Expression Levels Of Activation Markers Research Articles

Clinicoradiological Parameters and Biochemical and Molecular Alterations Predicting Remission and Recurrence After Surgical Treatment of Corticotroph Adenomas—Cushing Disease

ObjectivesEndonasal endoscopic transsphenoidal surgery (TSS) and resection of pituitary adenomas is considered to be the gold standard treatment for Cushing’s Disease (CD). Even with various recent advances in management, disease persistence and recurrences are common in these patients. The remission rate in the global population after surgery has been reported to vary widely from 64 to 93%. This study aims to determine various clinical, biochemical, radiological and histological factors that correlate with persistence and recurrence in patients with CD. This study also aims to understand the clinicopathological significance of EGFR-MAPK, NF-kB and SHH pathway activation and to study the protein expression levels of activation markers of these pathways viz. c-Fos , c-Jun, GLI-1 , pMEK, NR4A1 and p44 in functioning corticotroph pituitary adenomas. MethodsFrom January 2009 to September 2022, the clinical data of 167 patients who underwent surgical treatment (n=174 surgeries) for Cushing’s disease was ambispectively analysed with a median follow up of 8.1 years (1-13.29 years). The preoperative clinical, biochemical, radiological features, operative findings, postoperative clinical and biochemical data, histopathological and molecular profile, were retrieved from electronic records. The patients were followed up to assess the remission status. ResultsAmong the 174 surgeries performed, 140 were primary surgeries, 22 were revision surgeries, 24 surgeries were for paediatric patients and 12 surgeries were performed in patients with Nelson syndrome. In the primary surgery cohort, 74.3% were females and the average age was 28.73 years (SD 10.15). 75% of the patients experienced remission after primary surgery compared to 47.4% in revision surgery. The remission rate in paediatric patients was 55.5%. Post-operative day 1 plasma cortisol (p<0.001; AUC 0.8894 [0.8087-0.9701]) and ACTH (p<0.001; AUC 0.9 [0.7386-1]) levels were seen to be strong independent predictors of remission in the primary surgery cohort. Remission rates after Endoscopic TSS were more than that after microscopic TSS, in patients undergoing primary surgery (81.08% vs 57.14%; p=0.008). Presence of adenoma on histopathological examination (HPE) was also a strong predictor of disease remission (p=0.020). On categorising based on surgical approach and HPE, microscopically operated patients without histopathological evidence of adenoma had significantly higher odds of non-remission (OR 38.1 95% CI 4.2-348.3) compared to endoscopically operated patients with adenoma on HPE. Lower immune reactivity score (IRS) of NR4A1 was found to correlate with higher remission rates (p=0.074). However, none of the molecular markers studied viz. c-Fos , c-Jun, GLI-1, pMEK and p44 showed a significant correlation with the preoperative cortisol values. ConclusionRemission rate after primary surgery is higher as compared to that after revision surgery, and that in paediatric patients is lower as compared to adults. Post-operative day 1 plasma cortisol and ACTH levels are strong independent predictors of remission in the primary surgery cohort. Endoscopic approach with histopathological evidence of adenoma is associated with a higher remission rate and thus endoscopy should be the approach of choice in these patients with the goal of identification of an adenoma on histopathological analysis.

Interrelations between viral load and cellular immunity in patients with COVID-19 of varying severity

Assessment of viral load levels in various biological samples taken from the respiratory tract can be an indicator of an ongoing process of active viral replication and may be used to monitor severe respiratory viral infections. The study of the relationship between SARS-CoV-2 viral load and immunological laboratory parameters is an important step in the search for clinical markers of COVID-19.The aim of this research was to quantify viral load in patients with COVID-19 and to identify the relation-ship between viral load and changes in the parameters of the cellular component of the immune system.A laboratory examination was carried out on 74 patients diagnosed with COVID-19, they were divided into 3 groups based on the severity of the disease: mild, moderate, severe. Total viral load in clinical samples was determined by the number of SARS-CoV-2 RNA copies per 100 copies of the reference RNaseP gene. A comprehensive assessment of the cellular component of the immune system was performed using flow cytometry and direct monoclonal antibodies, and the IL-6, and C-reactive protein concentrations were determined.We revealed a relationship between the development of serious clinical conditions in the patients with COVID-19, and the levels of viral load. High levels of viral RNA in biological samples correlate with main indicators of the T cell component of the immune system associated with disease severity. In a subgroup of patients with an extremely high viral load, strong positive correlations were found between the relative numbers of cytotoxic lymphocytes (CD3+CD8+), activated T lymphocytes (CD3+HLA-DR+), as well as absolute and relative numbers of activated B lymphocytes and NK cells (CD3-CD25+).Laboratory monitoring of the cellular component of the immune system, along with the assessment of viral loads, should improve early assessment of clinical condition in the patients with COVID-19. Changes in expression levels of activation markers on immune cells can be potentially viewed as indicators of recovery during COVID-19.

Detection of alloreactive T cells from cryopreserved human peripheral blood mononuclear cells

Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.

The effect of BCG vaccination on alveolar macrophages obtained from induced sputum from healthy volunteers

The anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) is able to boost innate immune responses through a process called ‘trained immunity’. It is hypothesized that BCG-induced trained immunity contributes to protection against Mycobacterium tuberculosis infection. Since alveolar macrophages are the first cell type to encounter M. tuberculosis upon infection, we aimed to investigate the immunomodulatory effects of BCG vaccination on alveolar macrophages. Searching for a less-invasive method than bronchoalveolar lavage, we optimized the isolation of alveolar macrophages from induced sputum of healthy volunteers. Viable alveolar macrophages could be successfully isolated from induced sputum and showed signs of activation already upon retrieval. Further flow cytometric analyses revealed that at baseline, higher expression levels of activation markers were observed on the alveolar macrophages of smokers compared to non-smokers. In addition, BCG vaccination resulted in decreased expression of the activation markers CD11b and HLA-DR on alveolar macrophages. Future studies should evaluate the functional consequences of this reduced activation of alveolar macrophages after BCG vaccination.

Circulating monocyte subsets in human chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a major cause of late morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Monocytes/macrophages play a central role in inflammation, tissue repair, and fibrosis, which are the main clinical features of cGVHD. Here, we examined the expression levels of activation markers, chemokine receptors, and scavenger receptors for each circulating monocyte subset in 145 patients without disease recurrence at least 12 months after undergoing allogeneic HCT. There were no significant differences in the numbers and the proportions of each monocyte subset between patients without cGVHD and those with mild or moderate/severe cGVHD. Lower expression of CCR5 on classical monocytes, and higher expression of CD204 and lower expression of CX3CR1 on non-classical monocytes were associated with joint, and lung cGVHD, respectively. These data showed that alterations of activation markers and chemokine and scavenger receptors in each circulating monocyte subset were associated with the development of organ-specific cGVHD. Alterations of surface markers in each circulating monocyte subset may be candidate biomarkers for cGVHD.

Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis.

Background & AimsCurrently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA).MethodsHuman cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated.ResultsWe could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed.ConclusionHere, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.

Human IgG2- and IgG4-expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age.

The mechanisms involved in sequential immunoglobulin G (IgG) class switching are still largely unknown. Sequential IG class switching is linked to higher levels of somatic hypermutation (SHM) in vivo, but it remains unclear if these are generated temporally during an immune response or upon activation in a secondary response. We here aimed to uncouple these processes and to distinguish memory B cells from primary and secondary immune responses. SHM levels and IgG subclasses were studied with 454 pyrosequencing on blood mononuclear cells from young children and adults as models for primary and secondary immunological memory. Additional sequencing and detailed immunophenotyping with IgG subclass-specific antibodies was performed on purified IgG+ memory B-cell subsets. In both children and adults, SHM levels were higher in transcripts involving more downstream-located IGHG genes (esp. IGHG2 and IGHG4). In adults, SHM levels were significantly higher than in children, and downstream IGHG genes were more frequently utilized. This was associated with increased frequencies of CD27+IgG+ memory B cells, which contained higher levels of SHM, more IGHG2 usage, and higher expression levels of activation markers than CD27−IgG+ memory B cells. We conclude that secondary immunological memory accumulates with age and these memory B cells express CD27, high levels of activation markers, and carry high SHM levels and frequent usage of IGHG2. These new insights contribute to our understanding of sequential IgG subclass switching and show a potential relevance of using serum IgG2 levels or numbers of IgG2-expressing B cells as markers for efficient generation of memory responses.

Abstract 2300: Human CD133-specific chimeric antigen receptor (CAR) modified T cells target patient-derived glioblastoma brain tumors

Abstract Glioblastoma (GBM), an aggressive primary brain tumor in adults, is feared for its near uniformly fatal prognosis and is characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. We developed patient-derived brain tumor initiating cell (BTIC) early passage lines that describe the extent of intertumoral heterogeneity, presenting a powerful preclinical model of GBM. Numerous studies have implicated CD133+ BTICs as drivers of chemo- and radio-resistance in GBM. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of chimeric antigen receptor (CAR) T-cell strategy that specifically targets CD133+ GBM cells. From the previously generated CD133-specific humanized monoclonal antibody, we derived the single chain variable fragment (scFv) and cloned it into an antigen-specific second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR vector in two different orientations: Light chain-linker-Heavy chain (CD133 CAR-LH) and Heavy chain-linker-Light chain (CD133 CAR-HL). Following lentiviral preparation, the T cells isolated from PBMCs were transduced with CD133 CAR-LH and CD133 CAR-VH constructs. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. While expression of ΔNGFR was observed in all CAR T cells (including controls), we found expression of the myc tag in both variations of CARs, CD133 CAR-HL and CD133 CAR-LH. Furthermore, we used Presto Blue-based killing assays to test the ability of CD133 CARs to selectively bind and kill CD133+ GBM BTICs. Our data shows that CD133-specific CAR T cells not only recognized, but killed the CD133+ GBM cells selectively, validating this adoptive T-cell therapeutic strategy. CAR-expressing T cells were activated in presence of CD133high GBM cells showed increase surface expression of activation markers CD69 and CD25. Both, CD4+ and CD8+ CD133-specific CAR-T cells showed upregulation in surface expression levels of activation markers. This rigorously obtained data offers compelling evidence that CAR-T induced cytotoxicity against treatment-resistant and evasive CD133+ GBM BTICs could provide a very potent, specific and novel therapeutic strategy for GBM patients. Citation Format: Parvez Vora, Chitra Venugopal, Sujeivan Mahendram, Chirayu Chokshi, Maleeha Qazi, Minomi Subapanditha, Mohini Singh, David Bakhshinyan, Ksenia Bezverbnaya, Jarrett Adams, Nicole McFarlane, Sachdev Sidhu, Jason Moffat, Jonathan Bramson, Sheila Singh. Human CD133-specific chimeric antigen receptor (CAR) modified T cells target patient-derived glioblastoma brain tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2300.

MAIT cells are activated and accumulated in the inflamed mucosa of ulcerative colitis.

Ulcerative colitis (UC) is a chronic, relapsing and remitting, inflammatory disorder of the large intestine. Mucosal associated invariant T (MAIT) cells are a member of innate-like lymphocytes found abundantly in the mucosal tissue. The contribution of MAIT cells in the pathogenesis of UC is still unclear; therefore, this study aimed at investigating the role of these cells in patients with UC. The frequency of MAIT cells, as well as the production of cytokines and expression levels of activation markers by these cells in the peripheral blood of UC patients and healthy controls, was analyzed by flow cytometry. MAIT cells were also quantified in colon biopsies of UC patients using a confocal microscope. There was a significant reduction in MAIT cell frequency in the peripheral blood of UC patients compared with healthy controls (P < 0.0001). MAIT cells from UC patients secreted more interleukin (IL)-17 than healthy controls (P < 0.05). The expression levels of CD69 on these cells were correlated with disease activity and endoscopic scores and plasma levels of IL-18. Furthermore, MAIT cells increased in the inflamed mucosa, and their frequency was correlated with clinical and endoscopic disease activity in UC patients. The findings from this study indicate that MAIT cells could be associated with UC and may serve as potential biomarkers or therapeutic targets in UC.

Low-Dose Dexamethasone Does Not Abrogate the Immunomodulatory Effects of Lenalidomide and Both Reactivate the Impaired Immune System of High-Risk Smoldering Multiple Myeloma Patients

Introduction: While dexamethasone (Dex) has been commonly used in the treatment of multiple myeloma (MM), its immunosuppressive effect is becoming a matter of concern with the advent of immune-based therapies. One example is the combination of lenalidomide and Dex (LenDex) because it has been reported that Dex abrogates the immunomodulatory effects of Len; however, most of the studies have been performed in vitro, using high-doses of Dex, and in small series of relapsed patients previously exposed to other drugs.Methods: Because the potentially antagonist effect of Dex may represent a dilemma in the design of clinical trials, here we aim to shed light into the question about whether or not low-dose Dex abrogates the immunomodulatory effect of Len by studying the phenotypic profile of T-lymphocytes, NK-cells and dendritic cells (DCs) of 31 previously untreated high-risk smoldering MM (SMM) patients enrolled in the Quiredex trial at baseline, after 3 and 9 cycles of LenDex, and during maintenance with Len as single-agent.Results: Patients with high-risk SMM showed at baseline normal numbers of CD4 and CD8 T-lymphocytes as well as CD56dim and CD56bright NK-cells compared to age-matched healthy individuals. By contrast, they displayed an increment of TCRγδ positive T-lymphocytes (P =.02) and Tregs (P =.04), as well as an altered distribution of BDCA-1 positive myeloid DCs (P =.02) and tissue macrophages (P =.06). Moreover, the expression levels of activation markers (CD25, CD28 and CD54) as well as Th1-related markers (CD195, IFN-γ, TNF-α, or IL-2) were significantly inferior in T-lymphocytes from high-risk SMM patients. A significantdown-regulation of proliferation-related markers (CD119 and CD120b) was also noted. To assess the combined effect of LenDex in T-lymphocytes and NK-cells, we compared the immune status of the 31 high-risk SMM patients at baseline vs. after 3 and 9 cycles of LenDex. Interestingly, TCRγδ positive T-lymphocytes as well as Tregs were further increased with LenDex; conversely, CD4 T-lymphocytes were significantly decreased at the end of induction. There was a marked shift on the distribution of antigen-related maturation subsets induced by LenDex, and reflected by a significant increase of central memory CD4 (P<.001) and effector memory CD8 (P<.001) T-lymphocytes. Accordingly, CD4 and/or CD8 T-lymphocytes showed an increased expression of activation markers (CD69, CD25, CD28, and CD54), together with an up-regulation of the Th1 related chemokine CCR5 (CD195) and increased cytokine production of IFNγ, TNFα, and IL-2. NK-cells showed an up-regulation of the activation marker HLA-DR (P<.001), the ADCC associated receptor CD16 (P≤0.005), and the adhesion molecules CD11a (P≤0.001) and CD11b (P≤0.005) after 3 and 9 courses of LenDex. The percentage of cells in S-phase progressively increased from baseline vs. 3 and 9 cycles of LenDex for CD4 (P<0.001) and CD8 (P<0.001) T-lymphocytes as well as NK-cells (P<0.001). Most interestingly, high-risk SMM patients treated with LenDex and without disease progression showed higher numbers of functionally active T-lymphocytes as compared to those progressing to MM. To address the question whether Dex antagonizes Len, we compared the immune profile of 13 patients with PB samples collected at cycle 9 of induction vs. during maintenance (single-agent Len at least 3 months after Dex discontinuation). No significant differences were observed for the absolute numbers of all cell populations analyzed. From the total 63 phenotypic parameters analyzed, only 7 were found to be differently expressed. Namely, the expression of CD94, CD154 and CD212 positive T-lymphocytes as well as CD11a in T-lymphocytes and NK-cells were down-regulated during maintenance.Conclusions: Our results, obtained from a carefully selected population of patients without previous exposure to anti-MM therapy and with available longitudinal samples after consecutive cycles of LenDex, shed new light on the synergy between lenalidomide and dexamethasone which, at low doses, does not abrogate the immune modulatory effects of lenalidomide here analyzed. Accordingly, high-risk SMM patients have an impaired immune system that could be re-activated with LenDex, and support the value of therapeutic immunomodulation to delay the progression to MM. DisclosuresPaiva:Millenium: Consultancy; BD Bioscience: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Sanofi: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy. Mateos:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen-Cilag: Honoraria; Sanofi-Aventis: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Onyx: Honoraria.

In vitro reversion of activated primary human hepatic stellate cells.

BackgroundLiver fibrosis is characterized by the excessive formation and accumulation of matrix proteins as a result of wound healing in the liver. A main event during fibrogenesis is the activation of the liver resident quiescent hepatic stellate cell (qHSC). Recent studies suggest that reversion of the activated HSC (aHSC) phenotype into a quiescent-like phenotype could be a major cellular mechanism underlying fibrosis regression in the liver, thereby offering new therapeutic perspectives for the treatment of liver fibrosis. Whether human HSCs have the ability to undergo a similar reversion in phenotype is currently unknown. The aim of the present study is to identify experimental conditions that can revert the in vitro activated phenotype of primary human HSCs and consequently to map the molecular events associated with this reversion process by gene expression profiling.ResultsWe find that epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) synergistically downregulate the expression of ACTA2 and LOX in primary human aHSCs. Their combination with oleic acid, palmitic acid, and retinol further potentiates a more quiescent-like phenotype as demonstrated by the abundant presence of retinyl ester-positive intra-cytoplasmic lipid droplets, low expression levels of activation markers, and a reduced basal as well as cytokine-stimulated proliferation and matrix metalloproteinase activity. Gene expression profiling experiments reveal that these in vitro reverted primary human HSCs (rHSCs) display an intermediary phenotype that is distinct from qHSCs and aHSCs. Interestingly, this intermediary phenotype is characterized by the increased expression of several previously identified signature genes of in vivo inactivated mouse HSCs such as CXCL1, CXCL2, and CTSS, suggesting also a potential role for these genes in promoting a quiescent-like phenotype in human HSCs.ConclusionsWe provide evidence for the ability of human primary aHSCs to revert in vitro to a transitional state through synergistic action of EGF, FGF2, dietary fatty acids and retinol, and provide a first phenotypic and genomic characterization of human in vitro rHSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-015-0031-z) contains supplementary material, which is available to authorized users.

Modulation of HIVGP120 Antigen-Specific Immune Responses In Vivo by Δ9-Tetrahydrocannabinol

Approximately 25% of HIV patients use marijuana for its putative therapeutic benefit; however, it is unknown how cannabinoids affect the immune status of HIV patients. Previously, a surrogate in vitro mouse model was established, which induced CD8(+) T cell proliferation and gp120-specific IFNγ production. ∆(9)-Tetrahydrocannabinol (THC), the predominant psychoactive compound in marijuana, suppressed or enhanced the responses depending on the magnitude of cellular activation. The purpose of the current study was to investigate whether THC produced similar effects in vivo and therefore a mouse model to induce HIVgp120-specific immune responses was established. A gp120-expressing plasmid, pVRCgp120, or a vector plasmid, pVRC2000, was injected intramuscularly into mice, which were also dosed with THC orally. The gp120-specific IFNγ and IL-2 responses were detected when splenocytes were restimulated with gp120-derived peptide 81 (IIGDIRQAHCNISRA), which was identified as being immunodominant. Various cellular populations were activated in response to pVRCgp120 stimulation followed by peptide restimulation, as evidenced by increased expression levels of activation markers (e.g., CD69, CD80, and major histocompatibility complex II [MHC II]). TheIFNγ response and cellular activation were enhanced by THC in C57Bl/6 wild type (WT) mice but suppressed or not affected by THC in cannabinoid receptor 1 (CB1) and 2 (CB2) knockout (CB1 (-/-)CB2 (-/-)) mice. Furthermore, CB1 (-/-)CB2 (-/-) mice exhibited augmented IFNγ production when compared to WT mice in the absence of THC. Collectively, our findings demonstrate that under certain conditions, THC enhances HIV antigen-specific immune responses, which occurs through CB1/CB2-dependent and -independent mechanisms.

Chronice leptin exposure alters dendritic cell function (130.7)

Abstract Leptin is a pleiotropic cytokine/hormone that has been shown to play a critical role in immunity, including dendritic cells (DC). In vitro experiments in mice and humans have shown that short term leptin treatment results in enhanced DC function. We hypothesized that chronic exposure, as would occur in vivo, would lead to decreased DC function. Bone marrow (BM) stem cells were thus procured from female C57Bl/6 mice and were treated with leptin during the entire DC differentiation culture period. The chronic leptin-treated BM-DC and control BM-DC (no leptin) were analyzed for phenotype, activation, and phagocytosis. The data varied extensively where leptin treatment resulted in decreased, similar, or enhanced DC function in comparison to controls as determined by expression levels of activation markers, interleukin-12 production, and phagocytosis of fluorescently-labeled E. coli fragments. To evaluate the potential role of female hormones on this outcome, the experiments were repeated with male BM-DC. Interestingly, leptin-treatment consistently decreased DC function relative to controls. The data suggests that constitutive leptin exposure may negatively regulate DC function and is additionally affected by the stage of the estrous cycle in females. The results underscore the complexity of leptin activity on immune cells that is not readily ascertained with short term exposure experiments.

Exposure of murine macrophages to carbon nanoparticles promotes inflammation and inhibits phagocytosis (130.5)

Abstract Exposure to respirable ultrafine particles (2.5 μM) can adversely affect human health and have been implicated with episodes of increased respiratory diseases such as asthma and allergies; the same is predicted for nanoparticles, which can reach deeper regions of the lung. The objective of this investigation was to examine the effects of carbon nanoparticles on alveolar macrophages. Macrophages (MΦ) function as the first line of defense against invading pathogens and are likely to be amongst the first cells affected by nanoparticulates. We focused on two manufactured nanoparticles: multiwalled carbon nanotube aggregates and black-carbon. The two were tested against MΦ in a chronic contact model. We hypothesized that exposure to nanoparticles would decrease MΦ ability to effectively respond to immunological challenge. Production of interleukin (IL)-12 and nitric oxide (NO), as well as phagocytosis, and MΦ activation were examined in response to lipopolysaccharide (LPS) following a 144 hr exposure to the nanoparticles. Data demonstrated an increase in NO and IL-12 production, a decrease in phagocytosis, and a decrease in the expression levels of activation markers. The data suggests that carbon nanoparticle exposure alters MΦ responses to LPS marked by increased inflammation while potentially limiting clearance and ability to interact with effector T-cells. Thus, physiological exposure to carbon nanoparticles could potentially lead to ineffective pulmonary immunity.

Differentiation of primary adult microglia alters their response to TLR8‐mediated activation but not their capacity as APC

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses.