases; 42 patients with a CD4 count > or = 200 cells per microliter were selected as controls. Six-color flow cytometry was performed on whole blood. Cytokine levels in supernatants from whole blood stimulations were assessed. The case and control groups comprised 18 and 35 patients, respectively. Cases were older than controls (median: 54/46 years). The fraction of CD28+ cells was decreased among cases in the CD4+ and CD8+ T-cell subsets (P = 0.0014/P = 0.0349) and in the corresponding naive subsets (P = 0.0011/P , 0.0001). Cases had higher expression of human leukocyte antigen (HLA)-DR on naive CD4 and CD8 T cells (P = 0.0007/P = 0.0028). The production of interleukin (IL)-10 and IL-2 to phytohemagglutinin was decreased in cases (P < 0.0001/P = 0.019). Patients with impaired CD4 recovery shared a dysregulated T-cell phenotype with low CD28, high HLA-DR expression, and low IL-2 and IL-10 production.

Abstract 5504: Transient Decrease in Circulating Dendritic Cell Precursors After Acute Stroke - Potential Recruitment Into the Brain

Background: The role of dendritic cells (DC) as potent mediators of inflammation has not been sufficiently investigated in stroke. Methods: Circulating myeloid (mDCP), plasmacytoid (pDCP), and total DCP (tDCP) were flow cytometrically analyzed in healthy controls (n=29), patients with asymptomatic A. carotis interna stenosis (ACI-S, n=46), transient ischemic attack (TIA, n=39), acute ischemic stroke (AIS, n=73), and acute hemorrhagic stroke (AHS, n=31). The National Institutes of Health Stroke Scale (NIHSS) and the infarction size in CT scan were evaluated after stroke. In a patient subgroup, postmortem immunohistochemical brain analyses were performed to detect mDC (CD209), pDC (CD123), T cells (CD3), and HLA-DR. Results: In AIS and AHS, circulating mDCP, pDCP, and tDCP (each p&lt;0.005) were significantly reduced. A significant inverse correlation was found between the NIHSS and circulating DCP (p&lt;0.02), as well as between hsCRP and circulating DCP (p&lt;0.001). Patients with large stroke size in CT scan had significantly lower mDCP, pDCP, and tDCP (each p&lt;0.01) than those with smaller stroke. Follow-up analysis showed a significant recovery of circulating DCP in the first days after stroke. In the infarcted brain, a dense infiltration of mDC colocalized with T cells, single pDC, and high HLA-DR expression were observed. Conclusions: Acute stroke leads to a decrease in circulating DCP. Potentially, circulating DCP are recruited from the blood into the infarcted brain, and probably trigger cerebral immune reactions there.

Transient decrease in circulating dendritic cell precursors after acute stroke: potential recruitment into the brain

The role of DCs (dendritic cells) as potent mediators of inflammation has not been sufficiently investigated in stroke. Therefore, in the present study, circulating mDCPs (myeloid DC precursors), pDCPs (plasmacytoid DCPs) and tDCPs (total DCPs) were analysed by flow cytometry in (i) healthy controls (n=29), (ii) patients with ACI-S (asymptomatic cerebral infarction stenosis; n=46), (iii) patients with TIA (transient ischaemic attack; n=39), (iv) patients with AIS (acute ischaemic stroke; n=73), and (v) patients with AHS (acute haemorrhagic stroke; n=31). The NIHSS (National Institutes of Health Stroke Scale) and infarction size on a CT (computer tomography) scan were evaluated after stroke. In a patient subgroup, post-mortem immunohistochemical brain analyses were performed to detect mDCs (CD209), pDCs (CD123), T-cells (CD3) and HLA-DR. In AIS and AHS, the numbers of circulating mDCPs (P<0.005), pDCPs (P<0.005) and tDCPs (P<0.001) were significantly reduced. A significant inverse correlation was found between the NIHSS and circulating DCPs (P<0.02), as well as between hsCRP (high-sensitivity C-reactive protein) and circulating DCPs (P<0.001). Patients with large stroke sizes on a CT scan had significantly lower numbers of mDCPs (P=0.007), pDCPs (P=0.05) and tDCPs (P=0.01) than those with smaller stroke sizes. Follow-up analysis showed a significant recovery of circulating DCPs in the first few days after stroke. In the infarcted brain, a dense infiltration of mDCs co-localized with T-cells, single pDCs and high HLA-DR expression were observed. In conclusion, acute stroke leads to a decrease in circulating DCPs. Potentially, circulating DCPs are recruited from the blood into the infarcted brain and probably trigger cerebral immune reactions there.

In vivo Extravasated Human Monocytes have an Altered Expression of CD16, HLA‐DR, CD86, CD36 and CX3CR1

The phenotypic alterations in monocytes induced by extravasation in vivo are still largely unknown. We addressed the question whether a general phenotype of extravasated monocytes exists and whether this phenotype differs between healthy individuals and statin treated patients with coronary artery disease (CAD). In vivo extravasated monocytes from CAD patients and healthy controls were collected by use of the skin blister method and compared with peripheral circulating monocytes by flow cytometry. The number of CD14(+)CD16(+) monocytes were significantly higher in the skin blister compared with peripheral circulation in both patients (P < 0.001) and controls (P = 0.005). In vivo extravasated monocytes had in comparison with peripheral monocytes a lower expression of CX(3)CR1, a higher expression of HLA-DR, CD86 and CD36 and a higher binding of acetylated low density lipoprotein (acLDL) (significant for all markers). Skin blister fluid from CAD patients, compared with healthy controls, induced a 20% increase in monocyte CD36 expression (P = 0.008) following 18 h of in vitro incubation. The results indicate that the integrated response to the in vivo extravasation process is similar in statin treated stable CAD patients and healthy controls, with respect to phenotypic alterations. Such differences in CAD patients may, however, occur as a response to the inflammatory milieu.

Immunophenotypic, cytogenetic and clinical features of 192 AML patients in China

Immunophenotypic characterization of the leukemic cells has been widely used as a tool for diagnosis, classification and prognosis of leukaemia. A total of 192 Chinese patients with acute myeloid leukemia (AML) were immunophenotyped by flow cytometry using a panel of monoclonal antibodies. Among the 192 patients enrolled in this study, 125 cases were also subjected to karyotype analysis by G-banding technology. We found that CD33, CD13, MPO and CD117 were the most commonly expressed antigens in AML. A combination of intensive autofluorescence, both CD34(-) and HLA-DR(-), and high expression of CD13, CD33 and MPO had significant value for M3 diagnosis. CD14 was expressed only in M4 and M5, and both intensive positivity of CD64 and CD15 with high expression of HLA-DR may suggest great possibility for diagnosis of M5. Lymphoid markers expression was documented in 47.9% of the 192 AML cases analyzed. CD56 (26.0%) and CD7 (20.8%) were the most commonly expressed lymphoid markers in AML patients. Abnormal karyotypes were detected in 76 out of 125 (60.8%). Higher CD34 positivity was found in LymAg(+) group (77.2%) than in LymAg(-) group (48.0%). Our results indicate that immunophenotype analysis was useful for AML diagnosis and classification and the immunophenotype did have relevance to the abnormal cytogenetic changes and clinical features in AML.

Prognostic Utility of Immunophenotyping in Diffuse Large B-Cell and Follicular Lymphomas

Patients with Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphomas (FL) exhibit marked variability in survival, the international prognostic index (IPI) predicts the cure with chemotherapy as IPI is a well established predictor of outcome in DLBCL and FL. Biological prognostic markers including expression on of BCL2, BCL6, CD5, CD10, and HLA-DR have been described as IPI independent prognostic factors. The purpose of this study is to high light how these biomarkers might be incorporated into current risk – adjustment models for prognosis. Biological parameters were evaluated by flowcytometric immunophenotyping of 96 patients with DLBCL and FL.In our study, Superior overall survival (OS) was associated with high expression of HLA-DR in DLBCL either do novo or on top of FL (p < 0.001 for both group), and with low expression of CD 5 and BCL2 in de novo DLBCL (p < 0.001 for both ) independently of the IPI. On the other hand Inferior overall survival was established with high expression of BCL6 in DLBCL. However Inferior progression free survival (PFS) was independently correlated with high expression of BCL2 (p = 0.007 and 0.018 ) and lower expression of HLA-DR ( p=0.003 and 0.03 ) in both de novo DLBCL and on top of follicular, but not in FL and with high expression of CD10 in de novo DLBCL only ( p<0.001 ).In 96 patients with DLBCL and FL treated with risk adapted therapy, low HLA-DR expression is associated with poor outcome in DLBCL either de novo or on top of follicular but not in a FL group. the time is right to begin to consider how these biomarkers should be incorporated into current prognostic models to move beyond the clinically based IPI.

Peripheral Blood CD14high CD16+ Monocytes are Main Producers of IL‐10

Based on CD14 and CD16 expression, human peripheral blood monocytes (MO) can be divided into a major CD14(high) CD16(-) population and two minor CD14(high) CD16(+) and CD14(dim) CD16(+) subpopulations. CD14(dim) CD16(+) MO are well characterized and regarded as pro-inflammatory because upon stimulation produce TNF-alpha but little, if any, IL-10. By contrast, little is known about CD14(high) CD16(+) MO. We investigated the surface expression of selected determinants by CD16(+) MO subpopulations, cytokine production, phagocytosis and antigen presentation. We found that both CD16(+) subpopulations had a higher expression of HLA-DR, CD86, CD54 and a lower expression of CD64 than CD14(high) CD16(-) population. In addition, CD14(high) CD16(+) MO showed a higher expression of CD11b and TLR4 than CD14(dim) CD16(+) and CD14(high) CD16(-) subpopulations. CD14(high) CD16(+) MO exhibited an increased phagocytic activity and a decreased antigen presentation in comparison with CD14(dim) CD16(+). As expected, lipopolysaccharide (LPS)-stimulated CD14(dim) CD16(+) MO produced TNF-alpha but little IL-10. By contrast, LPS-stimulated CD14(high) CD16(+) subpopulation produced significantly more IL-10 than CD14(dim) CD16(+) and CD14(high) CD16(-) MO. In conclusion, our data show that human peripheral blood CD16(+) MO are heterogeneous in function and consist of two subpopulations: CD14(dim) CD16(+) pro-inflammatory and CD14(high) CD16(+) with anti-inflammatory potential.

Long-term interleukin-10 presence induces the development of a novel, monocyte-derived cell type.

Interleukin (IL)-10 is one of the most crucial immunoregulatory cytokines. Its short-term effects have been analysed extensively, but little is known about its long-term effects. This is of considerable importance, as high systemic IL-10 levels are present for long periods in patients with persistent viral infections, certain cancers and in critical care patients. Our study investigated the effects of the long-term presence of IL-10 on human peripheral blood monocytes. In vitro, IL-10 treatment of these cells for 7 days induced the development of a novel cell type characterized by unique phenotypical and functional characteristics. These cells showed high HLA-DR expression and low expression of CD86 and other co-stimulatory molecules on their surface. The mRNA levels of both HLA-DR and CD86 were high, but no intracellular accumulation of CD86 protein was observed. With respect to its function, these cells showed strongly diminished tumour necrosis factor-alpha production following lipopolysaccharide stimulation, strongly diminished allogenic CD4(+) T cell stimulatory capacity, and even induced a hyporesponsive state in CD4(+) T cells. The phenotype remained stable despite the removal of IL-10. In vivo, we found monocytic cells from patients exhibiting this phenotype after long-term IL-10 exposure. These results complement our knowledge further about the biological effects of IL-10 and may provide an explanation for the sustained immunodeficiency in cases of the persistent presence of systemic IL-10.

The presence of GM-CSF and IL-4 interferes with effect of TGF-β1 on antigen presenting cells in patients with multiple sclerosis and in rats with experimental autoimmune encephalomyelitis

The possibility to generate and expand tolerogenic dendritic cells (DC) with TGF-β1 in vitro opens new therapeutic perspectives for the treatment of autoimmune diseases. In the present study, GM-CSF+IL-4 induced the differentiation of DC from adherent peripheral blood mononuclear cells, which had a higher expression of HLA-DR, CD86 and CD1a and the capacity to stimulate T cells. TGF-β1 alone slightly promoted the generation of antigen presenting cells (APC) with higher expression of CD14, but did not differentiate them into E-cadherin + Langerhans cell (LC)-like DC. TGF-β1-driven APC exhibited the morphology, phenotypes and functions of tolerogenic immature DC, and had lower capacity to stimulate T cells. In vivo experiment demonstrates that TGF-β1-treated APC exhibited the therapeutic potential in Lewis rats with experimental autoimmune encephalomyelitis (EAE), followed by increase of IL-10 production in lymph nodes and decrease of inflammatory cells in spinal cords. Most importantly, GM-CSF/IL-4 used in DC preparation abolished the effect of TGF-β1 to induce tolerogenic APC in vitro and in vivo. The results reveal that the usage of GM-CSF for the generation of tolerogenic DC should not be copied from DC preparation for anti-tumor therapy.

Activated myeloid dendritic cells accumulate and co-localize with CD3 + T cells in coronary artery lesions in patients with Kawasaki disease

Emerging evidence implicating the participation of dendritic cells (DCs) and T cells in various vascular inflammatory diseases such as giant cell arteritis, Takayasu's arteritis, and atherosclerosis led us to hypothesize that they might also participate in the pathogenesis of coronary arteritis in Kawasaki disease (KD). Coronary artery specimens from 4 patients with KD and 6 control patients were obtained. Immunohistochemical and computer-assisted histomorphometric analyses were performed to detect all myeloid DCs (S-100 +, fascin +), all plasmacytoid DCs (CD123 +) as well as specific DC subsets (mature myeloid DCs [CD83 +], myeloid [BDCA-1 +] and plasmacytoid DC precursors [BDCA-2 +]), T cells (CD3 +), and all antigen-presenting cells (HLA-DR +). Co-localization of DCs with T cells was assessed using double immunostaining. Significantly more myeloid DCs at a precursor, immature or mature stage were found in coronary lesions of KD patients than in controls. Myeloid DC precursors were distributed equally in the intima and adventitia. Mature myeloid DCs were particularly abundant in the adventitia. There was a significant correlation between mature DCs and HLA-DR expression. Double immunostaining demonstrated frequent contacts between myeloid DCs and T cells in the outer media and adventitia. Plasmacytoid DC precursors were rarely found in the adventitia. In conclusion, coronary artery lesions of KD patients contain increased numbers of mature myeloid DCs with high HLA-DR expression and frequent T cell contacts detected immunohistochemically. This suggests that mature arterial myeloid DCs might be activating T cells in situ and may be a significant factor in the pathogenesis of coronary arteritis in KD.

Accumulation of immune cells and high expression of chemokines/chemokine receptors in the upstream shoulder of atherosclerotic carotid plaques

The presence of immune cells is important for plaque destabilization. Disturbed flow conditions were shown to enhance the recruitment of circulating immune cells. Thus, we analyzed in 54 atherosclerotic carotid plaques the frequency of different immune cells, HLA-DR, chemokines, and chemokine receptors, comparing the upstream with the downstream plaque shoulder. The presence of neovascularization and intraplaque hemorrhages was investigated by CD34 immunostaining and Mallory's iron stain. Immunohistochemical analyses were performed to detect smooth muscle cells (SMC: actin), macrophages (CD68), T cells (CD3), dendritic cells (DC: fascin), mature DC (CD83), and the expression of HLA-DR, chemokine receptors (CCR-2, CCR-6), and chemokines (MCP-1, MIP-3α). Significantly more SMC were detected downstream than upstream ( p < 0.001). In contrast, significantly more macrophages ( p = 0.01), DC ( p = 0.03), mature DC ( p = 0.007), and a higher expression of HLA-DR ( p = 0.004), CCR-2 ( p = 0.002), CCR-6 ( p < 0.001), MCP-1 ( p = 0.04), and MIP-3α ( p = NS) were observed upstream than downstream. Immune cells were strongly associated with neovascularization. The abundance of SMC downstream provides an explanation for distal plaque growth. Enhanced recruitment of immune cells through neovessels into the upstream shoulder might be contributing to plaque destabilization.

Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis

Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-γ, TNF-α, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin, LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-γ, TNF-α, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-γ was predominantly detected in CD3 +CD8 + T cells, whereas IL-2 and TNF-α were mainly expressed in CD3 +CD4 + cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection.