Direct Contact with Endoderm-Like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells

Rationale

Pluripotent stem cell–derived cardiac progenitor cells (CPCs) have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations.

Objective

Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells.

Method and Result

To test the hypothesis, we cocultured mouse embryonic stem (ES) cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1⁺ PDGFRa⁺ CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS) cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5⁺ and Isl1⁺ CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR⁺ PDGFRa⁺ CPCs from human ES cells.

Conclusions

Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.     

Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

Background

Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.

Objective

To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.

Methods

Human bronchial fibroblasts and CD4⁺T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4⁺T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.

Results

Co-culture of CD4⁺T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17⁺/CCR6⁺ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4⁺T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.

Conclusion

Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.     

Bone Mesenchymal Stem Cells Contributed to the Neointimal Formation after Arterial Injury

Objectives

Recent findings suggest that in response to repair-to-injury bone marrow mesenchymal stem cells (BMSCs) participate in the process of angiogenesis. It is unclear what role BMSCs play in the structure of the vessel wall. In present study, we aimed to determine whether BMSCs had the capacity of endothelial cells (ECs).

Methods

BMSCs were separated and cultured. FACS and RT-PCR analysis confirmed the gene expression phenotype. The capacity of migration and adhesion and the ultrastructure of BMSCs were examined. The effect of BMSCs transplantation on the vascular repair was investigated in a murine carotid artery-injured model.

Results

BMSCs could express some markers and form the tube-like structure. The migration and adhesion capacity of BMSCs increased significantly after stimulated. In addition, BMSCs had the intact cell junction. In vivo the local transfer of BMSCs differentiated into neo-endothelial cells in the injury model for carotid artery and contributed to the vascular remodeling.

Conclusion

These results showed that BMSCs could contribute to neointimal formation for vascular lesion and might be associated with the differentiation into ECs, which indicated the important therapeutic implications for vascular diseases.     

Gr1intCD11b+ Myeloid-Derived Suppressor Cells in Mycobacterium tuberculosis Infection

Background

Tuberculosis is one of the world’s leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1^intCD11b⁺ cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design.

Methodology/Principal Findings

We compared the bacterial burden, lung pathology and Gr1^intCD11b⁺ myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1⁺ cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1⁺CD11b⁺ cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1^hi and Gr1^int populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1^int populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1^hi and Gr1^int cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4⁺ T to Gr1⁺ cells increased. Our results illustrate a yet unrecognized interplay between Gr1⁺ cells and CD4⁺ T cells in tuberculosis.     

Role of Endothelial Progenitor Cells and Inflammatory Cytokines in Healing of Diabetic Foot Ulcers

Background

To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing.

Methods

We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.

Results

All EPC phenotypes except the kinase insert domain receptor (KDR)⁺CD133⁺ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34⁺KDR⁺ count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.

Conclusions

Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34⁺KDR⁺ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.     

Prognostic Value of Tumor-Infiltrating FoxP3+ T Cells in Gastrointestinal Cancers: A Meta Analysis

Purpose

Tumor-infiltrating FoxP3⁺ T cells have been reported in various human tumors, which impaired cell-mediated immunity and promoted disease progression. However, its prognostic value for survival in patients with different gastrointestinal cancers [hepatocellular carcinoma (HCC), colorectal cancer (CRC), gastric cancer (GC)] remains controversial.

Methods

Relevant literature was searched using PubMed, Embase, Cochrane, Ovid Medline and Chinese wanfang databases. A meta-analysis was conducted to estimate pooled survival and recurrence ratios. The odds ratio (OR) and 95% confidence intervals (CI) were calculated employing fixed- or random-effects models depending on the heterogeneity of the included trials.

Results

For HCC and GC, the overall survival at 1, 3 and 5-year of high FoxP3⁺ T cells infiltration patients were lower than low FoxP3⁺ T cells infiltration patients (P<0.05). The recurrences at 1, 3 and 5-year of high FoxP3⁺ T cells infiltration patients were higher than low FoxP3⁺ T cells infiltration patients (P<0.001). But for CRC, the overall survival at 1, 3 and 5-year of high FoxP3⁺ T cells infiltration patients were higher than low FoxP3⁺ T cells infiltration patients (P<0.001). There were no differences in 1, 3 and 5-year recurrences between high and low FoxP3⁺ T cells infiltration patients (P>0.05).

Conclusions

Our findings suggested that tumor-infiltrating FoxP3⁺ T cells were a factor for a poor prognosis for HCC and GC, but a good prognosis for CRC.     

A Mouse Model of Adoptive Immunotherapeutic Targeting of Autoimmune Arthritis Using Allo-Tolerogenic Dendritic Cells

Objective

Tolerogenic dendritic cells (tDCs) are immunosuppressive cells with potent tolerogenic ability and are promising immunotherapeutic tools for treating rheumatoid arthritis (RA). However, it is currently unknown whether allogeneic tDCs (allo-tDCs) induce tolerance in RA, and whether the numbers of adoptively transferred allo-tDCs, or the requirement for pulsing with relevant auto-antigens are important.

Methods

tDCs were derived from bone marrow precursors of C57BL/B6 mice, which were induced in vitro by GM-CSF, IL-10 and TGF-β1. Collagen-induced arthritis (CIA) was modeled in D1 mice by immunization with type II collagen (CII) to test the therapeutic ability of allo-tDCs against CIA. Clinical and histopathologic scores, arthritic incidence, cytokine and anti-CII antibody secretion, and CD4⁺Th subsets were analyzed.

Results

tDCs were characterized in vitro by a stable immature phonotype and a potent immunosuppressive ability. Following adoptive transfer of low doses (5×10⁵) of CII-loaded allo-tDCs, a remarkable anti-arthritic activity, improved clinical scores and histological end-points were found. Serological levels of inflammatory cytokines and anti-CII antibodies were also significantly lower in CIA mice treated with CII-pulsed allo-tDCs as compared with allo-tDCs. Moreover, treatment with allo-tDCs altered the proportion of Treg/Th17 cells.

Conclusion

These findings suggested that allo-tDCs, especially following antigen loading, reduced the severity of CIA in a dose-dependent manner. The dampening of CIA was associated with modulated cytokine secretion, Treg/Th17 polarization and inhibition of anti-CII secretion. This study highlights the potential therapeutic utility of allo-tDCs in autoimmune arthritis and should facilitate the future design of allo-tDC immunotherapeutic strategies against RA.     

Parasitic Nematode-Induced CD4+Foxp3+T Cells Can Ameliorate Allergic Airway Inflammation

Background

The recruitment of CD4⁺CD25⁺Foxp3⁺T (T_reg) cells is one of the most important mechanisms by which parasites down-regulate the immune system.

Methodology/Principal Findings

We compared the effects of T_reg cells from Trichinella spiralis-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced T_reg cells. After four weeks of T. spiralis infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4⁺Foxp3⁺ cells from T. spiralis-infected [Inf(+)Foxp3⁺] or uninfected [Inf(-)Foxp3⁺] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3⁺ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3⁺ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3⁺ cells migrated to inflammation sites in the lung and expressed higher levels of T_reg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3⁺ cells.

Conclusion/Significance

T. spiralis infection promotes the proliferation and functional activation of T_reg cells. Parasite-induced T_reg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced T_reg cells. The adoptive transfer of Inf(+)Foxp3⁺ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment of allergic airway diseases.     

Decreased Frequency of Intestinal Regulatory CD5+ B Cells in Colonic Inflammation

Background

CD5⁺ B cells are a type of regulatory immune cells, though the involvement of this B cell subset in intestinal inflammation and immune regulation is not fully understood.

Methods

We examined the distribution of CD5⁺ B cells in various mouse organs. Expression levels of CD11b, IgM, and toll-like receptor (TLR)-4 and -9 in B cells were evaluated. In vitro, TLR-stimulated IL-10 production by colonic lamina propria (LP) CD5⁺ and CD5^- B cells was measured. In vivo, mice with acute or chronic dextran sulfate sodium (DSS)-induced colonic injury were examined, and the frequency of colonic LP CD5⁺ B cells in those was assessed by flow cytometry.

Results

The expression level of TLR9 was higher in colonic LP CD5⁺ B cells as compared to CD5^- B cells. Colonic LP CD5⁺ B cells produced greater amounts of IL-10 following stimulation with TLR ligands, especially TLR9, as compared with the LP CD5^- B cells. Acute intestinal inflammation transiently decreased the frequency of colonic LP CD5⁺ B cells, while chronic inflammation induced a persistent decrease in colonic LP CD5⁺ B cells and led to a CD5^- B cell-dominant condition.

Conclusion

A persistent altered mucosal B cell population caused by chronic gut inflammation may be involved in the pathogenesis of inflammatory bowel diseases.     

CXCL17 Expression by Tumor Cells Recruits CD11b(+)Gr1(high)F4/80(-) Cells and Promotes Tumor Progression

Background

Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression.

Methodology/Principal Findings

CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b⁺Gr1⁺ myeloid-derived cells at tumor sites in mice and promoted CD31⁺ tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b⁺Gr1^highF4/80⁻ cells (∼90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b⁺Gr1⁺ cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation.

Conclusions/Significance

These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.     

Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

Background

T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.

Methods/ Findings

Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4⁺ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice.

Conclusions

The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.     

CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel

Background

Chronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection.

Methodology

CD4⁺CD25^hiFOXP3⁺ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE.

Principal Findings

S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only.

Conclusions

Schistosoma-associated CD4⁺CD25^hiFOXP3⁺Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.     

Impaired immunomodulatory function of chronic myeloid leukemia cancer stem cells and the possible mechanism involved in it

Backgroud

Cancer stem cells (CSCs) are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at CSCs holds hope for the improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. This is particularly true in chronic myeloid leukemia (CML).

Methods

In this study, we isolated fetal liver kinase-1-positive (Flk1⁺) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph⁺) patients with stem cells property. We examined their biological characteristics as well as immunological function and further detected the possible molecular mechanism involved in the leukemia genesis.

Results

We showed that CML patient-derived Flk1⁺CD31^?CD34^? MSCs had normal morphology, phenotype and karyotype but appeared impaired immunomodulatory function. The capacity of Flk1⁺CD31^?CD34^? MSCs from CML patients to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have dampening immunomodulatory functions, suggesting that the dysregulation of hematopoiesis and immune response might originate from MSCs rather than HSCs. These Ph⁺ putative CML hemangioblast upregulated TGF-β1 and resultantly activated matrix metalloproteinase-9 (MMP-9) to enhance s-KitL and s-ICAM-1 secretion, which activated c-kit⁺ HSCs from the quiescent state to proliferative state. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was involved in CML pathogenesis.

Conclusions

Flk1⁺CD31^?CD34^? MSCs that express BCR/ABL leukemia oncogene are CSCs of CML and they play a critical role in the progression of CML through PI3K/Akt/NF-κB/MMP-9/s-ICAM-1/s-KitL signaling pathway beyond HSCs.     

Adoptive Immunotherapy of Cytokine-Induced Killer Cell Therapy in the Treatment of Non-Small Cell Lung Cancer

Aim

The aim of this study was to systemically evaluate the therapeutic efficacy of cytokine-induced killer (CIK) cells for the treatment of non-small cell lung cancer.

Materials and Methods

A computerized search of randomized controlled trials for CIK cell-based therapy was performed. The overall survival, clinical response rate, immunological assessment and side effects were evaluated.

Results

Overall, 17 randomized controlled trials of non-small cell lung cancer (NSCLC) with a total of 1172 patients were included in the present analysis. Our study showed that the CIK cell therapy significantly improved the objective response rate and overall survival compared to the non-CIK cell-treated group. After CIK combined therapy, we observed substantially increased percentages of CD3⁺, CD4⁺, CD4⁺CD8⁺, CD3⁺CD56⁺ and NK cells, whereas significant decreases were noted in the percentage of CD8⁺ and regulatory T cell (Treg) subgroups. A significant increase in Ag-NORs was observed in the CIK-treated patient group (p = 0.00001), whereas carcinoembryonic antigen (CEA) was more likely to be reduced to a normal level after CIK treatment (p = 0.0008). Of the possible major side effects, only the incidence of fever in the CIK group was significantly higher compared to the group that received chemotherapy alone.

Conclusion

The CIK cell combined therapy demonstrated significant superiority in the overall survival, clinical response rate, and T lymphocytes responses and did not present any evidence of major adverse events in patients with NSCLC.     

Immune Surveillance by Rhinovirus-Specific Circulating CD4+ and CD8+ T Lymphocytes

Background

It is difficult to experimentally infect volunteers with RV strains to which the subject demonstrates serological immunity. However, in RV challenges, viral clearance begins before de novo adaptive immune responses would develop. We speculated that adaptive immunity to RV reflects heterologous immunity by effector memory cells.

Methods

DCs were generated from monocytes using GM-CSF and IL-4 and RV39 loading accomplished with a dose of ∼350 TCID₅₀/10⁵ cells. RV-induced maturation was established as modulation of MHC class II, CD80, CD83, and CD86. Circulating RV targeting CD4 and CD8 T cells were investigated as induction of RV-specific proliferation (CFSE-dilution).

Results

Maturation of DC by RV was confirmed as upregulation of MHC Class II (83.3±5.0% to 87.8±4.1%), CD80 (39.4±7.2% to 47.6±7.7%) and CD86 (78.4±4.7% to 84.1±3.4%). Both CD4 and CD8 memory T cells were recognized in the circulation of healthy subjects.

Conclusions

RV drives DC maturation and results in their ability to present RV antigens to both T helper and cytotoxic lymphocytes. Both CD4 and CD8 cells capable of recognizing RV-associated antigens are present in the circulation of healthy subjects where they are presumably involved in immune surveillance and explain the rapid recruitment of an adaptive immune response during RV infection.     

Reversal of Ischemic Cardiomyopathy with Sca-1+ Stem Cells Modified with Multiple Growth Factors

Background

We hypothesized that bone marrow derived Sca-1⁺ stem cells (BM Sca-1⁺) transduced with multiple therapeutic cytokines with diverse effects will induce faster angiomyogenic differentiation in the infarcted myocardium.

Methods and Results

BM Sca-1⁺ were purified from transgenic male mice expressing GFP. Plasmids encoding for select quartet of growth factors, i.e., human IGF-1, VEGF, SDF-1α and HGF were prepared and used for genetic modification of Sca-1⁺ cells (^GFSca-1⁺). Scramble transfected cells (^ScSca-1⁺) were used as a control. RT-PCR and western blotting showed significantly higher expression of the growth factors in ^GFSca-1⁺. Besides the quartet of the therapeutic growth factors, PCR based growth factor array showed upregulation of multiple angiogenic and prosurvival factors such as Ang-1, Ang-2, MMP9, Cx43, BMP2, BMP5, FGF2, and NGF in ^GFSca-1⁺ (p<0.01 vs ^ScSca-1⁺). LDH and TUNEL assays showed enhanced survival of ^GFSca-1⁺ under lethal anoxia (p<0.01 vs ^ScSca-1⁺). MTS assay showed significant increased cell proliferation in ^GFSca-1⁺ (p<0.05 vs ^ScSca-1⁺). For in vivo study, female mice were grouped to receive the intramyocardial injection of 15 μl DMEM without cells (group-1) or containing 2.5×10^{5 Sc}Sca-1⁺ (group-2) or ^GFSca-1⁺ (group-3) immediately after coronary artery ligation. As indicated by Sry gene, a higher survival of ^GFSca-1⁺ in group-3 on day4 (2.3 fold higher vs group-2) was observed with massive mobilization of stem and progenitor cells (cKit⁺, Mdr1⁺, Cxcr4⁺ cells). Heart tissue sections immunostained for actinin and Cx43 at 4 weeks post engraftment showed extensive myofiber formation and expression of gap junctions. Immunostaining for vWF showed increased blood vessel density in both peri-infarct and infarct regions in group-3. Infarct size was attenuated and the global heart function was improved in group-3 as compared to group-2.

Conclusions

Administration of BM Sca-1⁺ transduced with multiple genes is a novel approach to treat infarcted heart for its regeneration.     

Potential Role for HIV-Specific CD38?/HLA-DR+ CD8+ T Cells in Viral Suppression and Cytotoxicity in HIV Controllers

Background

HIV controllers (HIC) are rare HIV-1-infected patients who exhibit spontaneous viral control. HIC have high frequency of CD38⁻/HLA-DR⁺ HIV-specific CD8⁺ T cells. Here we examined the role of this subset in HIC status.

Materials and Methods

We compared CD38⁻/HLA-DR⁺ CD8⁺ T cells with the classical CD38⁺/HLA-DR⁺ activated phenotype in terms of 1) their activation status, reflected by CD69, CD25, CD71, CD40 and Ki67 expression, 2) functional parameters: Bcl-2 expression, proliferative capacity, and IFN-γ and IL-2 production, and 3) cytotoxic activity. We also investigated how this particular profile is generated.

Results

Compared to CD38⁺/HLA-DR⁺ cells, CD38⁻/HLA-DR⁺ cells exhibited lower expression of several activation markers, better survival capacity (Bcl-2 MFI, 367 [134–462] vs 638 [307–747], P = 0.001), higher frequency of polyfunctional cells (15% [7%–33%] vs 21% [16%–43%], P = 0.0003), greater proliferative capacity (0-fold [0–2] vs 3-fold [2]–[11], P = 0.007), and higher cytotoxicity in vitro (7% [3%–11%] vs 13% [6%–22%], P = 0.02). The CD38⁻/HLA-DR⁺ profile was preferentially generated in response to low viral antigen concentrations.

Conclusions

These data highlight the role of CD38⁻/HLA-DR⁺ HIV-specific CD8⁺ T cell cytotoxicity in HIC status and provide insights into the mechanism by which they are generated. Induction of this protective CD8⁺ subset may be important for vaccine strategies.     

Squamous Tissue Lymphocytes in the Esophagus of Controls and Patients with Reflux Esophagitis and Barrett’s Esophagus Are Characterized by a Non-Inflammatory Phenotype

Background and Objective

Reflux esophagitis (RE) is characterized by inflammation of the squamous epithelium (SQ) of the esophagus and may progress to Barrett’s esophagus (BE) characterized by intestinal metaplasia. The role of inflammation in this transition has been postulated but lacks experimental evidence. Here, the inflammatory responses in the esophagus of these patients were investigated.

Patients and Methods

Fifty-one esophageal biopsies from with patients BE (n = 19), RE (n = 8) and controls (n = 23) were analyzed. T-cells were analyzed before and after ex vivo expansion (14 days) by multicolor flow cytometric analysis. The following markers were studied: CD3, CD4, CD8 (T-cell markers), Granzyme B (marker of cytotoxicity), CD103 (αE/epithelial integrin) and NKg2a (inhibitory receptor on T-cells and NK-cells).

Results

Analysis of ex vivo cultures from normal looking SQ from controls, RE patients, and BE patients revealed no significant differences in the number and phenotypes of T-cells. In contrast, tissue from RE was different to normal SQ in four aspects: 1) higher percentages of CD3⁺CD4⁺-cells (72±7% vs 48±6%, p = 0.01) and 2) CD8⁺GranzymeB⁺ -cells (53±11% vs 26±4%, p<0.05), while 3) lower percentages of CD4⁺CD103⁺-cells (45±19% vs 80±3%, p = 0.02) and 4) CD8⁺NKg2a⁺- cells (31±12% vs 44±5%).

Conclusion

Despite the fact that both tissues are exposed to the same reflux associated inflammatory triggers, the immune response observed in RE is clearly distinct from that in SQ of BE. The differences in immune responses in BE tissue might contribute to its susceptibility for transformation into intestinal metaplasia.     

Pro-Inflammatory Activated Kupffer Cells by Lipids Induce Hepatic NKT Cells Deficiency through Activation-Induced Cell Death

Background

Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response.

Aims

The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells.

Methods

Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells.

Results

High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis.

Conclusion

High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.