Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells.

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8+ T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4+ T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.

Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and Siglec-3-expressing myeloid dendritic cells in systemic sclerosis patients.

OBJECTIVE: To investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients. METHODS: Spontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation. RESULTS: The frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function. CONCLUSIONS: These data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis.

Subset-specific alterations in frequencies and functional signatures of γδ T cells in systemic sclerosis patients.

OBJECTIVE AND DESIGN: Here, we evaluated the distribution and functional profile of circulating CD27+ and CD27- γδ T-cell subsets in systemic sclerosis (SSc) patients to assess their potential role in this disorder. MATERIALS AND METHODS: Peripheral blood from 39 SSc patients and 20 healthy individuals was used in this study. The TCR-γδ repertoire, cytokine production and cytotoxic signatures of circulating γδ T-cell subsets were assessed by flow cytometry. Gene expression of EOMES, NKG2D and GZMA was evaluated by quantitative RT-PCR in both purified γδ T-cell subsets. RESULTS: Absolute numbers of γδ T-cell subsets were significantly decreased in SSc groups, likely reflecting their mobilization to the inflamed skin. Both γδ T-cell subsets preserved their relative proportions and Th1-type cytokine responses. However, cytotoxic properties showed significant disease-associated and subset-specific changes. SSc patients exhibited increased percentages of CD27+ γδ T cells expressing granzyme B or perforin and upregulated GZMA expression in diffuse cutaneous SSc. Conversely, EOMES and NKG2D were downregulated in both SSc γδ T-cell subsets vs. normal controls. Interestingly, patients with pulmonary fibrosis showed a biased TCR repertoire, with a selected expansion of effector Vγ9+ γδ T cells associated with increased frequency of cells expressing granzyme B, but decreased IFN-γ production. CONCLUSIONS: Significant alterations on circulating γδ T-cell subsets suggest a deregulated (increased) cytotoxic activity and thus enhanced pathogenic potential of CD27+ γδ T cells in SSc.

Alterations on peripheral blood B cell subsets induced by allergic rhinitis.

OBJECTIVES AND DESIGN: Here we evaluated whether allergic rhinitis to Dermatophagoides pteronyssinus induces alterations on circulating B cell subsets. MATERIALS AND METHODS: Circulating B cell subsets and isotype expression on antigen-experienced B cells from allergic patients under conventional pharmacological treatment (NO-SIT, n = 15) and under subcutaneous immunotherapy (SCIT, n = 33), and non-allergic subjects (NC, n = 25) were analyzed by flow cytometry. RESULTS: In allergic patients, we found a significant decrease in IgM(+) and IgG(+) memory B cells and an increase in IgA(+) memory B cells. Additionally, the numbers of circulating IgA(+) plasmablasts in allergic patients were also increased, while those cells expressing IgM were reduced. CONCLUSIONS: Allergic patients have a disturbed B cell subsets distribution which seems to underlie rhinitis pathogenesis and remain unchanged after SCIT.

Changes in naïve and memory T-cells in elite swimmers during a winter training season.

High intensity training regimens appear to put athletes at a higher risk of illness. As these have been linked to alterations in the proportions of differentiated T cells, how training load affects these populations could have important implications for athlete susceptibility to disease. This study examined the effect of a winter training season on the proportions of circulating naïve and memory T cells subsets of high competitive level swimmers. Blood samples were taken at rest at 4 time-points during the season: before the start of the season (t0-September), after 7weeks of an initial period of gradually increasing training load (t1-November), after 6weeks of an intense training cycle (t2-February) and 48h after the main competition (t3-April) and from eleven non-athlete controls at 2 similar time-points (t2 and t3). CD4, CD8 and gamma-delta (γδ) T cells expressing the naïve (CCR7(+)CD45RA(+)), central-memory (CM-CCR7(+)CD45RA(-)), effector-memory (EM-CCR7(-)CD45RA(-)) and terminal effector (TEMRA-CCR7(-)CD45RA(+)) were quantified by flow cytometry. Statistical analyses were performed using multilevel modeling regression. Both T CD4(+) naïve and CM presented a linear increase in response to the first moment of training exposure, and had an exponential decrease until the end of the training exposure. As for TCD4(+) EM, changes were observed from t2 until the end of the training season with an exponential trend, while TCD4(+) TEMRA increased linearly throughout the season. TCD8(+) naïve increased at t1 and decreased exponentially thereafter. TCD8(+) TEMRA values decreased at t1 and increased exponentially until t3. γδT-EM had an increase at t1 and an exponential decrease afterwards. In contrast, γδT-TEMRA decreased at t1 and exponentially increased during the remaining 20weeks of training. An increase in TEMRA and EM T cells alongside a decrease in naïve T cells could leave athletes more susceptible to illness in response to variation in training stimulus during the season.

HLA frequency distribution of the Portuguese bone marrow donor registry.

Introduction: The Portuguese donor Registry of CEDACE was the fifth largest per capita bone marrow donor Registry of the WMDA as of 2019 and has yet to be thoroughly analyzed. We aimed to characterize its various aspects, including demographics and HLA allele and haplotype frequencies, to evaluate the genetic matching propensity score and ultimately further develop it. Methods: We described and compared characteristics of the donor population with census data and used an Expectation-Maximization algorithm and analyses of molecular variance to assess haplotype frequencies and establish phylogenetic distances between regions and districts within the country. Results: We identified 396545 donors, corresponding to 3.85% of the Portuguese population; the median donor age was 39 years, with 60.4% of female donors. Most donors were Portuguese nationals, although 40 other nationalities were present, with a significant proportion of donors from Brazil and Portuguese-speaking African Countries; almost all donors self-reported as Western, with the second largest group reporting African ancestry. There was an asymmetric contribution of donors from different districts and regions, with most coming from coastal districts and few from the southern districts and autonomous regions; foreign and self-declared non-Western donors were mainly located in the Metropolitan Area of Lisbon and the South. Although most donors were typed in three loci (HLA-A, HLA-B and HLA-DRB1), only 44% were also typed in HLA-C, 1.28% in HLA-DQB1 and only 0.77% in all five loci and in high-resolution. There were varying allele and haplotype frequencies across districts and regions, with the most common three loci, low-resolution haplotypes, being HLA-A*01~B*08~DRB1*03, A*29~B*44~DRB1*07 and HLA-A*02~B*44~DRB1*04; some haplotypes were more prevalent in the South, others in the North and a few in the autonomous regions; African and foreign donors presented relevant differences in haplotype frequency distributions, including rare haplotypes of potential interest. We also report on four loci, low-resolution frequency distributions. Using AMOVA, we compared genetic distances between districts and regions, which recapitulated the country's geography. Discussion: Our analysis showed potential paths to optimization of the Registry, including increasing the male donor pool and focusing on underrepresented districts and particular populations of interest, such as donors from Portuguese-speaking African countries.

Sialyl Lewisx-dependent binding of human monocyte-derived dendritic cells to selectins.

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.

Effects of bevacizumab on autocrine VEGF stimulation in bladder cancer cell lines.

INTRODUCTION: A functional vascular endothelial growth factor A (VEGF-A) autocrine loop is crucial for bladder cancer cell survival. We reasoned that treatment with the anti-VEGF antibody bevacizumab may result either in cell growth prevention or in the cell adaptation to compensate VEGF deprivation. METHODS: The cytotoxicity of different levels of bevacizumab and its effect on the gene expression was analyzed in human bladder cancer cell lines. RESULTS: Inhibition of bladder cancer cell proliferation was observed at >2.5 mg/ml of bevacizumab. Non-muscle-invasive bladder cancer cells expressed high concentrations of VEGF-A, and were less susceptible to bevacizumab inhibition. At 0.5 mg/ml (FDA approved concentration) of bevacizumab, cells increase their expression of VEGF-A, VEGF-A receptors and related growth factors. CONCLUSIONS: Bevacizumab cytotoxicity is only observed at high concentration, and it is inversely correlated with the basal VEGF-A expression of the bladder cancer cells. This is the first report showing that, at clinical bevacizumab concentrations, cancer cells compensate the VEGF-A blockade, by improving the expression of VEGF-A and related genes, highlighting the need to follow the patient's adaptation response to bevacizumab treatment.

Effect of sialic acid loss on dendritic cell maturation.

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I(-/-) and ST6Gal.I(-/-) mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I(-/-) and ST6Gal.I(-/-) strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading.

ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines.

BACKGROUND: The T antigen is a tumor-associated structure whose sialylated form (the sialyl-T antigen) involves the altered expression of sialyltransferases and has been related with worse prognosis. Since little or no information is available on this subject, we investigated the regulation of the sialyltransferases, able to sialylate the T antigen, in bladder cancer progression. METHODS: Matched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: ST3Gal.I, ST3Gal.II and ST3Gal.IV mRNA level by real-time PCR. Sialyl-T antigen was detected by dot blot and flow cytometry using peanut lectin. Sialyltransferase activity was measured against the T antigen in the cell lines. RESULTS: In nonmuscle-invasive bladder cancers, ST3Gal.I mRNA levels were significantly higher than corresponding urothelium (p < 0.001) and this increase was twice more pronounced in cancers with tendency for recurrence. In muscle-invasive cancers and matching urothelium, ST3Gal.I mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed ST3Gal.I mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the ST3Gal.I mRNA level, the ST activity (r = 0.99; p = 0.001) and sialyl-T antigen expression, demonstrating that sialylation of T antigen is attributable to ST3Gal.I. The expression of sialyl-T antigens was found in patients' bladder tumors and urothelium, although without a marked relationship with mRNA level. The two ST3Gal.I transcript variants were also equally expressed, independently of cell phenotype or malignancy. CONCLUSION: ST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of ST3Gal.I seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.

[Regulatory cells]. / Células reguladoras.

The role of regulatory T cells in the induction and maintenance of peripheral tolerance has received growing attention during the last years. Several subsets of regulatory T cells were described based on their surface markers and cytokine production, but nevertheless, there are no specific markers for any subsets and their classification relies on their suppression mechanism. It is unknown which of the subgroups of regulatory T cells is more important in the prevention and control of allergic diseases, being commonly accepted its importance in homeostasis.

A case of fulminant Type 1 diabetes following anti-PD1 immunotherapy in a genetically susceptible patient.

Programmed cell death-1 protein (PD-1) is an immune checkpoint that has gained popularity in the treatment of several advanced cancers. Inhibiting this checkpoint is known to enhance immune response, but is also known to diminish immune tolerance and to increase autoimmune toxicity. We discuss a case of rapid onset fulminant Type 1 diabetes induced by treatment with anti-programmed cell death-1 monoclonal antibody, nivolumab, in a patient with late-stage non-small-cell lung adenocarcinoma. The patient had no history of previous diabetes but did reveal a high-risk genotype for Type 1 diabetes development (DR3-DQ2; DR4-DQ8). This finding supports that acute Type 1 diabetes can be an important adverse effect of immunotherapies targeting T-cell activation regulation. Because of the severity of this adverse effect, physicians should be aware of it, and studies directed to the detection of new biomarkers for early risk stratification (e.g., HLA) should be sought.

The combined effect of platelet storage media and intercept pathogen reduction technology on platelet activation/activability and cellular apoptosis/necrosis: Lisbon-RBS experience.

Platelets are known to undergo shape change, activation, a release reaction and apoptosis/necrosis during processing and storage, all of which are collectively known as the platelet storage lesion. Any additional processing may have some deleterious impact on platelet activability and functional integrity, which need to be investigated. This preliminary investigation was undertaken to establish the combined effects of standard platelet storage media and the intercept pathogen reduction technology on platelet activation and activability during 7 day storage, using buffy-coat derived platelets in standard storage media containing 35% plasma (N=24). P-selectin (CD62p) expression, a classical marker of platelet activation, and phosphatidylserine (PS) exposure on the platelet surface membrane, a hallmark of cellular necrosis/apoptosis, were both measured by flow cytometry. The results reveal significant increases in activation, from an average of 22.7% on day 1 before treatment to 31.6% on day 2 after treatment and 58.7% at the end of storage. Concomitantly, the basal expression of PS was slightly increased from 1.9% to 2.8% at day 2 after treatment and 7.3% at the end of storage. However, the functional reserve of platelets during storage, which reflects their capability to undergo activation and the release reaction when platelets were challenged with either calcium ionophore or thrombin, was relatively well maintained. These preliminary data confirm the earlier data on the use of intercept, and for the first time, based on the assessment of platelet functional integrity, suggest that platelet functional reserve is relatively well maintained, with little change in the formation of apoptotic cells.

[Asthma genetic factors]. / Os factores genéticos da asma.

Asthma is a chronic inflammatory airways disease, with a rising prevalence, particularly in childhood, and is considered an important public health problem. Its familial transmission is recognised, while the description and identification of the genes implicated in this disease are a challenge. In this revision paper the authors give a comprehensive explanation of the associated genes as well as the laboratorial methods that allow their identification.

Mapping the HLA diversity of the Iberian Peninsula.

The polymorphism of HLA genes can be used to reconstruct human peopling history. However, this huge diversity impairs successful matching in stem cell transplantation, a situation which has led to the recruitment of millions of donors worldwide. In parallel to the increase of recruitment, registries are progressively relying on information from population genetics to optimize their donor pools in terms of HLA variability. In this study, the HLA data of 65,000 Spanish bone marrow donors were analyzed together with 60,000 Portuguese individuals to provide a comprehensive HLA genetic map of the Iberian Peninsula. The frequencies of many alleles were shown to vary continuously across the Peninsula, either increasing or decreasing from the Mediterranean coast to the Atlantic domain or from the Strait of Gibraltar to the Pyrenees and Bay of Biscay. Similar patterns were observed for several haplotypes. In addition, within some regions neighboring provinces share a close genetic similarity. These results outline the genetic landscape of the Iberian Peninsula, and confirm that the analysis of the HLA polymorphism may reveal relevant signatures of past demographic events even when data from donor registries are used. This conclusion stimulates future developments of the Spanish registry, presented here for the first time.

CD38, CD81 and BAFFR combined expression by transitional B cells distinguishes active from inactive systemic lupus erythematosus.

In view of its heterogeneous presentation and unpredictable course, clinical management of systemic lupus erythematosus (SLE) is difficult. There is a need for biomarkers and diagnostic aids to monitor SLE disease activity and severity prior to, during and after treatment. We undertook this study to search for unique phenotypic patterns in each peripheral blood (PB) B cell subset, capable of distinguishing SLE patients with inactive disease versus SLE patients with active disease versus controls by using an automated population separator (APS) visualization strategy. PB was collected from 41 SLE patients and 28 age- and gender-matched controls. We analyzed the cell surface markers (in a tube CD20/CD27/CD19/CD45/CD38/CD81/BAFFR combination) expression on PB B cell subsets using principal component analysis, implemented in the APS software tool. Overall, our analysis indicates that active SLE can be distinguished from inactive SLE on the basis of a single tube analysis, focused on the decreased expression of CD38, CD81 and BAFFR in transitional B cells. The cluster analysis of immunophenotypic profiles of B cell subsets highlighted disease-specific abnormalities on transitional B cells that emerge as promising surrogate markers for disease activity. Further validation is needed with larger samples and prospective follow-up of patients.

Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells.

The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-) α and macrophage inflammatory protein- (MIP-) 1ß protein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-α and MIP-1ß, whereas the reduction of TNF-α expression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1ß and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1ß and CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses.

Expression of CD44 and CD35 during normal and myelodysplastic erythropoiesis.

Erythroid dysplasia is a common feature of myelodysplastic syndromes (MDS). Currently available information about the immunophenotypic features of normal and dysplastic erythropoiesis is scarce and restricted to relatively few markers. Here we studied the expression of CD117, CD35 and CD44 throughout the normal (n=16) and dysplastic (n=48) bone marrow erythroid maturation. CD35 emerged as an early marker of CD34(+) erythroid-committed precursors, which is expressed before CD105 and remains positive thereafter. MDS patients (with and without morphologic dyserythropoiesis) displayed overall increased expression of CD44, associated with slight alterations on CD35 expression, suggesting that phenotypic alterations in MDS may precede morphologic dysplasia. In turn, MDS patients with anemia showed increased expression of CD117.

Effect of human bone marrow mesenchymal stromal cells on cytokine production by peripheral blood naive, memory, and effector T cells.

INTRODUCTION: The different distribution of T cells among activation/differentiation stages in immune disorders may condition the outcome of mesenchymal stromal cell (MSC)-based therapies. Indeed, the effect of MSCs in the different functional compartments of T cells is not completely elucidated. METHODS: We investigated the effect of human bone marrow MSCs on naturally occurring peripheral blood functional compartments of CD4(+) and CD8(+) T cells: naive, central memory, effector memory, and effector compartments. For that, mononuclear cells (MNCs) stimulated with phorbol myristate acetate (PMA) plus ionomycin were cultured in the absence/presence of MSCs. The percentage of cells expressing tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), and interleukin-2 (IL-2), IL-17, IL-9, and IL-6 and the amount of cytokine produced were assessed by flow cytometry. mRNA levels of IL-4, IL-10, transforming growth factor-beta (TGF-ß), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in purified CD4(+) and CD8(+) T cells, and phenotypic and mRNA expression changes induced by PMA + ionomycin stimulation in MSCs, were also evaluated. RESULTS: MSCs induced the reduction of the percentage of CD4(+) and CD8(+) T cells producing TNF-α, IFNγ, and IL-2 in all functional compartments, except for naive IFNγ(+)CD4(+) T cells. This inhibitory effect differentially affected CD4(+) and CD8(+) T cells as well as the T-cell functional compartments; remarkably, different cytokines showed distinct patterns of inhibition regarding both the percentage of producing cells and the amount of cytokine produced. Likewise, the percentages of IL-17(+), IL-17(+)TNF-α(+), and IL-9(+) within CD4(+) and CD8(+) T cells and of IL-6(+)CD4(+) T cells were decreased in MNC-MSC co-cultures. MSCs decreased IL-10 and increased IL-4 mRNA expression in stimulated CD4(+) and CD8(+) T cells, whereas TGF-ß was reduced in CD8(+) and augmented in CD4(+) T cells, with no changes for CTLA4. Finally, PMA + ionomycin stimulation did not induce significant alterations on MSCs phenotype but did increase indoleamine-2,3-dioxygenase (IDO), inducible costimulatory ligand (ICOSL), IL-1ß, IL-8, and TNF-α mRNA expression. CONCLUSIONS: Overall, our study showed that MSCs differentially regulate the functional compartments of CD4(+) and CD8(+) T cells, which may differentially impact their therapeutic effect in immune disorders. Furthermore, the influence of MSCs on IL-9 expression can open new possibilities for MSC-based therapy in allergic diseases.

Sera from patients with active systemic lupus erythematosus patients enhance the toll-like receptor 4 response in monocyte subsets.

BACKGROUND: Systemic Lupus Erythematosus (SLE) is an auto-immune disease whose complex pathogenesis remains unraveled. Here we aim to explore the inflammatory ability of SLE patients' sera upon peripheral blood (PB) monocyte subsets and myeloid dendritic cells (mDCs) obtained from healthy donors. METHODS: In this study we included 11 SLE patients with active disease (ASLE), 11 with inactive disease (ISLE) and 10 healthy controls (HC). PB from healthy donors was stimulated with patients' sera, toll-like receptor (TLR) 4 ligand - lipopolysaccharide or both. The intracellular production of TNF-α was evaluated in classical, non-classical monocytes and mDCs, using flow cytometry. TNF-α mRNA expression was assessed in all these purified cells, after sera treatment. RESULTS: We found that sera of SLE patients did not change spontaneous TNF-α production by monocytes or dendritic cells. However, upon stimulation of TLR4, the presence of sera from ASLE patients, but not ISLE, significantly increased the intracellular expression of TNF-α in classical and non-classical monocytes. This ability was related to titers anti-double stranded DNA antibodies in the serum. High levels of anti-TNF-α in the patients' sera were associated with increased TNF-α expression by co-cultured mDCs. No relationship was found with the levels of a wide variety of other pro-inflammatory cytokines. A slight increase of TNF-α mRNA expression was observed in these purified cells when they were cultured only in the presence of SLE serum. CONCLUSIONS: Our data suggest that SLE sera induce an abnormal in vitro TLR4 response in classical and non-classical monocytes, reflected by a higher TNF-α intracellular expression. These effects may be operative in the pathogenesis of SLE.