Importanza prognostica delle citochine e loro ruolo nell'immunomodulazione: risultati di studi sperimentali in vivo e in vitro

Citokines are proteins produced by several cell types and secreted in response to various stimuli. These molecules are able to modify the behaviour of other cells inducing activities like growth, differentiation and apoptosis. In the last years, veterinary scientists have investigated the role played by these factors; in fact, cytokines can act as intercellular communicative signals in immune response, cell damage repair and hematopoiesis. Up to date, various cytokines have been identified and in depth comprehension of their effects in physiology, pathology and therapy is an interesting field of research. This thesis aims to understand the role played by these mediators during natural or experimentally induced pathologies. In particular, it has been evaluated the genic and protein expressions of a large number of cytokines during several diseases and starting from different matrix. Considering the heterogeneity of materials used in experimentations, multiple methods and protocols of nucleic acids and proteins extractions have been standardized. Results on cytokines expression obtained from various in vitro and in vivo experimental studies have shown how important these mediators are in regulation and modulation of the host immune response also in veterinary medicine. In particular, the analysis of inflammatory and septic markers, like cytokines, has allowed a better understanding in the pathogenesis during horse Recurrent Airway Obstruction, foal sepsis, Bovine Viral Diarrhea Virus infection and dog Parvovirus infection and the effects of these agents on the host immune system. As experimentations with mice have shown, some pathologies of the respiratory and nervous system can be reduced or even erased by blocking cytokines inflammatory production. The in vitro cytokines expression evaluation in cells which are in vivo involved in the response to exogenous (like pathogens) or endogenous (as it happens during autoimmune diseases) inflammatory stimuli could represent a model for studying citokines effects during the host immune response. This has been analyzed using lymphocytes cultured with several St. aureus strains isolated from bovine mastitic milk and different colostrum products. In the first experiment different cytokines were expressed depending on enterotoxins produced, justifying a different behaviour of the microrganism in the mammal gland. In the second one, bone marrow cells derived incubated with murine lymphocytes with colostrum products have shown various cluster of differentiation expression , different proliferation and a modified cytokines profile. A better understanding of cytokine expression mechanisms will increase the know-how on immune response activated by several pathogen agents. In particular, blocking the cytokine production, the inhibition or catalyzation of the receptor binding mechanism and the modulation of signal transduction mechanism will represent a novel therapeutic strategy in veterinary medicine.

Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

Valutazione del rapporto urinario proteine totali/creatinina e albumina/creatinina in cani affetti da iperadrenocorticismo e da diabete mellito

INTRODUCTION – In human medicine, diabetes mellitus (DM), hypertension, proteinuria and nephropathy are often associated although it is still not clear whether hypertension is the consequence or the cause of nephropathy and albuminuria. Microalbuminuria, in humans, is an early and sensitive marker which permits timely and effective therapy in the early phase of renal damage. Conversely, in dogs, these relationships were not fully investigated, even though hypertension has been associated with many diseases (Bodey and Michell, 1996). In a previous study, 20% of diabetic dogs were found proteinuric based on a U:P/C > 1 and 46% were hypertensive; this latter finding is similar to the prevalence of hypertension in diabetic people (40-80%) (Struble et al., 1998). In the same canine study, hypertension was also positively correlated with the duration of the disease, as is the case in human beings. Hypertension was also found to be a common complication of hypercortisolism (HC) in dogs, with a prevalence which varies from 50 (Goy-Thollot et al., 2002) to 80% (Danese and Aron, 1994).The aim of our study was to evaluate the urinary albumin to creatinine ratio (U:A/C) in dogs affected by Diabetes Mellitus and HC in order to ascertain if, as in human beings, it could represent an early and more sensitive marker of renal damage than U:P/C. Furthermore, the relationship between proteinuria and hypertension in DM and HC was also investigated. MATERIALS AND METHODS – Twenty dogs with DM, 14 with HC and 21 healthy dogs (control group) were included in the prospective case-control study. Inclusion criteria were hyperglycaemia, glicosuria and serum fructosamine above the reference range for DM dogs and a positive ACTH stimulation test and/or low-dose dexamethasone test and consistent findings of HC on abdominal ultrasonography in HC dogs. Dogs were excluded if affected by urinary tract infections and if the serum creatinine or urea values were above the reference range. At the moment of inclusion, an appropriate therapy had already been instituted less than 1 month earlier in 12 diabetic dogs. The control dogs were considered healthy based on clinical exam and clinicopathological findings. All dogs underwent urine sample collection by cystocentesis and systemic blood pressure measurement by means of either an oscillometric device (BP-88 Next, Colin Corporation, Japan) or by Doppler ultrasonic traducer (Minidop ES-100VX, Hadeco, Japan). The choice of method depended on the dog’s body weight: Doppler ultrasonography was employed in dogs < 20 kg of body weight and the oscillometric method in the other subjects. Dogs were considered hypertensive whenever systemic blood pressure was found ≥ 160 mmHg. The urine was assayed for U:P/C and U:A/C (Gentilini et al., 2005). The data between groups were compared using the Mann-Whitney U test. The reference ranges for U:P/C and U:A/C had already been established by our laboratory as 0.6 and 0.05, respectively. U:P/C and U:A/C findings were correlated to systemic blood pressure and Spearman R correlation coefficients were calculated. In all cases, p < 0.05 was considered statistically significant. RESULTS – The mean ± sd urinary albumin concentration in the three groups was 1.79 mg/dl ± 2.18; 20.02 mg/dl ± 43.25; 52.02 mg/dl ± 98.27, in healthy, diabetic and hypercortisolemic dogs, respectively. The urine albumin concentration differed significantly between healthy and diabetic dogs (p = 0.008) and between healthy and HC dogs (p = 0.011). U:A/C values ranged from 0.00 to 0.34 (mean ± sd 0.02 ± 0.07), 0.00 to 6.72 (mean ± sd 0.62 ± 1.52) and 0.00 to 5.52 (mean ± sd 1.27 ± 1.70) in the control, DM and HC groups, respectively; U:P/C values ranged from 0.1 to 0.6 (mean ± sd 0.17 ± 0.15) 0.1 to 6.6 (mean ± sd 0.93 ± 1.15) and 0.2 to 7.1 (mean ± sd 1.90 ± 2.11) in the control, DM and HC groups, respectively. In diabetic dogs, U:A/C was above the reference range in 11 out of 20 dogs (55%). Among these, 5/20 (25%) showed an increase only in the U:A/C ratio while, in 6/20 (30%), both the U:P/C and the U:A/C were abnormal. Among the latter, 4 dogs had already undergone therapy. In subjects affected with HC, U:P/C and U:A/C were both increased in 10/14 (71%) while in 2/14 (14%) only U:A/C was above the reference range. Overall, by comparing U:P/C and U:A/C in the various groups, a significant increase in protein excretion in disease-affected animals compared to healthy dogs was found. Blood pressure (BP) in diabetic subjects ranged from 88 to 203 mmHg (mean ± sd 143 ± 33 mmHg) and 7/20 (35%) dogs were found to be hypertensive. In HC dogs, BP ranged from 116 to 200 mmHg (mean ± sd 167 ± 26 mmHg) and 9/14 (64%) dogs were hypertensive. Blood pressure and proteinuria were not significantly correlated. Furthermore, in the DM group, U:P/C and U:A/C were both increased in 3 hypertensive dogs and 2 normotensive dogs while the only increase of U:A/C was observed in 2 hypertensive and 3 normotensive dogs. In the HC group, the U:P/C and the U:A/C were both increased in 6 hypertensive and 2 normotensive dogs; the U:A/C was the sole increased parameter in 1 hypertensive dog and in 1 dog with normal pressure. DISCUSSION AND CONCLUSION- The findings of this study suggest that, in dogs affected by DM and HC, an increase in U:P/C, U:A/C and systemic hypertension is frequently present. Remarkably, some dogs affected by both DM and HC showed an U:A/C but not U:P/C above the reference range. In diabetic dogs, albuminuria was observed in 25% of the subjects, suggesting the possibility that this parameter could be employed for detecting renal damage at an early phase when common semiquantiative tests and even U:P/C fall inside the reference range. In HC dogs, a higher number of subjects with overt proteinuria was found while only 14% presented an increase only in the U:A/C. This fact, associated with a greater number of hypertensive dogs having HC rather than DM, could suggest a greater influence on renal function by the mechanisms involved in hypertension secondary to hypercortisolemia. Furthermore, it is possible that, in HC dogs, the diagnosis was more delayed than in DM dogs. However, the lack of a statistically significant correlation between hypertension and increased protein excretion as well as the apparently random distribution of proteinuric subjects in normotensive and hypertensive cases, imply that other factors besides hypertension are involved in causing proteinuria. Longitudinal studies are needed to further investigate the relationship between hypertension and proteinuria.

Impiego della spettrometria di massa nell'analisi di fumonisine in alimenti e matrici di origine animale

Mycotoxins are heterogeneous chemical compounds characterized by a low molecular weight and synthesized by the secondary metabolism of different molds. Fumonisins are water-soluble mycotoxins produced by Fusarium species spoiling corn and derived produc ts. These mycotoxins can be a health hazard when consuming contaminated cereals, but they can reach humans also indirectly through the consumption of food products derived from animals fed with contaminated feed. Fumonisins have been associated with several animal and human diseases: they are suspected risk factors for esophageal and liver cancers, neural tube defects and cardiovascular problems. Improved methods are needed to accurately assess fumonisins concentrations in food of vegetable and animal origin, in order to prevent acute and chronic human exposure. The aim of the present work was to evaluate the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis from foods and matrices of animal origin. Different methods for the identification and quantification of fumonisins and related products have been developed and validated to determine fumonisin B1 in milk, fumonisin B1, fumonisin B2 and their complete hydrolyzed products (HFB1 and HFB2) in pig liver and fumonisins B1 and B2 in complete and complementary dry dog food. The experimental procedures have been carefully studied, considering matrices features, number and type of molecules to detect. Therefore, several extraction, clean up and separation techniques were tested in order to obtain the better conditions of sample processing. The fit for purpose sample preparation, matched with high mass spectrometry sensibility and specificity, have allowed to achieve good results in any tested animal matrices. Hence, the developed methods were validated and have shown a high accuracy, sensibility and precision, fulfilling performance requirements of Decision 2002/657/EC and of European Project Standard, Measuring and Testing (SMT). In any developed method, the analytes were identified and quantified even at very low concentrations : the limits of quantification resulted lower than other similar works, performed with different detectors. These methods were applied to some commercial samples and to some samples collected for research projects in the Department of Veterinary Public Health and Animal Pathology (DVPHAP) of University of Bologna. Although the disclosed data must be considered completely preliminary and without statistical significance, they emphasize the presence of mycotoxins in animal products. The outcomes obtained from the processed samples (bovine milk, pig liver and dry dog food) suggest the efficacy of these methods also on other food matrices, confirming the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis. Moreover the results underline the need to set up a large scale monitoring in order to evaluate the presence of fumonisins in food of animal origin for human consumption.

Hepatobiliary diseases in small animals: a comparison of ultrasonography and multidetector-row computed tomography

Ultrasonography (US) is an essential imaging tool for identifying abnormalities of the liver parenchyma, biliary tract and vascular system. US has replaced radiography as the initial imaging procedure in screening for liver disease in small animals. There are few reports of the use of conventional and helical computed tomography (CT) to assess canine or feline parenchymal and neoplastic liver disease and biliary disorders. In human medicine the development of multidetector- row helical computed tomography (MDCT), with its superior spatial and temporal resolution, has resulted in improved detection and characterization of diffuse and focal liver lesions. The increased availability of MDCT in veterinary practice provides incentive to develop MDCT protocols for liver imaging in small animals. The purpose of this study is to assess the rule of MDCT in the characterization of hepatobiliary diseases in small animals; and to compare this method with conventional US. Candidates for this prospective study were 175 consecutive patients (dogs and cats) referred for evaluation of hepatobiliary disease. The patients underwent liver US and MDCT. Percutaneous needle biopsy was performed on all liver lesions or alterations encountered. As for gallbladder, histopatological evaluation was obtained from cholecystectomy specimens. Ultrasonographic findings in this study agreed well with those of previous reports. A protocol for dual-phase liver MDCT in small animals has been described. MDCT findings in parenchymal disorders of the liver, hepatic neoplasia and biliary disorders are here first described in dogs and cats and compared with the corresponding features in human medicine. The ability of MDCT in detection and characterization of hepatobiliary diseases in small animals is overall superior to conventional US. Ultrasonography and MDCT scanning, however, play complementary rules in the evaluation of these diseases. Many conditions have distinctive imaging features that may permit diagnosis. In most instances biopsy is required for definitive diagnosis.

Immunohistochemical and Molecular Prognostic/Predictive Markers in Neoplastic Diseases

Traditional morphological examinations are not anymore sufficient for a complete evaluation of tumoral tissue and the use of neoplastic markers is of utmost importance. Neoplastic markers can be classified in: diagnostic, prognostic and predictive markers. Three markers were analyzed. 1) Insulin-like growth factor binding protein 2 (IGFBP2) was immunohistochemically examined in prostatic tissues: 40 radical prostatectomies from hormonally untreated patients with their preoperative biopsies, 10 radical prostatectomies from patients under complete androgen ablation before surgery and 10 simple prostatectomies from patients with bladder outlet obstruction. Results were compared with α-methylacyl-CoA racemase (AMACR). IGFBP2 was expressed in the cytoplasm of untreated adenocarcinomas and, to a lesser extent, in HG-PIN; the expression was markedly lower in patients after complete androgen ablation. AMACR was similarly expressed in both adenocarcinoma and HG-PIN, the level being similar in both lesions; the expression was slightly lower in patients after complete androgen ablation. IGFBP2 may be used a diagnostic marker of prostatic adenocarcinomas. 2) Heparan surface proteoglycan immunohistochemical expression was examined in 150 oral squamous cell carcinomas. Follow up information was available in 93 patients (range: 6-34 months, mean: 19±7). After surgery, chemotherapy was performed in 8 patients and radiotherapy in 61 patients. Multivariate and univariate overall survival analyses showed that high expression of syndecan-1 (SYN-1) was associated with a poor prognosis. In patients treated with radiotherapy, such association was higher. SYN-1 is a prognostic marker in oral squamous cell carcinomas; it may also represent a predictive factor for responsiveness to radiotherapy. 3) EGFR was studied in 33 pulmonary adenocarcinomas with traditional DNA sequencing methods and with two mutation-specific antibodies. Overall, the two antibodies had 61.1% sensitivity and 100% specificity in detecting EGFR mutations. EGFR mutation-specific antibodies may represent a predictive marker to identify patients candidate to tyrosine kinase inhibitors therapy.

Atomic Force Microscopy Studies of the Amyloidogenic Processes of Intrinsically Unstructured Proteins related to Neurodegenerative Diseases

Protein aggregation and formation of insoluble aggregates in central nervous system is the main cause of neurodegenerative disease. Parkinson’s disease is associated with the appearance of spherical masses of aggregated proteins inside nerve cells called Lewy bodies. α-Synuclein is the main component of Lewy bodies. In addition to α-synuclein, there are more than a hundred of other proteins co-localized in Lewy bodies: 14-3-3η protein is one of them. In order to increase our understanding on the aggregation mechanism of α-synuclein and to study the effect of 14-3-3η on it, I addressed the following questions. (i) How α-synuclein monomers pack each other during aggregation? (ii) Which is the role of 14-3-3η on α-synuclein packing during its aggregation? (iii) Which is the role of 14-3-3η on an aggregation of α-synuclein “seeded” by fragments of its fibrils? In order to answer these questions, I used different biophysical techniques (e.g., Atomic force microscope (AFM), Nuclear magnetic resonance (NMR), Surface plasmon resonance (SPR) and Fluorescence spectroscopy (FS)).

Biomarkers in chronic obstructive pulmonary diseases. Clinical and functional correlations.

Asthma and chronic obstructive pulmonary disease (COPD) are two distinct lung diseases with distinctive clinical and inflammatory features. A proportion of asthmatic patients experience a fixed airflow obstruction that persists despite optimal pharmacologic treatment for reasons that are still largely unknown. We found that patients with asthma and COPD sharing a similar fixed airflow obstruction have an increased lung function decline and frequency of exacerbations. Nevertheless, the decline in lung function is associated with specific features of the underlying inflammation. Airway inflammation increases during asthma exacerbation and disease severity. Less is known about the correlations between symptoms and airway inflammation in COPD patients. We found that there is no correlation between symptoms and lung function in COPD patients. Nevertheless symptoms changes are associated with specific inflammatory changes: cough is associated with an increase of sputum neutrophils in COPD, dyspnoea is associated with an increase of eosinophils. The mechanisms of this correlation remain unknown. Neutrophils inflammation is associated with bacterial colonization in stable COPD. Is not known whether inhaled corticosteroids might facilitate bacterial colonization in COPD patients. We found that the use of inhaled corticosteroids in COPD patients is associated with an increase of airway bacterial load and with an increase of airway pathogen detection. Bacterial and viral infections are the main causes of COPD and asthma exacerbations. Impaired innate immune responses to rhinovirus infections have been described in adult patients with atopic asthma. Whether this impaired immune condition is present early in life and whether is modulated by a concomitant atopic condition is currently unknown. We found that deficient innate immune responses to rhinovirus infection are already present early in life in atopic patients without asthma and in asthmatic subjects. These findings generalize the scenario of increased susceptibility to viral infections to other Th2 oriented conditions.