28 research outputs found
Basi cellulari e molecolari della Graft-Versus-Host Desease
Get PDFBasi cellulari e molecolari della Graft-Versus-Host Disease
La chronic Graft-Versus-Host Disease (cGVHD) è la causa principale di mortalità tardiva in soggetti sottoposti a trapianto allogenico di cellule staminali. Tale condizione si presenta con quadri clinici proteiformi, che ricordano molte patologie autoimmuni; fra le tipologie clinico-patologiche più frequenti vi è la cosiddetta “cGVHD scleroderma-like”, che coinvolge principalmente il derma e più raramente di altri organi, ed è caratterizzata da lesioni simili a quelle presenti nella Sclerodermia Sistemica (SSc). Platelet-Derived Growth Factor (PDGF) e Transforming Growth Factorβ (TGFβ) sono le due citochine principali nell’induzione di processi infiammatori con esito fibrotico. Nei soggetti che sviluppano cGVHD, il nostro gruppo di ricerca ha precedentemente evidenziato il ruolo patogenetico svolto dall’asse PDGF-R e dall’attivazione intracellulare della sintesi del collagene associati alla presenza di anticorpi stimolanti anti-PDGF-R, analogamente a quanto avviene nei pazienti affetti da SSc. Gli inibitori delle tirosin-chinasi (TKI), come Imatinib e Nilotinib, bloccano sia dell’asse PDGF-R che del TGFβ ed esercitano un potente effetto antifibrotico in vitro; il nostro gruppo ne ha evidenziato l’attività in pazienti con cGVHD scleroderma-like. Oltre all’azione antifibrotica, il Nilotinib appare anche in grado di modulare l’immunità innata ed adattativa.
Il presente studio è stato condotto con lo scopo di approfondire due aspetti significativi per la comprensione della patogenesi della cGVHD cutanea: la caratterizzazione dei fibroblasti della cute patologica (GVHD-Fbs) e l’azione del Nilotinib sui fibroblasti e sulle cellule immunocompetenti.
I risultati ottenuti mostrano che i GVHD-Fbs in coltura, pur apparendo morfologicamente e fenotipicamente indistinguibili dai fibroblasti normali (n-Fbs), si espandono più lentamente ed arrivano alla senescenza cellulare dopo un minor numero di passaggi. L’analisi dell’espressione genica delle catene del collagene evidenzia che COL1α1 e COL1α2 vengono espressi rispettivamente 4 e 1,6 volte in più nei GVHD-Fbs rispetto ai n-Fbs (p=0,02). Contrariamente a quanto avviene dei n-Fbs, i GVHD-Fbs non mostrano variazioni significative nell’espressione dei geni del collagene se stimolati con la TGFβ. L’aggiunta di Nilotinib a dosi terapeutiche (1μM), riduce l’espressione di COL1α1 e di COL1α2 da parte dei GVHD-Fbs rispettivamente dell’86,5% e del 49% (p<0,01); la vitalità delle cellule T regolatorie e delle altre sottopopolazioni linfocitarie non risulta compromessa, ma la concentrazione delle citochine pro-infiammatorie nei surnatanti delle colture cellulari appare ridotta.
Il presente studio contribuisce pertanto a chiarire i meccanismi che governano lo sviluppo di fibrosi nei pazienti con cGVHD e suggerisce il potenziale utilizzo di Nilotinib come agente antifibrotico nella pratica clinica.Cellular and molecular basis of Graft-Versus-Host Disease
Chronic Graft Versus Host Disease (cGVHD) is a major complication of allogeneic stem-cell transplantation and is characterized by frequent multi-organ involvement that resembles the autoimmune diseases. Among the different clinical presentations of cGVHD, the "cGVHD scleroderma-like", is characterized by dermal fibrosis and sclerosis similar to Systemic Scleroderma (SSc). Our group previously demonstrated that SSc and cGVHD share common pathogenetic features; in both diseases stimulating anti-PDGF-R antibodies were found, leading to abnormal collagen production by fibroblasts eventually contributing to organ damage. Another pivotal cytokine involved in fibrosis is TGFβ that induces collagen production via c-Abl activation. Tyrosine kinase inhibitors (TKI) Imatinib and Nilotinib are potent inhibitor of these two cytokines and exert strong antifibrotic effects in vitro. In addition, Nilotinib regulates both innate and adaptive immune response by interacting with many cell population such as T-cells, B-cells, dendritic cells, mast cells and macrophages. The present study investigated two significant aspects of the pathogenesis of skin cGVHD: the cellular and molecular features of pathological skin fibroblasts (GVHD-Fbs) and the efficacy of Nilotinib on fibrosis modulation and on the lymphocyte regulation. Result showed that although GVHD-Fbs are morphologically and phenotypically similar to normal fibroblasts (n-FBS), they grow up slower and became senescent faster in culture. Gene expression analysis of collagen type 1 chains in GVHD-Fbs showed that express COL1α1 and COL1α2 is respectively 4 and 1,6 times highter compared to n-FBS (p = 0.02). Stimulation with TGFβ, did not change collagen expression in GVHD-Fbs, differently from what observed in n-FBS. Therapeutic doses of Nilotinib (1μM) induced 86,5% and 49% reduction of COL1α1 and COL1α2 expression, respectively (p <0.01). Finally, therapeutic doses of Nilotinib did not impair the function of regulatory T cells and other lymphocyte populations, but reduced the concentration of pro-inflammatory cytokines in cell cultures supernatants.
In conclusion, the present study provides some insights into the mechanisms underlying the development of fibrosis during cGVHD and suggests a potential use of Nilotinib as anti-fibrotic agent in this clinical setting
Bone Marrow Adipose Tissue
Get PDFBone marrow (BM) acts as a dynamic organ within the bone cavity, responsible for hematopoiesis, skeletal remodeling, and immune system control. Bone marrow adipose tissue (BMAT) was long simply considered a filler of space, but now it is known that it instead constitutes an essential element of the BM microenvironment that participates in homeostasis, influences bone health and bone remodeling, alters hematopoietic stem cell functions, contributes to the commitment of mesenchymal stem cells, provides effects to immune homeostasis and defense against infections, and participates in energy metabolism and inflammation. BMAT has emerged as a significant contributor to the development and progression of various diseases, shedding light on its complex relationship with health. Notably, BMAT has been implicated in metabolic disorders, hematological malignancies, and skeletal conditions. BMAT has been shown to support the proliferation of tumor cells in acute myeloid leukemia and niche adipocytes have been found to protect cancer cells against chemotherapy, contributing to treatment resistance. Moreover, BMAT's impact on bone density and remodeling can lead to conditions like osteoporosis, where high levels of BMAT are inversely correlated with bone mineral density, increasing the risk of fractures. BMAT has also been associated with diabetes, obesity, and anorexia nervosa, with varying effects on individuals depending on their weight and health status. Understanding the interaction between adipocytes and different diseases may lead to new therapeutic strategies
The value of networks in the governance of banks
No full textThe economic literature points out the impact of corporate networks on corporate decisions and firm performance. Corporate networks can arise from direct or indirect ownership connections among firms or from personal relationships of firm members, such as board interlocks or social interactions among managers and directors, due, for instance, to shared education or employment experiences. While the topic is of interest for any kind of corporation, only limited attention has been given to bank networks and their impact on bank governance and performance. Especially following the 2007-2008 financial crisis, such investigation has overwhelmingly become crucial given the importance of understanding the effect of governance mechanisms and network strategies on bank risk-appetite, performance and stability. Therefore, this paper provides a comprehensive review of the empirical evidence on how banks’ network affects their corporate choices and performance and, more importantly, points out the need of further empirical investigation in order to better assess the relevance of informal personal connections on banks’ performance in terms of both relationships within the board of directors, and between the board of directors and the CEO. Indeed, tracing the impact on banks’ performance of the strength and depth of personal connections could also support the supervisory authorities in controlling also for informal interlocking
Dose-dependent effects of curcumin on 22Rv1 prostate cancer cell line
Get PDFBackground: Prostate cancer (PCa) is the second most frequent cancer type in the male population over 66 years. Curcumin is a polyphenolic natural compound extract from the rhizomes of Curcuma longa Linn (Zingiberaceae family) which showed important anticancer effects by inhibiting cell proliferation and inducing apoptosis in several cancer types. Recently, some studies reported that oral curcumin lowered PSA levels, but it did not modify the clinical outcomes in patients with prostate cancer who received intermittent androgen deprivation (IAD). Other studies reported that high concentrations of curcumin were toxic for patients. Methods and results: In this study we showed that low doses of curcumin can induce senescence-like effects in 22Rv1 cell line while higher concentrations have cytotoxic effects. Five,15 and 30 μM curcumin blocked cell cycle in G2/M phase but only 15 and 30 μM curcumin induced cell death. In addition, an increased expression of p21, a known senescence marker, was detected in 22Rv1 cells treated with curcumin in every experimental condition. However, the expression of p16, another known senescence marker, increased only to 30 μM curcumin. Conclusion: In the context of personalized approach in PCa care, we suggest that the appropriate concentration of curcumin used in combination with radiotherapy or with androgen deprivation therapy (ADT) could be taken into consideration
In vitro comparison of different TKI activity in T-Cell populations: selective sparing of Treg by Nilotinib.
No full textIntroduction: Chronic Graft Versus Host Disease (cGVHD) is a major complication of allogeneic stem-cell transplantation and is characterized by frequent multi-organ involvement that resembles the autoimmune diseases. Donor-derived CD4+ and CD8+ T lymphocytes have classically been considered to be the main effector cells mediating GVHD pathogenesis. Indeed, removal of T cells from transplant inocula almost completely prevents GVHD developing, at the price of increased incidences of graft rejection and disease recurrence. However recent studies suggest that B cells might also play an important role in the biology of cGVHD. The role of Treg lymphocytes in the pathogenesis of cGVHD is still controversial and the tyrosine kinase inhibitor′s (TKI) role in the modulation of this pathway is not yet fully characterized. In vitro data confirm that TKIs regulates both innate and adaptive immune response by interacting with many cell population such as T-cells, B-cells, dendritic cells, mast cells and macrophages. According to these observations, we investigated the TKI′s immunomodulatory effects (Nilotinib, Dasatinib, Imatinib, Ponatinib) on lymphocyte populations.
Materials and Methods: Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Biocoll. Cells were cultured in RPMI 1640 at a concentration 1x106 cell/well. Nilotinib, Imatinib, Dasatinib and Ponatinib were added to cell cultures at serial concentration (Imatinib:1μM,10μM,50μM; Nilotinib:0.5μM,2μM,10μM; Dasatinib:50nM,100nM,200nM; Ponatinib:1nM,10nM,50nM,100nM) on the first day. Six-color flow cytometry analysis (Facs Canto II) was performed on the cells harvested after 96 h cultures using conjugated antibodies (CD3,CD4,CD16,CD56,CD3,CD25,CD19,CD45RA,FoxP3,CD127,7-Aminoactinomycin-D), for cell cycle analysis cells were stained with propidium iodide. For cytokine analysis, supernatants were collected and analyzed for cytokines according to the instruction of Bio-Plex Pro Human Cytokine 17-plex Assay with Bio-Plex (Bio-Rad).
Results: A significant decrease of cytotoxic T cells viability was observed when cells were cultured in presence of Imatinib (50μM,p<0.01), Ponatinib (10nM,p<0.05) and Dasatinib (100nM,p<0.01). On the contrary, exposure to Nilotinib didn′t induce cell death. Increasing concentrations of all the tested TKI significantly inhibited T cell proliferation in a dose-dependent manner; the effect become statistically significant starting from Imatinib (1μM,p<0.05), Dasatinib (50nM,p<0.01), Ponatinib (50nM,p<0.01) and Nilotinib (0.5μM,p<0.01). Exposure to Imatinib, Dasatinib and Ponatinib induced a statistically significant decrease (p<0.01) of Treg cells proportion, even at the lowest drug concentration in culture; Nilotinib induced Treg decrease only at concentrations exceeding 2μM (p<0.01), higher than those usually achieved in clinical practice. A significant increase of naive Treg apoptosis was observed after exposure to Dasatinib (50nmM,p<0.01), Ponatinib (50nM,p<0.01) and Imatinib (50μM,p<0.01); exposure to Nilotinib has no effect on this population. Both Nilotinib and Dasatinib induced a profound inhibition of pro-inflammatory cytokine production (in particular TNFα, IFNγ, IL13 and IL17) when added to the cell cultures (p<0.05); slower decrease in supernatant cytokine concentration was observed in presence of either Imatinib (50μM,p<0.05) and Ponatinib (50nM,p<0.05). Increasing concentrations of all TKIs except Nilotinib induced a significant decline of NK cells (p<0.01) and B cell (p<0.01).
Conclusion: The present study focuses the peculiar Nilotinib activity on lymphocyte′s regulation: this TKI, at therapeutic concentrations in vitro, interact with innate and adaptive immune response show anti-inflammatory properties. Unlike other TKIs, Nilotinib determine inflammatory cytokines reduction, preserving T cell population and Treg. These data support the potential use of Nilotinib in cGVH
Specific Anti-Nuclear Antibodies, Clinical Features and Outcome Following Treatment with Nilotinib In Patients with Chronic Graft-Versus-Host Disease. The 44th Annual Meeting of the European Society for Blood and Marrow Transplantation: Physicians Poster Sessions
No full textBackground: Clinical manifestations of chronic graft versus-host diseases (cGVHD) often resemble systemic autoimmune diseases. The association with autoantibodies, including anti-nuclear antibodies (ANA), has been reported, but the clinical and prognostic relevance of this association
is still unclear. Tyrosine kinase inhibitor nilotinib is currently under investigation for the treatment of cGVHD; we sought to investigate the relationship between presence of ANA and outcome of cGVHD following administration of nilotinib.
Methods: Fifteen patients with severe cGVHD following hematopoietic stem cell transplantation (HSCT) and nine transplanted patients without cGVHD (controls) were evaluated. Clinical data were collected by patients ́ files. A panel of 19 specific ANA was assessed by quantitative immunoblot assay (Alifax, Padova, Italy). Proportions of T cell subpopulations (including T helper, T cytotoxic and T regulatory), B lymphocytes, NK and NK/T cells were
assessed by flow cytometry (Bekton Dickinson, San Josè, CA). Data were compared by Student ́s t test; differences were considered statistically significant when p < 0.05.
Results: In patients who developed cGVHD, organs more commonly involved were skin (80%), eyes/mouth (Sicca Syndrome, 67%) and lungs (33%); ANA were detected in seven of these patients (47%, figure panel A). Most frequent specificity was anti-SSA/Ro (3 cases); other specificities were anti-SSB, anti-CENP A/B, anti-Nucleosome and anti-Pm-Scl (1 case each). Sicca Syndrome was the clinical manifestation more associated with the presence of ANA (7/10 cases, 70%), followed by skin disease (5/12 cases, 42%). Only one of the controls (11%) was tested ANA-positive (anti Histones/PCNA/DFS-70, figure panel A); of note, this patient suffered from isolated xerostomia. No difference in timing of cGVHD onset following HSCT was observed between ANA-positive and ANA-negative patients (267 vs. 280 days, p = 0.88). Compared to controls (figure panels BE), cGVHD patients had significantly higher proportions of T and NK/T cells (p < 0.05) and lower proportions of B (p=0.06) and NK cells (p < 0.05). CD4/CD8 ratio (not shown) as well as T regulatory cells (figure panel F) were comparable between the two groups; however, this latter subset showed a trend toward higher values among ANApositive compared to ANA-negative cGVHD patients (10,7±7.9% vs. 3.4±1.4%, p = 0.12). No difference in cGVHD progression nor in response to nilotinib was observed between ANA-positive and ANA-negative patients; after six months of treatment, eight (4 ANA-positive) remained stable and the other seven (3 ANA-positive) improved.
Conclusions: In agreement with published data, presence of ANA was frequent among patients who developed cGVHD and uncommon among transplanted patients without this condition. Most common cGVHD clinical features were skin disease and Sicca Syndrome; the latter was also the most frequent manifestation among ANA-positive cGVHD individuals. Although the frequency of B cells was not increased among patients developing cGVHD, these data suggest that B cells may play a pathogenic role in the onset of this condition and that presence of ANA may predict the development of particular clinical manifestations, such as Sicca Syndrome. Six-months course of nilotinib stabilized or improved cGVHD in all the study patients, independently of the presence of ANA. The evaluation of autoantibodies and immune population after nilotinib treatment is ongoing
Novel Translational Read-Through–Inducing Drugs as a Therapeutic Option for Shwachman-Diamond Syndrome
Get PDFShwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS protein expression in bone marrow stem cells isolated from SDS patients carrying the nonsense mutation K62X. In this study, the authors evaluated the effect of a panel of ataluren analogues in restoring SBDS protein resynthesis and function both in hematological and non-hematological SDS cells. Besides confirming that ataluren can efficiently induce SBDS protein re-expression in SDS cells, the authors found that another analogue, namely NV848, can restore full-length SBDS protein synthesis as well, showing very low toxicity in zebrafish. Furthermore, NV848 can improve myeloid differentiation in bone marrow hematopoietic progenitors, enhancing neutrophil maturation and reducing the number of dysplastic granulocytes in vitro. Therefore, these findings broaden the possibilities of developing novel therapeutic options in terms of nonsense mutation suppression for SDS. Eventually, this study may act as a proof of concept for the development of similar approaches for other IBMFS caused by nonsense mutations
Modulation of matrix metalloproteases by ciliary neurotrophic factor in human placental development
No full textCiliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor complex containing a specific subunit, CNTF receptor α (CNTFRα). The two molecules are constitutively expressed in key structures for human placental growth and differentiation. The possible role of CNTF in enhancing cell proliferation and/or invasion during placental development and remodelling was investigated using HTR-8/SVneo and BeWo cells, taken respectively as cytotrophoblast and syncytiotrophoblast models. In both cell lines, treatment with human recombinant (hr) CNTF activated JAK2/STAT3 signalling and inhibited the ERK pathway. Interestingly, in HTR-8/SVneo cells, 50 ng hrCNTF induced significant downregulation of matrix metalloprotease (MMP)-1 and significant upregulation of MMP-9. Moreover, pharmacological inhibition of JAK2/STAT3 signalling by AG490 and curcumin resulted in MMP-9 downregulation; it activated the ERK signalling pathway and upregulated MMP-1 expression. Collectively, these data suggest a role for CNTF signalling in extravillous cytotrophoblast invasion through the modulation of specific MMPs
Immunomodulatory Effects of Tyrosine Kinase Inhibitor In Vitro and In Vivo Study
Get PDFPathogenesis of chronic graft-versus-host disease (cGVHD) is incompletely defined, involving donor-derived CD4 and CD8-positive T lymphocytes as well as B cells. Standard treatment is lacking for steroid-dependent/refractory cases; therefore, the potential usefulness of tyrosine kinase inhibitors (TKIs) has been suggested, based on their potent antifibrotic effect. However, TKIs seem to have pleiotropic activity. We sought to evaluate the in vitro and in vivo impact of different TKIs on lymphocyte phenotype and function. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in the presence of increasing concentrations of nilotinib, imatinib, dasatinib, and ponatinib; in parallel, 44 PBMC samples from 15 patients with steroid-dependent/refractory cGVHD treated with nilotinib in the setting of a phase I/II trial were analyzed at baseline, after 90, and after 180 days of therapy. Flow cytometry was performed after labeling lymphocytes with a panel of monoclonal antibodies (CD3, CD4, CD16, CD56, CD25, CD19, CD45RA, FoxP3, CD127, and 7-amino actinomycin D). Cytokine production was assessed in supernatants of purified CD3+ T cells and in plasma samples from nilotinib-treated patients. Main T lymphocyte subpopulations were not significantly affected by therapeutic concentrations of TKIs in vitro, whereas proinflammatory cytokine (in particular, IL-2, IFN-γ, tumor necrosis factor-α, and IL-10) and IL-17 production showed a sharp decline. Frequency of T regulatory, B, and natural killer (NK) cells decreased progressively in presence of therapeutic concentrations of all TKIs tested in vitro, except for nilotinib, which showed little effect on these subsets. Of note, naive T regulatory cell (Treg) subset accumulated after exposure to TKIs. Results obtained in vivo on nilotinib-treated patients were largely comparable, both on lymphocyte subset kinetics and on cytokine production by CD3-positive cells. This study underlines the anti-inflammatory and immunomodulatory effects of TKIs and supports their potential usefulness as treatment for patients with steroid-dependent/refractory cGVHD. In addition, both in vitro and in vivo data point out that compared with other TKIs, nilotinib could better preserve the integrity of some important regulatory subsets, such as Treg and NK cells
