Responses of peripheral blood mononuclear cells to moderate exercise and hypoxia.

The purpose of this study was to analyze the physiological features of peripheral blood mononuclear cells (PBMCs) isolated from healthy female trekkers before and after physical activity carried out under both normoxia (low altitude, < 2000 m a.s.l.) and hypobaric hypoxia (high altitude, > 3700 m a.s.l.). The experimental design was to differentiate effects induced by exercise and those related to external environmental conditions. PBMCs were isolated from seven female subjects before and after each training period. The PBMCs were phenotypically and functionally characterized using fluorimetric and densitometric analyses, to determine cellular activation, and their intracellular Ca(2+) levels and oxidative status. After a period of normoxic physical exercise, the PBMCs showed an increase in fully activated T lymphocytes (CD3(+) CD69(+) ) and a reduction in intracellular Ca(2+) levels. On the other hand, with physical exercise performed under hypobaric hypoxia, there was a reduction in T lymphocytes and an increase in nonactivated B lymphocytes, accompanied by a reduction in O2 (-) levels in the mitochondria. These outcomes reveal that in women, low- to moderate-intensity aerobic trekking induces CD69 T cell activation and promotes anti-stress effects on the high-altitude-induced impairment of the immune responses and the oxidative balance.

Lactobacillus paracasei Lp6 favors immune modulation induced by allergoid treatment in ragweed sensitized mice.

It has been hypothesized that lactic acid bacteria (LAB) could be used as adjuvant for specific immunotherapy (SIT), as various studies conducted on humans and animals converge to define LAB as anti-Th2 modulators and Treg inducers. In the present study we evaluated the effects of LAB, in particular Lactobacillus paracasei Lp6 (Lp6), in a mouse model of ragweed (RW) allergy. Groups of Balb/c mice, experimentally sensitized towards ragweed, were treated by viable Lp6 or by RWallergoid with or without co-administration of Lp6. A control group was sham-sensitized with PBS and sham-treated with water and a group was sensitized with RW and treated with water. Serum IgE, RW-induced release of IFN-gamma, IL-4 and IL-10 from splenocytes and the frequency of CD4CD25 regulatory T cells (Tregs) expressing Foxp3 or IL-10 were evaluated in various groups. RW-allergoid treatment induced a reduction of serum IgE, with a decrease in RW-induced release of IL-4, and an increase in IL-10 and IFN-gamma, along with a significant change in the frequency of Tregs, both CD25+ and -. The joint RWallergoid+ Lp6 treatment induced the highest degree of suppression of allergen-driven IL-4, the greatest reduction of IL-4/IFN-gamma and IL-4/IL-10 ratios and the most significant increase of Foxp3 and IL-10 expressing Tregs. The study shows that Lp6 strengthens the immune modulation induced by allergoid-SIT in RW-sensitized mice, essentially characterized by a differential induction of Tregs associated to a reduction of IL-4; data converge to define a role of SIT adjuvant for Lp6.

Vascular endothelial growth factor enhances in vitro proliferation and osteogenic differentiation of human dental pulp stem cells.

Mesenchymal stem cells (MSC), isolated from dental tissues, are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from human dental pulp (DPSC) of normal impacted third molars that, when cultured in lineage-specific inducing media, differentiate into osteoblasts and adipocytes (evaluated by Alizarin Red S and Red Oil O stainings, respectively), thus showing a multipotency. We confirmed that DPSC, grown under undifferentiating conditions, are negative for hematopoietic (CD45, CD31, CD34, CD144) and positive for mesenchymal (CD29, CD90, CD105, CD166, CD146, STRO-1) markers, that underwent down-regulation when cells were grown in osteogenic medium for 3 weeks. In this condition, they also exhibit an increase in the expression of osteogenic markers (RUNX-2, alkaline phosphatase) and extracellular calcium deposition, whereas the expression of receptors (VEGFR-1 and -2) for vascular endothelial growth factors (VEGF) and related VEGF binding proteins was similar to that found in undifferentiated DPSC. Exposure of DPSC growing under undifferentiating or osteogenic conditions to VEGF-A165 peptide (10-40 ng/ml) for 8 days dose- and time-dependently increased the number of proliferating cells without inducing differentiation towards endothelial lineage, as evaluated by the lack of expression of specific markers (CD31, CD34, CD144). Additionally, exposure of DPSC cultured in osteogenic medium to VEGF-A165 for a similar period enhanced cell differentiation towards osteoblasts as evaluated after 14 and 21 days by Alizarin Red S staining and alkaline phosphatase activity quantification. These findings may have clinical implications possibly facilitating tissue repair and remodeling.

Corrigendum to "Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation".

[This corrects the article DOI: 10.1155/2019/5879616.].

Computational design of synthetic gene circuits with composable parts.

MOTIVATION: In principle, novel genetic circuits can be engineered using standard parts with well-understood functionalities. However, no model based on the simple composition of these parts has become a standard, mainly because it is difficult to define signal exchanges between biological units as unambiguously as in electrical engineering. Corresponding concepts and computational tools for easy circuit design in biology are missing. RESULTS: Taking inspiration from (and slightly modifying) ideas in the 'MIT Registry of Standard Biological Parts', we developed a method for the design of genetic circuits with composable parts. Gene expression requires four kinds of signal carriers: RNA polymerases, ribosomes, transcription factors and environmental 'messages' (inducers or corepressors). The flux of each of these types of molecules is a quantifiable biological signal exchanged between parts. Here, each part is modeled independently by the ordinary differential equations (ODE) formalism and integrated into the software ProMoT (Process Modeling Tool). In this way, we realized a 'drag and drop' tool, where genetic circuits are built just by placing biological parts on a canvas and by connecting them through 'wires' that enable flow of signal carriers, as it happens in electrical engineering. Our simulations of well-known synthetic circuits agree well with published computational and experimental results. AVAILABILITY: The code is available on request from the authors.

Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets.

Multi-colour flow cytometry is the only technological platform that can analyse the highly complex cellular composition of the immune system in parallel and at a single cell resolution. Analysis of the T cell compartment, in particular, requires the simultaneous measurement of multiple markers in order to account for lineage, phenotype and function. Flow cytometry also enables the analysis of intracellular signalling events. By combining the expression of surface markers, intracellular cytokines, phosphorylated versus unphosphorylated kinases, cell proliferation and DNA profile, mechanistic and kinetic information of subset-specific signalling may be obtained: this has not previously been achieved. Here we present a protocol which permits all of these aspects to be explored simultaneously. By comparing basic procedures previously described we were able to optimise different variables, including the choice of antibody/fluorochrome pairs, permeabilisation, fixation and labelling time, to obtain the best DNA staining of different cell types. We applied this method to study subset-specific signalling related to cytokine production and DNA synthesis in T cells responding to specific antigens.

Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation.

The recent introduction of the "precision medicine" concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326- EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

Expression and phosphorylation of protein kinase C isoforms in Abeta(1-42) activated T lymphocytes from Alzheimers disease.

The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction. There is evidence demonstrating altered activity of some PKC isoforms (PKC-alpha, PKC-delta and PKC-zeta) in the neurons of brains of Alzheimers Disease (AD) sufferers, but little is known about their involvement in the intracellular machinery of amyloid beta protein-reactive T lymphocytes in AD. By applying a modified, split-well culture system, for Abeta(1-42) reactivity, we carried out flow cytometry analysis and biochemical investigations on the possible involvement of PKC-alpha, PKC-delta and PKC-zeta in the signalling system activated in Abeta-reactive T cells purified from peripheral blood mononucleate cells (PBMC) from healthy subjects and patients with AD. Flow cytometry analysis of Abeta(1-42) activated T lymphocytes in the majority of AD patients highlighted a distinct cellular cluster highly expressing phospho-PKC-delta (P-PKC-delta), while most full-blown AD patients highly expressed two distinct P-PKC-delta and phospho-PKC-zeta (P-PKC-zeta) bright sub-populations. The same investigation performed in freshly purified peripheral T lymphocytes, did not highlight any subpopulation, suggesting that the detection of P-PKC-delta and P-PKC-zeta bright subpopulations is specifically linked to Abeta(1-42) activated T lymphocytes. The data presented here, therefore, suggest possible novel hallmarks to discriminate between healthy elderly subjects and beginning or full-blown Alzheimers Disease patients.

Nuclear protein kinase C-delta: a possible check-point of cell cycle progression.

Protein kinase Cs (PKCs) belong to a serine/threonine kinase family, ubiquitously expressed and claimed to be involved in physiological processes including apoptosis, cell growth and differentiation. The question of the subcellular localization and activity of PKCs remains to be clarified. Here we report that nuclear PKC-delta cooperates to regulate the S-G2/M phase transition of cell cycle, apparently being associated to chromosome condensation and alignment on the metaphase plate.

Phosphoinositide 3-kinase activity is essential for all-trans-retinoic acid-induced granulocytic differentiation of HL-60 cells.

Phosphoinositide 3-kinase (PI 3-K) activity increases in HL-60 cells that are induced to granulocytic differentiation by all-trans-retinoic acid. Immunochemical and immunocytochemical analyses by confocal microscopy also reveal an increase in the amount of the enzyme, which is particularly evident at the nuclear level. Inhibition of PI 3-K activity by nanomolar concentrations of wortmannin and of its expression by transfection with an antisense fragment of p85alpha prevented the differentiative process. The data obtained indicate that PI 3-K activity plays an essential role in promoting granulocytic differentiation.

Comparative proteomic profiling of Hodgkin lymphoma cell lines.

Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies.

Changes of nuclear PI-PLC gamma1 during rat liver regeneration.

We have previously demonstrated that rat liver nuclei contain PI-PLC beta1 and gamma1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669-678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (beta1, gamma1, delta1) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC beta1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear gamma1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC delta1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC gamma1 that paralleled the in situ observation whereas the beta1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP2, obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the gamma1 isoform of PI-PLC.

Stromal derived factor-1 alpha (SDF-1 alpha) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway.

Stromal-derived factor-1alpha (SDF-1alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF-1alpha-mediated Jurkat cell apoptosis was observed in serum-free or serum-containing cultures, peaked at SDF-1alpha concentrations of 10-100 ng/ml, required 3 days to take place, and was completely blocked by the z-VAD-fmk tripeptide caspase inhibitor. Although SDF-1alpha did not modify the expression of TNF-alpha or that of TNF-RI and TNF-RII, it increased the expression of surface Fas/APO-1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF-1alpha to induce apoptosis was inhibited by an anti-CD95 Fab' neutralizing antibody. These findings suggest a role for SDF-1alpha in the homeostatic control of CD4+ T-cell survival/apoptosis mediated by the CD95-CD95L pathway.

Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia.

Deregulation of the miR-15a/16-1 cluster has a key role in the pathogenesis of chronic lymphocytic leukemia (CLL), a clinically heterogeneous disease with indolent and aggressive forms. The miR-15a/16-1 locus is located at 13q14, the most frequently deleted region in CLL. Starting from functional investigations of a rare SNP upstream the miR cluster, we identified a novel allele-specific mechanism that exploits a cryptic activator region to recruit the RNA polymerase III for miR-15a/16-1 transcription. This regulation of the miR-15a/16- locus is independent of the DLEU2 host gene, which is often transcribed monoallellically by RPII. We found that normally one allele of miR-15a/16-1 is transcribed by RNAPII, the other one by RNAPIII. In our subset of CLL patients harboring 13q14 deletions, exclusive RNA polymerase III (RPIII)-driven transcription of the miR-15a/16-1 was the consequence of loss of the RPII-regulated allele and correlated with high expression of the poor prognostic marker ZAP70 (P=0.019). Thus, our findings point to a novel biological process, characterized by double allele-specific transcriptional regulation of the miR-15a/16-1 locus by alternative mechanisms. Differential usage of these mechanisms may distinguish at onset aggressive from indolent forms of CLL. This provides a basis for the clinical heterogeneity of the CLL patients carrying 13q14 deletions.

Hypertensive urgencies and emergencies. Prevalence and clinical presentation.

The prevalence and clinical picture of hypertensive urgencies and emergencies in an emergency department are poorly known. The aim of the present study was to evaluate the prevalence of hypertensive crises (urgencies and emergencies) in an emergency department during 12 months of observation and the frequency of end-organ damage with related clinical pictures during the first 24 hours after presentation. Hypertensive crises (76% urgencies, 24% emergencies) represented more than one fourth of all medical urgencies-emergencies. The most frequent signs of presentation were headache (22%), epistaxis (17%), faintness, and psychomotor agitation (10%) in hypertensive urgencies and chest pain (27%), dyspnea (22%), and neurological deficit (21%) in hypertensive emergencies. Types of end-organ damage associated with hypertensive emergencies included cerebral infarction (24%), acute pulmonary edema (23%), and hypertensive encephalopathy (16%) as well as cerebral hemorrhage, which accounted for only 4.5%. Age (67 +/- 16 versus 60 +/- 14 years [mean +/- SD], P < .001) and diastolic blood pressure (130 +/- 15 versus 126 +/- 10 mm Hg, P < .002) were higher in hypertensive emergencies than urgencies. Hypertension that was unknown at presentation was present in 8% of hypertensive emergencies and 28% of hypertensive urgencies. In conclusion hypertensive urgencies and emergencies are common events in the emergency department and differ in their clinical patterns of presentation. Cerebral infarction and acute pulmonary edema are the most frequent types of end-organ damage in hypertensive emergencies.

Nuclear association of tyrosine-phosphorylated Vav to phospholipase C-gamma1 and phosphoinositide 3-kinase during granulocytic differentiation of HL-60 cells.

The granulocytic differentiation of HL-60 cells induced by all-trans retinoic acid was accompanied by a progressive tyrosine phosphorylation of specific proteins in either cells or isolated nuclei. Among these phosphoproteins, we identified the Vav adaptor in whole cells as well as in the inner nuclear compartment, where the increase in its tyrosine phosphorylation level was more conspicuous. We also demonstrated the differentiation-dependent association of nuclear phosphorylated Vav to phospholipase C-gamma1 and to the p85 regulatory subunit of phosphoinositide 3-kinase. The role of the Vav/phospholipase C-gamma1/phosphoinositide 3-kinase phosphoprotein complexes in the nuclei of HL-60 induced to differentiate along the granulocytic lineage is discussed.

In vivo and in vitro modulatory effect of human immunodeficiency virus type-1 (HIV-1) Tat protein on protein kinase C activity.

Extracellular HIV-1 Tat protein shows a pleiotropic activity on the survival/proliferation of different cell types, which may be relevant to the pathogenesis of the immune suppression as well as of the frequent neoplastic disorders observed during the course of HIV-1 disease. Therefore, we investigated the effect of recombinant Tat on the protein kinase C (PKC) activity in Jurkat CD4(+) T lymphoma cells by using a serine substituted specific PKC peptide substrate, which allowed the evaluation of the whole catalytic activity of both Ca++-dependent and Ca++-independent PKC isoforms. High concentrations of recombinant Tat (1 mu g/ml) induced an early (5 min) stimulation followed by a secondary (30-60 min) inhibition of PKC in whole Jurkat cell homogenates. Immuno-localization experiments showed that recombinant Tat protein was rapidly taken up by Jurkat cells within the first 5 min from the addition in culture, thus suggesting the possibility that the secondary inhibitory phase of Tat on PKC activity in Jurkat cells could be due to a direct interaction between the two proteins. Consistently, PKC immunoprecipitated from Jurkat cells or purified from rat brain was significantly inhibited by the addition of high (0.1-1 mu g) but not low (1-10 ng) doses of Tat in a cell-free in vitro assay. The inhibition of PKC catalytic activity mediated by 1 mu g of Tat was at least partially due to competition among substrates. The present data may help in understanding the opposite effects on the survival/proliferation of different cell types observed in the presence of picomolar (stimulation) vs nanomolar (inhibition) concentrations of recombinant Tat.

Recent results on functional polymers and macromonomers of interest as biomaterials or for biomaterial modification.

Different families of functionalized polymers with potential as biomaterials, or for biomaterial modification, have been investigated. In particular, degradation studies have been performed on poly(amidoamines), a family of polymers obtained by polyaddition of amines to bisacrylamides, and endowed with heparin-complexing ability. Some new poly(amidoamines) with more resistance towards hydrolytic degradation than traditional ones have been discovered. Other ter-amino polymers deriving from the polyaddition of ter-amino functionalized bis-thiols to bis-acrylic esters, or other activated unsaturated compounds, have been studied. Their quaternarization products have been proven, in a parallel work, to act as powerful antimicrobial agents. By performing in situ the polyaddition reaction, semi-interpenetrated networks based on silicone rubber and the same polymers have been prepared. Finally, end-functionalized amphiphilic oligomers have been prepared by radical polymerization techniques, and their use for enzyme modification considered.

Heparin adsorbing capacities at physiological pH of three poly(amido-amine) resins, and of poly(amido-amine)-surface-grafted glass microspheres.

Three poly(amido-amine)s of similar structure in the form of highly hydrophilic crosslinked resins, have been prepared, and tested for their heparin-adsorbing capacity at physiological pH. They showed different capacities, and their capacities were related to their basicities. One of the same polymers was grafted on the surface of glass microspheres. After treatment, it was shown that the microspheres could adsorb significant amounts of heparin. In all cases most of the adsorbed heparin was hardly eluted with saline, plasma, or blood, but could be recovered by eluting with 0.1 M NaOH. The resins were found to have some haemolytic properties, but no haemolysis was observed with the grafted microspheres.

All-trans retinoic acid and induction of apoptosis in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) represents a highly effective treatment for acute promyelocytic leukemia (M3-ANLL). This compound induces the leukemic promyelocytes to differentiate into morphologically and phenotypically mature myeloid cells. The mechanism of action of ATRA is far from fully understood. It has recently been reported that, along with its differentiation activity, ATRA causes apoptosis in the acute promyelocytic leukemia cell line HL-60. In this study we attempted to test whether ATRA is also able to induce apoptosis in fresh leukemic cells from M3-ANLL patients. Our results indicated that although morphological differentiation was detectable in 9/9 M3-ANLL samples after in vitro exposure to ATRA 10(-6) M, the percentage of apoptotic cells in the treated samples did not significantly differ from that obtained in controls (13.1% vs 9.4% respectively, after 8 days exposure). These data suggest that apoptosis does not seem to be the key mechanism by which ATRA exerts its action in M3-ANLL, at least at the blast cell level.