49 research outputs found
Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation
Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field
A pH-sensitive stearoyl-PEG-poly(methacryloyl sulfadimethoxine)-decorated liposome system for protein delivery: an application for bladder cancer treatment
Stealth pH-responsive liposomes for the delivery of therapeutic proteins to the bladder epithelium were prepared using methoxy-poly(ethylene glycol)5kDa-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG5kDa-DSPE) and stearoyl-poly(ethylene glycol)-poly(methacryloyl sulfadimethoxine) copolymer (stearoyl-PEG-polySDM), which possesses an apparent pKa of 7.2. Liposomes of 0.2:0.6:100, 0.5:1.5:100 and 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/(soybean phosphatidylcholine + cholesterol) molar ratios were loaded with bovine serum albumin (BSA) as a protein model. The loading capacity was 1.3% w/w BSA/lipid. At pH 7.4, all liposome formulations displayed a negative zeta-potential and were stable for several days. By pH decrease or addition to mouse urine, the zeta potential strongly decreased, and the liposomes underwent a rapid size increase and aggregation. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) analyses showed that the extent of the aggregation depended on the stearoyl-PEG-polySDM/lipid molar ratio. Cytofluorimetric analysis and confocal microscopy showed that at pH 6.5, the incubation of MB49 mouse bladder cancer cells and macrophages with fluorescein isothiocyanate-labelled-BSA (FITC-BSA) loaded and N-(Lissamine Rhodamine B sulfonyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (rhodamine-DHPE) labelled 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/lipid molar ratio liposomes resulted in a time-dependent liposome association with the cells. At pH 7.4, the association of BSA-loaded liposomes with the MB49 cells and macrophages was remarkably lower than at pH 6.5. Confocal images of bladder sections revealed that 2 h after the instillation, liposomes at pH 7.4 and control non-responsive liposomes at pH 7.4 or 6.5 did not associate nor delivered FITC-BSA to the bladder epithelium. On the contrary, the pH-responsive liposome formulation set at pH 6.5 and soon administered to mice by bladder instillation showed that, 2 h after administration, the pH-responsive liposomes efficiently delivered the loaded FITC-BSA to the bladder epitheliu
Helicobacter pylori affects the antigen presentation activity of macrophages modulating the expression of the immune receptor CD300E through miR-4270
The Helicobacter cinaedi antigen CAIP participates in atherosclerotic inflammation by promoting the differentiation of macrophages in foam cells
Recent studies have shown that certain specific microbial infections participate in atherosclerosis by inducing inflammation and immune reactions, but how the pathogens implicated in this pathology trigger the host responses remains unknown. In this study we show that Helicobacter cinaedi (Hc) is a human pathogen linked to atherosclerosis development since at least 27% of sera from atherosclerotic patients specifically recognize a protein of the Hc proteome, that we named Cinaedi Atherosclerosis Inflammatory Protein (CAIP) (n = 71). CAIP appears to be implicated in this pathology because atheromatous plaques isolated from atherosclerotic patients are enriched in CAIP-specific T cells (10%) which, in turn, we show to drive a Th1 inflammation, an immunopathological response typically associated to atherosclerosis. Recombinant CAIP promotes the differentiation and maintenance of the pro-inflammatory profile of human macrophages and triggers the formation of foam cells, which are a hallmark of atherosclerosis. This study identifies CAIP as a relevant factor in atherosclerosis inflammation linked to Hc infection and suggests that preventing and eradicating Hc infection could reduce the incidence of atherosclerosis.Peer reviewe
Treponema pallidum (syphilis) antigen TpF1 induces angiogenesis through the activation of the IL-8 pathway
Over 10 million people every year become infected by Treponema pallidum and develop syphilis, a disease with broad symptomatology that, due to the difficulty to eradicate the pathogen from the highly vascularized secondary sites of infection, is still treated with injections of penicillin. Unlike most other bacterial pathogens, T. pallidum infection produces indeed a strong angiogenic response whose mechanism of activation, however, remains unknown. Here, we report that one of the major antigen of T. pallidum, the TpF1 protein, has growth factor-like activity on primary cultures of human endothelial cells and activates specific T cells able to promote tissue factor production. The growth factor-like activity is mediated by the secretion of IL-8 but not of VEGF, two known angiogenic factors. The pathogen's factor signals IL-8 secretion through the activation of the CREB/NF-\u3baB signalling pathway. These findings are recapitulated in an animal model, zebrafish, where we observed that TpF1 injection stimulates angiogenesis and IL-8, but not VEGF, secretion. This study suggests that the angiogenic response observed during secondary syphilis is triggered by TpF1 and that pharmacological therapies directed to inhibit IL-8 response in patients should be explored to treat this disease
Role of the protein NapA produced by Borrelia burgdorferi in the pathogenesis of Lyme arthritis
Borrelia burgdorferi is a spirochetal bacterium responsible for the human Lyme disease, a multisistemic illness transmitted by ticks. This pathology was described for the first time at the beginning of nineteens. Lyme borreliosis is due to three related species B. burgdorferi, B. afzelii and B. garinii; in Europe the disease is caused by the three species whereas in the U.S.A. B. burgdorferi is the sole cause. Lyme disease is a multisistemic illness and can be divided in three stages: the first one is characterized by a localized infection of the skin in the site of tick’s bite, the second one is the dissemination of the spirochetes and the last stage consists in a persistent infection. Lyme disease usually begins with a characteristic expanding skin lesion named erythema migrans. After days or weeks, the spirochetae may spread to different sites of the host involving heart, muscles and nervous system causing different clinical manifestation. Months later, in untreated patients, the infection could chronicize causing arthritis, affecting joints, in particular knee.
The aim of this project is to contribute in understanding which are the molecular and cellular mechanisms involved in the development of arthritis. Now it is established that the host immune response to B. burgdorferi influences the clinical outcome of the infection. In particular, until a few years ago, T lymphocytes, particularly IFN- secreting Th1 cells, were proposed to play a central role in Lyme arthritis. Recently, it was demonstrated, using a mouse model, that Th1 cells were not essential in inducing Lyme arthritis, suggesting the involvement of mediators different from IFN- and IL-12; consequentially a different T cell subsets is supposed to be involved in the pathogenesis of the desease. The attention was focused on a recent subset of T helper lymphocytes: Th17 cells. Th17 cells play a crucial role in the induction of autoimmune tissue injury via IL-17 release. IL-17 induces the release of several pro-inflammatory cytokines and chemokines by stromal cells, synoviocytes, chondrocytes, fibroblasts and macrophages, and recruits and activates neutrophils. Moreover, IL-17 is elevated in synovial fluids of patients with rheumatoid arthritis, where it promotes formation of osteoclasts and several experimental observations suggest its involvement in the pathogenesis of murine Lyme arthritis.
A major unanswered question remained that of the chemical nature of the bacterial factor(s) responsible for the induction of Th17 cells. Previously, in our laboratory, we have characterized a protein produced by Helicobacter pylori, a bacterium that causes chronic infections in humans, as B. burgdorferi does, termed HP-NAP (H. pylori Neutrophil Activating Protein). This protein is a strong immunogen and is endowed with immunomodulatory properties. Borrelia burgdorferi produces a protein which is homologue to HP-NAP, and the sole functional characterization had shown to be a protein essential for the persistence of spirochetae within ticks.
On the basis of this homology with HP-NAP, we decided to verify whether the product of the gene bb0690 of Borelia encoding for the protein NapA could play a role in the development of Lyme arthritis through a specific modulation of immune system.
In a first set of experiments we verify the presence of specific anti-NapA antobodies in serum samples from patients with Lyme arthritis, whereas, in samples from patients with erythema migrans and facial palsy, caracteristic of the early phase of the disease, anti-NapA specific antobodies were rarely detected.
Successively, considering the important role of Th17 cells in the development of Lyme arthritis, we decided to examine the functional profile of synovial T cells from patients with Lyme arthritis, and their specificity toward NapA. We have demonstrated that in synovial cavity of patients with Lyme arthritis there are Th17 lymphocytes NapA specific; moreover, when these cells, appropriately stimulated, were exposed to NapA in vitro, they were able to produce IL-17. On the basis of these observations, we evaluated whether the ability of NapA to polarize T helper lymphocytes toward a Th17 phenotype, could be the result of a cytokine environment rich in IL-6, TGF-, IL-1 and IL-23, tipically involved in the differentiation and proliferation of Th17 cells.
To this aim we used neutrophils and monocytes as experimental model, cells which are recruited in the synovial cavity during inflammation, and we have demonstrated that NapA is able to induce upregulation of synthesis of IL-6, TGF-, IL-1 and IL-23 in these cells.
A robust inflammatory response to B. burgdorferi is the hallmark of both early-stage (erythema migrans) and middle and late-stage (arthritis) manifestations of Lyme disease. These inflammatory responses are likely initiated by the expression of genes activated by the binding of borrelial components to Toll-like receptors (TLRs). To investigate the mechanism of the NapA activation of monocytes, we evaluated the possibility that NapA activates a TLR-mediated signalling. We demonstrate that NapA is a TLR2 agonist able to activate NF-B in HEK TLR2-transfected cells, and the involvement of TLR2 receptor in the activation of monocytes by NapA is also supported by the abrogation of IL-23 expression by a specific anti-TLR2 blocking antibody.
Altogether these results suggest that NapA is a B. burgdorferi virulence factor involved in the pathogenesis of Th17 inflammation in human Lyme arthritis (Codolo G. et al., 2008 Arthr andRheum).
Although NapA and HP-NAP sequences are identical to 28%, they are endowed with different immunological properties, being the former a pro-Th17 agonist and the latter an inducer of Th1-oriented responses.
The allignment between the protein NapA and HP-NAP from H.pylori, shows that NapA is 13 amino acids longer at the N-terminus and 20 amino acids longer at the C-terminus than HP-NAP. On the basis of these differences, we were concerned to investigate whether in these two portions of the molecule could reside the peculiar Th17-orienting property of NapA. To elucidate whether the immuno-modulating activity of NapA resides either in its N-terminal or in its C-terminal tail, we produced two NapA mutants lacking 13 residues at the N-terminus and 20 at the C-terminus, respectively, and assayed them for their Th17-orienting property. To this aim we analized the expression of IL-6, IL-1, TGF- and IL-23 in monocytes treated with NapA for different time points; surprisingly the exposure to both the deleted forms of NapA resulted in a even more pronounced induction of synthesis of these cytokines.
Finally, to further investigate whether NapA mutants retain the ability to engage the TLR2, which is crucial for the immuno-modulatory properties of the wild type protein, we evaluated the induction of IL-23 in the presence of a blocking anti-TLR2 antibody. The presence of the antibody significantly reduced the ability of NapA, both wt and mutant, to promote the synthesis and release of IL-23, suggesting that the portion of the protein responsible for the binding to the receptor does not localize within the two N- and C-terminal tails (Codolo G. et al., submitted).
Lyme arthritis is characterized by progressive joint damage that is mediated by several mechanisms. Early erosion of cartilage and bone is associated with the formation of a proliferating pannus.
The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to the proliferation of fibroblast-like synoviocytes (FLS) and the destruction of cartilage and bone.
Neutrophils extravasation into the infected joint is a key initial step in the development of joint inflammation, as reported in mouse model studies.
Migration of leukocytes into the synovium is a regulated multistep process involving interactions between leukocytes and endothelial cells and cellular adhesion molecules, as well as between leukocytes and chemokines and chemokine receptors. Synovial tissue and synovial fluid from Lyme arthritis patients, such as other chronic inflammatory arthritides, contain increased concentrations of several chemokines. The chemokine system is considered to be implicated in LA pathogenesis via the recruitment and retention of neutrophils, monocytes and T lymphocytes into the joints.
The last part of our work was aimed to understand whether NapA could be able to recruit immune cells involved in the inflammatory process. We first demonstrate, using a rat arthritis model, that NapA is able to recruit neutrophils and T lymphocytes able to produce IFN-. Moreover, analysis of cytokines and chemokines in synovial cavity of animals, reveals that NapA induces an increased production of chemokines such as CCL2, CCL20 and CXCL1, involved in the recruitment of neutrophils and T lymphocytes. These cells represent the major inflammatory cells recruited in joint of patients with Lyme arthritis..
On the basis of the results described above, we investigated the role of NapA in neutrophils and T lymphocytes recruitment. In a first set of experiments we observed that NapA, per se, is not able to recruit neutrophils and T lymphocytes. These observations suggest that, probably, NapA is not directly a chemotactict factor for immune cells. Therefore, it could be hypothesized that NapA acts indirectly inducing the production of factors able to attract neutrophils and T lymphocytes to the site of infection.
Particularly, on the basis of CCL2, CCL20 and CXCL1 accumulation in synovial fluids of rats treated with NapA, we wondered which cells could be the source of these mediators.
We performed preliminar experiments aimed to evaluate the production of chemokines in macrophages and neutrophils, after NapA stimulation. We focused our attention on these cells because macrophages are resident cells in synovia, and for this reason, once activated, macrophages are able to produce pro-inflammatory molecules involved in the recruitment of other cells to the site of infection. Neutrophils, instead, are the first cells recruited in synovial cavity of Lyme arthritis-affected patients.
From our preliminary observations it seems that both cell types might be able to produce chemokines involved in neutrophils and T lymphocytes recruitment. This is an interesting observation that we will deeply investigate. These data could correlate with those reported by Pellettier and colleagues who have recently demonstrated that IFN- plus LPS-activated neutrophils release chemokines, such as CCL2 and CCL20 which, in turn, are responsible for the recruitment of Th17 cells. Moreover, they showed that activated neutrophils, via the release of CXCL10 and CCL2, may also trigger the chemotaxis of Th1 cells. Finally, they demonstrated that, upon activation, human Th17 cells can directly chemoattract neutrophils through the release of CXCL8.
Therefore, according to Pellettier et al., neutrophils/Th cells interaction might create a pathogenic proinflammatory loop that ultimately amplifies the local accumulation of neutrophils and Th cells during inflammatory disease. We can speculate that such a loop could also occur during the inflammatory events in the pathogenesis of Lyme arthritis. In order to verify this hypothesis, we are currently performing experiments aimed to evaluate the phenotype of T lymphocytes recruited across a HUVEC-derived endothelium, under stimulation with supernatants harvested from either neutrophils or macrophages activated with NapA.Borrelia burgdorferi è l'agente eziologico della malattia di Lyme. La malattia di Lyme può essere causata da tre specie di Borrelia correlate tra loro: B. burgdorferi, B. afzelii and B. garinii. In Europa la malattia è causata da tutte e tre le specie, mentre, in America, Borrelia burgdorferi è l’unica causa.
La malattia di Lyme è una patologia multisistemica e può essere suddivisa in tre stadi: il primo è caratterizzato da un’infezione cutanea localizzata a livello del sito del morso della zecca; nella fase successiva, le spirochete disseminano in diversi tessuti, e l’ultimo stadio è caratterizzato dalla cronicizzazione dell’infezione. La malattia di Lyme ha inizio con una caratteristica lesione cutanea che, nelle ore e giorni successivi al morso, si espande, motivo per cui questo stadio della malattia viene chiamato Eritema migrans. Nelle settimane successive le spirochete disseminano in diversi siti. Dopo mesi, soprattutto nei pazienti che non vengono trattati con terapia antibiotica, l’infezione può cronicizzare causando artrite che colpisce le articolazioni, in particolare le ginocchia (artritie di Lyme).
Scopo di questo progetto è quello di contribuire alla comprensione dei meccansimi molecolari e cellulari coinvolti nello sviluppo dell’artrite. E’ oramai stabilito che la risposta immunitaria dell’ospite nei confronti di Borrelia burgdorferi influenza quella che sarà la manifestazione clinica dell’infezione. In particolare, fino a qualche anno fa si riteneva che i linfociti T di tipo Th1, secernenti IFN-, fossero il tipo cellulare principalmente coinvolto nell’artrite di Lyme. Recentemente , utilizzando un modello murino, è stato dimostrato che i linfociti Th1 non erano essenziali nell’indurre l’artrite di Lyme, suggerendo quindi il coinvolgimento di altri tipi cellulari e di mediatoridiversi da IFN- e IL-12 prodotti dalle cellule Th1. L’attenzione si è focalizzata su un nuovo sottotipo di linfociti T helper: i linfociti Th17. E’ riportato che queste cellule giocano un ruolo importante nell’induzione di danno tissutale in malattie autoimmuni attraverso la secrezione di interleuchina (IL)-17. La IL-17 induce il rilascio di varie citochine e chemochine ad attività pro-infiammatoria da parte di cellule stromali, sinoviociti, condrociti, fibroblasti e macrofagi, inoltre, sono in grado di reclutare neutrofili. Un’importante osservazione è costituita dal fatto che la IL-17 si trova a livelli elevati nei liquidi sinoviali di pazienti con artrite di Lyme, dove è in grado di promuovere la formazione di osteoclasti e diverse osservazioni sperimentali suggeriscono che IL-17 possa essere coinvolta nella patogenesi dell’artrite di Lyme.
Non era ancora noto, tuttavia, quali fossero i fattori batterici coinvolti nell’induzione di una risposta immunitaria Th17 mediata.
In precedenza, nel nostro laboratorio, era stata caratterizzata una proteina prodotta da Helicobacter pylori, batterio che nell’uomo provoca infezione cronica, così come è in grado di fare Borrelia burgdorferi, chiamata HP-NAP (Helicobacter pylori Neutrophil Activating Protein). Si tratta di una proteina fortemente immunogena dotata di specificihe proprietà immunomodulanti. Anche Borrelia possiede una proteina simile ad HP-NAP la cui unica caratterizzazione funzionale aveva portato alla dimostrazione che fosse essenziale per la persistenza della spirocheta nella zecca.
Sulla base dell’omologia con HP-NAP abbiamo voluto verificare se anche il prodotto del gene bb0690 di Borrelia e codificante per la proteina NapA potesse giocare un ruolo nello sviluppo dell’artite di Lyme attraverso una specifica modulazione del sistema immunitario.
In una prima fase dello studio abbiamo verificato la presenza di anticorpi specifici contro NapA nei sieri di pazienti affetti da artrite di Lyme, mentre, nei pazienti con eritema migrans o paralisi facciale, manifestazioni cliniche che caratterizzano gli stadi precoci della malattia, raramente sono stati trovati anticorpi contro NapA.
Successivamente, considerando l’emergente idea che i linfociti Th17 avessero un ruolo cruciale nello sviluppo dell’artrite di Lyme, abbiamo deciso di valutareo il profilo funzionale di linfociti T isolati da pazienti con artrite di Lyme e la eventuale loro specificità verso la proteina NapA. Abbiamo così dimostrato che nella cavità sinoviale di pazienti affetti da artrite di lyme ci sono linfociti Th17 specifici per NapA; inoltre, l’aggiunta della proteina ai suddetti linfociti opportunamente stimolati in vitro, si traduceva nella produzione di IL-17. Sulla base di questi risultati siamo passati a valutare se la capacità di NapA di polarizzare i linfociti T helper verso il profilo Th17, fosse il risultato dell’induzione di un particolare mielieu ricco in IL-6, IL-1, TGF- e IL-23, note citochine che insieme contribuiscono al differenziamento e alla proliferazione dei linfociti Th17. Utilizzando neutrofili e monociti umani come modello sperimentale, in quanto abbondantemente rappresentati nella cavità sinoviale in corso di infiammazione, abbiamo dimostrato che NapA è capace di indurre in queste cellule una maggiore sintesi e rilascio di IL-6, IL-1, TGF- e IL-23.
Una forte risposta infiammatoria a Borrelia è una caratteristica di tutti gli stadi della malattia di Lyme. A dare inizio a questa risposta infiammatoria è l’attivazione dell’espressione di geni dovuta al legame delle componenti batteriche ai Toll-like receptors (TLRs). Per tale ragione, al fine di indagare il meccanismo di attivazione dei monociti da parte di NapA, abbiamo considerato la possibilità che questa proteina attivasse un signaling mediato da un TLR.
Abbiamo dimostrato che NapA è un ligando del TLR2, e che questo recettore è coinvolto nell’attivazione dei monociti da parte di NapA; questo dato è stato supportato dall’osservazione che l’espressione e produzione di IL-23 veniva abrogata in presenza di un anticorpo specifico bloccante il TLR2.
Complessivamente i dati da noi ottenuti hanno dimostrato che NapA è un fattore di virulenza di Borrelia burgdorferi coinvolto nella patogenesi dell’infiammazione Th17 mediata dell’artrite di Lyme (Codolo G. et al., 2008).
Sebbene le sequenze di NapA e HP-NAP abbiano il 28% di identitĂ , esse hanno diverse attivitĂ immunomodulanti; la prima induce una risposta di tipo Th17, la seconda invece promuove risposte di tipo Th1.
L’allineamento tra NapA e HP-NAP ha mostrato che NapA è 13 aminoacidi più lunga all’estremità N-terminale e 20 aminoacidi più lunga all’estremità C-terminale. Sulla base di tali differenze, abbiamo voluto valutare se le proprietà pro-Th17 di NapA risiedessero in queste due porzioni della proteina. Sono state prodotte due proteine mutanti, una priva dei 13 residui all’N-terminale e una senza 20 aminoacidi al C-terminale, e abbiamo valutato le loro proprietà pro-Th17. A tale scopo è stata analizzata l’espressione di IL-6, IL-1, TGF- e IL-23 in monociti trattati con NapA a diversi tempi. Inaspettatamente, le due proteine delete hanno mostrato avere una capacità addirittura maggiore, rispetto a NapA intera, di indurre la sintesi delle citochine prese in esame.
Inoltre, siamo andati a vedere se le due proteine mutate fossero ancora in grado di legare il TLR2; per fare questo abbiamo analizzato l’induzione di IL-23 in presenza di uno specifico anticorpo bloccante il TLR2. Abbiamo osservato che la presenza dell’anticorpo bloccante riduce in modo significativo la capacità di NapA e delle due proteine delete la capacità di promuovere la sintesi e il rilascio di IL-23, suggerendo che la porzione di proteina responsabile del legame con il TLR2 e dotata di attività immunomodulante non sia localizzata alle estremità (Codolo G. et al. Submitted).
L’artrite di Lyme è caratterizzata dal progressivo danneggiamento dell’articolazione, che è mediato da diversi meccanismi. L’erosione della cartilagine e dell’osso è associata alla formazione del panno sinoviale proliferante.
Il processo infiammatorio è caratterizzato dall’infiltrazione di cellule infiammatorie nell’articolazione, che portano alla proliferazione dei sinoviociti simil-fibroblasti (fibroblast-like synoviocytes, FLS), e alla distruzione della cartilagine e dell’osso.
L’extravasazione dei neutrofili nell’articolazione è uno dei primi step chiave nello sviluppo dell’infiammazione delle articolazioni, come dimostrato utilizzando un modello murino.
La migrazione dei leucociti nella sinovia è un processo multistep finemente regolato che coinvolge l’interazione tra leucociti e cellule endoteliali e le molecole di adesione di superficie, e anche tra leucociti e chemochine e recettori per le chemochine. Il tessuto sinoviale e il liquido sinoviale di pazienti affetti da artrite di Lyme, così come in altre patologie legate ad artriti croniche, contengono elevate concentrazioni di chemochine. Il sistema delle chemochine si ritiene essere implicato nella patogenesi dell’artrite di Lyme attraverso il reclutamento di neutrofili, monociti e linfociti T nelle articolazioni.
L’ultima parte del lavoro che abbiamo svolto aveva lo scopo di capire se NapA fosse in grado di reclutare cellule infiammatorie ed immunitarie. Innanzitutto abbiamo dimostrato, utilizzando un modello di artrite indotta in ratto, che NapA è in grado di reclutare neutrofili e linfociti T capaci di produrre IFN-. Inoltre, abbiamo osservato che la proteina è in grado di indurre aumento dell’espressione di chemochine, quali CCL2, CCL20 e CXCL1, coinvolte nel reclutamento di neutrofili e linfociti T. Tali cellule rappresentano il maggior infiltrato di cellule infiammatorie richiamate a livello delle articolazioni di pazienti affetti da artrite di Lyme.
Sulla base dei risultati ottenuti da questi esperimenti, abbiamo voluto capire quale fosse il ruolo di NapA nel reclutamento di neutrofili e linfociti T. Da esperimenti preliminari, abbiamo osservato che la proteina NapA non è in grado, da sola, di reclutare tali cellule. Queste osservazioni suggeriscono che probabilmente NapA non possiede un’attività chemotattica diretta, perciò è possibile ipotizzare che NapA agisca in modo indiretto inducendo la produzione di fattori in grado di richiamare le cellule al sito di infezione.
In particolare, sulla base delle chemochine
Evaluation of the efficacy of the H. Pylori protein HP-NAP as a therapeutic tool for treatment of bladder cancer in an orthotopic murine model
Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu\ue9rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed. The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates. In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals
The effect of Helicobacter pylori on asthma and allergy
Amedeo Amedei1, Gaia Codolo2, Gianfranco Del Prete1, Marina de Bernard2, Mario M D’Elios11Policlinico AOU Careggi, Department Internal Medicine, University of Florence, Italy; 2Venetian Institute of Molecular Medicine, University of Padua, ItalyAbstract: Current evidence indicates an inverse association between Helicobacter pylori and asthma and allergy. H. pylori is a Gram-negative bacterium which represents the major cause of peptic ulcer and gastric cancer, and preferentially elicits a T helper (Th)-1 response. Many H. pylori factors, such as the neutrophil-activating factor of H. pylori (HP-NAP), are able to drive Th-1 polarization and to display a powerful inhibition of allergic Th-2 response. This article proposes an overview of the actual knowledge about the effects of H. pylori on asthma and allergy. Special attention has been drawn to HP-NAP as a potential novel strategy for the prevention and treatment of asthma and atopy.Keywords: Helicobacter pylori neutrophil-activating factor, protein, Th-1/Th-2, Treg, asthm