Recapitulating primary immunodeficiencies with expanded potential stem cells: Proof of concept with STAT1 gain of function.

BACKGROUND: Inborn errors of immunity (IEI) often lack specific disease models and personalized management. Signal transducer and activator of transcription (STAT)-1 gain of function (GoF) is such example of an IEI with diverse clinical phenotype with unclear pathomechanisms and unpredictable response to therapy. Limitations in obtaining fresh samples for functional testing and research further highlights the need for patient-specific ex vivo platforms. OBJECTIVE: Using STAT1-GoF as an example IEI, we investigated the potential of patient-derived expanded potential stem cells (EPSC) as an ex vivo platform for disease modeling and personalized treatment. METHODS: We generated EPSC derived from individual STAT1-GoF patients. STAT1 mutations were confirmed with Sanger sequencing. Functional testing including STAT1 phosphorylation/dephosphorylation and gene expression with or without Janus activating kinase inhibitors were performed. Functional tests were repeated on EPSC lines with GoF mutations repaired by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) editing. RESULTS: EPSC were successfully reprogrammed from STAT1-GoF patients and expressed the same pluripotent makers as controls, with distinct morphologic differences. Patient-derived EPSC recapitulated the functional abnormalities of index STAT1-GoF patients with STAT1 hyperphosphorylation and increased expression of STAT1 and its downstream genes (IRF1, APOL6, and OAS1) after IFN-γ stimulation. Addition of ruxolitinib and baricitinib inhibited STAT1 hyperactivation in STAT1-GoF EPSC in a dose-dependent manner, which was not observed with tofacitinib. Corrected STAT1 phosphorylation and downstream gene expression were observed among repaired STAT1-GoF EPSC cell lines. CONCLUSION: This proof-of-concept study demonstrates the potential of our patient-derived EPSC platform to model STAT1-GoF. We propose this platform when researching, recapitulating, and repairing other IEI in the future.

Children's exposure to size-fractioned particulate matter: Chemical composition and internal dose.

The health effects of the particulate matter (PM) depend not only on its aerodynamic diameter (AD) and chemical composition, but also on the time activity pattern of the individuals and on their age. The main objective of this work was to assess the exposure of children to aerosol particles by using personal instruments, to study the particle size and composition of the inhaled PM, and to estimate their transport and deposition into the human respiratory tract (HRT). The average daily PM2.5 exposure was 19 µg/m3 and the size fractions with the greatest contribution to PM2.5 concentrations were 1 < AD <2.5 µm and AD <0.25 µm. Results indicated a contribution of 9% from the mineral aerosol, 7.2% from anthropogenic sulphate, 6.7% from black carbon and 5% from anthropogenic trace elements to the daily exposure to PM2.5. The levels of mineral and marine elements increased with increasing particle size, while anthropogenic elements were present in higher concentrations in the finest particles. Particle size has been shown to influence the variability of daily dose deposited between the extrathoracic and alveolar-interstitial zones. On average, 3% of the PM deposited in the bronchial region, whereas 5% to 8% were found in the bronchiolar region. The level of physical activity had a significant contribution to the total daily dose.

Assessment of children's exposure to carbonaceous matter and to PM major and trace elements.

Particulate matter (PM) pollution is one of the major environmental concerns due to its harmful effects on human health. As children are particularly vulnerable to particle exposure, this study integrates the concentration of PM chemical compounds measured in the micro-environments (MEs) where children spend most of their time to assess the daily exposure and inhaled dose. PM samples were analysed for organic and elemental carbon and for major and trace elements. Results showed that the MEs that contribute most to the children's daily exposure (80%) and inhaled dose (65%) were homes and schools. Results indicated that the high contribution of particulate organic matter (POM) indoors indicate high contributions of indoor sources to the organic fraction of the particles. The highest concentrations of PM chemical compounds and the highest Indoor/Outdoor ratios were measured in schools, where the contribution of mineral elements stands out due to the resuspension of dust caused by the students and to the chalk used in blackboards. The contribution of the outdoor particles to inhaled dose (24%) was higher than to the exposure (12%), due to the highest inhalation rates associated with the activities performed outdoor. This study indicates the importance of indoor air quality for the children's exposure and health.

Particle exposure and inhaled dose while commuting in Lisbon.

While commuting, individuals are exposed to high concentrations of urban air pollutants that can lead to adverse health effects. This study aims to assess commuters' exposure to particulate matter (PM) when travelling by car, bicycle, metro and bus in Lisbon. Mass concentrations of PM2.5 and PM10 were higher in the metro. On the other hand, the highest BC and PN0.01-1 average concentrations were found in car and bus mode, respectively. In cars, the outdoor concentrations and the type of ventilation appeared to affect the indoor concentrations. In fact, the use of ventilation led to a decrease of PM2.5 and PM10 concentrations and to an increase of BC concentrations. The highest inhaled doses were mostly observed in bicycle journeys, due to the longest travel periods combined with enhanced physical activity and, consequently, highest inhalation rates.

Genetic parameters and path analysis in cowpea genotypes grown in the Cerrado/Pantanal ecotone.

Estimating genetic parameters in plant breeding allows us to know the population potential for selecting and designing strategies that can maximize the achievement of superior genotypes. The objective of this study was to evaluate the genetic potential of a population of 20 cowpea genotypes by estimating genetic parameters and path analysis among the traits to guide the selection strategies. The trial was conducted in randomized block design with four replications. Its morphophysiological components, components of green grain production and dry grain yield were estimated from genetic use and correlations between the traits. Phenotypic correlations were deployed through path analysis into direct and indirect effects of morphophysiological traits and yield components on dry grain yield. There were significant differences (P < 0.01) between the genotypes for most the traits, indicating the presence of genetic variability in the population and the possibility of practicing selection. The population presents the potential for future genetic breeding studies and is highly promising for the selection of traits dry grain yield, the number of grains per pod, and hundred grains mass. A number of grains per green pod is the main determinant trait of dry grain yield that is also influenced by the cultivar cycle and that the selection for the dry grain yield can be made indirectly by selecting the green pod mass and green pod length.

Exposure assessment of a cyclist to particles and chemical elements.

Cycle paths can be used as a route for active transportation or simply to cycle for physical activity and leisure. However, exposure to air pollutants can be boosted while cycling, in urban environments, due to the proximity to vehicular emissions and elevated breathing rates. The objective of this work was to assess the exposure of a cyclist to particles and to chemical elements by combining real-time aerosol mass concentration reading equipment and biomonitoring techniques. PM10 and PM2.5 were measured on three cycle paths located in Lisbon, during weekdays and weekends and during rush hours and off-peak hours resulting in a total of 60 campaigns. Lichens were exposed along cycle paths for 3 months, and their element contents were measured by instrumental neutron activation analysis using the k 0 methodology (k 0-INAA). Using a bicycle commute route of lower traffic intensity and avoiding rush hours or other times with elevated vehicular congestion facilitate a reduction in exposure to pollutants. The implementation of cycle paths in cities is important to stimulate physical activity and active transportation; however, it is essential to consider ambient air and pollutant sources to create safer infrastructures.

Timing of adjuvant chemotherapy in colorectal cancer.

AIM: Delay in commencing adjuvant therapy for colorectal cancer seems to impair survival in some retrospective studies. This study was planned to evaluate its impact on survival. METHODS: This was a retrospective study enrolling patients registered from 2000 to 2012 in two large cancer-dedicated institutions in Brazil. The primary outcome was overall survival according to early vs late chemotherapy initiation. The interval between the primary surgery and the start of adjuvant chemotherapy was calculated. Survival was estimated using the Kaplan-Meier method and the impact of multiple prognostic factors on survival by Cox regression analysis. RESULTS: By the end of 2012, a total of 1963 Stage II and III colorectal patients were identified and 1318 patients received adjuvant chemotherapy, with 22% and 46% of those starting adjuvant chemotherapy within 6 weeks and 8 weeks of surgery. The median period of follow-up was 41 months. Patients starting chemotherapy within 6-8 weeks of surgery had longer overall survival compared with those who started after (6 weeks vs later, hazard ratio 0.76, 95% CI 0.57-0.99, P = 0.046; 8 weeks vs later, hazard ratio 0.74, 95% CI 0.59-0.93, P = 0.011). In the multivariate analysis, age, stage, histological grade, angiolymphatic invasion, emergency surgery and preoperative therapy were independent prognostic factors, but the interval between surgery and start of adjuvant therapy was not. CONCLUSION: In this large retrospective study, the standard prognostic factors impacted on survival whereas the timing of adjuvant therapy did not. Patients with delayed adjuvant chemotherapy may have worse prognostic factors which could play a major role in their poor outcome.

Source apportionment of indoor PM10 in Elderly Care Centre.

Source contribution to atmospheric particulate matter (PM) has been exhaustively modelled. However, people spend most of their time indoors where this approach is less explored. This evidence worsens considering elders living in Elderly Care Centres, since they are more susceptible. The present study aims to investigate the PM composition and sources influencing elderly exposure. Two 2-week sampling campaigns were conducted-one during early fall (warm phase) and another throughout the winter (cold phase). PM10 were collected with two TCR-Tecora(®) samplers that were located in an Elderly Care Centre living room and in the correspondent outdoor. Chemical analysis of the particles was performed by neutron activation analysis for element characterization, by ion chromatography for the determination of water soluble ions and by a thermal optical technique for the measurement of organic and elemental carbon. Statistical analysis showed that there were no statistical differences between seasons and environments. The sum of the indoor PM10 components measured in this work explained 57 and 53 % of the total PM10 mass measured by gravimetry in warm and cold campaigns, respectively. Outdoor PM10 concentrations were significantly higher during the day than night (p value < 0.05), as well as Ca(2+), Fe, Sb and Zn. The contribution of indoor and outdoor sources was assessed by principal component analysis and showed the importance of the highways and the airport located less than 500 m from the Elderly Care Centre for both indoor and outdoor air quality.

Perfil de los pacientes rehabilitados por medio de prótesis atendidos en servicio público de salud en Ribeirão Preto, Brasil / Patients profile rehabilitated with dental prosthesis assisted by the health public service in Ribeirão Preto, Brazil

La prevalencia de sexo y el padrón de edad relacionado a los diferentes tipos de rehabilitaciones protésicas son variables importantes que deben ser estudiadas. De esta manera, el objetivo de este estudio fue evaluar el perfil de edad y género en pacientes rehabilitados con prótesis dentales en un curso de Odontología durante un periodo de cinco años. Fueron evaluados 638 fichas de pacientes que recibieron prótesis parcial fija (PPF), prótesis parcial removible (PPR) y prótesis total (PT). Las prótesis de los pacientes rehabilitados con PPR fueron clasificadas en dentomucosoportada (DM) o dentosoportadas (D). Para el análisis de los resultados, los pacientes fueron divididos en 2 grupos: A) >50 años y B) <50 años. Los datos fueron analizados estadísticamente por el test exacto de Fisher o chi-cuadrado (p<0,05). No se observaron diferencias significativas en relación a la edad y tipo de prótesis. Para la PPF 74% fueron <50 años, para la PPR 52%, >50 años y para la PT 92%, >50 años. En todas las prótesis, existió una mayor prevalencia del género femenino. Con relación a la clasificación de las PPRs, 47% fueron D y 53% DM, sin identificar diferencias estadísticamente significativas. De esta manera se puede concluir que la necesidad de rehabilitación de un mayor numero de elementos dentales aumenta con la edad, siendo prevalente en todos los periodos de la vida adulta, principalmente, en mujeres

The prevalence of gender and age related pattern of the different types of prosthetic restorations are important variables to be estimated. The aim of this study was to evaluate the profile of age and gender in patients rehabilitated with dental prostheses on School of Dentistry during five years. Were evaluated 638 records of patients who received fixed prosthesis (FP), removable partial (RP) or total dentures (T). Rehabilitated patients with RP had their prostheses classified into dental-mucous-supported (DM) or tooth-supported (D). To analyze the results, patients were divided into 2 groups: A) >50 years or B) <50 years. Data were statistically analyzed by Fisher's exact test or chi Square (p<0.05). No statistical significance relation to age and types of prostheses was observed. On RP, 47% was D and 53% DM, there was no statistically significant difference. It was possible to conclude that the rehabilitation of greater number of the teeth increases with age and is prevalent in all periods of adulthood, especially in women

A single resistance exercise session improves myocardial contractility in spontaneously hypertensive rats.

Resistance training evokes myocardial adaptation; however, the effects of a single resistance exercise session on cardiac performance are poorly understood or investigated. This study aimed to investigate the effects of a single resistance exercise session on the myocardial contractility of spontaneously hypertensive rats (SHRs). Male 3-month-old SHRs were divided into two groups: control (Ct) and exercise (Ex). Control animals were submitted to sham exercise. Blood pressure was measured in conscious rats before the exercise session to confirm the presence of arterial hypertension. Ten minutes after the exercise session, the animals were anesthetized and killed, and the hearts were removed. Cardiac contractility was evaluated in the whole heart by the Langendorff technique and by isometric contractions of isolated left ventricular papillary muscles. SERCA2a, phospholamban (PLB), and phosphorylated PLB expression were investigated by Western blot. Exercise increased force development of isolated papillary muscles (Ex=1.0±0.1 g/mg vs Ct=0.63±0.2 g/mg, P<0.05). Post-rest contraction was greater in the exercised animals (Ex=4.1±0.4% vs Ct=1.7±0.2%, P<0.05). Papillary muscles of exercised animals developed greater force under increasing isoproterenol concentrations (P<0.05). In the isolated heart, exercise increased left ventricular isovolumetric systolic pressure (LVISP; Δ +39 mmHg; P<0.05) from baseline conditions. Hearts from the exercised rats presented a greater response to increasing diastolic pressure. Positive inotropic intervention to calcium and isoproterenol resulted in greater LVISP in exercised animals (P<0.05). The results demonstrated that a single resistance exercise session improved myocardial contractility in SHRs.

New self-assembled supramolecular hydrogels based on dehydropeptides.

Supramolecular hydrogels rely on small molecules that self-assemble in water as a result of the cooperative effect of several relatively weak intermolecular interactions. Peptide-based low molecular weight hydrogelators have attracted enormous interest owing to the simplicity of small molecules combined with the versatility and biocompatibility of peptides. In this work, naproxen, a well known non-steroidal anti-inflammatory drug, was N-conjugated with various dehydrodipeptides to give aromatic peptide amphiphiles that resist proteolysis. Molecular dynamics simulations were used to obtain insight into the underlying molecular mechanism of self-assembly and to rationalize the design of this type of hydrogelators. The results obtained were in excellent agreement with the experimental observations. Only dehydrodipeptides having at least one aromatic amino acid gave hydrogels. The characterization of the hydrogels was carried out using transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy and also rheological assays.

Fungal contamination in two Portuguese wastewater treatment plants.

The presence of filamentous fungi was detected in wastewater and air collected at wastewater treatment plants (WWTP) from several European countries. The aim of the present study was to assess fungal contamination in two WWTP operating in Lisbon. In addition, particulate matter (PM) contamination data was analyzed. To apply conventional methods, air samples from the two plants were collected through impaction using an air sampler with a velocity air rate of 140 L/min. Surfaces samples were collected by swabbing the surfaces of the same indoor sites. All collected samples were incubated at 27°C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. For molecular methods, air samples of 250 L were also collected using the impinger method at 300 L/min airflow rate. Samples were collected into 10 ml sterile phosphate-buffered saline with 0.05% Triton X-100, and the collection liquid was subsequently used for DNA extraction. Molecular identification of Aspergillus fumigatus and Stachybotrys chartarum was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR Detection System (Corbett). Assessment of PM was also conducted with portable direct-reading equipment (Lighthouse, model 3016 IAQ). Particles concentration measurement was performed at five different sizes: PM0.5, PM1, PM2.5, PM5, and PM10. Sixteen different fungal species were detected in indoor air in a total of 5400 isolates in both plants. Penicillium sp. was the most frequently isolated fungal genus (58.9%), followed by Aspergillus sp. (21.2%) and Acremonium sp. (8.2%), in the total underground area. In a partially underground plant, Penicillium sp. (39.5%) was also the most frequently isolated, also followed by Aspergillus sp. (38.7%) and Acremonium sp. (9.7%). Using RT-PCR, only A. fumigatus was detected in air samples collected, and only from partial underground plant. Stachybotrys chartarum was not detected in any of the samples analyzed. The distribution of particle sizes showed the same tendency in both plants; however, the partially underground plant presented higher levels of contamination, except for PM2.5. Fungal contamination assessment is crucial to evaluating the potential health risks to exposed workers in these settings. In order to achieve an evaluation of potential health risks to exposed workers, it is essential to combine conventional and molecular methods for fungal detection. Protective measures to minimize worker exposure to fungi need to be adopted since wastewater is the predominant internal fungal source in this setting.

Triagem fitoquímica e atividade antioxidante de Costus spicatus (Jacq. ) S. w / Phytochemical screening and antioxidant activity of Costus spicatus (Jacq. ) Sw

O objetivo do trabalho foi avaliar o perfil fitoquímico e a atividade antioxidante comparando-se os resultados obtidos entre diferentes órgãos da Costus spicatus. Foram utilizados caules, folhas e flores da Costus spicatus, colhidos na Universidade Estadual do Norte Fluminense Darcy Ribeiro, no município de Campos dos Goytacazes, RJ. Os diferentes órgãos foram avaliados quanto ao perfil fitoquímico e atividade antioxidante nas frações hexânica, acetato de etila e butanólica. Os resultados obtidos comprovaram a presença de Triterpenos e Esteróides (Lieberman-Burchard), Flavonóides (Shinoda), Saponinas (Índice de espuma), Alcalóides (Drangendorff) e Taninos (Cloreto Férrico). Foi demonstrada a atividade antioxidante potencial. Concluiu-se que a Costus spicatus apresentou todas as classes de metabólitos pesquisadas, dentre as quais, algumas possuem atividade biológica já conhecida, fazendo-se necessária a realização de estudos quantitativos e pesquisas que demonstrem seus efeitos farmacológicos, contribuindo para o desenvolvimento de novos fármacos.

The objective of this work was to evaluate the phytochemical profile and the antioxidant activity comparing the results between different organs of Costus spicatus. We used the stems, leaves and flowers of Costus spicatus harvested at the State University of Norte Fluminense "Darcy Ribeiro" in the city of Campos dos Goytacazes, Rio de Janeiro, Brazil. The different organs were evaluated for antioxidant activity and phytochemical profile in the hexane, ethyl acetate and butanol fractions. The results obtained proved the presence of triterpenes and steroids (Liebermann-Burchard), flavonoids (Shinoda), saponins (foaming index), alkaloids (Dragendorff) and tannins (ferric chloride). We demonstrated the potential antioxidant activity. We concluded that Costus spicatus presented all classes of metabolites studied, among which some already have their biological activity known by the literature, being necessary the performance of quantitative and research studies that demonstrate their pharmacological effects, thus contributing to the development of new drugs.

Population structure and genetic diversity of wild Helianthus species from Mozambique.

The production of sunflower suffered a major decline in Mozambique after its independence in 1975. Civil war, human activities and environmental damage subjected the species to an ecological stress contributing to reduce the number and size of wild populations. As this reduction is often related to a loss of genetic variation we estimated the genetic diversity within and among populations of wild Helianthus from five districts of Mozambique using RAPD markers. The 44 accessions studied grouped into four major clusters exhibiting structured variability with regard to geographic origin. A high level of genetic diversity (He = 0.350 and I = 0.527) was retained at the population level. The genetic variation among populations was high (59.7%), which is consistent with low gene flow (Nm = 0.338). The proportion of total genetic diversity residing among these populations should be kept in mind to devise different conservation strategies in order to preserve these populations. Currently wild Helianthus genetic resources present in Maputo and Sofala are on the edge of extinction mainly due to excessive urbanization. Therefore, conservation of what remains of this plant genetic diversity is essential for sustainable utilization and can be useful for breeding programs.

Impact of prolonged physical training on the pituitary glucocorticoid sensitivity determined by very low dose intravenous dexamethasone suppression test.

The activity of the hypothalamic-pituitary-adrenal axis is usually modulated by several stress factors, including exercise. Different responses are induced by physical training according to duration and intensity of exercise. During prolonged training, cortisol remains normal or decreased as a consequence of altered cortisol secretion, metabolism and excretion, and possibly by changes in glucocorticoid sensitivity. The aim of this study was to evaluate the impact of prolonged physical training on the glucocorticoid sensitivity. Eighteen cadets of the Air Force Academy, mean (SD) age: 18.7 (1.0) years, underwent an intensive 6-week preparatory training-period considered adequate by inducing significant changes on body composition measured by bioelectrical impedance. Measurement of individual's pituitary glucocorticoid sensitivity was done by an intravenous very low dose dexamethasone suppression test (20 microg/m (2)) that was performed before and after the training period. Cortisol levels were obtained at basal condition and 120 minutes after the dexamethasone infusion. Basal cortisol showed a significant decrease after prolonged training. The percent cortisol suppression after dexamethasone tended to be lower after the training period. Overall, our data suggest that prolonged physical training is able to reduce glucocorticoid sensitivity, which can have a beneficial impact in chronic stress conditions.

Relationship between retroviral vector membrane and vector stability.

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.

Roughness of CdTe thin films grown on glass by hot wall epitaxy.

Cadmium telluride films were grown on glass substrates using the hot wall epitaxy (HWE) technique. The samples were polycrystalline with a preferential (111) orientation. Scanning electron micrographs reveal a grain size between 0.1 and 0.5 µm. The surface morphology of the samples was studied by measuring the roughness profile using a stylus profiler. The roughness as a function of growth time and scale size were investigated to determine the growth and roughness exponents, ß and α, respectively. From the results we can conclude that the growth surface has a self-affine character with a roughness exponent α equal to 0.69 ± 0.03 and almost independent of growth time. The growth exponent ß was equal to 0.38 ± 0.06. These values agree with that determined previously for CdTe(111) films grown on GaAs(100).

How plants cope with water stress in the field. Photosynthesis and growth.

Plants are often subjected to periods of soil and atmospheric water deficit during their life cycle. The frequency of such phenomena is likely to increase in the future even outside today's arid/semi-arid regions. Plant responses to water scarcity are complex, involving deleterious and/or adaptive changes, and under field conditions these responses can be synergistically or antagonistically modified by the superimposition of other stresses. This complexity is illustrated using examples of woody and herbaceous species mostly from Mediterranean-type ecosystems, with strategies ranging from drought-avoidance, as in winter/spring annuals or in deep-rooted perennials, to the stress resistance of sclerophylls. Differences among species that can be traced to different capacities for water acquisition, rather than to differences in metabolism at a given water status, are described. Changes in the root : shoot ratio or the temporary accumulation of reserves in the stem are accompanied by alterations in nitrogen and carbon metabolism, the fine regulation of which is still largely unknown. At the leaf level, the dissipation of excitation energy through processes other than photosynthetic C-metabolism is an important defence mechanism under conditions of water stress and is accompanied by down-regulation of photochemistry and, in the longer term, of carbon metabolism.

Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues.

This study sought to identify the spatial patterns of expression of peptide transporter 1 (PepT1), peptide transporter 3 (PTR3), peptide/histidine transporter 1 (PHT1), and the human peptide transporter 1 (HPT-1) mRNA in complementary DNA (cDNA) libraries of the human and rat gastrointestinal tracts (GIT), Caco-2 in vitro cell culture model, and in a human multiple tissue panel. Human PTR3 and PHT1 are putative peptide transporters recently discovered. Using sequence-specific primers designed to amplify regions of PepT1, PTR3, PHT1, and HPT-1, we were able to identify the expression of mRNA for each of these transporters in human cDNA panels (Clontech, Palo Alto, CA), the rat GIT, and in Caco-2 cDNA libraries by the polymerase chain reaction (PCR) and Southern Blot analysis. These studies suggest that in the human GIT, PepT1 appears to be localized predominantly in the duodenum, with decreasing expression in the jejunum and ileum. In contrast, PTR3 and HPT-1 were widely expressed in the human GIT, with predominant expression in the different regions of the colon. PHT1 appeared to be expressed in low levels throughout the human GI tract. Interestingly, the mRNAs for all 4 peptide transporters were expressed in Caco-2 cells throughout 30 days of culture. PepT1, PTR3, PHT1, and HPT-1 were also widely expressed in the rat GIT. Human tissue cDNA panel screening suggests that PTR3 and PHT1 are more uniformly expressed, whereas PepT1 and HPT-1 demonstrated site-specific expression. These results suggest that PepT1, PTR3, PHT1, and HPT-1 all may act to facilitate the diffusion of peptides and peptide-based pharmaceuticals in the GIT. PTR3, PHT1, and HPT-1 expressions in Caco-2 cell monolayers strongly suggest that their function needs to be further elucidated and their contribution to peptide transport not ignored. Taken together, these results demonstrate the potential for molecular biological characterization in localizing active transporter systems that can potentially be targeted for enhancing the absorption of peptide-based pharmaceuticals.

BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation.

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.