Studio delle citochine nella distrofia muscolare di Emery-Dreifuss: Possibili marker patogenetici e bersagli di cura della malattia

La distrofia muscolare di Emery-Dreifuss (EDMD) è una miopatia degenerativa ereditaria caratterizzata da debolezza e atrofia dei muscoli senza coinvolgimento del sistema nervoso. Individui EDMD presentano, inoltre, cardiomiopatia con difetto di conduzione che provoca rischio di morte improvvisa. Diversi studi evidenziano un coinvolgimento di citochine in diverse distrofie muscolari causanti infiammazione cronica, riassorbimento osseo, necrosi cellulare. Abbiamo effettuato una valutazione simultanea della concentrazione di citochine, chemochine, fattori di crescita, presenti nel siero di un gruppo di 25 pazienti EDMD. L’analisi effettuata ha evidenziato un aumento di citochine quali IL-17, TGFβ2, INF-γ e del TGFβ1. Inoltre, una riduzione del fattore di crescita VEGF e della chemochina RANTES è stata rilevata nel siero dei pazienti EDMD rispetto ai pazienti controllo. Ulteriori analisi effettuate tramite saggio ELISA hanno evidenziato un aumento dei livelli di TGFβ2 e IL-6 nel terreno di coltura di fibroblasti EDMD2. Per testare l’effetto nei muscoli, di citochine alterate, abbiamo utilizzato terreno condizionante di fibroblasti EDMD per differenziare mioblasti murini C2C12. Una riduzione del grado di differenziamento è stata osservata nei mioblasti condizionati con terreno EDMD. Trattando queste cellule con anticorpi neutralizzanti contro TGFβ2 e IL-6 si è avuto un miglioramento del grado di differenziamento. In C2C12 che esprimevano la mutazione H222P del gene Lmna,non sono state osservate alterazioni di citochine e benefici di anticorpi neutralizzanti. I dati mostrano un effetto patogenetico delle citochine alterate come osservato in fibroblasti e siero di pazienti, suggerendo un effetto sul tessuto fibrotico di muscoli EDMD. Un effetto intrinseco alla mutazione della lamina A è stato rilevato sul espressione di caveolina 3 in mioblasti differenziati EDMD. I risultati si aggiungono a dati forniti sulla patogenesi dell' EDMD confermando che fattori intrinseci ed estrinseci contribuiscono alla malattia. Utilizzo di anticorpi neutralizzanti specifici contro fattori estrinseci potrebbe rappresentare un approccio terapeutico come mostrato in questo studio.

Outcome a lungo termine dei pazienti pediatrici con rene singolo. Valutazione di marker laboratoristici e strumentali di danno renale

Obiettivi: valutare in pazienti con rene singolo congenito la correlazione tra il filtrato glomerulare misurato con il DTPA (DTPA-VFG) e 1) marker laboratoristici di danno renale (creatinina, cistatinaC, proteinuria) 2) formule per stimare il filtrato glomerulare 3) parametri di valutazione della crescita renale ecografica. Materiali e metodi: Sono stati arruolati 118 pazienti con rene singolo congenito tra 0 e 18 anni. Sono stati valutati a ogni visita altezza, creatinina, cistatinaC, proteinuria e lunghezza ecografica renale. E’ stato calcolato il filtrato stimato con formule basate sulla creatinina (Schwartz), sulla cistatina C (Zappitelli, Filler, Grubb e Bokenkamp) e su entrambe (equazione di Zappitelli). La crescita renale è stata valutata come rapporto lunghezza ecografica/altezza corporea (USL/H), differenza percentuale tra lunghezza renale misurata e attesa per età (delta%) e presenza o meno d’ipertrofia compensatoria. In 74 bambini è stata misurata la DTPA-VFG. Risultati: Il follow-up è di 2.1 ± 0.9 anni. Il 65% sono maschi. Nessun paziente ha sviluppato danno renale cronico. La media del DTPA-VFG era di 135±44ml/min/1.73m², il valore medio della creatinina 0.47±0.17mg/dl e di cistatinaC di 1±0.4mg/L. La lunghezza ecografica renale media era di 100±17 mm, il rapporto USL/H medio di 0.8±0,1 e il delta% di 1,13±11,4, il 66% presentava ipertrofia renale. Le uniche correlazioni significative con DTPA-VFG sono inversa con la creatinina (p=<.001) e lineare con USL/H (p=<.001). Discussione: Lo studio ha mostrato che come per altre nefrouropatie, la creatina e l’ecografia renale siano due strumenti validi per il follow-up dei pazienti con rene singolo congenito. Il limite principale è dovuto al fatto che nessuno dei pazienti ha sviluppato danno renale cronico e pertanto non è stato possibile stabilire dei cutt-off di rischio per parametri quali USL/H.

A novel marker for heart failure: Cross sectional area assessment in a large vein by conductance catheter.

The present study investigates the feasibility of a new application able to check the heart failure status in a patient through the estimation of the venous distension. In this way it would be possible to follow up patients, avoiding invasive or expensive exams such as cardiac catheterization and echocardiography. Moreover, the devices would also be able to diagnose the decline of the disease, in order to allow a new adaptation to therapy, and vice versa to check the improvement in the patient’s conditions after the CRT device implant. This thesis is essentially divided into three parts: an analytical model was used to obtain an estimation of the error committed for the calculation of the CSA and to understand how the accuracy and sensitivity depend on the different configurations of the electrodes and the catheter position inside the vein; secondly, an in-vitro experiment was carried out in order to verify the practical feasibility for these kinds of measurements, in a very simplified model; in the end, several animal experiments were done to test the in-vivo practicability of the proposed method. The obtained results showed the feasibility of this approach. In fact, the error committed in the estimation of CSA, during the animal experiments, can be considered acceptable (CSAerror_max ≈ -14%). Moreover, it has been demonstrated that the conductance catheter allows assessing, not only the vein CSA, but also the breathing of the animal.

Dynamic characterisation of four nine-story large-panel R.C. buildings in Bishkek (Kyrgyzstan): A comparison between experimental ambient vibration analysis and numerical finite element modeling

With the outlook of improving seismic vulnerability assessment for the city of Bishkek (Kyrgyzstan), the global dynamic behaviour of four nine-storey r.c. large-panel buildings in elastic regime is studied. The four buildings were built during the Soviet era within a serial production system. Since they all belong to the same series, they have very similar geometries both in plan and in height. Firstly, ambient vibration measurements are performed in the four buildings. The data analysis composed of discrete Fourier transform, modal analysis (frequency domain decomposition) and deconvolution interferometry, yields the modal characteristics and an estimate of the linear impulse response function for the structures of the four buildings. Then, finite element models are set up for all four buildings and the results of the numerical modal analysis are compared with the experimental ones. The numerical models are finally calibrated considering the first three global modes and their results match the experimental ones with an error of less then 20%.

The use of flat panel angioCT (DynaCT) for navigation through a deformed and fractured carotid stent

Navigation through a previously deployed and deformed stent is a difficult interventional task. Inadvertent navigation through the struts of a stent can potentially lead to incomplete secondary stent extension and vessel occlusion. Better visualisation of the pathway through the stent can reduce the risks of the procedural complications and reduce the reluctance of the interventionalist to navigate through a previously deployed stent. We describe a technique of visualisation of the pathway navigated by a guidewire through a previously deployed deformed and fractured carotid stent by the use of DynaCT. Three-dimensional reconstruction of the stent/microwire allows excellent visualisation of the correct pathway of the microwire within the stent.

Lysyl oxidase expression is an independent marker of prognosis and a predictor of lymph node metastasis in oral and oropharyngeal squamous cell carcinoma (OSCC)

Proteins of the lysyl oxidase (LOX) family are important modulators of the extracellular matrix. However, they have an important role in the tumour development as well as in tumour progression. To evaluate the diagnostic and prognostic value of the LOX protein in oral and oropharyngeal squamous cell carcinoma (OSCC) we performed QRT-PCR and immunohistochemical analysis on two tissue microarrays (622 tissue samples in total). Significantly higher LOX expression was detected in high grade dysplastic oral mucosa as well as in OSCC when compared to normal oral mucosa (P < 0.001). High LOX expression was correlated with clinical TNM stage (P = 0.020), lymph node metastases for the entire cohort (P < 0.001), as well as in the subgroup of small primary tumours (T1/T2, P < 0.001). Moreover, high LOX expression was correlated with poor overall survival (P = 0.004) and disease specific survival (P = 0.037). In a multivariate analysis, high LOX expression was an independent prognostic factor, predicting unfavourable overall survival. In summary, LOX expression is an independent prognostic biomarker and a predictor of lymph node metastasis in OSCC. Moreover, LOX overexpression may be an early phenomenon in the pathogenesis of OSCC and thus an attractive novel target for chemopreventive and therapeutic strategies.

Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma

We have investigated the use of hierarchical clustering of flow cytometry data to classify samples of conventional central chondrosarcoma, a malignant cartilage forming tumor of uncertain cellular origin, according to similarities with surface marker profiles of several known cell types. Human primary chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines from chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers. For clustering, eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z-score normalization, were systematically evaluated. By selecting clustering approaches shown to give reproducible results for cluster recovery of known cell types, primary conventional central chondrosacoma cells could be grouped in two main clusters with distinctive marker expression signatures: one group clustering together with mesenchymal stem cells (CD49b-high/CD10-low/CD221-high) and a second group clustering close to fibroblasts (CD49b-low/CD10-high/CD221-low). Hierarchical clustering also revealed substantial differences between primary conventional central chondrosarcoma cells and established chondrosarcoma cell lines, with the latter not only segregating apart from primary tumor cells and normal tissue cells, but clustering together with cell lines from epithelial lineage. Our study provides a foundation for the use of hierarchical clustering applied to flow cytometry data as a powerful tool to classify samples according to marker expression patterns, which could lead to uncover new cancer subtypes.

Atypical expression and distribution of embryonic stem cell marker, OCT4, in human lung adenocarcinoma

Lung cancer is one of the leading causes of cancer-related deaths in the world. Although the origin still remains to be resolved, a prevailing hypothesis implies the involvement of cancer stem cells (CSCs) responsible for tumor initiation, maintenance, and progression. Embryonic stem cell marker, OCT4, encoding the spliced variants OCT4A and OCT4B, has recently been shown to have a dual role; as a potential adult stem cell marker and as a CSC marker in germline and somatic tumors.

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events

Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

The alphavbeta6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are common primary hepatic malignancies. Their immunohistological differentiation using specific markers is pivotal for treatment and prognosis. We found alphavbeta6 integrin strongly upregulated in biliary fibrosis, but its expression in primary and secondary liver tumours is unknown. Here, we aimed to evaluate the diagnostic applicability of alphavbeta6 integrin in differentiating primary liver cancers.

Smoothelin is a specific and robust marker for distinction of muscularis propria and muscularis mucosae in the gastrointestinal tract

As tumour specimens and biopsy specimens become smaller, recognition of anatomical structures relevant for staging is increasingly challenging. So far no marker is known that reliably discriminates between muscularis propria (MP) and muscularis mucosae (MM) of the gastrointestinal tract. Recently, smoothelin expression has been shown to differ in MP and MM of the urinary bladder. We aimed to analyse the expression of smoothelin in the gastrointestinal tract in MP and MM in order to define a novel diagnostic tool to identify MM bundles.

Differential cell cycle and proliferation marker expression in ductal pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia (PanIN)

Pancreatic cancer is an aggressive tumour following a multistep progression model through precursors called pancreatic intraepithelial neoplasia (PanIN). Identification of reliable prognostic markers would help in improving survival. The aim of this study was to investigate the role as well as the prognostic significance of different cell cycle and proliferation markers, namely p21, p27, p53 and Ki-67, in pancreatic carcinogenesis.

Loss of pace capture on the ablation line: a new marker for complete radiofrequency lesions to achieve pulmonary vein isolation

Catheter ablation procedures for atrial fibrillation (AF) often involve circumferential antral isolation of pulmonary veins (PV). Inability to reliably identify conduction gaps on the ablation line necessitates placing additional lesions within the intended lesion set.