Structural and cellular characterization of Idiopathic Pulmonary Fibrosis

La Fibrosi Polmonare Idiopatica (IPF) è una malattia polmonare cronica, irreversibile la cui eziologia risulta essere ignota, caratterizzata da un processo fibrotico progressivo che inizia nel tratto respiratorio inferiore. Le persone affette da IPF presentano età media compresa tra 55 e 77 anni. L’incidenza annuale di IPF è stata recentemente stimata tra 14 e 42,7 casi per 100.000 persone e tale dato risulta essere in aumento. IPF fa parte delle malattie Polmonari Idiopatiche Interstiziali (IIP) che comprendono patologie con quadri istologici e clinici differenti. Le affezioni su cui si concentrerà questo studio sono: UIP (Usual Interstitial Pneumonia) caratterizzata da fibrosi interstiziale e dalla presenza di foci fibrotici connessi alla pleura e corrispondente al quadro anatomopatologico della maggior parte dei casi di IPF; NSIP (Non Specific Interstitial Pneumonia) simile alla UIP ma con maggiore uniformità temporale e spaziale delle manifestazioni; Sarcoidosi, malattia granulomatosa ad eziologia ignota. Attualmente la gravità della IPF, che implica una mortalità del 50% dei pazienti a 5 anni dall’esordio, e la scarsa efficacia farmacologica nel rallentarne la progressione vedono il trapianto polmonare come unica possibilità di sopravvivenza nelle forme più severe. Al momento non è chiaro il meccanismo patogenetico di insorgenza e progressione della IPF anche se sono stati individuati alcuni fattori scatenanti quali fumo di sigaretta, infezioni respiratorie e inquinanti atmosferici; tuttavia nessuno di tali elementi può da solo determinare un così esteso e progressivo rimodellamento del parenchima polmonare. Numerose sono le evidenze di come il substrato genetico, le alterazioni del rapporto morte/proliferazione cellulare e le citochine svolgano un ruolo nella genesi e nella progressione della malattia, ma non sono ancora chiari i fenomeni biologico-cellulari che la sostengono e, quindi, quali siano i punti di attacco per poter incidere terapeuticamente nel modificare l’evoluzione della IPF. Poiché il nostro laboratorio ha partecipato alla scoperta dell’esistenza di cellule staminali nel polmone umano normale, uno degli obiettivi finali di questo progetto si basa sull’ipotesi che un’alterazione del compartimento staminale svolga un ruolo cruciale nella eziopatogenesi di IPF. Per questo in precedenti esperienze abbiamo cercato di identificare nella IPF cellule che esprimessero antigeni associati a staminalità quali c-kit, CD34 e CD133. Questo lavoro di tesi si è proposto di condurre un’indagine morfometrica ed immunoistochimica su biopsie polmonari provenienti da 9 pazienti affetti da UIP, 3 da NSIP e 5 da Sarcoidosi al fine di valutare le alterazioni strutturali principali imputabili alle patologie. Preparati istologici di 8 polmoni di controllo sono stati usati come confronto. Come atteso, è stato osservato nelle tre patologie esaminate (UIP, NSIP e Sarcoidosi) un significativo incremento nella sostituzione del parenchima polmonare con tessuto fibrotico ed un ispessimento dei setti alveolari rispetto ai campioni di controllo. L’analisi dei diversi pattern di fibrosi presenti fa emergere come vi sia una netta differenza tra le patologie con una maggiore presenza di fibrosi di tipo riparativo e quindi altamente cellulata nei casi di UIP, e NSIP mentre nelle Sarcoidosi il pattern maggiormente rappresentato è risultato essere quello della fibrosi replacement o sostitutiva. La quantificazione delle strutture vascolari è stata effettuata tenendo separate le aree di polmone alveolare rispetto a quelle occupate da focolai sostitutivi di danno (componente fibrotica). Nei campioni patologici analizzati era presente un significativo riarrangiamento di capillari, arteriole e venule rispetto al polmone di controllo, fenomeno principalmente riscontrato nel parenchima fibrotico. Tali modifiche erano maggiormente presenti nei casi di NSIP da noi analizzati. Inoltre le arteriole subivano una diminuzione di calibro ed un aumento dello spessore in special modo nei polmoni ottenuti da pazienti affetti da Sarcoidosi. Rispetto ai controlli, nella UIP e nella Sarcoidosi i vasi linfatici risultavano inalterati nell’area alveolare mentre aumentavano nelle aree di estesa fibrosi; quadro differente si osservava nella NSIP dove le strutture linfatiche aumentavano in entrambe le componenti strutturali. Mediante indagini immunoistochimiche è stata documentata la presenza e distribuzione dei miofibroblasti, positivi per actina muscolare liscia e vimentina, che rappresentano un importante componente del danno tissutale nella IPF. La quantificazione di questo particolare fenotipo è attualmente in corso. Abbiamo inoltre analizzato tramite immunoistochimica la componente immunitaria presente nei campioni polmonari attraverso la documentazione dei linfociti T totali che esprimono CD3, andando poi a identificare la sottopopolazione di T citotossici esprimenti la glicoproteina CD8. La popolazione linfocitaria CD3pos risultava notevolmente aumentata nelle tre patologie analizzate soprattutto nei casi di UIP e Sarcoidosi sebbene l`analisi della loro distribuzione tra i vari distretti tissutali risultasse differente. Risultati simili si sono ottenuti per l`analisi dei linfociti CD8pos. La componente monocito-macrofagica è stata invece identificata tramite la glicoproteina CD68 che ha messo in evidenza una maggiore presenza di cellule positive nella Sarcoidosi e nella UIP rispetto ai casi di NSIP. I dati preliminari di questo studio indicano che il rimodellamento strutturale emo-linfatico e cellulare infiammatorio nella UIP si differenziano rispetto alle altre malattie interstiziali del polmone, avanzando l’ipotesi che il microambiente vascolare ed immunitario giochino un ruolo importante nella patogenesi della malattia

Mechanisms of Mycoplasma-induced inflammation and cellular transformation

There is a growing interest in “medical gasses” for their antibacterial and anti-inflammatory properties. Hydrogen sulfide (H2S), a member of the family of gasotransmitters, is in fact increasingly being recognized as an important signaling molecule, but its precise role in the regulation of the inflammatory response is still not clear. For this reason, the aim of the first part of this thesis was to investigate the effects of H2S on the expression of pro-inflammatory cytokines, such as MCP-1, by using an in vitro model composed by both primary monocytes-derived macrophages cultures and the human monocytic cell line U937 infected with Mycoplasma fermentans, a well-known pro-inflammatory agent. In our experiments, we observed a marked increase in the production of pro-inflammatory cytokines in infected cells. In particular, MCP-1 was induced both at the RNA and at the protein level. To test the effects of H2S on infected cells, we treated the cells with two different H2S donors (NaHS and GYY4137), showing that both H2S treatments had anti-inflammatory effects in Mycoplasma-infected cells: the levels of MCP-1, both mRNA expression and protein production, were reduced. Our subsequent studies aimed at understanding the molecular mechanisms responsible for these effects, focused on two specific molecular pathways, both involved in inflammation: the NF-κB and the Nrf2 pathway. After treatment with pharmacological inhibitors, we demonstrated that Mycoplasma fermentans induces MCP-1 expression through the TLR-NF-κB pathway with the nuclear translocation of its subunits, while treatment with H2S completely blocked the nuclear translocation of NF-κB heterodimer p65/p50. Then, once infected cells were treated with H2S donors, we observed an increased protective effect of Nrf2 and also a decrease in ROS production. These results highlight the importance of H2S in reducing the inflammatory process caused by Mycoplasma fermentans. To this regard, it should be noted that several projects are currently ongoing to develop H2S-releasing compounds as candidate drugs capable of alleviating cell deterioration and to reduce the rate of decline in organ function. In the second part of this study, we investigated the role of Mycoplasma infection in cellular transformation. Infectious agents are involved in the etiology of many different cancers and a number of studies are still investigating the role of microbiota in tumor development. Mycoplasma has been associated with some human cancers, such as prostate cancer and non-Hodgkin’s lymphoma in HIV-seropositive people, and its potential causative role and molecular mechanisms involved are being actively investigated. To this regard, in vitro studies demonstrated that, upon infection, Mycoplasma suppresses the transcriptional activity of p53, key protein in the cancer suppression. As a consequence, infected cells were less susceptible to apoptosis and proliferated more than the uninfected cells. The mechanism(s) responsible for the Mycoplasma-induced inhibitory effect on p53 were not determined. Aim of the second part of this thesis was to better understand the tumorigenic role of the microorganism, by investigating more in details the effect(s) of Mycoplasma on p53 activity in an adenocarcinoma HCT116 cell line. Treatment of Mycoplasma-infected cells with 5FU or with Nutlin, two molecules that induce p53 activity, resulted in cellular proliferation comparable to untreated controls. These results suggested that Mycoplasma infection inhibited p53 activity. Immunoprecipitation of p53 with specific antibodies, and subsequent Gas Chromatography and Mass Spectroscopy (GC-MS) assays, allowed us to identify several Mycoplasma-specific proteins interacting with p53, such as DnaK, a prokaryotic heat shock protein and stress inducible chaperones. In cells transfected with DnaK we observed i) reduced p53 protein levels; ii) reduced activity and expression of p21, Bax and PUMA, iii) a marked increase in cells leaving G1 phase. Taken together, these data show an interaction between the human p53 and the Mycoplasma protein DnaK, with the consequent decreased p53 activity and decreased capability to respond to DNA damage and prevent cell proliferation. Our data indicate that Mycoplasma could be involved in cancer formation and the mechanism(s) has the potential to be a target for cancer diagnosis and treatment(s).

Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

Signal processing for molecular and cellular biological physics:an emerging field

Recent advances in our ability to watch the molecular and cellular processes of life in action-such as atomic force microscopy, optical tweezers and Forster fluorescence resonance energy transfer-raise challenges for digital signal processing (DSP) of the resulting experimental data. This article explores the unique properties of such biophysical time series that set them apart from other signals, such as the prevalence of abrupt jumps and steps, multi-modal distributions and autocorrelated noise. It exposes the problems with classical linear DSP algorithms applied to this kind of data, and describes new nonlinear and non-Gaussian algorithms that are able to extract information that is of direct relevance to biological physicists. It is argued that these new methods applied in this context typify the nascent field of biophysical DSP. Practical experimental examples are supplied.

Synthesis and characterization of dual-functionalized core-shell fluorescent microspheres for bioconjugation and cellular delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins, as described in the accompanying paper.

Elevated ε-(γ-glutamyl)lysine in human diabetic nephropathy results from increased expression and cellular release of tissue transglutaminase

Introduction: Diabetic nephropathy (DN) is the leading cause of chronic kidney failure, however the mechanisms underlying the characteristic expansion of the extracellular matrix (ECM) in diabetic kidneys remain controversial and unclear. In non-diabetic kidney scarring the protein crosslinking enzyme tissue transglutaminase (tTg) has been implicated in this process by the formation of increased ε-(γ-glutamyl)lysine bonds between ECM components in both experimental and human disease. Studies in db/db diabetic mice and in streptozotocin-treated rats have suggested a similar mechanism, although the relevance of this to human disease has not been addressed. Methods: We have undertaken a retrospective analysis of renal biopsies from 16 DN patients with type 2 diabetes mellitus using an immunohistochemical and immunofl uorescence approach, with tTg and ε-(γ-glutamyl)lysine crosslink quantified by confocal microscopy. Results: Immunofl uorescent analysis of human biopsies (confocal microscopy) showed increases in levels of tTg (+1,266%, p <0.001) and ε-(γ-glutamyl)lysine (+486%, p <0.001) in kidneys with DN compared to normal. Changes were predominantly in the extracellular periglomerular and peritubular areas. tTg staining correlated with e-(?-glutamyl)lysine (r = 0.615, p <0.01) and renal scarring (Masson's trichrome, r = 0.728, p <0.001). Significant changes in e-(?-glutamyl)lysine were also noted intracellularly in some (=5%) tubular epithelial cells. This is consistent with cells undergoing a novel transglutaminase-mediated cell death process in response to Ca influx and subsequent activation of intracellular tTg. Conclusion: Changes in tTg and ε-(γ- glutamyl)lysine occur in human DN. Cellular export of tTg may therefore be a factor in the perpetuation of DN by crosslinking and stabilisation of the ECM, while intracellular activation may lead to cell death contributing towards tubular atrophy. Copyright © 2004 S. Karger AG, Basel.

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.

Self-organizing Map and Cellular Automata Combined Technique for Advanced Mesh Generation in Urban and Architectural Design

This paper presents a technique for building complex and adaptive meshes for urban and architectural design. The combination of a self-organizing map and cellular automata algorithms stands as a method for generating meshes otherwise static. This intends to be an auxiliary tool for the architect or the urban planner, improving control over large amounts of spatial information. The traditional grid employed as design aid is improved to become more general and flexible.

Phytoestrogen-ind uced glucose uptake and cellular proliferation in L6 myotubesadipocyte function

Aims: Oestrogens are known to act on a number of tissues throughout the body via classical oestrogen receptors, alpha (ER-a) and beta (ER-beta). Previous research has shown that oestrogens can regulate skeletal muscle glucose uptake cellular proliferation. Thus, oestrogens and related molecules provide an interesting focus for research into possible therapies for the treatment of metabolic disorders and sarcopenia. Enterodiol and enterolactone are plant derived mammalian enterolignans which share a struc- tural similarity to the human oestrogen oestradiol. Methods: In the present study we incubated the differentiated rat skeletal muscle cell line L6 concentration ranges of both com- pounds in the presence/absence of oestrogen receptor antagonists and measured glucose uptake using the non-metabolised glucose analogue 2-NBDG. Cellular proliferation was also measured using a modified MTS assay. Results: Enterolactone was seen to cause a significant increase in cellular proliferation after 48h (a maximal 25% at 0.1nmol/l), in an ER-a dependent mechanism. Incubation with 10nmol/l and 100nmol/l enterodiol caused significant increases in 2-NBDG (5000% compared with control, p < 0.001) and 2h glucose depletion from media (15% increase compared with control, p < 0.05), also in an ER-a dependent way. These results suggest these dietary derived oestrogen-like molecules might be of potential use in targeting metabolic disorders or sarcopenia. Conclusion: We can report here that the phytoestrogen derived molecules enterodiol and enterolactone interact with ER-a in the myotubes to regulate glucose uptake and cellular proliferation respectively.

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

Mechanical properties and cellular response of novel electrospun nanofibers for ligament tissue engineering: Effects of orientation and geometry

Electrospun nanofibers are a promising material for ligamentous tissue engineering, however weak mechanical properties of fibers to date have limited their clinical usage. The goal of this work was to modify electrospun nanofibers to create a robust structure that mimics the complex hierarchy of native tendons and ligaments. The scaffolds that were fabricated in this study consisted of either random or aligned nanofibers in flat sheets or rolled nanofiber bundles that mimic the size scale of fascicle units in primarily tensile load bearing soft musculoskeletal tissues. Altering nanofiber orientation and geometry significantly affected mechanical properties; most notably aligned nanofiber sheets had the greatest modulus; 125% higher than that of random nanofiber sheets; and 45% higher than aligned nanofiber bundles. Modifying aligned nanofiber sheets to form aligned nanofiber bundles also resulted in approximately 107% higher yield stresses and 140% higher yield strains. The mechanical properties of aligned nanofiber bundles were in the range of the mechanical properties of the native ACL: modulus=158±32MPa, yield stress=57±23MPa and yield strain=0.38±0.08. Adipose derived stem cells cultured on all surfaces remained viable and proliferated extensively over a 7 day culture period and cells elongated on nanofiber bundles. The results of the study suggest that aligned nanofiber bundles may be useful for ligament and tendon tissue engineering based on their mechanical properties and ability to support cell adhesion, proliferation, and elongation.