Experimental models of autoimmune inflammatory ocular diseases

Ocular inflammation is one of the leading causes of blindness and loss of vision. Human uveitis is a complex and heterogeneous group of diseases characterized by inflammation of intraocular tissues. The eye may be the only organ involved, or uveitis may be part of a systemic disease. A significant number of cases are of unknown etiology and are labeled idiopathic. Animal models have been developed to the study of the physiopathogenesis of autoimmune uveitis due to the difficulty in obtaining human eye inflamed tissues for experiments. Most of those models are induced by injection of specific photoreceptors proteins (e.g., S-antigen, interphotoreceptor retinoid-binding protein, rhodopsin, recoverin, phosducin). Non-retinal antigens, including melanin-associated proteins and myelin basic protein, are also good inducers of uveitis in animals. Understanding the basic mechanisms and pathogenesis of autoimmune ocular diseases are essential for the development of new treatment approaches and therapeutic agents. The present review describes the main experimental models of autoimmune ocular inflammatory diseases.

Prior history of sexually transmitted diseases in women living with AIDS in São Paulo, Brazil

OBJECTIVES: To describe the epidemiological profile, risk behaviors, and the prior history of sexually transmitted diseases (STDs) in women living with acquired immunodeficiency syndrome (AIDS). METHODS: Cross-sectional study, performed at the Centro de Referência e Treinamento em DST/AIDS of São Paulo. The social, demographic, behavioral, and clinical data such as age, schooling, marital status, age at first sexual intercourse, number of sexual partners, parity, use of drugs, time of HIV diagnosis, CD4 count, and viral load determination were abstracted from the medical records of women living with AIDS who had gynecological consultation scheduled in the period from June 2008 to May 2009. RESULTS: Out of 710 women who were scheduled to a gynecological consultation during the period of the study, 598 were included. Previous STD was documented for 364 (60.9%; 95% CI: 56.9%-64.8%) women. The associated factors with previous STDs and their respective risks were: human development index (HDI) < 0.50 (ORaj = 5.5; 95% CI: 2.8-11.0); non-white race (ORaj = 5.2; 95% CI: 2.5-11.0); first sexual intercourse at or before 15 years of age (ORaj = 4.4; 95% CI: 2.3-8.3); HIV infection follow-up time of nine years or more (ORaj = 4.2; 95% CI: 2.3-7.8)]; number of sexual partners during the entire life between three and five partners (ORaj = 2.2; 95% CI: 1.1-4.6), and six or more sexual partners (ORaj = 3.9; 95% CI: 1.9-8.0%); being a sex worker (ORaj = 1.9; 95% CI: 1.1-3.1). CONCLUSIONS: A high prevalence of a prior history of STDs in the studied population was found. It is essential to find better ways to access HIV infection prevention, so that effective interventions can be more widely implemented.

The humankind genome: from genetic diversity to the origin of human diseases

Genome-wide association studies have failed to establish common variant risk for the majority of common human diseases. The underlying reasons for this failure are explained by recent studies of resequencing and comparison of over 1200 human genomes and 10 000 exomes, together with the delineation of DNA methylation patterns (epigenome) and full characterization of coding and noncoding RNAs (transcriptome) being transcribed. These studies have provided the most comprehensive catalogues of functional elements and genetic variants that are now available for global integrative analysis and experimental validation in prospective cohort studies. With these datasets, researchers will have unparalleled opportunities for the alignment, mining, and testing of hypotheses for the roles of specific genetic variants, including copy number variations, single nucleotide polymorphisms, and indels as the cause of specific phenotypes and diseases. Through the use of next-generation sequencing technologies for genotyping and standardized ontological annotation to systematically analyze the effects of genomic variation on humans and model organism phenotypes, we will be able to find candidate genes and new clues for disease’s etiology and treatment. This article describes essential concepts in genetics and genomic technologies as well as the emerging computational framework to comprehensively search websites and platforms available for the analysis and interpretation of genomic data.

Impact of exercise training on redox signaling in cardiovascular diseases

Reactive oxygen and nitrogen species regulate a wide array of signaling pathways that governs cardiovascular physiology. However, oxidant stress resulting from disrupted redox signaling has an adverse impact on the pathogenesis and progression of cardiovascular diseases. In this review, we address how redox signaling and oxidant stress affect the pathophysiology of cardiovascular diseases such as ischemia-reperfusion injury, hypertension and heart failure. We also summarize the benefits of exercise training in tackling the hyperactivation of cellular oxidases and mitochondrial dysfunction seen in cardiovascular diseases

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.

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.

Human papillomavirus (HPV) and associated diseases: between applied diagnostic and basic research

Human Papillomavirus (HPV) is the cause of cervical cancers (among these, adenocarcinoma, AdCa) and is associated to a subgroup of oropharyngeal carcinomas (OPSCCs). Even if the risk for cancer development is linked to the infection by some viral genotypes, mainly HPV16 and 18, viral DNA alone seems not to be sufficient for diagnosis. Moreover, the role of the virus in OPSCCs has not been totally clarified yet. In the first part of the thesis, the performances concerning viral genotyping in clinical cervical samples of a new pyrosequencing-based test and a well-known hybridization-based assay have been compared. Similar results between the methods have been obtained. However, the former showed advantages in detecting intratype variants, higher specificity and a broader spectrum of detectable HPV types. The second part deals with the evaluation of virological markers (genotyping, viral oncoproteins expression, viral load, physical state and CpG methylation of HPV16 genome) in the diagnosis/prognosis of cervical AdCa and HPV-associated OPSCCs. HPV16 has been confirmed the most prevalent genotype in both the populations. Interestingly, the mean methylation frequency of viral DNA at the early promoter showed the tendency to be associated to invasion for cervical AdCa and to a worse prognosis for OPSCCs, suggesting a promising role as diagnostic/prognostic biomarker. The experiments of the third part were performed at the DKFZ in Heidelberg (Germany) and dealt with the analysis of the response to IFN-k transfection in HPV16-positive cervical cancer and head&neck carcinoma cell lines to evaluate its potential role as new treatment. After 24h, we observed increased IFN-b expression which lead to the up-regulation of genes involved in the antigens presentation pathway (MHC class I and immunoproteasome) and antiviral response as well, in particular in cervical cancer cell lines. This fact suggested also the presence of different HPV-mediated carcinogenic pathways between the two anatomical districts.

Development of new therapeutic approaches in the treatments of Inflammatory Bowel Diseases: functional food and nutraceuticals vs synthetic peptides based vaccines

Inflammatory Bowel Diseases (IBD) are intestinal chronic relapsing diseases which ethiopathogenesis remains uncertain. Several group have attempted to study the role of factors involved such as genetic susceptibility, environmental factors such as smoke, diet, sex, immunological factors as well as the microbioma. None of the treatments available satisfy several criteria at the same time such as safety, long-term remission, histopatological healing, and specificity. We used two different approaches for the development of new therapeutic treatment for Inflammatory Bowel Disease. The first is focused on the understanding of the potential role of functional food and nutraceuticals nutrients in the treatment of IBD. To do so, we investigated the role of Curcuma longa in the treatment of chemical induced colitis in mice model. Since Curcma Longa has been investigated for its antinflammatory role related to the TNFα pathway as well investigators have reported few cases of patients with ulcerative colites treated with this herbs, we harbored the hypothesis of a role of Curcuma Longa in the treatment f IBD as well as we decided to assess its role in intestinal motility. The second part is based on an immunological approach to develop new drugs to induce suppression in Crohn’s disease or to induce mucosa immunity such as in colonrectal tumor. The main idea behind this approach is that we could manipulate relevant cell-cell interactions using synthetic peptides. We demonstrated the role of the unique interaction between molecules expressed on intestinal epithelial cells such as CD1d and CEACAM5 and on CD8+ T cells. In normal condition this interaction has a role for the expansion of the suppressor CD8+ T cells. Here, we characterized this interaction, we defined which are the epitope involved in the binding and we attempted to develop synthetic peptides from the N domain of CEACAM5 in order to manipulate it.