Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

Background

Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.

Methodology/Principal Findings

EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34⁺/CD45⁻ cells/1×10⁵ mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34⁺ cells/HPF, P<0.001) 3 days after the last injection.

Conclusions/Significance

Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.     

CD133+ Anaplastic Thyroid Cancer Cells Initiate Tumors in Immunodeficient Mice and Are Regulated by Thyrotropin

Background

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. Its rapid onset and resistance to conventional therapeutics contribute to a mean survival of six months after diagnosis and make the identification of thyroid-cancer-initiating cells increasingly important.

Methodology/Principal Findings

In prior studies of ATC cell lines, CD133⁺ cells exhibited stem-cell-like features such as high proliferation, self-renewal and colony-forming ability in vitro. Here we show that transplantation of CD133⁺ cells, but not CD133⁻ cells, into immunodeficient NOD/SCID mice is sufficient to induce growth of tumors in vivo. We also describe how the proportion of ATC cells that are CD133⁺ increases dramatically over three months of culture, from 7% to more than 80% of the total. This CD133⁺ cell pool can be further separated by flow cytometry into two distinct populations: CD133^+/high and CD133^+/low. Although both subsets are capable of long-term tumorigenesis, the rapidly proliferating CD133^+/high cells are by far the most efficient. They also express high levels of the stem cell antigen Oct4 and the receptor for thyroid stimulating hormone, TSHR. Treating ATC cells with TSH causes a three-fold increase in the numbers of CD133⁺ cells and elicits a dose-dependent up-regulation of the expression of TSHR and Oct4 in these cells. More importantly, immunohistochemical analysis of tissue specimens from ATC patients indicates that CD133 is highly expressed on tumor cells but not on neighboring normal thyroid cells.

Conclusions/Significance

To our knowledge, this is the first report indicating that CD133⁺ ATC cells are solely responsible for tumor growth in immunodeficient mice. Our data also give a unique insight into the regulation of CD133 by TSH. These highly tumorigenic CD133⁺ cells and the activated TSH signaling pathway may be useful targets for future ATC therapies.     

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.     

Prioritizing CD4 Count Monitoring in Response to ART in Resource-Constrained Settings: A Retrospective Application of Prediction-Based Classification

Background

Global programs of anti-HIV treatment depend on sustained laboratory capacity to assess treatment initiation thresholds and treatment response over time. Currently, there is no valid alternative to CD4 count testing for monitoring immunologic responses to treatment, but laboratory cost and capacity limit access to CD4 testing in resource-constrained settings. Thus, methods to prioritize patients for CD4 count testing could improve treatment monitoring by optimizing resource allocation.

Methods and Findings

Using a prospective cohort of HIV-infected patients (n = 1,956) monitored upon antiretroviral therapy initiation in seven clinical sites with distinct geographical and socio-economic settings, we retrospectively apply a novel prediction-based classification (PBC) modeling method. The model uses repeatedly measured biomarkers (white blood cell count and lymphocyte percent) to predict CD4⁺ T cell outcome through first-stage modeling and subsequent classification based on clinically relevant thresholds (CD4⁺ T cell count of 200 or 350 cells/µl). The algorithm correctly classified 90% (cross-validation estimate = 91.5%, standard deviation [SD] = 4.5%) of CD4 count measurements <200 cells/µl in the first year of follow-up; if laboratory testing is applied only to patients predicted to be below the 200-cells/µl threshold, we estimate a potential savings of 54.3% (SD = 4.2%) in CD4 testing capacity. A capacity savings of 34% (SD = 3.9%) is predicted using a CD4 threshold of 350 cells/µl. Similar results were obtained over the 3 y of follow-up available (n = 619). Limitations include a need for future economic healthcare outcome analysis, a need for assessment of extensibility beyond the 3-y observation time, and the need to assign a false positive threshold.

Conclusions

Our results support the use of PBC modeling as a triage point at the laboratory, lessening the need for laboratory-based CD4⁺ T cell count testing; implementation of this tool could help optimize the use of laboratory resources, directing CD4 testing towards higher-risk patients. However, further prospective studies and economic analyses are needed to demonstrate that the PBC model can be effectively applied in clinical settings. Please see later in the article for the Editors'' Summary     

APC Activation Restores Functional CD4+CD25+ Regulatory T Cells in NOD Mice that Can Prevent Diabetes Development

Background

Defects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4⁺CD25⁺ regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4⁺CD25⁺ regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease.

Methodology/Principal Findings

To test these hypotheses, we used the well-documented ability of complete Freund''s adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4⁺CD25⁺ regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4⁺CD25⁺ cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4⁺CD25⁺Foxp3⁺ cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4⁺CD25⁺ cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4⁺CD25⁺ cells in vitro compared to APC from untreated NOD mice.

Conclusions/Significance

These data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC.     

FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Background

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.

Methodology

FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.

Principal Findings

HIV infected individuals had significantly higher frequencies of CD4⁺FOXP3⁺ T cells (median of 8.11%; range 1.33%–26.27%) than healthy controls (median 3.72%; range 1.3–7.5%; P = 0.002), despite having lower absolute counts of CD4⁺FOXP3⁺ T cells. There was a significant positive correlation between the frequency of CD4⁺FOXP3⁺ T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = −0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/µl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%–26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4⁺ T cells following antigenic or other stimulation.

Conclusions/Significance

FOXP3 expression in the CD4⁺ T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression.     

Preserved MHC Class II Antigen Processing in Monocytes from HIV-Infected Individuals

Background

MHC-II restricted CD4+ T cells are dependent on antigen presenting cells (APC) for their activation. APC dysfunction in HIV-infected individuals could accelerate or exacerbate CD4+ T cell dysfunction and may contribute to increased levels of immunodeficiency seen in some patients regardless of their CD4+ T cell numbers. Here we test the hypothesis that APC from HIV-infected individuals have diminished antigen processing and presentation capacity.

Methodology/Principal Findings

Monocytes (MN) were purified by immuno-magnetic bead isolation techniques from HLA-DR1.01+ or DR15.01+ HIV-infected and uninfected individuals. MN were analyzed for surface MHC-II expression and for antigen processing and presentation capacity after overnight incubation with soluble antigen or peptide and HLA-DR matched T cell hybridomas. Surface expression of HLA-DR was 20% reduced (p<0.03) on MN from HIV-infected individuals. In spite of this, there was no significant difference in antigen processing and presentation by MN from 14 HIV-infected donors (8 HLA-DR1.01+ and 6 HLA-DR15.01+) compared to 24 HIV-uninfected HLA-matched subjects.

Conclusions/Significance

We demonstrated that MHC class II antigen processing and presentation is preserved in MN from HIV-infected individuals. This further supports the concept that this aspect of APC function does not further contribute to CD4+ T cell dysfunction in HIV disease.     

Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol

Aims

Circulating endothelial progenitor cells (EPC), involved in endothelial regeneration, neovascularisation, and determination of prognosis in cardiovascular disease can be characterised with functional assays or using immunofluorescence and flow cytometry. Combinations of markers, including CD34+KDR+ or CD133+KDR+, are used. This approach, however may not consider all characteristics of EPC. The lack of a standardised protocol with regards to reagents and gating strategies may account for the widespread inter-laboratory variations in quantification of EPC. We, therefore developed a novel protocol adapted from the standardised so-called ISHAGE protocol for enumeration of haematopoietic stem cells to enable comparison of clinical and laboratory data.

Methods and Results

In 25 control subjects, 65 patients with coronary artery disease (CAD; 40 stable CAD, 25 acute coronary syndrome/acute myocardial infarction (ACS)), EPC were quantified using the following approach: Whole blood was incubated with CD45, KDR, and CD34. The ISHAGE sequential strategy was used, and finally, CD45^dimCD34⁺ cells were quantified for KDR. A minimum of 100 CD34⁺ events were collected. For comparison, CD45⁺CD34⁺ and CD45⁻CD34⁺ were analysed simultaneously. The number of CD45^dimCD34⁺KDR⁺ cells only were significantly higher in healthy controls compared to patients with CAD or ACS (p = 0.005 each, p<0.001 for trend). An inverse correlation of CD45^dimCD34⁺KDR⁺ with disease activity (r = −0.475, p<0.001) was confirmed. Only CD45^dimCD34⁺KDR⁺ correlated inversely with the number of diseased coronaries (r = −0.344; p<0.005). In a second study, a 4-week de-novo treatment of atorvastatin in stable CAD evoked an increase only of CD45^dimCD34⁺KDR⁺ EPC (p<0.05). CD45⁺CD34⁺KDR⁺ and CD45⁻CD34⁺KDR⁺ were indifferent between the three groups.

Conclusion

Our newly established protocol adopted from the standardised ISHAGE protocol achieved higher accuracy in EPC enumeration confirming previous findings with respect to the correlation of EPC with disease activity and the increase of EPC during statin therapy. The data of this study show the CD45^dim fraction to harbour EPC.     

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.     

A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency

Introduction

Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4⁺CD25^high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.

Objective

In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.

Materials and Methods

Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).

Results

A significantly lower proportion of CD4⁺CD25^highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8⁺ T cells from CVID patients expressing the activation markers, CD38⁺ and HLA-DR⁺ (P<0.05), we observed no significant correlation between Tregs and immune activation.

Conclusion

Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.     

Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment

Background

Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4⁺, murine CD4^−/−, HLA-DR3⁺ mice.

Methodology/Principal Findings

Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45⁺ showed depletion of CD4⁺, CD8⁺, CD19⁺, and CD117⁺ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4⁺ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death.

Conclusions/Significance

NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.     

Evaluation of portable point-of-care CD4 counter with high sensitivity for detecting patients eligible for antiretroviral therapy

Background

Accurate, inexpensive point-of-care CD4+ T cell testing technologies are needed that can deliver CD4+ T cell results at lower level health centers or community outreach voluntary counseling and testing. We sought to evaluate a point-of-care CD4+ T cell counter, the Pima CD4 Test System, a portable, battery-operated bench-top instrument that is designed to use finger stick blood samples suitable for field use in conjunction with rapid HIV testing.

Methods

Duplicate measurements were performed on both capillary and venous samples using Pima CD4 analyzers, compared to the BD FACSCalibur (reference method). The mean bias was estimated by paired Student''s t-test. Bland Altman plots were used to assess agreement.

Results

206 participants were enrolled with a median CD4 count of 396 (range; 18–1500). The finger stick PIMA had a mean bias of −66.3 cells/µL (95%CI −83.4−49.2, P<0.001) compared to the FACSCalibur; the bias was smaller at lower CD4 counts (0–250 cells/µL) with a mean bias of −10.8 (95%CI −27.3−+5.6, P = 0.198), and much greater at higher CD4 cell counts (>500 cells/µL) with a mean bias of −120.6 (95%CI −162.8, −78.4, P<0.001). The sensitivity (95%CI) of the Pima CD4 analyzer was 96.3% (79.1–99.8%) for a <250 cells/ul cut-off with a negative predictive value of 99.2% (95.1–99.9%).

Conclusions

The Pima CD4 finger stick test is an easy-to-use, portable, relatively fast device to test CD4+ T cell counts in the field. Issues of negatively-biased CD4 cell counts especially at higher absolute numbers will limit its utility for longitudinal immunologic response to ART. The high sensitivity and negative predictive value of the test makes it an attractive option for field use to identify patients eligible for ART, thus potentially reducing delays in linkage to care and ART initiation.     

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein

Background

To initiate infection, Bacillus anthracis needs to overcome the host innate immune system. Anthrax toxin, a major virulence factor of B. anthracis, impairs both the innate and adaptive immune systems and is important in the establishment of anthrax infections.

Methodology/Principal Findings

To measure the ability of anthrax toxin to target immune cells, studies were performed using a fusion of the anthrax toxin lethal factor (LF) N-terminal domain (LFn, aa 1–254) with β-lactamase (LFnBLA). This protein reports on the ability of the anthrax toxin protective antigen (PA) to mediate LF delivery into cells. Primary immune cells prepared from mouse spleens were used in conjunction with flow cytometry to assess cleavage and resulting FRET disruption of a fluorescent β-lactamase substrate, CCF2/AM. In spleen cell suspensions, the macrophages, dendritic cells, and B cells showed about 75% FRET disruption of CCF2/AM due to cleavage by the PA–delivered LFnBLA. LFnBLA delivery into CD4+ and CD8+ T cells was lower, with 40% FRET disruption. When the analyses were done on purified samples of individual cell types, similar results were obtained, with T cells again having lower LFnBLA delivery than macrophages, dendritic cells, and B cells. Relative expression levels of the toxin receptors CMG2 and TEM8 on these cells were determined by real-time PCR. Expression of CMG2 was about 1.5-fold higher in CD8+ cells than in CD4+ and B cells, and 2.5-fold higher than in macrophages.

Conclusions/Significance

Anthrax toxin entry and activity differs among immune cells. Macrophages, dendritic cells, and B cells displayed higher LFnBLA activity than CD4+ and CD8+ T cells in both spleen cell suspension and the purified samples of individual cell types. Expression of anthrax toxin receptor CMG2 is higher in CD4+ and CD8+ T cells, which is not correlated to the intracellular LFnBLA activity.     

CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

Background

Although the etiology of idiopathic pulmonary fibrosis (IPF) remains perplexing, adaptive immune activation is evident among many afflicted patients. Repeated cycles of antigen-induced proliferation cause T-cells to lose surface expression of CD28, and we hypothesized this process might also occur in IPF.

Methodology/Principal Findings

Peripheral blood CD4 T-cells from 89 IPF patients were analyzed by flow cytometry and cytokine multiplex assays, and correlated with clinical events. In comparison to autologous CD4⁺CD28⁺cells, the unusual CD4⁺CD28^null lymphocytes seen in many IPF patients had discordant expressions of activation markers, more frequently produced cytotoxic mediators perforin (2.4±0.8% vs. 60.0±7.4%, p<0.0001) and granzyme B (4.5±2.8% vs.74.9±6.5%, p<0.0001), produced greater amounts of many pro-inflammatory cytokines, and less frequently expressed the regulatory T-cell marker FoxP3 (12.9±1.1% vs. 3.3±0.6% p<0.0001). Infiltration of CD4⁺CD28^null T-cells in IPF lungs was confirmed by confocal microscopy. Interval changes of CD28 expression among subjects who had replicate studies were correlated with conterminous changes of their forced vital capacities (r_s = 0.49, p = 0.012). Most importantly, one-year freedom from major adverse clinical events (either death or lung transplantation) was 56±6% among 78 IPF patients with CD4⁺CD28⁺/CD4_total≥82%, compared to 9±9% among those with more extensive CD28 down-regulation (CD4⁺CD28⁺/CD4_total<82%) (p = 0.0004). The odds ratio for major adverse events among those with the most extensive CD28 down-regulation was 13.0, with 95% confidence intervals 1.6-111.1.

Conclusions/Significance

Marked down-regulation of CD28 on circulating CD4 T-cells, a result of repeated antigen-driven proliferations, is associated with poor outcomes in IPF patients. The CD4⁺CD28^null cells of these patients have potentially enhanced pathogenic characteristics, including increased productions of cytotoxic mediators and pro-inflammatory cytokines. These findings show proliferative T-cell responses to antigen(s) resulting in CD28 down-regulation are associated with progression and manifestations of IPF, and suggest assays of circulating CD4 T-cells may identify patients at greatest risk for clinical deterioration.     

Clinical Reactivations of Herpes Simplex Virus Type 2 Infection and Human Immunodeficiency Virus Disease Progression Markers

Background

The natural history of HSV-2 infection and role of HSV-2 reactivations in HIV disease progression are unclear.

Methods

Clinical symptoms of active HSV-2 infection were used to classify 1,938 HIV/HSV-2 co-infected participants of the Women''s Interagency HIV Study (WIHS) into groups of varying degree of HSV-2 clinical activity. Differences in plasma HIV RNA and CD4+ T cell counts between groups were explored longitudinally across three study visits and cross-sectionally at the last study visit.

Results

A dose dependent association between markers of HIV disease progression and degree of HSV-2 clinical activity was observed. In multivariate analyses after adjusting for baseline CD4+ T cell levels, active HSV-2 infection with frequent symptomatic reactivations was associated with 21% to 32% increase in the probability of detectable plasma HIV RNA (trend p = 0.004), an average of 0.27 to 0.29 log10 copies/ml higher plasma HIV RNA on a continuous scale (trend p<0.001) and 51 to 101 reduced CD4+ T cells/mm³ over time compared to asymptomatic HSV-2 infection (trend p<0.001).

Conclusions

HIV induced CD4+ T cell loss was associated with frequent symptomatic HSV-2 reactivations. However, effect of HSV-2 reactivations on HIV disease progression markers in this population was modest and appears to be dependent on the frequency and severity of reactivations. Further studies will be necessary to determine whether HSV-2 reactivations contribute to acceleration of HIV disease progression.     

Regulatory T Cell Expansion in HTLV-1 and Strongyloidiasis Co-infection Is Associated with Reduced IL-5 Responses to Strongyloides stercoralis Antigen

Background

Human strongyloidiasis varies from a chronic but limited infection in normal hosts to hyperinfection in patients treated with corticosteroids or with HTLV-1 co-infection. Regulatory T cells dampen immune responses to infections. How human strongyloidiasis is controlled and how HTLV-1 infection affects this control are not clear. We hypothesize that HTLV-1 leads to dissemination of Strongyloides stercoralis infection by augmenting regulatory T cell numbers, which in turn down regulate the immune response to the parasite.

Objective

To measure peripheral blood T regulatory cells and Strongyloides stercoralis larval antigen-specific cytokine responses in strongyloidiasis patients with or without HTLV-1 co-infection.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated from newly diagnosed strongyloidiasis patients with or without HTLV-1 co-infection. Regulatory T cells were characterized by flow cytometry using intracellular staining for CD4, CD25 and FoxP3. PBMCs were also cultured with and without Strongyloides larval antigens. Supernatants were analyzed for IL-5 production.

Results

Patients with HTLV-1 and Strongyloides co-infection had higher parasite burdens. Eosinophil counts were decreased in the HTLV-1 and Strongyloides co-infected subjects compared to strongyloidiasis-only patients (70.0 vs. 502.5 cells/mm³, p = 0.09, Mann-Whitney test). The proportion of regulatory T cells was increased in HTLV-1 positive subjects co-infected with strongyloidiasis compared to patients with only strongyloidiasis or asymptomatic HTLV-1 carriers (median = 17.9% vs. 4.3% vs. 5.9 p<0.05, One-way ANOVA). Strongyloides antigen-specific IL-5 responses were reduced in strongyloidiasis/HTLV-1 co-infected patients (5.0 vs. 187.5 pg/ml, p = 0.03, Mann-Whitney test). Reduced IL-5 responses and eosinophil counts were inversely correlated to the number of CD4+CD25+FoxP3+ cells.

Conclusions

Regulatory T cell counts are increased in patients with HTLV-1 and Strongyloides stercoralis co-infection and correlate with both low circulating eosinophil counts and reduced antigen-driven IL-5 production. These findings suggest a role for regulatory T cells in susceptibility to Strongyloides hyperinfection.     

Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

Background

Diabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR α and β/δ) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.

Methodology/Principal Findings

Streptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR α and β/δ gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (−14%, P<0.05) associated with a decrease in myosin ATPase activity (−49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARβ/δ (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.

Conclusions/Significance

Type I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARβ/δ that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms.     

rBCG Induces Strong Antigen-Specific T Cell Responses in Rhesus Macaques in a Prime-Boost Setting with an Adenovirus 35 Tuberculosis Vaccine Vector

Background

BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals).

Methods and Findings

Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4⁺, CD8alpha/beta⁺, and CD8alpha/alpha⁺ T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha⁺ T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha⁺ T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity.

Conclusion

AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials     

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.