Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and Elisa tests for the diagnosis of Chagas disease

Using the indirect hemagglutination (IH), indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) tests for the diagnosis of Chagas disease, 4000 serum samples were examined. This study was conducted with different purposes: clinical interest, research support and parasitological monitoring of those patients with Chagas disease who were treated with heart transplantations. The tests occurred without patient selection and in accordance with the medical requests. The results showed discrepancies and brought about several questions, considering the different results that all three methods showed when considered together. What was found brought about concerns and we suggest the adoption of different measures, aiming to avoid these mismatches in the context of this disease.

Children with Type 1 Diabetes Mellitus and their friends: the influence of this interaction in the management of the disease

Friends' support is a key element in the management of Type 1 Diabetes Mellitus. This study describes the influence of friends on the lives of children with Diabetes Mellitus and its implications for managing the disease. Empirical data were collected through semi-structured interviews, supported with the use of puppets, with 19 children aged between seven and 12 years old. The qualitative analysis of the testimonies allowed understanding the phenomenon from two perspectives: the attitude of friends towards the child, positively or negatively affecting the disease's management, and the attitude of the child toward friends. The knowledge of those involved and the interaction between the children with DM1 and their friends impacts the management of the disease. Understanding the implications of these interactions contributes to the delivery of qualified nursing care to this population.

Prevalence of celiac disease among blood donors in São Paulo: the most populated city in Brazil

OBJECTIVE: Celiac disease is a permanent enteropathy caused by the ingestion of gluten, which leads to an immunemediated inflammation of the small intestine mucosa. The prevalence of celiac disease varies among different nations and ethnic backgrounds, and its diversity is determined by genetic and environmental factors. São Paulo city is one of the largest cities in the world, with a vast population and an important history of internal migratory flow from other Brazilian regions, as well as immigration from other, primarily European, countries, resulting in significant miscegenation. The aim of the present study was to estimate the prevalence of adults with undiagnosed celiac disease among blood donors of São Paulo by collecting information on the ancestry of the population studied. METHODS: The prevalence of celiac disease was assessed by screening for positive IgA transglutaminase and IgA endomysium antibodies in 4,000 donors (volunteers) in the Fundação Pró-Sangue Blood Center of São Paulo, São Paulo, Brazil. The antibody-positive subjects were asked to undergo a small bowel biopsy. RESULTS: Of the 4,000 subjects, twenty-four had positive tests, although both antibody tests were not always concordant. For example, ten subjects were positive for IgA tissue transglutaminase only. In twenty-one positive patients, duodenal biopsies were performed, and the diagnosis of celiac disease was confirmed in fourteen patients (Marsh criteria modified by Oberhuber). In this group, 67% claimed to have European ancestry, mainly from Italy, Portugal and Spain. CONCLUSION: The prevalence of celiac disease is at least 1:286 among supposedly healthy blood bank volunteers in São Paulo, Brazil.

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 105 tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34th and 35th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.

Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor gene

During my PhD I have been involved in several projects regarding the morphogenesis of the follicular epithelium, such as the analysis of the pathways that correlate follicular epithelium patterning and eggshell genes expression. Moreover, I used the follicular epithelium as a model system to analyze the function of the Drosophila homolog of the human von Hippel-Lindau (d-VHL) during oogenesis, in order to gain insight into the role of h-VHL for the pathogenesis of VHL disease. h-VHL is implicated in a variety of processes and there is now a greater appreciation of HIF-independent h-VHL functions that are relevant to tumour development, including maintenance and organization of the primary cilium, maintenance of the differentiated phenotype in renal cells and regulation of epithelial-mesenchymal transition. However, the function of h-VHL gene during development has not been fully understood. It was previously shown that d-VHL down-regulates the motility of tubular epithelial cells (tracheal cells) during embryogenesis. Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. Therefore, to examine whether d-VHL has a role in epithelial morphogenesis and maintenance, I performed genetic and molecular analyses by using in vivo and in vitro approaches. From my analysis, I determined that d-VHL binds to and stabilizes microtubules. Loss of d-VHL depolymerizes the microtubule network during oogenesis, leading to a possible deregulation in the subcellular trafficking transport of polarity markers from Golgi apparatus to the different domains in which follicle cells are divided. The analysis carried out has allowed to establish a significant role of d-VHL in the maintenance of the follicular epithelium integrity.

Evaluation of cardiovascular disease markers in patients submitted to carotid artery stenting or endarterectomy

Introduction. Microembolization during the carotid artery revascularization procedure may cause cerebral lesions. Elevated C-Reactive Protein (hsCRP), Vascular endothelial growth factor (VEGF) and serum amyloid A protein (SAA) exert inflammatory activities thus promoting carotid plaque instability. Neuron specific enolase (NSE) is considered a marker of cerebral injury. Neoangiogenesis represents a crucial step in atherosclerosis, since neovessels density correlates with plaque destabilization. However their clinical significance on the outcome of revascularization is unknown. This study aims to establish the correlation between palque vulnerabilty, embolization and histological or serological markers of inflammation and neoangiogenesis. Methods. Serum hsCRP, SAA, VEGF, NSE mRNA, PAPP-A mRNA levels were evaluated in patients with symptomatic carotid stenosis who underwent filter-protected CAS or CEA procedure. Cerebral embolization, presence of neurologicals symptoms, plaque neovascularization were evaluated testing imaging, serological and histological methods. Results were compared by Fisher’s, Student T test and Mann-Whitney U test. Results. Patients with hsCRP<5 mg/l, SAA<10mg/L and VEGF<500pg/ml had a mean PO of 21.5% versus 35.3% (p<0.05). In either group, embolic material captured by the filter was identified as atherosclerotic plaque fragments. Cerebral lesions increased significantly in all patients with hsCRP>5mg/l and SAA>10mg/l (16.5 vs 2.8 mean number, 3564.6 vs 417.6 mm3 mean volume). Discussion. High hsCRP, SAA and VEGF levels are associated with significantly greater embolization during CAS and to the vulnerabiliy of the plaque. This data suggest CAS might not be indicated as a method of revascularization in this specific group of patients.

Pathogenesis of cell toxicity by plant toxins

Ribosome inactivating proteins (RIPs) are a family of plant proteins that depurinate the major rRNA, inhibiting the protein synthesis. RIPs are divided into type 1, single chain proteins with enzymatic activity, and type 2 RIPs (toxic and non-toxic), with the enzymatic chain linked to a binding chain. RIPs have been used alone or as toxic component of immunotoxins for experimental therapy of many diseases. The knowledge of cell death pathway(s) induced by RIPs could be useful for clarifying the mechanisms induced by RIPs and for designing specific immunotherapy. The topic of the current study was (i) the determination of the amino acid sequence of the type 2 RIP stenodactylin. The comparison with other RIPs showed that the A chain is related to other toxic type 2 RIPs. whereas the B chain is more related to the non-toxic type 2 RIPs. This latter result is surprising because stenodactylin is actually the most toxic type 2 RIP known; (ii) the study of the cell death mechanisms induced by stenodactylin in human neuroblastoma cells (NB100). High doses of stenodactylin can activate the effector caspases (perhaps through the DNA damage and/or intrinsic/extrinsic pathways) and also cause ROS generation. Low doses cause a caspase-dependent apoptosis, mainly via extrinsic pathway. Moreover, the activation of caspases precedes the inhibition of protein synthesis; (iii) the investigation of the cell death pathway induced by the non-toxic type 2 RIPs ebulin l and nigrin b. These RIPs demonstrated high enzymatic activity in a cell-free system, but they lack high cytotoxicity. These preliminary studies demonstrate that the cell death mechanism induced by the two non-toxic RIPs is partially caspase-dependent apoptosis, but other mechanisms seem to be involved

Molecular mechanisms involved in the pathogenesis of beet soil-borne viruses.

The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses. BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so I developed agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins. Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of post-transcriptional gene silencing sharing common features with BNYVV p14.

The impact of cardiac rehabilitation on lifestyles, psychological correlates and the clinical course of cardiac disease

Objectives: The aim of this research was to evaluate the impact of Cardiac Rehabilitation (CR) on risky lifestyles, quality of life, psychopathology, psychological distress and well-being, considering the potential moderating role of depression, anxiety and psychosomatic syndromes on lifestyles modification. The influence of CR on cardiac morbidity and mortality was also evaluated. Methods: The experimental group (N=108), undergoing CR, was compared to a control group (N=85) of patients affected by cardiovascular diseases, not undergoing CR, at baseline and at 1-month, 6- and 12-months follow-ups. The assessment included: the Structured Clinical Interview for DSM-IV, the structured interview based on Diagnostic Criteria for Psychosomatic Research (DCPR), GOSPEL questionnaire on lifestyles, Pittsburgh Sleep Quality Index, Morisky Medication Adherence Scale, MOS 36-Item Short Form Health Survey, Symptom Questionnaire, Psychological Well-Being Scale and 14-items Type D Scale. Results: Compared to the control group, CR was associated to: maintenance of the level of physical activity, improvement of correct dietary behaviors and stress management, enhancement of quality of life and sleep; reduction of the most frequently observed psychiatric diagnoses and psychosomatic syndromes at baseline. On the contrary, CR was not found to be associated with: healthy dietary habits, weight loss and improvement on medications adherence. In addition, there were no relevant effects on sub-clinical psychological distress and well-being, except for personal growth and purpose in life (PWB). Also, CR did not seem to play a protective role against cardiac recurrences. The presence of psychosomatic syndromes and depressive disorders was a mediating factor on the modification of specific lifestyles. Conclusions: The findings highlight the need of a psychosomatic assessment and an evaluation of psychological sub-clinical symptomatology in cardiac rehabilitation, in order to identify and address specific factors potentially associated with the clinical course of the heart disease.

Gene therapy for a novel mouse model of Canavan disease

Canavan disease (CD) is a rare leukodystrophy caused by loss-of-function mutations in the gene encoding aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme. It is characterised by the accumulation of the ASPA substrate N-acetylaspartate (NAA) in brain, blood and urine, leading to a spongiform vacuolisation of the brain, severe motoric and cognitive impairments and premature death. To date, no therapy is available due to the lack of a gene-transfer system allowing transgene expression in oligodendrocytes (OLs) and the restoration of the missing enzyme. Hence, the aim of this study was to establish a novel gene-transfer system and its preclinical evaluation in a CD animal model.rnIn the first part of this thesis, a novel ASPA mouse mutant was generated. A βgeo cassette (including the genes encoding β-galactosidase and neomycin) flanked by frt sites was inserted into intron 1 of the intact aspa gene. Additionally, exon 2 was flanked by loxP sites for optional conditional deletion of the targeted locus. The resulting ASPA-deficient aspalacZ/lacZ-mouse was found to be an accurate model of CD and an important tool to identify novel aspects of its complex pathology. Homozygous mutants showed a CD-like histopathology, neurological impairment, behavioural deficits as well as a reduced body weight. Additionally, MRI data revealed changes in brain metabolite composition. rnRecombinant adeno-associated viral (rAAV) vectors have become a versatile tool for gene transfer to the central nervous system because they are efficient, non-toxic and replication-deficient. Based on the natural neurotropism of AAV vectors, AAV-based gene delivery has entered the clinics for the treatment of neurodegenerative diseases. However, the lack of AAV vectors with oligodendroglial tropism has precluded gene therapy for leukodystrophies. In the second part of this work, it was shown that the transduction profile of established AAV serotypes can be targeted towards OLs in a transcriptional approach, using the oligodendrocyte-specific myelin basic protein (MBP) promoter to drive transgene expression in OLs.rnIn the last part of this work, the therapeutic efficacy of AAV-mediated aspa gene transfer to OLs of juvenile aspalacZ/lacZ mice was evaluated. AAV-aspa injections into multiple sites of the brain parenchyma resulted in transduction of OLs in the grey and white matter throughout the brain. Histological abnormalities in the brain of ASPA-deficient mice were ameliorated and accompanied by a reduction of NAA levels. Furthermore, the treatment resulted in normalisation of body weight, motor function and nest-building behaviour. These data provide a proof-of-concept for a successful gene therapy of Canavan disease. This might pave the way towards translation into clinical application and serve as the basis for the genetic treatment of other leukodystrophies.

The role of DNA damage in the pathogenesis of nitrate tolerance - nitrosative versus oxidative DNA damage

6. Summary Despite the lack of direct evidence from large clinical trials for mutagenic and genotoxic effects of GTN therapy, the present study show s the induction of pre-mutagenic lesions, such as 8- oxo - G and O 6 - me - G by GTN t reatment as well as increased formation of DNA strand breaks. These results were obtained in an in vitro (EA.hy 926 – human endothelial cell line) and in vivo (Wistar rats and C57BL/6 mice) setting. However, GTN - induced DNA damage had no effect on the degr ee of nitrate tolerance but only on other pathological side effects such as oxidative stress, as confirmed by studies in MGMT knockout mice. Of clinical importance , this study establishes potent apoptotic properties of organic nitrates, which has been demo nstrated by the levels of the novel apoptotic marker and caspase - 3 substrate, fractin, as well as levels of cleaved caspase - 3 , the activated form of this pro - apoptotic enzyme . The p rotein analy tical data ha ve been confirmed by an independent assay for the apoptosis , Cell death detection assay (TUNEL) . First, these GTN - mediated apoptotic effects may account for the previously reported anti - cancer effects of GTN therapy (probably based on induction of apoptosis in tumor cells). Second, these GTN - mediated apop totic effects may account for the increased mortality rates observed in the group of organic nitrate - treated patients as reported by two independent meta - analysis (probably due to induction of apoptosis in highly beneficial endothelial progenitor cells as well as in cardiomyocytes during wound healing and cardiac remodeling) . Summary of the current investigations can be seen in Figure 18.

Significance of Periodontal Risk Assessment in the recurrence of periodontitis and tooth loss

Aim: To investigate the association of the Periodontal Risk Assessment (PRA) model categories with periodontitis recurrence and tooth loss during supportive periodontal therapy (SPT) and to explore the role of patient compliance. Material and Methods: In a retrospective cohort, PRA was performed for 160 patients after active periodontal therapy (APT) and after 9.5 ± 4.5 years of SPT. The recurrence of periodontitis and tooth loss were analysed according to the patient's risk profile (low, moderate or high) after APT and compliance with SPT. The association of risk factors with tooth loss and recurrence of periodontitis was investigated using logistic regression analysis. Results: In 18.2% of patients with a low-risk profile, in 42.2% of patients with a moderate-risk profile and in 49.2% of patients with a high-risk profile after APT, periodontitis recurred. During SPT, 1.61 ± 2.8 teeth/patient were lost. High-risk profile patients lost significantly more teeth (2.59 ± 3.9) than patients with moderate- (1.02 ± 1.8) or low-risk profiles (1.18 ± 1.9) (Kruskal–Wallis test, p=0.0229). Patients with erratic compliance lost significantly (Kruskal–Wallis test, p=0.0067) more teeth (3.11 ± 4.5) than patients compliant with SPT (1.07 ± 1.6). Conclusions: In multivariate logistic regression analysis, a high-risk patient profile according to the PRA model at the end of APT was associated with recurrence of periodontitis. Another significant factor for recurrence of periodontitis was an SPT duration of more than 10 years.

Influence of polyvascular disease on cardiovascular event rates. Insights from the REACH Registry

Cardiovascular event rates have been shown to increase substantially with the number of symptomatic disease locations. We sought to assess the risk profile, management and subsequent event rates of polyvascular disease patients. Consecutive outpatients were assessed for atherosclerotic risk factors and medications in the REACH Registry. A total of 19,117 symptomatic patients in Europe completed a 2-year follow-up: 77.2% with single arterial bed disease (coronary artery or cerebrovascular or peripheral arterial disease) and 22.8% with polyvascular disease (>/= 1 disease location). Polyvascular disease patients were older (68.5 +/- 9.4 vs 66.3 +/- 9.9 years, p < 0.0001), more often current or former smokers (64.9% vs 58.7%, p < 0.0001), and more often suffered from hypertension (59.5% vs 46.6%, p < 0.0001) and diabetes (34.5% vs 25.9%, p < 0.0001) than single arterial bed disease patients. Despite more intense medical therapy, risk factors (smoking, hypertension, low fasting glucose, and low fasting total cholesterol) were less often controlled in polyvascular disease patients. This was associated with substantially more events over 2 years compared with single arterial bed disease patients (cMACCE [cardiovascular death/non-fatal stroke/non-fatal MI] odds ratio, 1.63 [95% CI, 1.45-1.83], p < 0.0001). In conclusion, polyvascular disease patients have more cardiovascular risk factors, and the prognosis for these patients is significantly worse than for patients with single arterial bed disease. This suggests a need to improve detection and consequent medical treatment of polyvascular disease.

[Pathogenesis of functional gastrointestinal disorders - an interdisciplinary perspective]

Functional gastrointestinal disorders (FGD) are highly prevalent worldwide. Recent research demonstrates that complex and interacting biological and behavioral mechanisms contribute particularly to the pathogenesis of irritable bowel syndrome and functional dyspepsia. Dysregulation of the enteral, neuroenteric, visceral-autonomic, and central nervous systems are important biological contributors, whereas the psychological state of a patient may evidently modulate aspects related to biological stress reactivity and somatic perception both playing a role in the clinical manifestation of FGD. Our overview clearly shows that an interdisciplinary perspective of the pathogenesis of FGD may best serve clinicians and patients.