48 research outputs found
Aspetti immunologici di pazienti pediatrici con immunodeficienze primitive
La sindrome di DiGeorge (DGS) è causata da una delezione in emizigosi del locus 22q11.2 responsabile di difetti embriogenetici che determinano l’alterazione del timo e delle ghiandole paratiroidee, difetti cardiaci e anomalie facciali caratteristiche. In molti pazienti affetti da questa sindrome, il difetto immunitario è principalmente del compartimento T, sebbene siano state riportate anche anomalie quali disgammaglobulinemia, deficit di IgA e di cellule B della memoria. Sulla base dei dati immunologici, i pazienti DGS sono classificati in DGS completi (cDGS) e DGS parziali (pDGS): i cDGS corrispondono ad una rara forma di immunodeficienza grave combinata (0,5-1%), mentre i pDGS presentano una forma variabile di linfopenia T (da leggera a moderata). Dal punto di vista clinico, questi pazienti mostrano un ampio spettro d’infezioni insieme ad una disregolazione del sistema immunitario con manifestazioni atopiche e autoimmunitarie. I livelli cellulari T, sebbene ridotti, non sono predittivi del rischio di infezioni o autoimmunità .
Le alterazioni della distribuzione del repertorio del recettore delle cellule T (TCR) e il ridotto apporto timico nei pazienti pDGS è stato variabilmente associato ad un maggior rischio di infezioni o autoimmunità .
Per meglio definire alcuni aspetti della patogenesi e delle caratteristiche immunologiche, abbiamo studiato la cinetica della ricostituzione immunologica in una coorte di pazienti pDGS e in pazienti affetti da altre immunodeficienze primitive non direttamente riconducibili ad alterazioni del compartimento di cellule T (come pazienti con Malattia Granulomatosa Cronica - CGD). A tal fine sono state analizzate le cellule mononucleate del sangue periferico valutando:
1. Il fenotipo e le funzioni immunologiche tramite tecniche convenzionali.
2. La distribuzione del repertorio del TCR dei compartimenti cellulari T CD4+ e T CD8+ (TCRBV spectratyping).
3. L’immunofenotipo di maturazione delle cellule B.
4. La frequenza delle cellule nTreg.
La correlazione tra il profilo immunologico in vitro e le caratteristiche cliniche può aiutare a chiarire alcuni aspetti della patogenesi e del difetto immunologico, al fine di identificare dei possibili indicatori di suscettibilità verso le infezioni o verso lo sviluppo di autoimmunità .
Risultati e Discussione: I pazienti pDGS mostravano un ridotto numero di cellule T, sebbene non sia stata trovata una correlazione tra i bassi valori di cellule T e le infezioni ricorrenti. Il numero totale di cellule B nei pazienti pDGS e CGD era normale, sebbene sia stato osservato un significativo decremento di cellule B della memoria. La frequenza delle cellule T regolatorie naturali dei pazienti non mostrava differenze se confrontata con i valori dei controlli sani di pari età .
La risposta linfoproliferativa verso ogni stimolo era statisticamente diminuita in tutti i pazienti.
La distribuzione delle famiglie dei TCRBV è risultata perturbata, con maggiore rilevanza nelle cellule T CD8+ sia nei pazienti pDGS che in quelli CGD. In particolare, l’alterazione delle famiglie TCRBV ha mostrato una tendenza di normalizzazione nella distribuzione del repertorio delle cellule T (sia CD4+ che CD8+), come osservato anche in pazienti infetti da CMV. Le infezioni ricorrenti correlavano con una maggiore frequenza di alterazioni delle famiglie TCRBV nei pazienti pDGS ma non nei pazienti CGD.
Sebbene nella sindrome DiGeorge i bassi valori di cellule T non siano predittivi delle infezioni, le maggiori alterazioni delle famiglie TCRBV, così come l’immunodeficienza umorale e la ridotta frequenza di cellule B della memoria CD27+, risultavano essere associati a un maggiore rischio di infezioni in questi pazienti.
L’analisi dei pazienti CGD ha mostrato delle alterazioni significative sia nel compartimento cellulare T che in quello B, suggerendo che le alterazioni quantitative e qualitative trovate, possono contribuire alla eterogeneità del fenotipo clinico.
Ulteriori studi sono necessari per delucidare come il sistema della NADPH ossidasi è coinvolto nell’alterazione immunologica dei pazienti CGD.
In conclusione, le nostre analisi nelle PID hanno rilevato che altri compartimenti cellulari, oltre a quelli noti, possono essere coinvolti in queste malattie. Pertanto alcuni parametri come la distribuzione delle famiglie del TCRBV e la valutazione della maturazione delle cellule B dovrebbero essere usati come indicatori prognostici del rischio d’infezioni nel corso del follow-up per intraprendere eventuali terapie specifiche.DiGeorge syndrome (DGS) is caused by a deletion in hemizygosis of 22q11.2 locus responsible for embryogenesis defects causing alterations of thymus and parathyroid glands, cardiac defects and abnormal facial features. In most patients, the immune defect is basically in T cell subset although abnormalities such as dysgammaglobulinemia, IgA and memory B cells deficiency have been also reported.
On the basis of the immunologic findings, DGS patients are divided in complete DGS (cDGS), a rare form of severe combined immune deficiency (0.5-1%), and partial DGS (pDGS) presenting mild/moderate T cell lymphopenia. Clinically, these patients display a wide spectrum of infections together with a dysregulation of immune system as atopic and autoimmune manifestations. T cell levels, although reduced, are not predictive of the risk to develop infections or autoimmunity.
The alterations of the T cell receptor (TCR) repertoire distribution and impaired thymic output in pDGS have been variably associated to a higher risk of infections or autoimmunity.
In order to better define some aspects of the pathogenesis and immunological features we studied the kinetic of immune reconstitution in a cohort of pDGS patients and in patients affected by other primary immunodeficiencies not directly affecting T-cell compartments (as Chronic Granulomatous Disease patients) through peripheral blood mononuclear cells (PBMC) analysis of:
1. Phenotype and immunologic functions through standard techniques.
2. TCR repertoire distribution of T CD4+ and T CD8+ subsets (TCRVB spectratyping).
3. Immunophenotypic B-cell maturation.
4. Frequency of T regulatory cells.
The correlation between the in vitro immunological profile and the clinical features might help to clarify some aspects of the pathogenesis of the immunological defects, in order to identify possible prognostic markers of increased risk of susceptibility to infections or of development of autoimmunity.
Results and Discussion: pDGS patients exhibited decreased T-cell numbers, although no correlation was found between low T-cell values and recurrent infections. Total B-cell numbers in pDGS and CGD patients were normal, although a significantly decreased proportion of memory B cells was observed. No difference in natural T regulatory cells frequency was evident when compared with healthy controls in any groups.
A statistical reduced lymphoproliferative response to stimuli (PHA, OKT3 and PWM) in all patients was observed.
TCRBV family distribution resulted perturbed, with higher degree in CD8+ T-cell subset in both CGD and pDGS patients. Particularly, TCRBV family alterations in pDGS patients showed a trend of normalization in T cell repertoire distribution (both CD4+ and CD8+ T-cell), as observed in CMV congenital infected patients. Recurrent infections correlated with a high frequency of TCRBV family alterations in pDGS but not in CGD patients.
Although low T-cell values were not predictive of recurrent infections in DiGeorge syndrome, higher TCRBV family alterations, as well as humoral immunodeficiency and reduced CD27+ B cell memory frequency, were associated to an increased risk of infections in these patients.
Interestingly analysis of CGD patients showed significative alterations in both T and B cells compartments, suggesting that quantitative and qualitative alterations found, might contribute to the heterogeneity in the clinical phenotype.
Further studies are needed to elucidate how NADPH oxidase system is involved in CGD patients immune alterations.
In conclusion, our analysis on PID patients with well known defective cell compartments revealed that other subsets were also involved, therefore some parameters such as TCRBV family distribution and B-cell maturation could be used as further prognostic markers for follow-up and specific treatment
Ultrastructural Alterations of the Glomerular Filtration Barrier in Fish Experimentally Exposed to Perfluorooctanoic Acid
Per- and polyfluoroalkyl substances can be referred to as the most critical group of contaminants of emerging concern. They can accumulate in high concentration in the kidney and are known to potentially affect its function. Nonetheless, there is a lack of knowledge about their morphopathological effect on the glomerular filtration barrier. Since previous research suggests perfluorooctanoic acid (PFOA) induces glomerular protein leakage, the glomerular filtration barrier of 30 carp from the same parental stock (10 unexposed; 10 exposed to 200 ng L−1 of PFOA; and 10 exposed to 2 mg L−1 of PFOA for 56 days) was screened for possible PFOA-induced ultrastructural lesions in order to shed light on the related pathophysiology. PFOA exposure affected the glomerular filtration barrier in carp experimentally exposed to 2 mg L−1, showing ultrastructural alterations compatible with glomerulonephrosis: podocyte effacement, reduction of filtration slits and filtration slit diaphragms, basement membrane disarrangement, and occurrence of proteinaceous material in the urinary space.
The results of the present research confirm the glomerular origin of the PFOA-induced protein leakage and can contribute to the mechanistic comprehension of PFOA’s impact on renal function and to the assessment of the exposure effect of environmental pollutants on animals and humans, according to the One Health approach
CCR4+ skin-tropic phenotype as a feature of central memory CD8+ T cells in healthy subjects and psoriasis patients
The chemokine receptor CCR4 has emerged as a skin-homing molecule important for the migration of T cells from the blood to the dermis. From our previous data on psoriasis patients, CCR4+ memory T cells emerged as a putative recirculating population between skin and blood. Here we focused our attention on the expression of CCR4 and skin-tropic molecules in the different stages of memory T cell differentiation. We analyzed the chemokine receptor profile in CD8+ and CD4+ CD45RA−CCR7+ (TCM) and CD45RA−CCR7− (TEM) cells. Subpopulations were further divided on the basis of CD62L expression, and the distribution among the subsets of the skin-homing molecule CLA (Cutaneous Lymphocyte Antigen) was evaluated. The characterization was performed on peripheral blood mononuclear cells isolated from 21 healthy subjects and 24 psoriasis patients. The results indicate that (i) the skin-homing CCR4 marker is mainly expressed in TCM cells, (ii) CCR4+ TCM cells also express high level of CLA and that (iii) the more differentiated phenotype TEM expresses CXCR3 and CCR5 but lower level of CCR4 and CLA. This indicates that progressive stages of memory T cell differentiation have profoundly different chemokine receptor patterns, with CD8+ TCM displaying a marked skin-tropic phenotype CLA+CCR4+. Differential skin-tropic phenotype between TCM and TEM cells was observed in both healthy subjects and psoriasis patients. However, patients showed an expanded circulating population of CD8+ TCM cells with phenotype CCR4+CXCR3+ that could play a role in the pathophysiology of psoriasis and possibly in disease recurrence
Cell cycle block by p53 activation reduces SARS-CoV-2 release in infected alveolar basal epithelial A549-hACE2 cells
SARS-CoV viruses have been shown to downregulate cellular events that control antiviral defenses. They adopt several strategies to silence p53, key molecule for cell homeostasis and immune control, indicating that p53 has a central role in controlling their proliferation in the host. Specific actions are the stabilization of its inhibitor, MDM2, and the interference with its transcriptional activity. The aim of our work was to evaluate a new approach against SARS-CoV-2 by using MDM2 inhibitors to raise p53 levels and activate p53-dependent pathways, therefore leading to cell cycle inhibition. Experimental setting was performed in the alveolar basal epithelial cell line A549-hACE2, expressing high level of ACE2 receptor, to allow virus entry, as well as p53 wild-type. Cells were treated with several concentrations of Nutlin-3 or RG-7112, two known MDM2 inhibitors, for the instauration of a cell cycle block steady-state condition before and during SARS-CoV-2 infection, and for the evaluation of p53 activation and impact on virus release and related innate immune events. The results indicated an efficient cell cycle block with inhibition of the virion release and a significant inhibition of IL-6, NF-kB and IFN-λ expression. These data suggest that p53 is an efficient target for new therapies against the virus and that MDM2 inhibitors deserve to be further investigated in this field
Purinergic Signaling in Controlling Macrophage and T Cell Functions During Atherosclerosis Development
Atherosclerosis is a hardening and narrowing of arteries causing a reduction of blood flow. It is a leading cause of death in industrialized countries as it causes heart attacks, strokes, and peripheral vascular disease. Pathogenesis of the atherosclerotic lesion (atheroma) relies on the accumulation of cholesterol-containing low-density lipoproteins (LDL) and on changes of artery endothelium that becomes adhesive for monocytes and lymphocytes. Immunomediated inflammatory response stimulated by lipoprotein oxidation, cytokine secretion and release of pro-inflammatory mediators, worsens the pathological context by
amplifying tissue damage to the arterial lining and increasing flow-limiting stenosis. Formation of thrombi upon rupture of the endothelium and the fibrous cup may also occur, triggering thrombosis often threatening the patient’s life. Purinergic signaling, i.e., cell responses induced by stimulation of P2 and P1 membrane receptors for the extracellular nucleotides (ATP, ADP, UTP, and UDP) and nucleosides (adenosine), has been implicated in modulating the immunological response in atherosclerotic cardiovascular disease. In this review we will describe advancements in the understanding of purinergic modulation of the two main immune cells involved in
atherogenesis, i.e., monocytes/macrophages and T lymphocytes, highlighting modulation of pro- and anti-atherosclerotic mediated responses of purinergic signaling in these cells and providing new insights to point out their potential clinical significance
Purinergic Signaling and Inflammasome Activation in Psoriasis Pathogenesis
Psoriasis is a chronic inflammatory disease of the skin associated with systemic and joint manifestations and accompanied by comorbidities, such as metabolic syndrome and increased risk of cardiovascular disease. Psoriasis has a strong genetic basis, but exacerbation requires additional signals that are still largely unknown. The clinical manifestations involve the interplay between dendritic and T cells in the dermis to generate a self-sustaining inflammatory loop around the TNFα/IL-23/IL-17 axis that forms the psoriatic plaque. In addition, in recent years, a critical role of keratinocytes in establishing the interplay that leads to psoriatic plaques’ formation has re-emerged. In this review, we analyze the most recent evidence of the role of keratinocytes and danger associates molecular patterns, such as extracellular ATP in the generation of psoriatic skin lesions. Particular attention will be given to purinergic signaling in inflammasome activation and in the initiation of psoriasis. In this phase, keratinocytes’ inflammasome may trigger early inflammatory pathways involving IL-1β production, to elicit the subsequent cascade of events that leads to dendritic and T cell activation. Since psoriasis is likely triggered by skin-damaging events and trauma, we can envisage that intracellular ATP, released by damaged cells, may play a role in triggering the inflammatory response underlying the pathogenesis of the disease by activating the inflammasome. Therefore, purinergic signaling in the skin could represent a new and early step of psoriasis; thus, opening the possibility to target single molecular actors of the purinome to develop new psoriasis treatments
Role of vitamin D in the pathogenesis of atheromatosis
Background and Aims
Cardiovascular disease is the main cause of death worldwide, but the collective efforts to prevent this pathological condition are directed exclusively to individuals at higher risk due to hypercholesterolemia, hypertension, obesity, diabetes. Recently, vitamin D deficiency was identified as a risk factor for cardiovascular disease in healthy people, as it predisposes to different vascular dysfunctions that can result in plaque development and fragility. In this scenario, the fundamental aim of the study was to reproduce a disease model inducing vitamin D deficiency and atheromatosis in ApoE-/- mice and then to evaluate the impact of this vitamin D status on the onset/progression of atheromatosis, focusing on plaque formation and instability.
Methods and Results
In our murine disease model, vitamin D deficiency was achieved by 3 weeks of vitamin D deficient diet along with intraperitoneal paricalcitol injections, while atheromatosis by western-type diet. Under these experimental conditions, vitamin D deficient mice developed more unstable atheromatous plaques with reduced or absent fibrotic cap. Since calcium and phosphorus metabolism and also cholesterol and triglycerides systemic concentration were not affected by vitamin D level, our results highlighted the role of vitamin D deficiency in the formation/instability of atheromatous plaque and, although further studies are needed, suggested a possible intervention with vitamin D to prevent or delay the atheromatous disease.
Conclusions
The data obtained open the question about the potential role of the vitamins in the pharmacological treatments of cardiovascular disorders as coadjutant of the primary drugs used for these pathologies
Anticancer Activity of Aqueous Extracts from Asparagus officinalis L. Byproduct on Breast Cancer Cells
Cultivation of asparagus (Asparagus officinalis L.; Asp) for food and medicinal use has taken place since the early Roman Empire. Today, Asp represents a worldwide diffuse perennial crop. Lower portions of the spears represent a food industry waste product that can be used to extract bioactive molecules. In this study, aqueous extracts derived from the non-edible portion of the plant (hard stem) were prepared and characterized for chemical content. Furthermore, the biocompatibility and bioactivity of Asp aqueous extracts were assessed in vitro on normal fibroblasts and on breast cancer cell lines. Results showed no interference with fibroblast viability, while a remarkable cytostatic concentration-dependent activity, with significant G1/S cell cycle arrest, was specifically observed in breast cancer cells without apoptosis induction. Asp extracts were also shown to significantly inhibit cell migration. Further analyses showed that Asp extracts were characterized by specific pro-oxidant activity against tumoral cells, and, importantly, that their combination with menadione resulted in a significant enhancement of oxidants production with respect to menadione alone in breast cancer cells but not in normal cells. This selectivity of action on tumoral cells, together with the easiness of their preparation, makes the aqueous Asp extracts very attractive for further investigation in breast cancer research, particularly to investigate their role as possible co-adjuvant agents of clinical drug therapies
HelixComplex snail mucus exhibits pro-survival, proliferative and pro-migration effects on mammalian fibroblasts.
Snail mucus is a mixture of active substances commonly thought to have healthy properties for the treatment of skin disorders. Although snail mucus is an ingredient of several cosmetic and para-pharmaceutic products, a comprehensive characterization of chemical composition and biological effects are still missing. Crude purified extracts from Helix aspersa muller mucus (HelixComplex®) were prepared and, after chemical characterization, tested on in vitro experimental models. Differently from what expected, HelixComplex® was characterized by the presence of small amounts of glycolic acid and allantoin. By using different in vitro assays on fibroblast cultures, we found that HelixComplex® lacked of cytotoxicity, protected cells from apoptosis (p<0.05) and, importantly, was able to significantly induce cell proliferation and migration through direct and indirect mechanisms. These effects were associated to morphological changes, cytoskeleton re-organization and release of cytokines. In conclusion, our findings suggest that snail mucus biological effects are attributable to cell proliferation and migration, and pave the way for further investigating snail mucus potential as therapeutic agent
Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1
Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH2++•2DCA- cocrystal exhibited enhanced in vitro anti-leukemic activity, with respect to the treatment with the mix consisting of Metformin plus DCA. In particular, the treatment with the cocrystal MetH2++•2DCA- induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL