Characteristics of circulating endothelial cells obtained from non-ST-segment elevation myocardial infarction patients with additional diastolic dysfunction of left ventricle observed in echocardiography.

BACKGROUND: Circulating endothelial cells (CEC) may be used to find new strategies for the early di-agnosis of cardiovascular diseases. The major objective of the project is to broaden knowledge of CEC biology by determining their phenotypic characteristics. The additional aim is to clarify whether on the basis of these information it is possible to identify the origin of CEC release (from various cardiovascular compartments). METHODS: Circulating endothelial cells were collected from arterial blood prior to angiography, as well as from arterial and venous blood obtained after angiography/coronary angioplasty, from 18 patients with non-ST-segment elevation myocardial infarction (NSTEMI). CECs were quantified by flow cytometry and defined as Syto16 (dye)+, CD45dim/neg, CD31+ and CD146+. The additional CD36+ was establish as a marker of endothelial cells released from small vessels of the microcirculation. RESULTS: The total number of CECs increased significantly after the percutaneous transluminal coronary angioplasty (PTCA) in the arterial system. Number of CECs isolated at similar time points (after invasive procedure) did not differ significantly between arteries and veins, but the number of CD36+ CECs after coronary angioplasty was significantly higher in the venous system, than in the arterial system. CONCLUSIONS: The number of CD36+ in artery samples obtained after coronary angioplasty (PTCA) had tendency to be decreased (in comparison to the sample obtained before angiography). It was major difference between those who had PTCA performed vs. those who had not.

Circulating endothelial cells as biomarker for cardiovascular diseases.

BACKGROUND: Endothelial dysfunction is involved in several cardiovascular diseases. Elevated levels of circulating endothelial cells (CECs) and low levels of endothelial progenitor cells (EPCs) have been described in different cardiovascular conditions, suggesting their potential use as diagnostic biomarkers for endothelial dysfunction. Compared to typical peripheral blood leukocyte subsets, CECs and EPCs occur at very low frequency. The reliable identification and characterization of CECs and EPCs is a prerequisite for their clinical use, however, a validated method to this purpose is still missing but a key for rare cell events. OBJECTIVES: To establish a validated flow cytometric procedure in order to quantify CECs and EPCs in human whole blood. METHODS: In the establishment phase, the assay sensitivity, robustness, and the sample storage conditions were optimized as prerequisite for clinical use. In a second phase, CECs and EPCs were analyzed in heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction, in arterial hypertension (aHT), and in diabetic nephropathy (DN) in comparison to age-matched healthy controls. RESULTS: The quantification procedure for CECs and EPCs showed high sensitivity and reproducibility. CEC values resulted significantly increased in patients with DN and HFpEF in comparison to healthy controls. CEC quantification showed a diagnostic sensitivity of 90% and a sensitivity of 68.0%, 70.4%, and 66.7% for DN, HFpEF, and aHT, respectively. CONCLUSION: A robust and precise assay to quantify CECs and EPCs in pre-clinical and clinical studies has been established. CEC counts resulted to be a good diagnostic biomarker for DN and HFpEF.

Communication between Human Dendritic Cell Subsets in Tuberculosis: Requirements for Naive CD4(+) T Cell Stimulation.

Human primary dendritic cells (DCs) are heterogeneous by phenotype, function, and tissue localization and distinct from inflammatory monocyte-derived DCs. Current information regarding the susceptibility and functional role of primary human DC subsets to Mycobacterium tuberculosis (Mtb) infection is limited. Here, we dissect the response of different primary DC subsets to Mtb infection. Myeloid CD11c(+) cells and pDCs (C-type lectin 4C(+) cells) were located in human lymph nodes (LNs) of tuberculosis (TB) patients by histochemistry. Rare CD141(hi) DCs (C-type lectin 9A(+) cells) were also identified. Infection with live Mtb revealed a higher responsiveness of myeloid CD1c(+) DCs compared to CD141(hi) DCs and pDCs. CD1c(+) DCs produced interleukin (IL)-6, tumor necrosis factor α, and IL-1ß but not IL-12p70, a cytokine important for Th1 activation and host defenses against Mtb. Yet, CD1c(+) DCs were able to activate autologous naïve CD4(+) T cells. By combining cell purification with fluorescence-activated cell sorting and gene expression profiling on rare cell populations, we detected in responding CD4(+) T cells, genes related to effector-cytolytic functions and transcription factors associated with Th1, Th17, and Treg polarization, suggesting multifunctional properties in our experimental conditions. Finally, immunohistologic analyses revealed contact between CD11c(+) cells and pDCs in LNs of TB patients and in vitro data suggest that cooperation between Mtb-infected CD1c(+) DCs and pDCs favors stimulation of CD4(+) T cells.

Crosstalk between human DC subsets promotes antibacterial activity and CD8+ T-cell stimulation in response to bacille Calmette-Guérin.

To date, little is known about the unique contributions of specialized human DC subsets to protection against tuberculosis (TB). Here, we focus on the role of human plasmacytoid (p)DCs and myeloid (m)DCs in the immune response to the TB vaccine bacille Calmette-Guérin (BCG). Ex vivo DC subsets from human peripheral blood were purified and infected with BCG expressing GFP to distinguish between infected and noninfected cells. BDCA-1(+) myeloid DCs were more susceptible than BDCA-3(+) mDCs to BCG infection. Plasmacytoid DCs have poor phagocytic activity but are equipped with endocytic receptors and can be activated by bystander stimulation. Consequently, the mutual interaction of the two DC subsets in response to BCG was analyzed. We found that pDCs were activated by BCG-infected BDCA-1(+) mDCs to upregulate maturation markers and to produce granzyme B, but not IFN-α. Reciprocally, the presence of activated pDCs enhanced mycobacterial growth control by infected mDCs and increased IL-1ß availability. The synergy between the two DC subsets promoted BCG-specific CD8(+) T-cell stimulation and the role of BCG-infected BDCA-1(+) mDCs could not be efficiently replaced by infected BDCA-3(+) mDCs in the crosstalk with pDCs. We conclude that mDC-pDC crosstalk should be exploited for rational design of next-generation TB vaccines.

MicroRNA-223 controls susceptibility to tuberculosis by regulating lung neutrophil recruitment.

The molecular mechanisms that control innate immune cell trafficking during chronic infection and inflammation, such as in tuberculosis (TB), are incompletely understood. During active TB, myeloid cells infiltrate the lung and sustain local inflammation. While the chemoattractants that orchestrate these processes are increasingly recognized, the posttranscriptional events that dictate their availability are unclear. We identified microRNA-223 (miR-223) as an upregulated small noncoding RNA in blood and lung parenchyma of TB patients and during murine TB. Deletion of miR-223 rendered TB-resistant mice highly susceptible to acute lung infection. The lethality of miR-223(­/­) mice was apparently not due to defects in antimycobacterial T cell responses. Exacerbated TB in miR-223(­/­) animals could be partially reversed by neutralization of CXCL2, CCL3, and IL-6, by mAb depletion of neutrophils, and by genetic deletion of Cxcr2. We found that miR-223 controlled lung recruitment of myeloid cells, and consequently, neutrophil-driven lethal inflammation. We conclude that miR-223 directly targets the chemoattractants CXCL2, CCL3, and IL-6 in myeloid cells. Our study not only reveals an essential role for a single miRNA in TB, it also identifies new targets for, and assigns biological functions to, miR-223. By regulating leukocyte chemotaxis via chemoattractants, miR-223 is critical for the control of TB and potentially other chronic inflammatory diseases.

The recombinant tuberculosis vaccine rBCG ΔureC::hly(+) induces apoptotic vesicles for improved priming of CD4(+) and CD8(+) T cells.

BACKGROUND: The recombinant BCG ΔureC::hly(+) (rBCG) vaccine candidate is more efficient than parental BCG (pBCG) against tuberculosis (TB) in preclinical models. Evidence exists for superior CD4 and CD8 T cell stimulation. Although the responsible immune mechanisms are incompletely understood, crosspriming of CD8 T cells has been proposed as a major mechanism underlying better protection of rBCG over pBCG. The present study investigates the role of apoptotic vesicles from pBCG- and rBCG-infected macrophages in crosspriming. METHODS: Apoptotic vesicles were isolated from pBCG- and rBCG-infected mouse macrophages. The priming potential of the isolated vesicles was evaluated in terms of dendritic cell activation and specific T cell stimulation. RESULTS: Apoptotic vesicles from both pBCG- and rBCG-infected macrophages activated dendritic cells but to a different degree. Overall, rBCG-infected apoptotic vesicles induced more profound CD4 and CD8 T cell responses as compared to pBCG. CONCLUSIONS: These data support the notion that the improved vaccine efficacy of rBCG rests on enhanced crosspriming as a consequence of stronger apoptosis.

Cells as factories for humanized encapsulation.

Biocompatibility is of paramount importance for drug delivery, tumor labeling, and in vivo application of nanoscale bioprobes. Until now, biocompatible surface processing has typically relied on PEGylation and other surface coatings, which, however, cannot minimize clearance by macrophages or the renal system but may also increase the risk of chemical side effects. Cell membranes provide a generic and far more natural approach to the challenges of encapsulation and delivery in vivo. Here we harness for the first time living cells as "factories" to manufacture cell membrane capsules for encapsulation and delivery of drugs, nanoparticles, and other biolabels. Furthermore, we demonstrate that the built-in protein channels of the new capsules can be utilized for controlled release of encapsulated reagents.

Effects of interferon-α on the inflammatory response of swine peripheral blood mononuclear cells.

Interferon-α (IFN-α) at low concentrations had been previously shown to control the expression of inflammatory cytokine genes in swine pulmonary alveolar macrophages. In the first part of this study, cultured swine peripheral blood mononuclear cells (PBMCs) were supplemented with IFN-α at low/moderate concentrations, and then stimulated with lipopolysaccharide (LPS). The expression of IFN-α, IFN-γ, IL-1ß, TNF-α, and IL-6 genes was determined by real-time PCR. IFN-α at low/moderate concentrations did not significantly reduce the expression of any cytokine gene under study, with clear trends though to a concentration-dependent reduction of IL-1ß gene expression and to a concentration-dependent increase of IFN-γ gene expression. In vivo, orally administered IFN-α was shown instead to modulate the inflammatory response to early weaning in uncultured PBMCs of specific pathogen-free piglets. As opposed to the in vitro model, the oral IFN-α treatment reduced after weaning the expression of the IFN-γ gene (P < 0.08) and increased that of the IL-1ß gene (P < 0.05). There was also a trend to a reduced expression of both IL-6 and TNF-α. The above modulation of cytokine genes expression and the greater daily mean weight gain of treated piglets highlight important regulatory properties of oral IFN-α in the response to the weaning stress.

Modulation of ovine neutrophil function and apoptosis by standardized extracts of Echinacea angustifolia, Butea frondosa and Curcuma longa.

Impaired neutrophil function has been associated with increased infectious diseases in ruminants. Attachment of neutrophils to endothelium and superoxide production is critical features of their immune activity. Once the infection is cleared, programmed cell death ensures the rapid resolution of inflammation. To develop new natural therapeutics for ruminants, standard extracts of Echinacea angustifolia (Polinacea), Butea frondosa and Curcuma longa (Curcuvet) were first evaluated on ovine neutrophil functions. Curcuvet strongly reduced PMA-stimulated adhesion and superoxide production. Polinacea and B. frondosa extract also reduced these functions, but with less efficacy than Curcuvet. We analyzed the effect of extracts on spontaneous apoptosis and gene expression in neutrophils aged in vitro for up to 22h. IL8 is critical for neutrophil recruitment and the immune response; Bcl2-related proteins, Bcl2A1 and Bax, are key regulators of neutrophil fate. Spontaneous apoptosis strongly increased in ovine neutrophils cultured for 22h (T22), accompanied by an upregulation of IL8 and a decreased Bcl2A1:Bax ratio. Curcuvet stimulated spontaneous apoptosis and inhibited IL8 and Bcl2A1 gene expression at T22, whereas Polinacea and B. frondosa extract inhibited spontaneous apoptosis and stimulated IL8 expression at T22. These results suggest that Curcuvet has antiinflammatory activity, whereas Polinacea and B. frondosa have an immunomodulatory action on sheep neutrophils.

Immunomodulatory activity of plant residues on ovine neutrophils.

Neutrophils play an essential role in host defense and inflammation. Plants have long been used to improve the immune function, but for most of them specific investigations on animal health are lacking. In the present study, water and hydroethanolic extracts from 11 plant wastes have been screened on immune responses of ovine neutrophils. Eight sheep clinically healthy, not lactating, non-pregnant were selected and used for the experiment. Freshly isolated neutrophils were incubated with the extracts of the residues at increasing doses, and then they were tested for adhesion and superoxide production induced with PMA. The residues of Larix decidua, Thymus vulgaris, Salix alba, Sinupret, Helianthus annuus, Mangifera indica modulated the neutrophil immune functions, moreover, Larix decidua, Thymus vulgaris and Salix alba presented the highest anti-inflammatory activity.

Improved apoptosis detection in ovine neutrophils by annexin V and carboxyfluorescein diacetate staining.

Neutrophil apoptosis is critical for final resolution of the inflammation in the tissues and for maintenance of neutrophil homeostasis under normal condition. An early hallmark of apoptotic cells is translocation of phosphatidylserine (PS) residues, normally located in the inner leaflet of cellular membrane, to the external cell surface; exposed PS is recognized by specific PS receptors on disposing cells. Here we report an improved procedure to detect neutrophil apoptosis by simultaneous staining for exposed PS with Cy3-labeled annexin V (Cy3) and for membrane integrity with the vital dye 6-carboxyfluorescein diacetate (6-CFDA) based on the APOAC apoptosis detection kit (Sigma). Spontaneous apoptosis was evaluated in ovine neutrophils cultured ex vivo for 18 h. We investigated the multiple parameters involved in the assay, i.e. the type of fixative (methanol, paraformaldehyde, or no fixation) and the type of slide (coated with Vectabond, polylysine or Parafilm((R))). Results indicated that both the adhesion to the slide and the fixation can modify neutrophil functional status and morphology, which result in misleading apoptosis detection. In order to minimize these artifacts, we have developed an improved APOAC assay procedure, staining cells while in suspension and using Parafilm((R)) coated slides.

Expression and localization studies of hSDA, the human ortholog of the yeast SDA1 gene.

The yeast SDA1 gene was reported to play a critical role in G(1) events and to be involved in 60S ribosome biogenesis. Although the basic cellular mechanisms appear conserved from yeast to man, the human genes may have more diversified functions. In this view we obtained the first experimental evidences about the human ortholog of the yeast SDA1, i.e., hSDA. The gene is localized at the chromosomal region 4q21 and encodes for a 627a.a. long protein highly homologous to the yeast Sda1. Subcellular localization experiments indicate that the human protein behaves similarly to nucleolar proteins involved in rRNA processing machinery but not in RNA PolI transcriptional events. hSda appears localized in the granular component of the nucleolus and in the nucleoplasm, which is consistent with a role in early-intermediate steps of ribosome biogenesis. hSDA appears preferentially expressed in fetal tissues, pinpointing its role during development. Different expression levels in different tumor cell lines might suggest that the gene is involved also in tumorigenesis. However our preliminary results indicate that hSDA does not behave like a proapoptotic gene and its involvement in tumorigenesis is still to be clarified.