Differential Role for CD80 and CD86 in the Regulation of the Innate Immune Response in Murine Polymicrobial Sepsis

Background

Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86^−/− mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis.

Methodology/Principal Findings

CD80^−/− mice had improved survival after CLP when compared to WT or CD86^−/− mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80^−/− mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-κB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model.

Conclusions

In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80.     

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.

Objective

The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.

Methods and Results

We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84^−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84^−/− mice. In vivo, Cd84^−/− mice exhibited unaltered hemostatic function and arterial thrombus formation.

Conclusion

These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.     

Blunted Response to Combination Antiretroviral Therapy in HIV Elite Controllers: An International HIV Controller Collaboration

Objective

HIV “elite controllers” (ECs) spontaneously control viral load, but some eventually require combination antiretroviral treatment (cART), due to a loss of viral control or a decline in CD4 T-cell counts. Here we studied the CD4 T-cell count dynamics after cART initiation among 34 ECs followed in U.S. and European cohorts, by comparison with chronically viremic patients (VIRs).

Methods

ECs were defined as patients with at least ≥5 viral load (VL) measurements below 400 copies/mL during at least a 5-year period despite never receiving ART and were selected from the French ANRS CO18 cohort, the U.S. SCOPE cohort, the International HIV Controllers study and the European CASCADE collaboration. VIRs were selected from the ANRS COPANA cohort of recently-diagnosed (<1 year) ART-naïve HIV-1-infected adults. CD4 T-cell count dynamics after cART initiation in both groups were modelled with piecewise mixed linear models.

Results

After cART initiation, CD4 T-cell counts showed a biphasic rise in VIRs with: an initial rapid increase during the first 3 months (+0.63/month), followed by +0.19/month. This first rapid phase was not observed in ECs, in whom the CD4Tc count increased steadily, at a rate similar to that of the second phase observed in VIRs. After cART initiation at a CD4 T-cell count of 300/mm³, the estimated mean CD4 T-cell gain during the first 12 months was 139/mm³ in VIRs and 80/mm³ in ECs (p = 0.048).

Conclusions

cART increases CD4 T-cell counts in elite controllers, albeit less markedly than in other patients.     

Central Muscarinic Cholinergic Activation Alters Interaction between Splenic Dendritic Cell and CD4+CD25- T Cells in Experimental Colitis

Background

The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4⁺/CD25⁻T cell activation in the context of experimental colitis.

Methods

The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4⁺CD25⁻T cell co-culture were determined.

Results

McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4⁺CD25⁻T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy.

Conclusions

Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.     

Vasoprotective Effects of Urocortin 1 against Atherosclerosis In Vitro and In Vivo

Aim

Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis.

Methods

We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe ^−/−) mice.

Results

Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe ^−/− mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions.

Conclusions

This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.     

Cross Talk with Hematopoietic Cells Regulates the Endothelial Progenitor Cell Differentiation of CD34 Positive Cells

Introduction

Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear.

Methods

In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34⁺ and CD34⁻ cells, and determined the optimal concentrations of CD34⁺ cells and CD34⁻ cells for spindle-shaped EPC differentiation.

Results

Functionally, the coculture of CD34⁺ and CD34⁻ cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34⁺ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34⁺ and CD34⁻ cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3⁺) markedly accelerated the early EPC status of CD34⁺ cells, while macrophages (CD11b⁺) or megakaryocytes (CD41⁺) specifically promoted large EPC colonies.

Conclusion

Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34⁺ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy.     

Period2 Deficiency Blunts Hypoxia-Induced Mobilization and Function of Endothelial Progenitor Cells

Background

In the clinic, variations in circadian rhythm are evident in patients with cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. In this study, we focused on the role of the circadian gene period2 (per2) in mobilization and function of endothelial progenitor cells (EPCs) in vitro and in vivo after myocardial infarction (MI) in mice.

Methods and Results

MI was produced by surgical ligation of the left anterior descending coronary artery in mice with and without per2 deficiency. Trans-thoracic echocardiography was used to evaluate cardiac function in mice. Per2^−/− mice with MI showed decreased cardiac function and increased infarct size. The number of CD34+ cells and capillary density were decreased in the myocardium of per2^−/− mice on immunohistochemistry. Flow cytometry revealed decreased number of circulating EPCs in per2^−/− mice after MI. In vitro, per2^−/− EPCs showed decreased migration and tube formation capacity under hypoxia. Western blot analysis revealed inhibited activation of extracellular signal-regulated kinase and Akt signaling in the bone marrow of per2^−/− mice and inhibited PI3K/Akt expression in per2^−/− EPCs under hypoxia.

Conclusions

Per2 modulates EPC mobilization and function after MI, which is important to recovery after MI in mice.     

Detection and Validation of Circulating Endothelial Cells,a Blood-based Diagnostic Marker of Acute Myocardial Infarction

Background

Circulating endothelial cells (CECs) are markers of vascular damage that have clinical relevance in many diseases, including acute myocardial infarction (AMI), and may be predictors of treatment responses. Herein, we investigated the diagnostic and prognostic value of CEC monitoring in AMI patients and a murine model.

Methodology/Principal Findings

CECs were defined as Hoechst 33342⁺/CD45^−/CD31⁺/CD146⁺/CD133⁻ in human blood samples and Hoechst 33342⁺/CD45^−/CD31⁺/KDR⁺/CD117⁻ in murine samples. To evaluate the validity and variability of our CEC detection system, peripheral blood samples of vascular endothelial growth factor-treated athymic nude mice and AMI patients were collected and subjected to intra-assay analysis. CEC detection by flow cytometry and real-time PCR were compared. Blood samples were obtained from 61 AMI patients, 45 healthy volunteers and 19 samples of the original AMI patients accepted one month treatment, via flow cytometry and expressed as a percentage of peripheral blood mononuclear cells.

Results

Our CEC detection method was validated and had limited variability. CEC concentrations were higher in AMI patients compared to healthy controls. One month post-treatment, CECs levels decreased significantly.

Conclusions/Significance

CEC levels may be useful as a diagnostic and prognostic biomarker in AMI patients.     

Human CD4+ Memory T Cells Can Become CD4+IL-9+ T Cells

Background

IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-β directs mouse CD4⁺CD25⁻CD62L⁺ T cells to commit to inflammatory IL-9 producing CD4⁺ T cells.

Methodology/Principal Findings

Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-β induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4⁺CD25⁻CD45RO⁺ T cells as compared to naïve CD4⁺CD25⁻CD45RA⁺ T cells. In addition, as compared to pbCD3/sCD28 plus TGF-β stimulation, IL-4+TGF-β stimulated memory CD4⁺CD25⁻CD45RO⁺ T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4⁺IL-9⁺ T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNγ or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-β stimulated resting memory CD4⁺ T cells demonstrated that the addition of IL-1β, IL-12, and IL-21 further enhance IL-9 production.

Conclusions/Significance

Taken together these data show both the differences and similarities between mouse and human CD4⁺IL9⁺ T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4⁺ T cells to antigen.     

VR09 Cell Line: An EBV-Positive Lymphoblastoid Cell Line with In Vivo Characteristics of Diffuse Large B Cell Lymphoma of Activated B-Cell Type

Background

small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro.

Design and Method

we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features.

Results

VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2^−/− γ-chain^−/− mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/− CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53.

Conclusion

This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features.     

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu,Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

Purpose

The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 ^−/−) mouse.

Methods

Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 ^−/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed.

Results

Corneal vital staining scores in the Sod1 ^−/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 ^−/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 ^−/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 ^−/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 ^−/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 ^−/− mice.

Conclusions

Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 ^−/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.     

Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24? Subpopulations of Breast Cancer Cells

Background

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH⁺), or cell surface molecule CD44-positive (CD44⁺) but CD24-negative (CD24⁻) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells is unknown.

Methods and Results

We examined STAT3 activation in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH⁺) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH⁻) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH⁺ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH⁺ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH⁺ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH⁺ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH⁺ cells were further selected for the stem cell markers CD44⁺ and CD24⁻.

Conclusion

These studies demonstrate an important role for STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.     

18F-FDG PET Imaging of Murine Atherosclerosis: Association with Gene Expression of Key Molecular Markers

Aim

To study whether ¹⁸F-FDG can be used for in vivo imaging of atherogenesis by examining the correlation between ¹⁸F-FDG uptake and gene expression of key molecular markers of atherosclerosis in apoE^−/− mice.

Methods

Nine groups of apoE^−/− mice were given normal chow or high-fat diet. At different time-points, ¹⁸F-FDG PET/contrast-enhanced CT scans were performed on dedicated animal scanners. After scans, animals were euthanized, aortas removed, gamma counted, RNA extracted from the tissue, and gene expression of chemo (C-X-C motif) ligand 1 (CXCL-1), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, cluster of differentiation molecule (CD)-68, osteopontin (OPN), lectin-like oxidized LDL-receptor (LOX)-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGF), and tissue factor (TF) was measured by means of qPCR.

Results

The uptake of ¹⁸F-FDG increased over time in the groups of mice receiving high-fat diet measured by PET and ex vivo gamma counting. The gene expression of all examined markers of atherosclerosis correlated significantly with ¹⁸F-FDG uptake. The strongest correlation was seen with TF and CD68 (p<0.001). A multivariate analysis showed CD68, OPN, TF, and VCAM-1 to be the most important contributors to the uptake of ¹⁸F-FDG. Together they could explain 60% of the ¹⁸F-FDG uptake.

Conclusion

We have demonstrated that ¹⁸F-FDG can be used to follow the progression of atherosclerosis in apoE^−/− mice. The gene expression of ten molecular markers representing different molecular processes important for atherosclerosis was shown to correlate with the uptake of ¹⁸F-FDG. Especially, the gene expressions of CD68, OPN, TF, and VCAM-1 were strong predictors for the uptake.     

Cortisol Patterns Are Associated with T Cell Activation in HIV

Objective

The level of T cell activation in untreated HIV disease is strongly and independently associated with risk of immunologic and clinical progression. The factors that influence the level of activation, however, are not fully defined. Since endogenous glucocorticoids are important in regulating inflammation, we sought to determine whether less optimal diurnal cortisol patterns are associated with greater T cell activation.

Methods

We studied 128 HIV-infected adults who were not on treatment and had a CD4⁺ T cell count above 250 cells/µl. We assessed T cell activation by CD38 expression using flow cytometry, and diurnal cortisol was assessed with salivary measurements.

Results

Lower waking cortisol levels correlated with greater T cell immune activation, measured by CD38 mean fluorescent intensity, on CD4⁺ T cells (r = −0.26, p = 0.006). Participants with lower waking cortisol also showed a trend toward greater activation on CD8⁺ T cells (r = −0.17, p = 0.08). A greater diurnal decline in cortisol, usually considered a healthy pattern, correlated with less CD4⁺ (r = 0.24, p = 0.018) and CD8⁺ (r = 0.24, p = 0.017) activation.

Conclusions

These data suggest that the hypothalamic-pituitary-adrenal (HPA) axis contributes to the regulation of T cell activation in HIV. This may represent an important pathway through which psychological states and the HPA axis influence progression of HIV.     

Double Negative (CD3+4?8?) TCRαβ Splenic Cells from Young NOD Mice Provide Long-Lasting Protection against Type 1 Diabetes

Background

Double negative CD3⁺4⁻8⁻ TCRαβ splenic cells (DNCD3) can suppress the immune responses to allo and xenografts, infectious agents, tumors, and some autoimmune disorders. However, little is known about their role in autoimmune diabetes, a disease characterized by the reduction of insulin production subsequent to destruction of pancreatic β-cells by a polyclonal population of self-reactive T-cells. Herein, we analyzed the function and phenotype of DNCD3 splenic cells in young NOD mice predisposed to several autoimmune disorders among which, the human-like autoimmune diabetes.

Methodology/Principal Findings

DNCD3 splenic cells from young NOD mice (1) provided long-lasting protection against diabetes transfer in NOD/Scid immunodeficient mice, (2) proliferated and differentiated in the spleen and pancreas of NOD/Scid mice and pre-diabetic NOD mice into IL-10-secreting T_R-1 like cells in a Th2-like environment, and (3) their anti-diabetogenic phenotype is CD3⁺(CD4⁻CD8⁻)CD28⁺CD69⁺CD25^low Foxp3⁻ iCTLA-4⁻TCRαβ⁺ with a predominant Vβ13 gene usage.

Conclusions/Significance

These findings delineate a new T regulatory component in autoimmune diabetes apart from that of NKT and CD4⁺CD25^high Foxp3⁺T-regulatory cells. DNCD3 splenic cells could be potentially manipulated towards the development of autologous cell therapies in autoimmune diabetes.     

Functional Improvement of Regulatory T Cells from Rheumatoid Arthritis Subjects Induced by Capsular Polysaccharide Glucuronoxylomannogalactan

Objective

Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal).

Methods

Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.

Results

GXMGal was able to: i) induce strong increase of FOXP3 on CD4⁺ T cells without affecting the number of CD4⁺CD25⁺FOXP3⁺ Treg cells with parallel increase in the percentage of non-conventional CD4⁺CD25⁻FOXP3⁺ Treg cells; ii) increase intracellular levels of TGF-β1 in CD4⁺CD25⁻FOXP3⁺ Treg cells and of IL-10 in both CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells; iii) enhance the suppressive activity of CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-γ and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production.

Conclusion

These data show that GXMGal improves Treg functions and increases the number and function of CD4⁺CD25⁻FOXP3⁺ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases.     

Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction

We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl⁻) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.

Methods

Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR^−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl⁻ transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca²⁺]_i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.

Results

Sinupret-mediated Cl⁻ secretion [ΔI_SC(µA/cm²)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR^−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca²⁺ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl⁻ secretion. Sinupret also enhanced CBF and ASL depth.

Conclusion

Sinupret stimulates CBF, promotes transepithelial Cl⁻ secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca²⁺-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl⁻ secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.     

Small-Size Circulating Endothelial Microparticles in Coronary Artery Disease

Objectives

Several recent lines of evidence indicate that endothelial microparticles are a new biomarker that can be used to monitor endothelial dysfunction in coronary artery disease (CAD). However, data concerning the detection of small microparticles (diameter <0.5 µm) are lacking. The aim of this study was to detect small-size endothelial microparticles (SEMPs) in CAD patients to monitor endothelial dysfunction.

Methods

In total, 19 CAD patients and 14 healthy subjects were recruited. The absolute numbers and percentages of CD31⁺/CD42b⁻ SEMPs and CD62E⁺ SEMPs were determined by flow cytometry. Clinical parameters were also recorded.

Results

The mean percentage of CD62E⁺ SEMPs was higher in the CAD patient group than in the healthy subject group. The area under the receiver operating characteristic curve of the percentage of CD62E⁺ SEMPs was 0.795, and the cut-off value was 1.35. There was no correlation between the percentage of CD62E⁺ SEMPs and various clinical parameters.

Conclusion

The percentage of CD62E⁺ SEMPs is a potential biomarker for monitoring endothelial function in CAD.