Humoral autoimmune response heterogeneity in the spectrum of primary biliary cirrhosis

Objective To compare autoantibody features in patients with primary biliary cirrhosis (PBC) and individuals presenting antimitochondria antibodies (AMAs) but no clinical or biochemical evidence of disease. Methods A total of 212 AMA-positive serum samples were classified into four groups: PBC (definite PBC, n = 93); PBC/autoimmune disease (AID; PBC plus other AID, n = 37); biochemically normal (BN) individuals (n = 61); and BN/AID (BN plus other AID, n = 21). Samples were tested by indirect immunofluorescence (IIF) on rat kidney (IIF-AMA) and ELISA [antibodies to pyruvate dehydrogenase E2-complex (PDC-E2), gp-210, Sp-100, and CENP-A/B]. AMA isotype was determined by IIF-AMA. Affinity of anti-PDC-E2 IgG was determined by 8 M urea-modified ELISA. Results High-titer IIF-AMA was more frequent in PBC and PBC/AID (57 and 70 %) than in BN and BN/AID samples (23 and 19 %) (p < 0.001). Triple isotype IIF-AMA (IgA/IgM/IgG) was more frequent in PBC and PBC/AID samples (35 and 43 %) than in BN sample (18 %; p = 0.008; p = 0.013, respectively). Anti-PDC-E2 levels were higher in PBC (mean 3.82; 95 % CI 3.36–4.29) and PBC/AID samples (3.89; 3.15–4.63) than in BN (2.43; 1.92–2.94) and BN/AID samples (2.52; 1.54–3.50) (p < 0.001). Anti-PDC-E2 avidity was higher in PBC (mean 64.5 %; 95 % CI 57.5–71.5 %) and PBC/AID samples (66.1 %; 54.4–77.8 %) than in BN samples (39.2 %; 30.9–37.5 %) (p < 0.001). PBC and PBC/AID recognized more cell domains (mitochondria, nuclear envelope, PML/sp-100 bodies, centromere) than BN (p = 0.008) and BN/AID samples (p = 0.002). Three variables were independently associated with established PBC: high-avidity anti-PDC-E2 (OR 4.121; 95 % CI 2.118–8.019); high-titer IIF-AMA (OR 4.890; 2.319–10.314); antibodies to three or more antigenic cell domains (OR 9.414; 1.924–46.060). Conclusion The autoantibody profile was quantitatively and qualitatively more robust in definite PBC as compared with AMA-positive biochemically normal individuals.

Modelli di scambio termico in pozzo sulla base della caratterizzazione geotermica di un reservoir a bassa entalpia: studio di sensitività sulla efficienza dei sistemi di condizionamento a pompa di calore

Tra i temi di attualità, quello del risparmio energetico è tra i più dibattuti negli ultimi anni; tale tema è strettamente correlato al problema del riscaldamento globale, infatti, mentre sul prossimo esaurimento delle risorse energetiche tradizionali non vi sono ancora certezze assolute, per quanto riguarda l’azione nociva dei gas serra, la Comunità Scientifica Internazionale si ritrova d’accordo su una netta presa di posizione contro l’emissione di tali sostanze, provocata in larga parte dall’utilizzo dei combustibili fossili. In questo contesto, l’Unione Europea sta promuovendo la diffusione di tecnologie che non prevedano l’utilizzo di gas, petrolio o carbone, soprattutto per il settore dell’edilizia, ove una corretta progettazione e l’utilizzo di tecnologie non convenzionali può portare alla riduzione anche dell’80% dei consumi, con conseguente abbattimento delle emissioni. Tra questi interventi innovativi, il più comune e conosciuto è sicuramente quello del solare termico e fotovoltaico; ma ne esistono anche di altri, ancora non molto pubblicizzati in Italia, ma ampiamente conosciuti e utilizzati in altri paesi dell’Unione. Tra questi, vi è il sistema di riscaldamento analizzato in questa tesi: la pompa di calore geotermica. Tale sistema, come verrà spiegato nell’elaborato di laurea, ha indubbi vantaggi economici, energetici ed ambientali, a fronte di una non trascurabile spesa iniziale. Attualmente, nel Nord Italia, si incominciano a vedere impianti di questo tipo, sulla scia del successo riscontrato nei paesi confinanti (in particolare Austria e Svizzera). La progettazione si basa attualmente su modelli statici, sviluppati dall’Università Svizzera del Canton Ticino, per l’utilizzo della pompa di calore nel territorio alpino. Obiettivo della tesi, è la verifica di tali modelli, di cui si è venuto a conoscenza grazie alla collaborazione con l’Università SUPSI, sulle condizioni idrogeologiche della Pianura Padana, soffermandosi su alcuni parametri fondamentali della progettazione di una pompa di calore geotermica, quali la conduttività e la capacità termica volumetrica dei terreni incontrati, la presenza di falde, ed i parametri geometrici del pozzo, al fine di dare una valutazione tecnica ed economica dell’impianto. Tali analisi è stata infatti fino ad ora affrontata in maniera sommaria dai perforatori, che eseguono generalmente sempre lo stesso modello di pozzo geotermico, sulla base degli esempi consolidati di Svizzera e Germania. Alcune misure di temperatura in situ sono state rilevate in collaborazione con la società Geotermia SRL di Mantova, ditta specializzata nella perforazione di pozzi geotermici (tale esperienza è parte centrale dell’estratto “Laboratorio di Tesi Ls”), mentre la parte modellistica della tesi è stata sviluppata in collaborazione con lo studio di progettazione Studio Seta SRL di Faenza, il cui stabile è climatizzato in parte con una pompa di calore geotermica.

Marcatura di molecole biologiche a funzione antigenica per lo studio e la caratterizzazione di protocolli di vaccinoterapia in oncologia medica

Dendritic Cells (DCs) derived from human blood monocytes that have been nurtured in GM-CSF and IL-4, followed by maturation in a monocyte-conditioned medium, are the most potent APCs known. These DCs have many features of primary DCs, including the expression of molecules that enhance antigen capture and selective receptors that guide DCs to and from several sites in the body, where they elicit the T cell mediated immune response. For these features, immature DCs (iDC) loaded with tumor antigen and matured (mDC) with a standard cytokine cocktail, are used for therapeutic vaccination in clinical trials of different cancers. However, the efficacy of DCs in the development of immunocompetence is critically influenced by the type (whole lysate, proteins, peptides, mRNA), the amount and the time of exposure of the tumor antigens used for loading in the presentation phase. The aim of the present study was to create instruments to acquire more information about DC antigen uptake and presentation mechanisms to improve the clinical efficacy of DCbased vaccine. In particular, two different tumor antigen were studied: the monoclonal immunoglobulin (IgG or IgA) produced in Myeloma Multiple, and the whole lysate obtained from melanoma tissues. These proteins were conjugated with fluorescent probe (FITC) to evaluate the kinetic of tumor antigen capturing process and its localization into DCs, by cytofluorimetric and fluorescence microscopy analysis, respectively. iDC pulsed with 100μg of IgG-FITC/106 cells were monitored from 2 to 22 hours after loading. By the cytofluorimetric analysis it was observed that the monoclonal antibody was completely captured after 2 hours from pulsing, and was decreased into mDC in 5 hours after maturation stimulus. To monitor the lysate uptake, iDC were pulsed with 80μg of tumor lysate/106 cells, then were monitored in the 2h to 22 hours interval time after loading. Then, to reveal difference between increasing lysate concentration, iDC were loaded with 20-40-80-100-200-400μg of tumor lysate/106 cells and monitored at 2-4-8-13h from pulsing. By the cytofluorimetric analysis, it was observed that, the 20-40-80-100μg uptake, after 8 hours loading was completed reaching a plateau phase. For 200 and 400μg the mean fluorescence of cells increased until 13h from pulsing. The lysate localization into iDC was evaluated with conventional and confocal fluorescence microscopy analysis. In the 2h to 8h time interval from loading an intensive and diffuse fluorescence was observed within the cytoplasmic compartment. Moreover, after 8h, the lysate fluorescence appeared to be organized in a restricted cloudy-shaded area with a typical polarized aspect. In addition, small fluorescent spots clearly appeared with an increment in the number and fluorescence intensity. The nature of these spot-like formations and cloudy area is now being investigated detecting the colocalization of the fluorescence lysate and specific markers for lysosomes, autophagosomes, endoplasmic reticulum and MHCII positive vesicles.

Adaptive T-spline refinement for isogeometric analysis in planar geometries

[EN]We present a new strategy, based on the meccano method [1, 2, 3], to construct a T-spline parameterization of 2D geometries for the application of isogeometric analysis. The proposed method only demands a boundary representation of the geometry as input data. The algorithm obtains, as a result, high quality parametric transformation between 2D objects and the parametric domain, the unit square. The key of the method lies in defining an isomorphic transformation between the parametric and physical T-mesh finding the optimal position of the interior nodes by applying a new T-mesh untangling and smoothing procedure. Bivariate T-spline representation is calculated by imposing the interpolation conditions on points sited both on the interior and on the boundary of the geometry…

Some advances in open problems of isogeometric analysis

[EN]Isogeometric analysis (IGA) has arisen as an attempt to unify the fields of CAD and classical finite element methods. The main idea of IGA consists in using for analysis the same functions (splines) that are used in CAD representation of the geometry. The main advantage with respect to the traditional finite element method is a higher smoothness of the numerical solution and more accurate representation of the geometry. IGA seems to be a promising tool with wide range of applications in engineering. However, this relatively new technique have some open problems that require a solution. In this work we present our results and contributions to this issue…

TNFRSF13B Genetic variability an anthropological - evolutionary approach to Biomedical Research

In the recent years TNFRSF13B coding variants have been implicated by clinical genetics studies in Common Variable Immunodeficiency (CVID), the most common clinically relevant primary immunodeficiency in individuals of European ancestry, but their functional effects in relation to the development of the disease have not been entirely established. To examine the potential contribution of such variants to CVID, the more comprehensive perspective of an evolutionary approach was applied in this study, underling the belief that evolutionary genetics methods can play a role in dissecting the origin, causes and diffusion of human diseases, representing a powerful tool also in human health research. For this purpose, TNFRSF13B coding region was sequenced in 451 healthy individuals belonging to 26 worldwide populations, in addition to 96 control, 77 CVID and 38 Selective IgA Deficiency (IgAD) individuals from Italy, leading to the first achievement of a global picture of TNFRSF13B nucleotide diversity and haplotype structure and making suggestion of its evolutionary history possible. A slow rate of evolution, within our species and when compared to the chimpanzee, low levels of genetic diversity geographical structure and the absence of recent population specific selective pressures were observed for the examined genomic region, suggesting that geographical distribution of its variability is more plausibly related to its involvement also in innate immunity rather than in adaptive immunity only. This, together with the extremely subtle disease/healthy samples differences observed, suggests that CVID might be more likely related to still unknown environmental and genetic factors, rather than to the nature of TNFRSF13B variants only.

Use of sub-lethal high pressure homogenization (HPH) treatments to enhance functional properties of lactic acid bacteria probiotic strains

The aim of this PhD thesis was to evaluate the effect of a sub-lethal HPH treatment on some probiotic properties and on cell response mechanisms of already-known functional strains, isolated from Argentinean dairy products. The results achieved showed that HPH treatments, performed at a sub-lethal level of 50 MPa, increased some important functional and technological characteristics of the considered non intestinal probiotic strains. In particular, HPH could modify cell hydrophobicity, autoaggregation and resistance to acid gastric conditions (tested in in vitro model), cell viability and cell production of positive aroma compounds, during a refrigerate storage in a simulated dairy product. In addition, HPH process was able to increase also some probiotic properties exerted in vivo and tested for two of the considered strains. In fact, HPH-treated cells were able to enhance the number of IgA+ cells more than other not treated cells, although this capacity was time dependent. On the other hand, HPH treatment was able to modify some important characteristics that are linked to the cell wall and, consequently, could alter the adhesion capacity in vivo and the interaction with the intestinal cells. These modifications, involving cell outermost structures, were highlighted also by Trasmission Electron Microscopy (TEM) analysis. In fact, the micrographs obtained showed a significant effect of the pressure treatment on the cell morphology and particularly on the cell wall. Moreover, the results achieved showed that composition of plasma membranes and their level of unsaturation are involved in response mechanisms adopted by cells exposed to the sub-lethal HPH treatment. Although the response to the treatment varied according to the characteristics of individual strains, time of storage and suspension media employed, the results of present study, could be exploited to enhance the quality of functional products and to improve their organoleptic properties.

Impatto della glomerulopatia cronica da trapianto sulla disfunzione tardiva del rene trapiantato

INTRODUCTION. Late chronic allograft disfunction (CAD) is one of the more concerning issues in the management of patients (pts) with renal transplant (tx). Humoral immune response seems to play an important role in CAD pathogenesis. AIM OF THE STUDY. To identify the causes of late chronic allograft disfunction. METHODS. This study (march 2004-august 2011) enrolled pts who underwent renal biopsy (BR) because of CAD (increase of creatininemia (s-Cr) >30% and/or proteinuria >1g/day at least one year after tx). BR were classified according to 1997/2005 Banff classification. Histological evaluation of C4d (positive if >25%), glomerulitis, tubulitis, intimal arteritis, atrophy/fibrosis and arteriolar-hyalinosis were performed. Ab anti-HLA research at BR was an inclusion criteria. Pts were divided into two groups: with or without transplant glomerulopathy (CTG). RESULTS. Evaluated BR: 93/109. BR indication: impaired s-Cr (52/93), proteinuria (23/93), both (18/93). Time Tx-BR: 7.4±6.3 yrs; s-Cr at BR: 2.7±1.4 mg/dl. CTG group(n=49) not-CTG group(n=44) p Time tx-BR (yrs) 9.3±6.7 5.3±5.2 0.002 Follow-up post-BR (yrs) 2.7±1.8 4.1±1.4 0.0001 s-Cr at BR (mg/dl) 2.9±1.3 2.4±1.5 NS Rate (%) of pts: Proteinuria at BR 61% 25% 0.0004 C4d+ 84% 25% <0.0001 Ab anti-HLA+ 71% 30% 0.0001 C4d+ and/or Ab antiHLA 92% 43% 0.0001 Glomerulitis 76% 16% <0.0001 Tubulitis 6% 32% 0.0014 Intimal arteritis 18% 0% 0.002 Arteriolar hyalinosis 65% 50% NS Atrophy/fibrosis 80% 77% NS Graft survival 45% 86% 0.00005 Histological Diagnosis: CTG group (n=49:Chronic rejection 94%;IgA recurrence + humoral activity 4%;IIA acute rejection + humoral activity 2%. Not-CTG group (n=44: GN recurrence 27%;IF/TA 23%; acute rejection 23%;BKV nephritis 9%; mild not specific alterations 18%. CONCLUSIONS: CTG is the morphological lesion mainly related to CAD. In the 92% of the cases it is associated with markers of immunological activity. It causes graft failure within five years after diagnosis in 55% of pts.

Reproducibility and validity of video screen measurements of gait in children with spastic cerebral palsy

PURPOSE: To determine the reproducibility and validity of video screen measurement (VSM) of sagittal plane joint angles during gait. METHODS: 17 children with spastic cerebral palsy walked on a 10m walkway. Videos were recorded and 3d-instrumented gait analysis was performed. Two investigators measured six sagittal joint/segment angles (shank, ankle, knee, hip, pelvis, and trunk) using a custom-made software package. The intra- and interrater reproducibility were expressed by the intraclass correlation coefficient (ICC), standard error of measurements (SEM) and smallest detectable difference (SDD). The agreement between VSM and 3d joint angles was illustrated by Bland-Altman plots and limits of agreement (LoA). RESULTS: Regarding the intrarater reproducibility of VSM, the ICC ranged from 0.99 (shank) to 0.58 (trunk), the SEM from 0.81 degrees (shank) to 5.97 degrees (trunk) and the SDD from 1.80 degrees (shank) to 16.55 degrees (trunk). Regarding the interrater reproducibility, the ICC ranged from 0.99 (shank) to 0.48 (trunk), the SEM from 0.70 degrees (shank) to 6.78 degrees (trunk) and the SDD from 1.95 degrees (shank) to 18.8 degrees (trunk). The LoA between VSM and 3d data ranged from 0.4+/-13.4 degrees (knee extension stance) to 12.0+/-14.6 degrees (ankle dorsiflexion swing). CONCLUSION: When performed by the same observer, VSM mostly allows the detection of relevant changes after an intervention. However, VSM angles differ from 3d-IGA and do not reflect the real sagittal joint position, probably due to the additional movements in the other planes.

Lymphangiogenesis is upregulated in kidneys of patients with multiple myeloma

Neolymphangiogenesis has recently been demonstrated in transplanted kidneys as well as in chronic interstitial nephritis and IgA nephropathy. However, its significance in kidney disease remains to be defined and a systematic study of renal lymphangiogenesis is warranted. We investigated patients with multiple myeloma (MM) presenting in the great majority with acute renal insufficiency. Controls were allograft kidney donors and patients with renal insufficiency due to acute renal failure (ARF). Lymph vessel length density (LVD) was quantified immunohistochemically by means of antipodoplanin staining followed by computer-assisted stereology. The mean LVD in kidneys of patients with MM (23.19 mm(-2)) was higher when compared with allograft donors (7.42 mm(-2), P = 0.0003) and patients with ARF (6.78 mm(-2), P = 0.0002). The higher LVD was significantly associated with interstitial inflammation, and the newly formed lymph vessels were accompanied by diffuse and nodular interstitial infiltrates composed mainly of CD20(+) B cells and CD27(+) plasma cells. The infiltrates in patients with MM also displayed a higher expression of the B-cell chemoattractant CXCL13. These results demonstrate for the first time that lymphangiogenesis is a prominent feature in MM kidneys and that it is associated with a significant accumulation of macrophages, CD20(+) and CD27(+) B lymphocytes. Further studies should clarify whether these changes represent a beneficial or detrimental factor in the progression of the myeloma-related kidney damage.

In remembrance of commensal intestinal microbes

Mammals contain an enormous load of commensal microbes in the lower intestine, which induce adaptive responses in the host immune system that ensure mutual coexistence of the host and its microbial passengers. The main way of studying how the host responds to commensal colonization has been to compare animals kept in entirely germ-free conditions and their colonized counterparts. We present an overview of our development of a reversible colonization system, whereby germ free animals can be treated with live commensal bacteria that do not persist in the host, so it becomes germ free again. We describe how this system has been used to demonstrate that there is little or no immune memory for specific IgA induction in the intestinal mucosal immune system by commensal intestinal bacteria.

Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut

Using a systems biology approach, we discovered and dissected a three-way interaction between the immune system, the intestinal epithelium and the microbiota. We found that, in the absence of B cells, or of IgA, and in the presence of the microbiota, the intestinal epithelium launches its own protective mechanisms, upregulating interferon-inducible immune response pathways and simultaneously repressing Gata4-related metabolic functions. This shift in intestinal function leads to lipid malabsorption and decreased deposition of body fat. Network analysis revealed the presence of two interconnected epithelial-cell gene networks, one governing lipid metabolism and another regulating immunity, that were inversely expressed. Gene expression patterns in gut biopsies from individuals with common variable immunodeficiency or with HIV infection and intestinal malabsorption were very similar to those of the B cell-deficient mice, providing a possible explanation for a longstanding enigmatic association between immunodeficiency and defective lipid absorption in humans.

Immunoglobulin A: a bridge between innate and adaptive immunity

The review summarizes the recent progress that has been made in understanding the function of immunoglobulin A (IgA) in promoting a healthy mutualism with the commensal microbiota and protecting against pathogens. Although IgA is by far the most abundant antibody produced by mammals, direct experimental evidence for its function is still lacking.

SNPs in DNA repair genes associated to meningitis and host immune response

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis. The patient genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. A higher frequency (P<0.05) of APE1 Glu allele in bacterial meningitis (BM) and aseptic meningitis (AM) patients was observed. The genotypes Asn/Asn in control group and Asn/Glu in BM group was also higher. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs is significantly higher in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 Glu allele or OGG1 Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1 Asn148Glu, OGG1 Ser326Cys or PARP-1 Val762Ala. Moreover, reduction in the levels of IL-6, IL-1Ra, MCP-1/CCL2 and IL-8/CXCL8 was observed in the presence of APE1 Glu allele in BM patients. In conclusion, we obtained indications of an effect of SNPs in DNA repair genes on the regulation of immune response in meningitis.