Molecular basis for the development of diagnostic methods and specific treatment modalities for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown aetiology and poor prognosis. IPF is characterized by alveolar epithelial damage that leads tissue remodelling and ultimately to the loss of normal lung architecture and function. Treatment has been focused on anti-inflammatory therapies, but due to their poor efficacy new therapeutic modalities are being sought. There is a need for early diagnosis and also for differential diagnostic markers for IPF and other interstitial lung diseases. The study utilized patient material obtained from bronchoalveolar lavage (BAL), diagnostic biopsies or lung transplantation. Human pulmonary fibroblast cell cultures were propagated and asbestos-induced pulmonary fibrosis in mice was used as an experimental animal model of IPF. The possible markers for IPF were scanned by immunohistochemistry, RT-PCR, ELISA and western blot. Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in tissue remodelling. Microarray studies have introduced potential markers that could serve as additional tools for the assessment of IPF and one of the most promising was MMP 7. MMP-7 protein levels were measured in the BAL fluid of patients with idiopathic interstitial lung diseases or idiopathic cough. MMP-7 was however similarly elevated in the BAL fluid of all these disorders and thus cannot be used as a differential diagnostic marker for IPF. Activation of transforming growth factor (TGF)-ß is considered to be a key element in the progression of IPF. Bone morphogenetic proteins (BMP) are negative regulators of intracellular TGF-ß signalling and BMP-4 signalling is in turn negatively regulated by gremlin. Gremlin was found to be highly upregulated in the IPF lungs and IPF fibroblasts. Gremlin was detected in the thickened IPF parenchyma and endothelium of small capillaries, whereas in non-specific interstitial pneumonia it localized predominantly in the alveolar epithelium. Parenchymal gremlin immunoreactivity might indicate IPF-type interstitial pneumonia. Gremlin mRNA levels were higher in patients with end-stage fibrosis suggesting that gremlin might be a marker for more advanced disease. Characterization of the fibroblastic foci in the IPF lungs showed that immunoreactivity to platelet-derived growth factor (PDGF) receptor-α and PDGF receptor-β was elevated in IPF parenchyma, but the fibroblastic foci showed only minor immunoreactivity to the PDGF receptors or the antioxidant peroxiredoxin II. Ki67 positive cells were also observed predominantly outside the fibroblastic foci, suggesting that the fibroblastic foci may not be composed of actively proliferating cells. When inhibition of profibrotic PDGF-signalling by imatinib mesylate was assessed, imatinib mesylate reduced asbestos-induced pulmonary fibrosis in mice as well as human pulmonary fibroblast migration in vitro but it had no effect on the lung inflammation.

Myotonic dystrophy type 2 (DM2) : Diagnostic methods and molecular pathology

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are the most common forms of muscular dystrophy affecting adults. They are autosomal dominant diseases caused by microsatellite tri- or tetranucleotide repeat expansion mutations in transcribed but not translated gene regions. The mutant RNA accumulates in nuclei disturbing the expression of several genes. The more recently identified DM2 disease is less well known, yet more than 300 patients have been confirmed in Finland thus far, and the true number is believed to be much higher. DM1 and DM2 share some features in general clinical presentation and molecular pathology, yet they show distinctive differences, including disease severity and differential muscle and fiber type involvement. However, the molecular differences underlying DM1 and DM2 muscle pathology are not well understood. Although the primary tissue affected is muscle, both DMs show a multisystemic phenotype due to wide expression of the mutation-carrying genes. DM2 is particularly intriguing, as it shows an incredibly wide spectrum of clinical manifestations. For this reason, it constitutes a real diagnostic challenge. The core symptoms in DM2 include proximal muscle weakness, muscle pain, myotonia, cataracts, cardiac conduction defects and endocrinological disturbations; however, none of these is mandatory for the disease. Myalgic pains may be the most disabling symptom for decades, sometimes leading to incapacity for work. In addition, DM2 may cause major socio-economical consequences for the patient, if not diagnosed, due to misunderstanding and false stigmatization. In this thesis work, we have (I) improved DM2 differential diagnostics based on muscle biopsy, and (II) described abnormalities in mRNA and protein expression in DM1 and DM2 patient skeletal muscles, showing partial differences between the two diseases, which may contribute to muscle pathology in these diseases. This is the first description of histopathological differences between DM1 and DM2, which can be used in differential diagnostics. Two novel high-resolution applications of in situ -hybridization have been described, which can be used for direct visualization of the DM2 mutation in muscle biopsy sections, or mutation size determination on extended DNA-fibers. By measuring protein and mRNA expression in the samples, differential changes in expression patterns affecting contractile proteins, other structural proteins and calcium handling proteins in DM2 compared to DM1 were found. The dysregulation at mRNA level was caused by altered transciption and abnormal splicing. The findings reported here indicate that the extent of aberrant splicing is higher in DM2 compared to DM1. In addition, the described abnormalities to some extent correlate to the differences in fiber type involvement in the two disorders.

Occupational chronic solvent encephalopathy in Finland 1995 - 2007 : incidence and diagnostic methods

The occurrence of occupational chronic solvent encephalopathy (CSE) seems to decrease, but still every year reveals new cases. To prevent CSE and early retirement of solvent-exposed workers, actions should focus on early CSE detection and diagnosis. Identifying the work tasks and solvent exposure associated with high risk for CSE is crucial. Clinical and exposure data of all the 128 cases diagnosed with CSE as an occupational disease in Finland during 1995-2007 was collected from the patient records at the Finnish Institute of Occupational Health (FIOH) in Helsinki. The data on the number of exposed workers in Finland were gathered from the Finnish Job-exposure Matrix (FINJEM) and the number of employed from the national workforce survey. We analyzed the work tasks and solvent exposure of CSE patients and the findings in brain magnetic resonance imaging (MRI), quantitative electroencephalography (QEEG), and event-related potentials (ERP). The annual number of new cases diminished from 18 to 3, and the incidence of CSE decreased from 8.6 to 1.2 / million employed per year. The highest incidence of CSE was in workers with their main exposure to aromatic hydrocarbons; during 1995-2006 the incidence decreased from 1.2 to 0.3 / 1 000 exposed workers per year. The work tasks with the highest incidence of CSE were floor layers and lacquerers, wooden surface finishers, and industrial, metal, or car painters. Among 71 CSE patients, brain MRI revealed atrophy or white matter hyperintensities or both in 38% of the cases. Atrophy which was associated with duration of exposure was most frequently located in the cerebellum and in the frontal or parietal brain areas. QEEG in a group of 47 patients revealed increased power of the theta band in the frontal brain area. In a group of 86 patients, the P300 amplitude of auditory ERP was decreased, but at individual level, all the amplitude values were classified as normal. In 11 CSE patients and 13 age-matched controls, ERP elicited by a multimodal paradigm including an auditory, a visual detection, and a recognition memory task under single and dual-task conditions corroborated the decrease of auditory P300 amplitude in CSE patients in single-task condition. In dual-task conditions, the auditory P300 component was, more often in patients than in controls, unrecognizable. Due to the paucity and non-specificity of the findings, brain MRI serves mainly for differential diagnostics in CSE. QEEG and auditory P300 are insensitive at individual level and not useful in the clinical diagnostics of CSE. A multimodal ERP paradigm may, however, provide a more sensitive method to diagnose slight cognitive disturbances such as CSE.

A boarding school outbreak of pertussis in adolescents : value of laboratory diagnostic methods

Culture for Bordetella pertussis (B. pertussis) is the traditional gold standard for laboratory diagnosis of pertussis but is insensitive, especially later in the course of illness and in vaccinated persons. Interpretation of serology is limited by the lack of an appropriate reference standard. An outbreak of pertussis in a crowded boarding-school dormitory allowed evaluation of laboratory correlates of infection. Questionnaires, serum samples and throat swabs were collected from members of the exposed group. Serum samples from unexposed controls of a similar age group were used for comparison. B. pertussis PCR was performed on throat swabs, and sera were tested for IgA antibodies against whole-cell (WC) B. pertussis antigen and IgG antibodies to pertussis toxin (PT). The Centers for Disease Control and Prevention definition for pertussis was used to define clinical cases. We evaluated the use of a previously published cut-off for PT IgG of 125 EIA units (EU)/ml. Completed questionnaires were obtained from 115 students, of whom 85 (74%) reported coughing symptoms, including 32 (28%) who met the clinical case definition for pertussis. B. pertussis was detected by PCR in 17 (15%) and WC IgA in 22 (19%) students; neither correlated with symptoms, but dormitory of residence strongly predicted PCR status. The mean PT IgG geometric mean concentration, in this situation of high pertussis exposure, correlated with severity of symptoms and was significantly higher in both symptomatic and asymptomatic children exposed during the outbreak (P<0·001) than in control children. A cut-off for PT IgG of 125 EU/ml was too high in an outbreak situation to be sensitive enough to identify pertussis cases. A case of pertussis in a crowded boarding-school dormitory resulted rapidly in an outbreak. Serology and PCR were useful in identifying the outbreak and commencing disease control measures. The use of serology has mostly been evaluated in community serosurveys, where it is not possible to determine if immunity reflects vaccination, asymptomatic disease or symptomatic disease. This outbreak gave us the opportunity to evaluate the value of serology and PCR in the presence of confirmed exposure to pertussis.

Molecular diagnostic methods for identifying Serrasalmid fish (Pacu, Pirapitinga, and Tambaqui) and their hybrids in the Brazilian aquaculture industry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development of diagnostic methods and study of the immunoreactivity of a mixture of recombinant core and E2 proteins fused to GST with control serum positive for hepatitis C

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Doppler and Elastography as complementary diagnostic methods for mammary neoplasms in female cats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Malnutrition in patients with gastrointestinal cancer: effectiveness of different diagnostic methods

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Proximal caries lesion detection in primary teeth: does this justify the association of diagnostic methods?

The aim of this clinical study was to evaluate and compare the performance of visual exam with use of the Nyvad criteria (visual examination - (VE)), interproximal radiography (BW), laser fluorescence device (DIAGNOdent Pen-DDPen), and their association in the diagnosis of proximal lesions in primary teeth. For this purpose, 45 children (n = 59 surfaces) of both sexes, aged between 5 and 9 years were selected, who presented healthy primary molars or primary molars with signs suggestive of the presence of caries lesions. The surfaces were clinically evaluated and coded according to the Nyvad criteria and immediately afterwards with the DDPen. Radiographic exam was performed only on the surfaces coded with Nyvad scores 2, 3, 5, or 6. Active caries lesions and/or those with discontinuous surfaces were restored, considering the depth of lesion as reference standard. Sensitivity, specificity, accuracy, and area under ROC curve were calculated for each technique and its associations. Visual exam with Nyvad criteria presented the highest specificity, accuracy, and area under ROC curve values. The DDPen presented the highest sensitivity values. Association with one or more methods resulted in an increase in specificity. The performance of visual, radiographic, and DDpen exams and their associations were good; however, the clinical examination with the Nyvad criteria was sufficient for the diagnosis of interproximal lesions in primary teeth.

Diagnostic methods in sepsis: the need of speed

Objective: Sepsis is a common condition encountered in hospital environments. There is no effective treatment for sepsis, and it remains an important cause of death at intensive care units. This study aimed to discuss some methods that are available in clinics, and tests that have been recently developed for the diagnosis of sepsis. Methods: A systematic review was performed through the analysis of the following descriptors: sepsis, diagnostic methods, biological markers, and cytokines. Results: The deleterious effects of sepsis are caused by an imbalance between the invasiveness of the pathogen and the ability of the host to mount an effective immune response. Consequently, the host's immune surveillance fails to eliminate the pathogen, allowing it to spread. Moreover, there is a pro-inflammatory mediator release, inappropriate activation of the coagulation and complement cascades, leading to dysfunction of multiple organs and systems. The difficulty achieve total recovery of the patient is explainable. There is an increased incidence of sepsis worldwide due to factors such as aging population, larger number of surgeries, and number of microorganisms resistant to existing antibiotics. Conclusion: The search for new diagnostic markers associated with increased risk of sepsis development and molecules that can be correlated to certain steps of sepsis is becoming necessary. This would allow for earlier diagnosis, facilitate patient prognosis characterization, and prediction of possible evolution of each case. All other markers are regrettably constrained to research units.

Diagnostic methods in sepsis: the need of speed

OBJECTIVE: Sepsis is a common condition encountered in hospital environments. There is no effective treatment for sepsis, and it remains an important cause of death at intensive care units. This study aimed to discuss some methods that are available in clinics, and tests that have been recently developed for the diagnosis of sepsis. METHODS: A systematic review was performed through the analysis of the following descriptors: sepsis, diagnostic methods, biological markers, and cytokines. RESULTS: The deleterious effects of sepsis are caused by an imbalance between the invasiveness of the pathogen and the ability of the host to mount an effective immune response. Consequently, the host's immune surveillance fails to eliminate the pathogen, allowing it to spread. Moreover, there is a pro-inflammatory mediator release, inappropriate activation of the coagulation and complement cascades, leading to dysfunction of multiple organs and systems. The difficulty achieve total recovery of the patient is explainable. There is an increased incidence of sepsis worldwide due to factors such as aging population, larger number of surgeries, and number of microorganisms resistant to existing antibiotics. CONCLUSION: The search for new diagnostic markers associated with increased risk of sepsis development and molecules that can be correlated to certain steps of sepsis is becoming necessary. This would allow for earlier diagnosis, facilitate patient prognosis characterization, and prediction of possible evolution of each case. All other markers are regrettably constrained to research units.

Chapter II: Diagnostic methods

Non-invasive vascular studies can provide crucial information on the presence, location, and severity of critical limb ischaemia (CLI), as well as the initial assessment or treatment planning. Ankle-brachial index with Doppler ultrasound, despite limitations in diabetic and end-stage renal failure patients, is the first-line evaluation of CLI. In this group of patients, toe-brachial index measurement may better establish the diagnosis. Other non-invasive measurements, such as segmental limb pressure, continuous-wave Doppler analysis and pulse volume recording, are of limited accuracy. Transcutaneous oxygen pressure (TcPO(2)) measurement may be of value when rest pain and ulcerations of the foot are present. Duplex ultrasound is the most important non-invasive tool in CLI patients combining haemodynamic evaluation with imaging modality. Computed tomography angiography (CTA) and magnetic resonance angiography (MRA) are the next imaging studies in the algorithm for CLI. Both CTA and MRA have been proven effective in aiding the decision-making of clinicians and accurate planning of intervention. The data acquired with CTA and MRA can be manipulated in a multiplanar and 3D fashion and can offer exquisite detail. CTA results are generally equivalent to MRA, and both compare favourably with contrast angiography. The individual use of different imaging modalities depends on local availability, experience, and costs. Contrast angiography represents the gold standard, provides detailed information about arterial anatomy, and is recommended when revascularisation is needed.

Similarities and differences between physician and non-physician acupuncturists in using diagnostic methods - an explorative study

Purpose: Acupuncture is one of the complementary medicine therapies with the greatest demand in Switzerland and many other countries in the West and in Asia. Over the past decades, the pool of scientific literature in acupuncture has markedly increased. The diagnostic methods upon which acupuncture treatment is based, have only been addressed sporadically in scientific journals. The goal of this study is to assess the use of different diagnostic methods in the acupuncture practices and to investigate similarities and differences in using these diagnostic methods between physician and non-physician acupuncturists. Methods: 44 physician acupuncturists with certificates of competence in acupuncture – traditional chinese medicine (TCM) from ASA (Assoziation Schweizer Ärztegesellschaften für Akupunktur und Chinesische Medizin: the Association of Swiss Medical Societies for Acupuncture and Chinese Medicine) and 33 non-physician acupuncturists listed in the EMR (Erfahrungsmedizinisches Register: a national register, which assigns a quality label for CAM therapists in complementary and alternative medicine) in the cantons Basel-Stadt and Basel-Land were asked to fill out a questionnaire on diagnostic methods. The responder rate was 46.8% (69.7% non-physician acupuncturists and 29, 5% physician acupuncturists). Results: The results show that both physician and non-physician acupuncturists take patients’ medical history (94%), use pulse diagnosis (89%), tongue diagnosis (83%) and palpation of body and ear acupuncture points (81%) as diagnostic methods to guide their acupuncture treatments. Between the two groups, there were significant differences in the diagnostic tools being used. Physician acupuncturists do examine their patients significantly more often with western medical methods (p<.05) than this is the case for nonphysician acupuncturists. Non-physician acupuncturists use pulse diagnosis more often than physicians (p<.05). A highly significant difference was observed in the length of time spent with collecting patients’ medical history, where nonphysician acupuncturists clearly spent more time (p<.001). Conclusion: Depending on the educational background of the acupuncturist, different diagnostic methods are used for making the diagnosis. Especially the more time consuming methods like a comprehensive anamnesis and pulse diagnosis are more frequently employed by non-physician practitioners. Further studies will clarify if these results are valid for Switzerland in general, and to what extent the differing use of diagnostic methods has an impact on the diagnosis itself and on the resulting treatment methods, as well as on the treatment success and the patients’ satisfaction.

Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.