Comprehensive analysis of proteins secreted by dermatophytes in a protein medium which to some extent mimic "in vivo" growth parameters

RESUME : Les dermatophytes sont les agents infectieux les plus fréquents responsables de la plupart des mycoses superficielles chez les humains et chez les animaux. Ces infections, dermatophytoses, également appelées tineas ou teignes, sont fréquentes et causent des problèmes de santé publique au niveau mondial. La capacité d'envahir et de progresser au sein des structures kératinisées est probablement liée à la sécrétion de différentes enzymes kératinolytiques, qui sont considérées comme la principale caractéristique liée à la pathogénicité de ces champignons. L'objectif de ma thèse a été premièrement de progresser dans l'identification et la caractérisation des nouvelles protéines sécrétées, afin de mieux comprendre a) la capacité globale des dermatophytes à envahir les structures kératinisées, et b) les différences dans la virulence et la spécificité d'hôte que présentent les espèces étudiées .Pour progresser dans l'identification et la caractérisation de ces nouvelles protéines, les secretomes de six espèces de dermatophytes (Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii et Trichophyton tonsurans) ont été étudiés. Bien qu'il y ait un niveau globalement élevé de similitude entre les protéases sécrétées, les différentes espèces de dermatophytes sécrètent des profiles protéiques distincts lorsqu'elles sont cultivées dans les mêmes conditions de culture, et donc une signature spécifique a pu être associé à chaque espèce. Ces profiles ont été un outil avantageux pour identifier et cartographier les protéines orthologues aux six espèces et ont aussi permit la discrimination d'espèces très proches comme T. tonsurans et T. equinum qui ne peuvent pas être différenciées par l'ADN ribosomal. Ce travail également présente ce que l'on croit être la première identification global des protéines sécrétées par les dermatophytes dans des conditions de culture que incitent l'activité protéolytique extracellulaire. Ce catalogue de protéines, comprenant des endo- and exo- proteases, autres hydrolases, oxydoreductases et des protéines avec fonction inconnue, représente probablement le spectre d'enzymes qui permettent la dégradation des tissus kératinisés en composés qui peuvent être assimilés par le champignon. Les résultats suggèrent qu'un changement écologique pourrait être associé à une expression différentielle des gènes codant les protéines sécrétées, en particulier, les protéases, plutôt qu'à des divergences génétiques au niveau des gènes codant les protéines orthologues. Une sécrétion différentielle des protéines par les dermatophytes pourrait également être responsable de la variabilité inflammatoire qui causent ces agents infectieux chez les différents hôtes. Par conséquent, les protéines identifiées ici sont également importantes pour faire la lumière sur la réponse immunitaire de l'hôte au cours du processus infectieux. SUMMARY : Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Dermatophytoses, also called tineas or ringworm, are frequent and cause public health problems worldwide. The secretion of different keratinolytic enzymes is believed to be a key pathogenicity-related characteristic of these fungi. The aim of this work was first to progress in the identification and characterization of novel secreted proteins, in order to better understand a) the overall capability of dermatophytes to invade keratinised structures, and b) differences in virulence and host-specificity of the investigated species. To progress in the identification and characterization of novel proteins, the secretomes from Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii and Trichophyton tonsurans were studied. Although there is a high global level of similarity among the secreted proteases, different dermatophyte species produce distinct patterns of proteins when grown in the same culture medium, and so a specific signature could be associated to each species. These patterns were useful to identify and map orthologous proteins among the six species, as well as to discriminate the closely related species T. tonsurans and T. equinum, which cannot be differentiated by ribosomal DNA. This work also presents the first in-depth identification of the major proteins secreted by dermatophytes growing under conditions promoting extracellular proteolytic activity. This catalogue of proteins, which include several endo- and exo- proteases, other hydrolases, oxydoreductases, and proteins of unknown function, probably represents the spectrum of enzymes that allow the degradation of keratinized tissues into compounds which can be assimilated by the fungus. The results suggest that ecological switching could be related to a differential expression of genes encoding secreted proteins, particularly, proteases, rather than genetic divergences of the genes encoding orthologous proteins. Differential secretion of proteins by Dermatophyte species could also be responsible for the variable inflammation caused by the infectious agent within the host. Therefore, the proteins here identified are also important to shed light into the immune response of the host during the infection process.

Outcome evaluation of ankle osteoarthritis treatments using spatio-temporal gait parameters and plantar pressure during unconstrained long distance walking : P25

Introduction: Ankle arthrodesis (AD) and total ankle replacement (TAR) are typical treatments for ankle osteoarthritis (AO). Despite clinical interest, there is a lack of their outcome evaluation using objective criteria. Gait analysis and plantar pressure assessment are appropriate to detect pathologies in orthopaedics but they are mostly used in lab with few gait cycles. In this study, we propose an ambulatory device based on inertial and plantar pressure sensors to compare the gait during long-distance trials between healthy subjects (H) and patients with AO or treated by AD and TAR. Methods: Our study included four groups: 11 patients with AO, 9 treated by TAR, 7 treated by AD and 6 control subjects. An ambulatory system (Physilog®, CH) was used for gait analysis; plantar pressure measurements were done using a portable insole (Pedar®-X, DE). The subjects were asked to walk 50 meters in two trials. Mean value and coefficient of variation of spatio-temporal gait parameters were calculated for each trial. Pressure distribution was analyzed in ten subregions of foot. All parameters were compared among the four groups using multi-level model-based statistical analysis. Results: Significant difference (p <0.05) with control was noticed for AO patients in maximum force in medial hindfoot and forefoot and in central forefoot. These differences were no longer significant in TAR and AD groups. Cadence and speed of all pathologic groups showed significant difference with control. Both treatments showed a significant improvement in double support and stance. TAR decreased variability in speed, stride length and knee ROM. Conclusions: In spite of a small sample size, this study showed that ankle function after AO treatments can be evaluated objectively based on plantar pressure and spatio-temporal gait parameters measured during unconstrained walking outside the lab. The combination of these two ambulatory techniques provides a promising way to evaluate foot function in clinics.

Cooperation of human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR.

Efficient immune attack of malignant disease requires the concerted action of both CD8+ CTL and CD4+ Th cells. We used human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic mice, in which the mouse CD8 molecule cannot efficiently interact with the alpha3 domain of A2.1, to generate a high-affinity, CD8-independent T cell receptor (TCR) specific for a commonly expressed, tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the human p53 tumor suppressor protein. Retroviral expression of this CD8-independent, p53-specific TCR into human T cells imparted the CD8+ T lymphocytes with broad tumor-specific CTL activity and turned CD4+ T cells into potent tumor-reactive, p53A2.1-specific Th cells. Both T cell subsets were cooperative and interacted synergistically with dendritic cell intermediates and tumor targets. The intentional redirection of both CD4+ Th cells and CD8+ CTL by the same high-affinity, CD8-independent, tumor-specific TCR could provide the basis for novel broad-spectrum cancer immunotherapeutics.

Chemotherapy in patients with advanced pancreatic cancer: too close to death?

PURPOSE: We evaluated the attitude in using chemotherapy near the end of life in advanced pancreatic adenocarcinoma (PAC). Clinical and laboratory parameters recorded at last chemotherapy administration were analyzed, in order to identify risk factors for imminent death. METHODS: Retrospective analysis of patients who underwent at least one line of palliative chemotherapy was made. Data concerning chemotherapy (regimens, lines, and date of last administration) were collected. Clinical and laboratory factors recorded at last chemotherapy administration were: performance status, presence of ascites, hemoglobin, white blood cell (WBC), platelets, total bilirubin, albumin, LDH, C-reactive protein (C-rp), and Ca 19.9. RESULTS: We analyzed 231 patients: males/females, 53/47 %; metastatic/locally advanced disease, 80/20 %; and median age, 66 years (range 32-85). All patients died due to disease progression. Median overall survival was 6.1 months (95 % CI 5.1-7.2). At the last chemotherapy delivery, performance status was 0-1 in 37 % and 2 in 63 %. Fifty-nine percent of patients received one chemotherapy line, while 32, 8, and 1 % had second-, third-, and fourth line, respectively. The interval between last chemotherapy administration and death was <4 weeks in 24 %, ≥4-12 in 47 %, and >12 in 29 %. Median survival from last chemotherapy to death was 7.5 weeks (95 % CI 6.7-8.4). In a univariate analysis, ascites, elevated WBC, bilirubin, LDH, C-rp and Ca 19.9, and reduced albumin were found to predict shorter survival; however, none of them remained significant in a multivariate analysis. CONCLUSIONS: A significant proportion of patients with advanced PAC received chemotherapy within the last month of life. The clinical and laboratory parameters recorded at last chemotherapy delivery did not predict shorter survival.

Identification of Wax Esters in Latent Print Residues by Gas Chromatography-Mass Spectromertry and Their Potential Use as Aging Parameters

Recent studies show that the composition of fingerprint residue varies significantly from the same donor as well as between donors. This variability is a major drawback in latent print dating issues. This study aimed, therefore, at the definition of a parameter that is less variable from print to print, using a ratio of peak area of a target compound degrading over time divided by the summed area of peaks of more stable compounds also found in latent print residues.Gas chromatography-mass spectrometry (GC/MS) analysis of the initial lipid composition of latent prints identifies four main classes of compounds that can be used in the definition of an aging parameter: fatty acids, sterols, sterol precursors, and wax esters (WEs). Although the entities composing the first three groups are quite well known, those composing WEs are poorly reported. Therefore, the first step of the present work was to identify WE compounds present in latent print residues deposited by different donors. Of 29 WEs recorded in the chromatograms, seven were observed in the majority of samples.The identified WE compounds were subsequently used in the definition of ratios in combination with squalene and cholesterol to reduce the variability of the initial composition between latent print residues from different persons and more particularly from the same person. Finally, the influence of a latent print enhancement process on the initial composition was studied by analyzing traces after treatment with magnetic powder, 1,2-indanedione, and cyanoacrylate.

Exclusion of pulmonary embolism using C-reactive protein and D-dimer. A prospective comparison.

Our goal was to evaluate the diagnostic utility of C-reactive protein (CRP) alone or combined with clinical probability assessment in patients with suspected pulmonary embolism (PE), and to compare its performance to a D-dimer assay. We conducted a prospective study in which we performed a common immuno-turbidimetric CRP test and a rapid enzyme-linked immunosorbent assay (ELISA) D-dimer test in 259 consecutive outpatients with suspected PE at the emergency department of a teaching hospital. We assessed clinical probability of PE by a validated prediction rule overridden by clinical judgment. Patients with D-dimer levels &gt; or = 500 microg/l underwent a work-up consisting of lower-limb venous ultrasound, spiral computerized tomography, ventilation-perfusion scan, or pulmonary angiography. Patients were followed up for three months. Seventy-seven (30%) of the patients had PE. The CRP alone had a sensitivity of 84% (95% confidence interval [CI).: 74 to 92%) and a negative predictive value (NPV) of 87% (95% CI: 78 to 93%) at a cutpoint of 5 mg/l. Overall, 61 (24%) patients with a low clinical probability of PE had a CRP &lt; 5 mg/l. Due to the low prevalence of PE (9%) in this subgroup, the NPV increased to 97% (95% CI: 89 to 100%). The D-dimer (cutpoint 500 micro g/l) showed a sensitivity of 100% (95% CI: 95 to 100%) and a NPV of 100% (95% CI: 94 to 100%) irrespective of clinical probability and accurately rule out PE in 56 (22%) patients. Standard CRP tests alone or combined with clinical probability assessment cannot safely exclude PE.

Continuous monitoring and quantification of multiple parameters of daily physical activity in ambulatory Duchenne muscular dystrophy patients.

Multiple motor function and strength assessment tools exist for the evaluation of neuromuscular diseases, but most do not directly assess functional ability in the patients' daily physical activity in their home environment. In this study our aim was to assess: 1) the feasibility and accuracy of physical activity monitoring during two days in a home environment of five DMD patients using a non-commercialized monitor containing a 3D accelerometer and a gyroscope, 2) if a difference in the physical activity parameters could be measured before and one month after starting prednisolone. We reliably quantified the time spend sitting, standing, lying, walking, the number of steps taken, the cadence, the number of walking episodes and their duration as well as how these were distributed over the day. Parameters possibly reflecting endurance, such as the duration of the walking episodes or the succession of two or three walking episodes lasting more than 30 s were the most improved after prednisolone treatment. This degree of detailed determination of physical activity in a home environment has not been previously reported in neuromuscular disorders to our knowledge and some of the reported parameters are potential new outcome measures in clinical trials.

Pharmacokinetic parameters of artesunate and dihydroartemisinin in rats infected with Fasciola hepatica

OBJECTIVES: The pharmacokinetic (PK) parameters of artesunate, recently discovered to possess promising trematocidal activity, and its main metabolite dihydroartemisinin (DHA) were determined in rats infected with hepatic and biliary stages of Fasciola hepatica and compared with uninfected rats after single intragastric and intravenous (iv) doses. METHODS: Rats infected with F. hepatica for 25 and 83 days and uninfected rats were cannulated in the right jugular vein and blood samples were withdrawn at selected timepoints following 10 mg/kg of iv and a single 100 mg/kg oral dose of artesunate. Plasma was analysed for artesunate and DHA by liquid chromatography coupled to tandem mass spectrometry. RESULTS: Rats harbouring juvenile and adult F. hepatica infections revealed considerable changes in PK parameters of artesunate and DHA. Following oral administration, maximum plasma concentrations (C(max)) of artesunate and DHA were 1.8-2.3-fold higher in infected rats [artesunate: 1334 +/- 1404 ng/mL (no infection) versus 2454 +/- 1494 ng/mL (acute infection) and 2768 +/- 538 ng/mL (chronic infection); DHA: 3802 +/- 2149 ng/mL (no infection) versus 6507 +/- 3283 ng/mL (acute infection) and 9093 +/- 884 ng/mL (chronic infection)]. The AUCs of artesunate and DHA were 2.1-4.4-fold greater in infected rats. An opposite trend was observed after iv injection. C(max) and AUC of artesunate and DHA following iv dosing were 5784 +/- 3718 and 140 938 +/- 128 783 ng.min/mL and 3849 +/- 3060 and 86 107 +/- 41 863 ng.min/mL, respectively, in uninfected rats versus 2623 +/- 1554 and 21 617 +/- 12 230 ng.min/mL and 2835 +/- 980 and 64 290 +/- 29 057 ng.min/mL, respectively, in rats harbouring a chronic infection. The elimination half-lives (t(1/2)) of artesunate and DHA were considerably altered in infected rats following oral and iv administration of artesunate. CONCLUSIONS: F. hepatica infections strongly influence the disposition kinetics of artesunate and its metabolite in rats. The clinical implications of this finding need to be carefully studied.

Reaktive Thymushyperplasie infolge der Therapie ACTH-produzierender Tumoren [Reactive thymus dysplasia following therapy for ACTH-producing tumors]

Surgical or conservative treatment of ACTH-producing tumors results in acute drop of the previously excessively high cortisol levels. The following associated pathophysiological changes also occur in the organism's recovery from stress, such as trauma, operation or chemotherapy of tumors. Both cases result in a regeneration of the immune system, which might even be exalted. The corresponding radiographic feature is the "rebound" enlargement of the thymus occurring about six months after remission of hypercortisolism. Histological examination reveals benign thymus hyperplasia. Especially in cases of still unknown primary tumor the appearance of this anterior mediastinal mass can lead to misdiagnosis. We present the cases of two patients with diffuse thymic hyperplasia following surgical and medical correction of hypercortisolism. One patient suffered from classic Cushing's disease responding to transsphenoidal resection of an ACTH-secreting pituitary microadenoma. Six months later CT of the chest incidentally demonstrated an anterior mediastinal mass known as thymic hyperplasia. The second patient presented with an ectopic, still unkown source of ACTH-production. Six months after medical correction of hypercortisolism CT of the thorax showed an enlargement of the anterior mediastinum. Thymectomy was performed in order to exclude thymus carcinoid. Histological examination revealed benign thymus hyperplasia with negative immunostaining. CONCLUSION: Radiologists and clinicians should be familiar with the pathophysiological changes resulting from precipitously dropping cortisol levels in order to prevent diagnostic errors and unnecessary operations.

In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells.

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vbeta elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR alphabeta P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+ SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

Reduced cardiorespiratory fitness, low physical activity and an urban environment are independently associated with increased cardiovascular risk in children.

AIMS/HYPOTHESIS: To assist in the development of preventive strategies, we studied whether the neighbourhood environment or modifiable behavioural parameters, including cardiorespiratory fitness (CRF) and physical activity (PA), are independently associated with obesity and metabolic risk markers in children. METHODS: We carried out a cross-sectional analysis of 502 randomly selected first and fifth grade urban and rural Swiss schoolchildren with regard to CRF, PA and the neighbourhood (rural vs urban) environment. Outcome measures included BMI, sum of four skinfold thicknesses, homeostasis model assessment of insulin resistance (HOMA-IR) and a standardised clustered metabolic risk score. RESULTS: CRF and PA (especially total PA, but also the time spent engaged in light and in moderate and vigorous intensity PA) were inversely associated with measures of obesity, HOMA-IR and the metabolic risk score, independently of each other, and of sociodemographic and nutritional parameters, media use, sleep duration, BMI and the neighbourhood environment (all p < 0.05). Children living in a rural environment were more physically active and had higher CRF values and reduced HOMA-IR and metabolic risk scores compared with children living in an urban environment (all p < 0.05). These differences in cardiovascular risk factors persisted after adjustment for CRF, total PA and BMI. CONCLUSIONS/INTERPRETATION: Reduced CRF, low PA and an urban environment are independently associated with an increase in metabolic risk markers in children.

Effects of computing parameters and measurement locations on the estimation of 3D NPS in non-stationary MDCT images.

The goal of this study was to investigate the impact of computing parameters and the location of volumes of interest (VOI) on the calculation of 3D noise power spectrum (NPS) in order to determine an optimal set of computing parameters and propose a robust method for evaluating the noise properties of imaging systems. Noise stationarity in noise volumes acquired with a water phantom on a 128-MDCT and a 320-MDCT scanner were analyzed in the spatial domain in order to define locally stationary VOIs. The influence of the computing parameters in the 3D NPS measurement: the sampling distances bx,y,z and the VOI lengths Lx,y,z, the number of VOIs NVOI and the structured noise were investigated to minimize measurement errors. The effect of the VOI locations on the NPS was also investigated. Results showed that the noise (standard deviation) varies more in the r-direction (phantom radius) than z-direction plane. A 25 × 25 × 40 mm(3) VOI associated with DFOV = 200 mm (Lx,y,z = 64, bx,y = 0.391 mm with 512 × 512 matrix) and a first-order detrending method to reduce structured noise led to an accurate NPS estimation. NPS estimated from off centered small VOIs had a directional dependency contrary to NPS obtained from large VOIs located in the center of the volume or from small VOIs located on a concentric circle. This showed that the VOI size and location play a major role in the determination of NPS when images are not stationary. This study emphasizes the need for consistent measurement methods to assess and compare image quality in CT.

Reactive rise in blood pressure upon cuff inflation: cuff inflation at the arm causes a greater rise in pressure than at the wrist in hypertensive patients.

OBJECTIVE: Cuff inflation at the arm is known to cause an instantaneous rise in blood pressure, which might be due to the discomfort of the procedure and might interfere with the precision of the blood pressure measurement. In this study, we compared the reactive rise in blood pressure induced by cuff inflation when the cuff was placed at the upper arm level and at the wrist. PARTICIPANTS AND METHODS: The reactive rise in systolic and diastolic blood pressure to cuff inflation was measured in 34 normotensive participants and 34 hypertensive patients. Each participant was equipped with two cuffs, one around the right upper arm (OMRON HEM-CR19, 22-32 cm) and one around the right wrist (OMRON HEM-CS 19, 17-22 cm; Omron Health Care Europe BV, Hoofddorp, The Netherlands). The cuffs were inflated in a double random order (maximal cuff pressure and position of the cuff) with two maximal cuff pressures: 180 and 240 mmHg. The cuffs were linked to an oscillometric device (OMRON HEM 907; Omron Health Care). Simultaneously, blood pressure was measured continuously at the middle finger of the left hand using photoplethysmography. Three measurements were made at each level of blood pressure at the arm and at the wrist, and the sequence of measurements was randomized. RESULTS: In normotensive participants, no significant difference was observed in the reactive rise in blood pressure when the cuff was inflated either at the arm or at the wrist irrespective of the level of cuff inflation. Inflating a cuff at the arm, however, induced a significantly greater rise in blood pressure than inflating it at the wrist in hypertensive participants for both systolic and diastolic pressures (P<0.01), and at both levels of cuff inflation. The blood pressure response to cuff inflation was independent of baseline blood pressure. CONCLUSIONS: The results show that in hypertensive patients, cuff inflation at the wrist produces a smaller reactive rise in blood pressure. The difference between the arm and the wrist is independent of the patient's level of blood pressure.