Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury

Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1⁺CD45^- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1⁺CD45^- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1⁺CD45^- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/Significance

These studies demonstrate that cloned Sca-1⁺CD45^- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1⁺) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.     

Peripheral T cell cytokine responses for diagnosis of active tuberculosis

Background

A test for diagnosis of active Tuberculosis (TB) from peripheral blood could tremendously improve clinical management of patients.

Methods

Of 178 prospectively enrolled patients with possible TB, 60 patients were diagnosed with pulmonary and 27 patients with extrapulmonary TB. The frequencies of Mycobacterium tuberculosis (MTB) specific CD4⁺ T cells and CD8⁺ T cells producing cytokines were assessed using overnight stimulation with purified protein derivate (PPD) or early secretory antigenic target (ESAT)-6, respectively.

Results

Among patients with active TB, an increased type 1 cytokine profile consisting of mainly CD4⁺ T cell derived interferon (IFN)-γ was detectable. Despite contributing to the cytokine profile as a whole, the independent diagnostic performance of one cytokine producing T cells as well as polyfunctional T cells was poor. IFN-γ/Interleukin(IL)-2 cytokine ratios discriminated best between active TB and other diseases.

Conclusion

T cells producing one cytokine and polyfunctional T cells have a limited role in diagnosis of active TB. The significant shift from a “memory type” to an “effector type” cytokine profile may be useful for further development of a rapid immune-diagnostic tool for active TB.     

Inhaled NO contributes to lung repair in piglets with acute respiratory distress syndrome via increasing circulating endothelial progenitor cells

Background

Nitric oxide (NO) plays an important role in mobilization of endothelial progenitor cells (EPCs). We hypothesized that inhaled NO (iNO) would induce EPC mobilization and therefore promote lung repair in acute respiratory distress syndrome (ARDS).

Methodology/Principal Findings

Healthy piglets were randomized into four groups (n = 6): Control (Con; mechanical ventilation only); ARDS (established by oleic acid infusion and mechanical ventilation); ARDS plus granulocyte-colony stimulating factor (G-CSF; 10 µg/kg/d subcutaneously); ARDS plus NO inhalation (iNO; 10 ppm). EPCs and mobilizing cytokines were assayed at different time points (baseline, 0, 24, 72 and 168 h) and injury reparation was assessed at 168 h. Compared to the Con group, the levels of EPCs were increased in bone marrow but not in blood in the ARDS group at 24 h. Compared to the ARDS group, inhaled NO induced a rapid elevation in the number of CD34⁺KDR⁺, KDR⁺CD133⁺ and CD34⁺KDR⁺CD133⁺ EPCs in blood (2163±454 vs. 1094±416, 1302±413 vs. 429±244, 1140±494 vs. 453±273 cells/ml, respectively, P<0.05), and a reduction in the percentage of KDR⁺CD133⁺ cells in bone marrow. Lung CD34, CD133, VEGF, VEGF receptor 2, endothelial NO synthase mRNA, and VEGF and VEGF receptor 2 protein expression levels were augmented in the iNO group, but not in the G-CSF group, compared to ARDS. Furthermore, iNO treatment reduced vascular permeability, increased pulmonary vessel density, and alleviated pulmonary edema and inflammation compared to ARDS treatment. Plasma VEGF, stromal cell-derived factor-1 (SDF-1) and bone marrow NO₂ ⁻/NO₃ ⁻ were significantly higher in the iNO group compared to the ARDS group at 72 h.

Conclusions

These results suggest that iNO induces mobilization of EPCs from bone marrow into circulation, contributes to vascular repair, and thereby alleviates lung damage.     

CD34+ cells represent highly functional endothelial progenitor cells in murine bone marrow

Background

Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow.

Methodology/Principal Findings

CD34⁺ cells, c-Kit⁺/Sca-1⁺/Lin⁻ (KSL) cells, c-Kit⁺/Lin⁻ (KL) cells and Sca-1⁺/Lin⁻ (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34⁺ cells showed the lowest EPC colony forming activity, CD34⁺ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34⁺ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34⁺ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34⁺ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others.

Conclusion

These findings suggest that mouse CD34⁺ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.     

Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells

Background

Bisphosphonates are a class of pharmacologic compounds that are commonly used to treat postmenopausal osteoporosis and malignant osteolytic processes. Studies have shown that bone marrow-derived endothelial progenitor cells (EPCs) play a significant role in postnatal neovascularization. Whether the nitrogen-containing bisphosphonate zoledronate inhibits ischemia-induced neovascularization by modulating EPC functions remains unclear.

Methodology/Principal Findings

Unilateral hindlimb ischemia was surgically induced in wild-type mice after 2 weeks of treatment with vehicle or zoledronate (low-dose: 30 μg/kg; high-dose: 100 μg/kg). Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio was significantly lower in wild-type mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in controls 4 weeks after ischemic surgery (control vs. low-dose vs. high-dose: 87±7% vs. *61±18% vs. **49±17%, *p<0.01, **p<0.005 compared to control). Capillary densities were also significantly lower in mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in control mice. Flow cytometry analysis showed impaired mobilization of EPC-like cells (Sca-1⁺/Flk-1⁺) after surgical induction of ischemia in mice treated with zoledronate but normal levels of mobilization in mice treated with vehicle. In addition, ischemic tissue from mice that received zoledronate treatment exhibited significantly lower levels of the active form of MMP-9, lower levels of VEGF, and lower levels of phosphorylated eNOS and phosphorylated Akt than ischemic tissue from mice that received vehicle. Results of the in vitro studies showed that incubation with zoledronate inhibited the viability, migration, and tube-forming capacities of EPC.

Conclusions/Significance

Zoledronate inhibited ischemia-induced neovascularization by impairing EPC mobilization and angiogenic functions. These findings suggest that administration of zoledronate should be withheld in patients with ischemic events such as acute limb ischemia.     

Circulating endothelial progenitor cells in kidney transplant patients

Background

Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs.

Methods

We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry.

Results

Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1.

Conclusions

We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.     

"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Background

Cooperation of CD4⁺ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4⁺ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal Findings

Here we have developed a protocol to selectively reduce the CD4⁺ T cell response against viral CD4⁺ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4⁺ T cells without affecting CD4⁺ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4⁺ T cell help, tolerisation did not impair CD8⁺ T cell responses.

Conclusions

This result reveals a novel “negative vaccination” strategy where specific CD4⁺ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.     

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose

Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice.

Methods

The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8 ^+/− mice expressing ß-galactosidase. Aged Mfge8 ^+/− and Mfge8 ^−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV.

Results

rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8^−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch''s membrane (BM) was slightly but significantly thicker in Mfge8^−/− mice as compared to controls.

Conclusions

Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8^−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.     

PlGF repairs myocardial ischemia through mechanisms of angiogenesis, cardioprotection and recruitment of myo-angiogenic competent marrow progenitors

Rationale

Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected.

Objective

We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity.

Methods and Results

Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1⁺/Lin⁻ (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage.

Conclusions

Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease.     

Phloroglucinol inhibits the bioactivities of endothelial progenitor cells and suppresses tumor angiogenesis in LLC-tumor-bearing mice

Background

There is increasing evidence that phloroglucinol, a compound from Ecklonia cava, induces the apoptosis of cancer cells, eventually suppressing tumor angiogenesis.

Methodology/Principal Findings

This is the first report on phloroglucinol''s ability to potentially inhibit the functional bioactivities of endothelial progenitor cells (EPCs) and thereby attenuate tumor growth and angiogenesis in the Lewis lung carcinoma (LLC)-tumor-bearing mouse model. Although Phloroglucinol did not affect their cell toxicity, it specifically inhibited vascular endothelial growth factor (VEGF) dependent migration and capillary-like tube formation of EPCs. Our matrigel plug assay clearly indicated that orally injected phloroglucinol effectively disrupts VEGF-induced neovessel formation. Moreover, we demonstrated that when phloroglucinol is orally administered, it significantly inhibits tumor growth and angiogenesis as well as CD45⁻/CD34⁺ progenitor mobilization into peripheral blood in vivo in the LLC-tumor-bearing mouse model.

Conclusions/Significance

These results suggest a novel role for phloroglucinol: Phloroglucinol might be a modulator of circulating EPC bioactivities, eventually suppressing tumorigenesis. Therefore, phloroglucinol might be a candidate compound for biosafe drugs that target tumor angiogenesis.     

Pre-procedural atorvastatin mobilizes endothelial progenitor cells: clues to the salutary effects of statins on healing of stented human arteries

Objectives

Recent clinical trials suggest an LDL-independent superiority of intensive statin therapy in reducing target vessel revascularization and peri-procedural myocardial infarctions in patients who undergo percutaneous coronary interventions (PCI). While animal studies demonstrate that statins mobilize endothelial progenitor cells (EPCs) which can enhance arterial repair and attenuate neointimal formation, the precise explanation for the clinical PCI benefits of high dose statin therapy remain elusive. Thus we serially assessed patients undergoing PCI to test the hypothesis that high dose Atorvastatin therapy initiated prior to PCI mobilizes EPCs that may be capable of enhancing arterial repair.

Methods and Results

Statin naïve male patients undergoing angiography for stent placement were randomized to standard therapy without Atorvastatin (n = 10) or treatment with Atorvastatin 80 mg (n = 10) beginning three days prior to stent implantation. EPCs were defined by flow cytometry (e.g., surface marker profile of CD45dim/34+/133+/117+). As well, we also enumerated cultured angiogenic cells (CACs) by standard in vitro culture assay. While EPC levels did not fluctuate over time for the patients free of Atorvastatin, there was a 3.5-fold increase in EPC levels with high dose Atorvastatin beginning within 3 days of the first dose (and immediately pre-PCI) which persisted at 4 and 24 hours post-PCI (p<0.05). There was a similar rise in CAC levels as assessed by in vitro culture. CACs cultured in the presence of Atorvastatin failed to show augmented survival or VEGF secretion but displayed a 2-fold increase in adhesion to stent struts (p<0.05).

Conclusions

High dose Atorvastatin therapy pre-PCI improves EPC number and CAC number and function in humans which may in part explain the benefit in clinical outcomes seen in patients undergoing coronary interventions.     

Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15

Background

NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated.

Methodology/Principal Findings

Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of “NK-ireg” cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34⁺ PB-HP. Finally, a small subset of NKp46⁺ HLA-G⁺ IL-10⁺ is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells.

Conclusions/Significance

In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56⁺ CD16⁺ NKp30⁺ NKp44⁺ NKp46⁺ CD94⁺ CD69⁺ CCR7⁺) generated from specific pSTAT6⁺ GATA3⁺ precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.     

Decreased circulating endothelial progenitor cell levels and function in patients with nonalcoholic fatty liver disease

Objectives

Nonalcoholic fatty liver disease (NAFLD) is associated with advanced atherosclerosis and a higher risk of cardiovascular disease. Increasing evidence suggests that injured endothelial monolayer is regenerated by circulating bone marrow derived-endothelial progenitor cells (EPCs), and levels of circulating EPCs reflect vascular repair capacity. However, the relation between NAFLD and EPC remains unclear. Here, we tested the hypothesis that patients with nonalcoholic fatty liver disease (NAFLD) might have decreased endothelial progenitor cell (EPC) levels and attenuated EPC function.

Methods and Results

A total of 312 consecutive patients undergoing elective coronary angiography because of suspected coronary artery disease were screened and received examinations of abdominal ultrasonography between July 2009 and November 2010. Finally, 34 patients with an ultrasonographic diagnosis of NAFLD, and 68 age- and sex-matched controls without NAFLD were enrolled. Flow cytometry with quantification of EPC markers (defined as CD34⁺, CD34⁺KDR⁺, and CD34⁺KDR⁺CD133⁺) in peripheral blood samples was used to assess circulating EPC numbers. The adhesive function, and migration, and tube formation capacities of EPCs were also determined in NAFLD patients and controls. Patients with NAFLD had a significantly higher incidence of metabolic syndrome, previous myocardial infarction, hyperuricemia, and higher waist circumference, body mass index, fasting glucose and triglyceride levels. In addition, patients with NAFLD had significantly decreased circulating EPC levels (all P<0.05), attenuated EPC functions, and enhanced systemic inflammation compared to controls. Multivariate logistic regression analysis showed that circulating EPC level (CD34⁺KDR⁺ [cells/10⁵ events]) was an independent reverse predictor of NAFLD (Odds ratio: 0.78; 95% confidence interval: 0.69–0.89, P<0.001).

Conclusions

NAFLD patients have decreased circulating EPC numbers and functions than those without NAFLD, which may be one of the mechanisms to explain atherosclerotic disease progression and enhanced cardiovascular risk in patients with NAFLD.     

Granulysin-expressing CD4+ T cells as candidate immune marker for tuberculosis during childhood and adolescence

Background

Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB.

Methods

Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4⁺ CD45RO⁺ memory T cells.

Results

CD4⁺ CD45RO⁺ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSE_lowCD4⁺CD45RO⁺) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4⁺ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells.

Conclusions

Our data suggest granulysin expression by CD4⁺ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence.     

Visceral adipose inflammation in obesity is associated with critical alterations in tregulatory cell numbers

Background

The development of insulin resistance (IR) in mouse models of obesity and type 2 diabetes mellitus (DM) is characterized by progressive accumulation of inflammatory macrophages and subpopulations of T cells in the visceral adipose. Regulatory T cells (Tregs) may play a critical role in modulating tissue inflammation via their interactions with both adaptive and innate immune mechanisms. We hypothesized that an imbalance in Tregs is a critical determinant of adipose inflammation and investigated the role of Tregs in IR/obesity through coordinated studies in mice and humans.

Methods and Findings

Foxp3-green fluorescent protein (GFP) “knock-in” mice were randomized to a high-fat diet intervention for a duration of 12 weeks to induce DIO/IR. Morbidly obese humans without overt type 2 DM (n = 13) and lean controls (n = 7) were recruited prospectively for assessment of visceral adipose inflammation. DIO resulted in increased CD3⁺CD4⁺, and CD3⁺CD8⁺ cells in visceral adipose with a striking decrease in visceral adipose Tregs. Treg numbers in visceral adipose inversely correlated with CD11b⁺CD11c⁺ adipose tissue macrophages (ATMs). Splenic Treg numbers were increased with up-regulation of homing receptors CXCR3 and CCR7 and marker of activation CD44. In-vitro differentiation assays showed an inhibition of Treg differentiation in response to conditioned media from inflammatory macrophages. Human visceral adipose in morbid obesity was characterized by an increase in CD11c⁺ ATMs and a decrease in foxp3 expression.

Conclusions

Our experiments indicate that obesity in mice and humans results in adipose Treg depletion. These changes appear to occur via reduced local differentiation rather than impaired homing. Our findings implicate a role for Tregs as determinants of adipose inflammation.     

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish

Background

Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood.

Methodology/Principal Findings

In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp ⁺ cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli ⁺ etsrp ⁺ hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp ⁺ cell numbers at the LPM.

Conclusions/Significance

This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.     

An MSI tumor specific frameshift mutation in a coding microsatellite of MSH3 encodes for HLA-A0201-restricted CD8+ cytotoxic T cell epitopes

Background

Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI⁺ colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies.

Methodology/Principal Findings

FSP-specific CD8⁺ T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI⁺ colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity.

Conclusions

We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were ³⁸⁶-FLLALWECSL (FSP18) and ³⁸⁷-LLALWECSL (FSP19) as well as ⁴⁰³-IVSRTLLLV (FSP23) and ⁴⁰²-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI⁺-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or –even more important– preventive purposes.     

Accelerated vascular aging in CuZnSOD-deficient mice: impact on EPC function and reparative neovascularization

Objective

Aging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. CuZnSOD has an important role to limit oxidative stress in the vasculature. Here we investigated the role of CuZnSOD for the modulation of ischemia-induced neovascularisation during aging.

Methods and Results

Hindlimb ischemia was surgically induced in young (2- month-old) or older (8-month-old) wild type (WT) and CuZnSOD^−/− mice. We found that blood flow recovery after ischemia and vascular density in ischemic muscles were significantly reduced in older compared to young WT mice. Both in young and older mice, CuZnSOD deficiency led to a further reduction of neovascularization. Accordingly, the resulting neovascularisation potential in a young CuZnSOD^−/− mouse was similar to that of an older WT mouse. Oxidative stress levels were also increased to similar levels in the ischemic muscles of young CuZnSOD^−/− and older WT mice. To identify potential mechanisms involved, we investigated the effect of aging and CuZnSOD deficiency on the number and the function of endothelial progenitor cells (EPCs). Both aging and CuZnSOD deficiency were associated with reduced number of bone marrow and peripheral EPCs. The effect of moderate aging alone on specific functional activities of EPCs (migration, integration into tubules) was modest. However, CuZnSOD deficiency was associated with severe age-dependent defects in EPC functional activities.

Conclusions

CuZnSOD deficiency is associated with accelerated vascular aging and impaired ischemia-induced neovascularization. Our results suggest that in the context of aging, CuZnSOD has an essential role to protect against excessive oxidative stress in ischemic tissues and preserve the function of EPCs.     

Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation

Background

The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8⁺ T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8⁺ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons.

Methodology/Principal Findings

Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8⁺ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8⁺ T cells is dependent on CD4⁺ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis.

Conclusions/Significance

B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.     

Fetal microchimeric cells in blood of women with an autoimmune thyroid disease

Context

Hashimoto''s thyroiditis (HT) and Graves'' disease (GD), two autoimmune thyroid diseases (AITD), occur more frequently in women than in men and show an increased incidence in the years following parturition. Persisting fetal cells could play a role in the development of these diseases.

Objective

Aim of this study was to detect and characterize fetal cells in blood of postpartum women with and without an AITD.

Participants

Eleven patients with an AITD and ten healthy volunteers, all given birth to a son maximum 5 years before analysis, and three women who never had been pregnant, were included. None of them had any other disease of the thyroid which could interfere with the results obtained.

Methods

Fluorescence in situ hybridization (FISH) and repeated FISH were used to count the number of male fetal cells. Furthermore, the fetal cells were further characterized.

Results

In patients with HT, 7 to 11 fetal cells per 1.000.000 maternal cells were detected, compared to 14 to 29 fetal cells in patients with GD (p = 0,0061). In patients with HT, mainly fetal CD8⁺ T cells were found, while in patients with GD, fetal B and CD4⁺ T cells were detected. In healthy volunteers with son, 0 to 5 fetal cells were observed, which was significantly less than the number observed in patients (p<0,05). In women who never had been pregnant, no male cells were detected.

Conclusion

This study shows a clear association between fetal microchimeric cells and autoimmune thyroid diseases.