Multielemental Analysis of Low-Volume Samples Reveals Cancer-Specific Profile in Serum and Sorted Immune Cells.

We developed and validated a reliable, robust, and easy-to-implement quantitative method for multielemental analysis of low-volume samples. Our ICP-MS-based method comprises the analysis of 20 elements (Mg, P, S, K, Ca, V, Cr, Mn, Fe, Co, Cu, Zn, Se, Br, Rb, Sr, Mo, I, Cs, and Ba) in 10 µL of serum and 12 elements (Mg, S, Mn, Fe, Co, Cu, Zn Se, Br, Rb, Mo, and Cs) in less than 250 000 cells. As a proof-of-concept, we analyzed the elemental profiles of serum and sorted immune T cells derived from naïve and tumor-bearing mice. The results indicate a tumor systemic effect on the elemental profiles of both serum and T cells. Our approach highlights promising applications of multielemental analysis in precious samples such as rare cell populations or limited volumes of biofluids that could provide a deeper understanding of the essential role of elements as cofactors in biological and pathological processes.

Progression of brain white matter hyperintensities in asymptomatic patients with carotid atherosclerotic plaques and no indication for revascularization.

BACKGROUND AND AIMS: Brain white matter hyperintensities (WMHs) have been associated with an increased risk of ischemic stroke and considered as markers of brain ischemia. Progression of WMHs in asymptomatic patients with non-hemodynamically significant carotid plaque could represent a putative marker of plaque vulnerability. We prospectively evaluate progression and determinants of WMHs in this population. METHODS: This prospective study included 51 asymptomatic patients with carotid stenosis <70% that underwent brain magnetic resonance imaging scans at baseline and after a median follow up of 595 days (interquartile range 553-641 days). Patients (mean age of 69 years and 45% females) underwent baseline carotid computed tomography angiography, contrast-enhanced ultrasound for carotid plaque characterization and analysis of subsets of circulating lymphocytes and monocytes by flow cytometry. RESULTS: Seventeen subjects (33.3%) had carotid stenoses of 50-70% (Doppler flow velocity) while the rest had stenoses of <50%. In 25 (49.0%) patients, new WMHs, with 5 new lesions on average and a median volume of 134 mm3, were detected at follow-up. None of the plaque characteristics or of the circulating cellular biomarkers investigated were associated with the global and ipsilateral occurrence of new WMHs whereas, at multivariate analysis, female sex, hypercholesterolemia, and lower glomerular filtration rate (GFR) emerged as independent variables associated with new WMHs. CONCLUSIONS: Half of the patients with carotid plaques of intermediate severity had evidence of WMH progression at follow up. Female gender and systemic factors such as hypercholesterolemia, and lower GFR, but not plaque characteristics or circulating cellular biomarkers, are associated with WMH progression.

Macroencapsulated Human iPSC-Derived Pancreatic Progenitors Protect against STZ-Induced Hyperglycemia in Mice.

In type 1 diabetes, a renewable source of human pancreatic ß cells, in particular from human induced pluripotent stem cell (hiPSC) origin, would greatly benefit cell therapy. Earlier work showed that pancreatic progenitors differentiated from human embryonic stem cells in vitro can further mature to become glucose responsive following macroencapsulation and transplantation in mice. Here we took a similar approach optimizing the generation of pancreatic progenitors from hiPSCs. This work demonstrates that hiPSCs differentiated to pancreatic endoderm in vitro can be efficiently and robustly generated under large-scale conditions. The hiPSC-derived pancreatic endoderm cells (HiPECs) can further differentiate into glucose-responsive islet-like cells following macroencapsulation and in vivo implantation. The HiPECs can protect mice from streptozotocin-induced hyperglycemia and maintain normal glucose homeostasis and equilibrated plasma glucose concentrations at levels similar to the human set point. These results further validate the potential use of hiPSC-derived islet cells for application in clinical settings.

Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors.

Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro-adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis.

Carotid artery plaque uptake of 11C-PK11195 inversely correlates with circulating monocytes and classical CD14++CD16- monocytes expressing HLA-DR.

BACKGROUND: We explored the relation between blood concentrations of monocyte/lymphocyte subsets and carotid artery plaque macrophage content, measured by positron emission tomography (PET) with 11C-PK11195. METHODS AND RESULTS: In 9 patients with carotid plaques we performed 11C-PK11195-PET/computed tomography angiography imaging and measurement of absolute concentrations and frequencies of circulating monocytes and T-cell subsets. Plaque standardized uptake value (SUV) for 11C-PK11195 was negatively correlated with concentrations of total monocytes (r = -0.58, p = 0.05) and CD14++CD16-HLA-DR+ classical subset (r = -0.82, p = 0.005). These correlations hold true also in relation to plaque target to background ratio. No correlation was observed between plaque SUV and CD3+T lymphocytes, CD4+T lymphocytes nor with activated CD3+CD4+T cells expressing HLA-DR. CONCLUSIONS: We first demonstrated a reduction in the absolute concentration of monocytes and particularly in classical monocytes expressing HLA-DR in the presence of an increased uptake of 11C-PK11195 in carotid plaques. The present work, despite being a pilot study comprising only a small number of subjects provides new insights in the search for specific cellular biomarkers with potential diagnostic and prognostic value in patients with a known carotid plaque.

PPARγ Controls Ectopic Adipogenesis and Cross-Talks with Myogenesis During Skeletal Muscle Regeneration.

Skeletal muscle is a regenerative tissue which can repair damaged myofibers through the activation of tissue-resident muscle stem cells (MuSCs). Many muscle diseases with impaired regeneration cause excessive adipose tissue accumulation in muscle, alter the myogenic fate of MuSCs, and deregulate the cross-talk between MuSCs and fibro/adipogenic progenitors (FAPs), a bi-potent cell population which supports myogenesis and controls intra-muscular fibrosis and adipocyte formation. In order to better characterize the interaction between adipogenesis and myogenesis, we studied muscle regeneration and MuSC function in whole body Pparg null mice generated by epiblast-specific Cre/lox deletion (PpargΔ/Δ). We demonstrate that deletion of PPARγ completely abolishes ectopic muscle adipogenesis during regeneration and impairs MuSC expansion and myogenesis after injury. Ex vivo assays revealed that perturbed myogenesis in PpargΔ/Δ mice does not primarily result from intrinsic defects of MuSCs or from perturbed myogenic support from FAPs. The immune transition from a pro- to anti-inflammatory MuSC niche during regeneration is perturbed in PpargΔ/Δ mice and suggests that PPARγ signaling in macrophages can interact with ectopic adipogenesis and influence muscle regeneration. Altogether, our study demonstrates that a PPARγ-dependent adipogenic response regulates muscle fat infiltration during regeneration and that PPARγ is required for MuSC function and efficient muscle repair.

Evaluation of Lymphocyte Response to the Induced Oxidative Stress in a Cohort of Ageing Subjects, including Semisupercentenarians and Their Offspring.

The production of reactive oxygen species (ROS) may promote immunosenescence if not counterbalanced by the antioxidant systems. Cell membranes, proteins, and nucleic acids become the target of ROS and progressively lose their structure and functions. This process could lead to an impairment of the immune response. However, little is known about the capability of the immune cells of elderly individuals to dynamically counteract the oxidative stress. Here, the response of the main lymphocyte subsets to the induced oxidative stress in semisupercentenarians (CENT), their offspring (OFF), elderly controls (CTRL), and young individuals (YO) was analyzed using flow cytometry. The results showed that the ratio of the ROS levels between the induced and noninduced (I/NI) oxidative stress conditions was higher in CTRL and OFF than in CENT and YO, in almost all T, B, and NK subsets. Moreover, the ratio of reduced glutathione levels between I/NI conditions was higher in OFF and CENT compared to the other groups in almost all the subsets. Finally, we observed significant correlations between the response to the induced oxidative stress and the degree of methylation in specific genes on the oxidative stress pathway. Globally, these data suggest that the capability to buffer dynamic changes in the oxidative environment could be a hallmark of longevity in humans.

Encapsulation of Insulin-Secreting Cells Expressing a Genetically Encoded Fluorescent Calcium Indicator for Cell-Based Sensing In Vivo.

The development of cell-based biosensors that give insight into cell and tissue function in vivo is an attractive technology for biomedical research. Here, the development of a cell line expressing a fluorescent calcium sensor for the study of beta-cell function in vivo is reported. The bioresponsive cell model is based on INS-1E pancreatic beta-cells, stably expressing the genetically encoded cameleon-based fluorescent sensor YC3.6cyto . Following single-cell selection and expansion, functional testing and in vitro encapsulation experiments are used to identify a suitable clone of INS-1E cells expressing the calcium sensor. This clone is transplanted subcutaneous in mouse using a cell macroencapsulation system based on flat sheet porous membranes. Cells in the implanted capsules are able to respond to glucose in vivo by secreting insulin and thereby contributing to the regulation of glycaemia in the mice. Furthermore, fluorescence imaging of explanted devices shows that encapsulated cells maintain high level expression of YC3.6cyto in vivo. In conclusion, these data show that encapsulated INS-1E cells stably expressing a genetically encoded calcium sensor can be successfully implanted in vivo, and therefore serve as biosensing element or in vivo model to longitudinally monitor the function of pancreatic beta-cells.

Nuclear Proteomics Uncovers Diurnal Regulatory Landscapes in Mouse Liver.

Diurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus.

Circulating CD14+ and CD14highCD16- classical monocytes are reduced in patients with signs of plaque neovascularization in the carotid artery.

BACKGROUND AND AIMS: Monocytes are known to play a key role in the initiation and progression of atherosclerosis and contribute to plaque destabilization through the generation of signals that promote inflammation and neoangiogenesis. In humans, studies investigating the features of circulating monocytes in advanced atherosclerotic lesions are lacking. METHODS: Patients (mean age 69 years, 56% males) with intermediate asymptomatic carotid stenosis (40-70% in diameter) were evaluated for maximal stenosis in common carotid artery, carotid bulb and internal carotid artery, overall disease burden as estimated with total plaque area (TPA), greyscale and neovascularization in 244 advanced carotid plaques. Absolute counts of circulating CD14+ monocytes, of classical (CD14highCD16-), intermediate (CD14highCD16+) and non-classical (CD14lowCD16+) monocytes and HLA-DR+ median fluorescence intensity for each subset were evaluated with flow cytometry. RESULTS: No correlation was found between monocytes and overall atherosclerotic burden, nor with high sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6). In contrast, plaque signs of neovascularization were associated with significantly lower counts of circulating CD14+ monocytes (297 versus 350 cells/mm3, p = 0.039) and of classical monocytes (255 versus 310 cells/mm3, p = 0.029). CONCLUSIONS: Neovascularized atherosclerotic lesions selectively associate with lower blood levels of CD14+ and CD14highCD16- monocytes independently of systemic inflammatory activity, as indicated by normal hsCRP levels. Whether the reduction of circulating CD14+ and CD14highCD16- monocytes is due to a potential redistribution of these cell types into active lesions remains to be explored.

Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice.

Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.

Quantifying Ocular Surface Inflammation and Correlating It With Inflammatory Cell Infiltration In Vivo: A Novel Method.

PURPOSE: The purpose of this study was to develop a novel, objective, and semiautomated method to quantify conjunctival redness by correlating measured redness with standard clinical redness and symptom scales and inflammatory cell infiltration. METHODS: Eleven outpatients presenting with mild to severe conjunctival hyperemia were included in the study. Clinical examination included patient history; visual analogue score (VAS) for ocular symptoms; 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ 25) for quality of life/vision; photographs of the anterior segment graded for conjunctival hyperemia, using Efron, relative redness of image (RRI), and edge feature (EF) scales; and conjunctival impression cytology analyzed by flow cytometry. Differences between affected and unaffected eyes were evaluated, and correlations among questionnaire scores, ocular hyperemia grading scores, and assessment of biological markers were performed. RESULTS: Visual analogue score (P < 0.0001), Efron scale (P = 0.0003), RRI scores (P = 0.0004), and EF scores (P < 0.0001) and the percentage of granulocytes (defined as cluster of differentiation [CD] 45dim; P = 0.0080) were significantly higher in affected eyes. Conversely, the percentage of CD45bright leukocytes was reduced in affected eyes (P = 0.0054). Both the RRIs and EFs were positively correlated with VAS, Efron scale, percentages of conjunctival granulocytes, and CD45brightCD3neg cells, whereas they were negatively correlated with the percentage of CD45brightCD3pos cells. Edge feature and RRI were correlated (Spearman r = 0.78, P < 0.0001). CONCLUSIONS: Ocular redness is a cardinal sign driving clinical judgment in highly prevalent ocular disorders; hence, we suggest that our semiautomated and reproducible method may represent a helpful tool in the follow-up of these patients. Italian Abstract.

Endothelial progenitor cells and response to ranibizumab in age-related macular degeneration.

BACKGROUND: Choroidal neovascularization (CNV) is the main cause of vision loss in age-related macular degeneration (AMD). In experimental CNV, endothelial progenitor cells (EPCs) contribute to the formation of new vessels. The aim of this study was to investigate whether the behavior of EPCs in patients with AMD supports a role for EPCs in human CNV. METHODS: The number of circulating EPCs that are considered pure endothelial precursors and EPCs with monocytic characteristics, and the plasma levels of regulatory cytokines were evaluated in 23 patients with AMD with active CNV and 20 matched controls. In the patients, this profile was re-evaluated after ranibizumab. RESULTS: When compared with controls, the patients with AMD showed a lower number of both EPC types (P = 0.03) and higher plasma levels (P = 0.03) of stromal cell-derived factor 1. Three monthly injections of ranibizumab returned to control levels the number of circulating EPCs considered pure endothelial precursors and of stromal cell-derived factor 1, but not of monocytic EPCs. CONCLUSION: The observations indicate responsiveness of circulating EPCs to the CNV process in AMD. They suggest the hypothesis that increased stromal cell-derived factor 1 production at the CNV site (reflected in higher plasma levels) recruits EPCs from the circulation, and that antivascular endothelial growth factor therapy selectively decreases the recruitment of cells to be incorporated into new vessels.

Effect of normalization of fasting glucose by intensified insulin therapy and influence of eNOS polymorphisms on the incidence of restenosis after peripheral angioplasty in patients with type 2 diabetes: a randomized, open-label clinical trial.

Primary objective was to evaluate whether an intensified insulin therapy (IIT) incorporating the target of normal fasting glucose and HbA1c levels could halve the incidence of restenosis/amputation/SCA/death at 6 months after peripheral angioplasty compared with standard care (SC) in patients with type 2 diabetes (DMT2) affected by critical limb ischemia (CLI). Forty-six consecutive patients with DMT2 and CLI were randomly assigned to a parallel, open-label study with IIT (basal-bolus glulisine + glargine administrations) or SC (glargine administration + oral antidiabetic drugs). A SNP of eNOS (rs753482-A>C) and circulating CD34(+) and CD34(+)KDR(+) progenitor cells were determined. At the end of the study, although HbA1c levels were lower in IIT than in SC (6.9 ± 1.3 % vs. 7.6 ± 1.2 %, p < 0.05), IIT did not reduce the cumulative incidence of restenosis/amputation/SCA/death (52 and 65 %, respectively, odd ratio 0.59; CI 95 %: 0.21-1.62, p = 0.59). rs753482AC+CC as compared with rs753482AA increased the cumulative incidence of restenosis/amputation/SCA/death (79 and 42 %; odd ratio 5.3; CI 95 %: 1.41-19.5, p < 0.02). Baseline CD34(+)KDR(+) were higher in rs753482AA (166.2 ± 154.0 × 10(6) events) than in rs753482AC+CC (63.1 ± 26.9 × 10(6) events, p < 0.01). At the end of the study, the highest circulating CD34(+)KDR(+) were found in IIT rs753482AA (246.9 ± 194.0 × 10(6) events) while the lowest levels were found in SC rs753482AC+CC (70.9 ± 45.0 × 10(6) events). IIT did not decrease the cumulative incidence of restenosis/amputation/SCA/death in DMT2 and CLI patients. These patients correspond to a class of fragile subjects at high risk of cardiovascular events, and new predictors of restenosis should be contemplated, such as of eNOS polymorphism, (rs753482-A>C SNP) and circulating endothelial progenitor cells.

Effector Memory T cells Are Associated With Atherosclerosis in Humans and Animal Models.

BACKGROUND#ENTITYSTARTX02014;: Adaptive T-cell response is promoted during atherogenesis and results in the differentiation of naïve CD4(+)T cells to effector and/or memory cells of specialized T-cell subsets. Aim of this work was to investigate the relationship between circulating CD4(+)T-cell subsets and atherosclerosis. METHODS AND RESULTS#ENTITYSTARTX02014;: We analyzed 57 subsets of circulating CD4(+)T cells by 10-parameter/8-color polychromatic flow cytometry (markers: CD3/CD4/CD45RO/CD45RA/CCR7/CCR5/CXCR3/HLA-DR) in peripheral blood from 313 subjects derived from 2 independent cohorts. In the first cohort of subjects from a free-living population (n=183), effector memory T cells (T(EM): CD3(+)CD4(+)CD45RA(-)CD45RO(+)CCR7(-) cells) were strongly related with intima-media thickness of the common carotid artery, even after adjustment for age (r=0.27; P<0.001). Of note, a significant correlation between T(EM) and low-density lipoproteins was observed. In the second cohort (n=130), T(EM) levels were significantly increased in patients with chronic stable angina or acute myocardial infarction compared with controls. HLA-DR(+)T(EM) were the T(EM) subpopulation with the strongest association with the atherosclerotic process (r=0.37; P<0.01). Finally, in animal models of atherosclerosis, T(EM) (identified as CD4(+)CD44(+)CD62L(-)) were significantly increased in low-density lipoprotein receptor and apolipoprotein E deficient mice compared with controls and were correlated with the extent of atherosclerotic lesions in the aortic root (r=0.56; P<0.01). CONCLUSIONS#ENTITYSTARTX02014;: Circulating T(EM) cells are associated with increased atherosclerosis and coronary artery disease in humans and in animal models and could represent a key CD4(+)T-cell subset related to the atherosclerotic process. (J Am Heart Assoc. 2012;1:27-41.).

Identification and predictive value of interleukin-6+ interleukin-10+ and interleukin-6- interleukin-10+ cytokine patterns in ST-elevation acute myocardial infarction.

RATIONALE: At the onset of ST-elevation acute myocardial infarction (STEMI), patients can present with very high circulating interleukin-6 (IL-6(+)) levels or very low-IL-6(-) levels. OBJECTIVE: We compared these 2 groups of patients to understand whether it is possible to define specific STEMI phenotypes associated with outcome based on the cytokine response. METHODS AND RESULTS: We compared 109 patients with STEMI in the top IL-6 level (median, 15.6 pg/mL; IL-6(+) STEMI) with 96 in the bottom IL-6 level (median, 1.7 pg/mL; IL-6(-) STEMI) and 103 matched controls extracted from the multiethnic First Acute Myocardial Infarction study. We found minimal clinical differences between IL-6(+) STEMI and IL-6(-) STEMI. We assessed the inflammatory profiles of the 2 STEMI groups and the controls by measuring 18 cytokines in blood samples. We exploited clustering analysis algorithms to infer the functional modules of interacting cytokines. IL-6(+) STEMI patients were characterized by the activation of 2 modules of interacting signals comprising IL-10, IL-8, macrophage inflammatory protein-1α, and C-reactive protein, and monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and monokine induced by interferon-γ. IL-10 was increased both in IL-6(+) STEMI and IL-6(-) STEMI patients compared with controls. IL-6(+)IL-10(+) STEMI patients had an increased risk of systolic dysfunction at discharge and an increased risk of death at 6 months in comparison with IL-6(-)IL-10(+) STEMI patients. We combined IL-10 and monokine induced by interferon-γ (derived from the 2 identified cytokine modules) with IL-6 in a formula yielding a risk index that outperformed any single cytokine in the prediction of systolic dysfunction and death. CONCLUSIONS: We have identified a characteristic circulating inflammatory cytokine pattern in STEMI patients, which is not related to the extent of myocardial damage. The simultaneous elevation of IL-6 and IL-10 levels distinguishes STEMI patients with worse clinical outcomes from other STEMI patients. These observations could have potential implications for risk-oriented patient stratification and immune-modulating therapies.

Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation.

The recent hypothesis that postnatal microglia are maintained independently of circulating monocytes by local precursors that colonize the brain before birth has relevant implications for the treatment of various neurological diseases, including lysosomal storage disorders (LSDs), for which hematopoietic cell transplantation (HCT) is applied to repopulate the recipient myeloid compartment, including microglia, with cells expressing the defective functional hydrolase. By studying wild-type and LSD mice at diverse time-points after HCT, we showed the occurrence of a short-term wave of brain infiltration by a fraction of the transplanted hematopoietic progenitors, independently from the administration of a preparatory regimen and from the presence of a disease state in the brain. However, only the use of a conditioning regimen capable of ablating functionally defined brain-resident myeloid precursors allowed turnover of microglia with the donor, mediated by local proliferation of early immigrants rather than entrance of mature cells from the circulation.

Endothelial progenitor cells carrying monocyte markers are selectively abnormal in type 1 diabetic patients with early retinopathy.

Endothelial progenitor cells (EPCs) enter the systemic circulation in response to cues related to vascular damage and need for neovascularization. Thus, EPCs could become readily accessible informers of vascular status and enable the survey of vascular pathologies during preclinical stages. To identify EPC changes with biomarker potential, we investigated whether discrete EPC abnormalities were associated with early nonproliferative diabetic retinopathy (NPDR). Two EPC subtypes with different functions have been characterized to date-one solely committed to the endothelial lineage and the other carrying both endothelial and monocytic markers. We found that only the latter, colony-forming units (CFU)-Hill cells, manifested abnormalities in type 1 diabetic patients with NPDR compared with control subjects. The abnormalities consisted in an increased number of colonies formed in vitro and downregulation of the molecules that facilitate homing at sites of vascular injury. The abnormalities were absent in type 1 diabetic patients free of retinopathy and other complications, despite long diabetes duration, but were detected in some of the patients without clinical retinopathy after short diabetes duration. CFU-Hill cells are potential informers of diabetic microangiopathy but may be preempted from carrying out reparative functions if the molecular abnormalities compromise interactions with the damaged vascular wall.

Joint production of prime/boost pairs of Fowlpox Virus and Modified Vaccinia Ankara recombinants carrying the same transgene.

Pairs of recombinant MVA (Modified Vaccinia Ankara) and FPV (Fowlpox Virus) expressing the same transgene are reasonable candidates for prime/boost regimens, because cross-reacting immune responses between the two vectors, both non-replicative in mammalian hosts, are very limited. The acceptor virus FPD-Red, a derivative of FPV, carrying a red fluorescent protein gene flanked by the homology regions of MVA deletion III, was constructed. The same MVA Transfer Plasmid Green, designed to insert transgenes into the MVA deletion III locus, can therefore be used to transfer transgenes into both acceptor viruses MVA-Red and FPD-Red with the described recently Red-to-Green gene swapping method. Cells infected by either recombinant virus can be sorted differentially by a simple and reliable FACS-based purification protocol. The procedure is carried out in primary chick embryo fibroblasts grown in serum-free media and was applied to the production of three rMVA/rFPV pairs expressing the H5N1 avian influenza antigens M1, M2 and NP. The viral genes were human codon-optimized and expressed at high levels in both chick and mammalian cells. Both single-step and multiple-step growth analyses showed no significant differences in growth due to the transgenes in either rMVA or rFPV derivatives.

Circulating CD4+CD25hiCD127lo regulatory T-Cell levels do not reflect the extent or severity of carotid and coronary atherosclerosis.

OBJECTIVE: Regulatory T (Treg) cells play a protective role in experimental atherosclerosis. In the present study, we investigated whether the levels of circulating Treg cells relate to the degree of atherosclerosis in carotid and coronary arteries. METHODS AND RESULTS: We studied 2 distinct populations: (1) 113 subjects, selected from a free-living population (carotid study), in which we measured the intima-media thickness of the common carotid artery, as a surrogate marker of initial atherosclerosis; and (2) 75 controls and 125 patients with coronary artery disease (coronary study): 36 with chronic stable angina, 50 with non-ST-elevation acute coronary syndrome, 39 with ST-elevation acute myocardial infarction. Treg-cell levels were evaluated by flow cytometry (Treg cells identified as CD3(+)CD4(+)CD25(high)CD127(low)) and by mRNA expression of forkhead box P3 or of Treg-associated cytokine interleukin 10. In the carotid study, no correlation was observed between Treg-cell levels and intima-media thickness. No differences in Treg-cell levels were observed comparing rapid versus slow intima-media thickness progressors from a subgroup of patients (n=65), in which prospective data on 6-year intima-media thickness progression were available. In the coronary group, Treg-cell levels were not altered in chronic stable angina patients. In contrast, nonunivocal variations were observed in patients suffering an acute coronary syndrome (with a Treg-cell increase in ST-elevation acute myocardial infarction and a Treg-cell decrease in non-ST-elevation acute coronary syndrome patients). CONCLUSIONS: The results suggest that determination of circulating Treg-cell levels based on flow cytometry or mRNA assessment is not a useful indicator of the extent or severity of atherosclerosis.