Organization of Home Mechanical Ventilation in Portugal: Characterization of current centers and a pathway to uniformization.

INTRODUCTION: Home Mechanical Ventilation (HMV) is increasing worldwide. OBJECTIVE: Characterization of the Portuguese HMV Units. METHODS: The HMV Team Group of the Portuguese Pulmonology Society prepared a questionnaire that was sent by e-mail addressed to Pneumology Department Directors throughout the country, and the responses were then analyzed. The results enabled a provisional classification of the Units, which followed specific criteria. RESULTS: Thirty centers were surveyed, of which 60% (18) sent the answers to the questionnaire. As for the results obtained, only one center was considered as a basic unit. Most centers (14/18) were considered specialized units. 3/18 centers were classified as highly complex multidisciplinary units. Of the 12 centers that did not answer the questionnaire, one refused to do it and another center was in transition period. CONCLUSIONS: Analysis of the results reveals the high number of patients treated with HMV in Portugal, supports the importance of creating protocols to standardize HMV countrywide, and audit its practice through the creation of a national register.

Clinical and molecular markers in COPD.

Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous disease, and there is a clinical need for validated markers and biomarkers that can contribute to the assessment of patients, risk prediction, treatment guidance, and assessment of response. Although according to the 2018 GOLD guidelines clinically useful biomarkers for COPD patients in stable condition have yet to be identified, several clinical markers and biomarkers have been proposed for COPD. These include isolated clinical markers, such as symptoms and Health Status assessment, exercise tests, function tests and imaging, and also composite scores and molecular markers. However, and despite strong efforts to identify useful markers in an attempt to improve prognostic and therapeutic approaches, results have not been consistent and expectations of relying on these markers in near future are faint. Current approaches to COPD have shifted from treating the disease to treating the individual patient. There is a clear need to identify treatable traits, focusing more on the patient and not on the disease, in order to implement an increasingly personalized treatment of COPD in the clinic, leading to true precision medicine. There is a need to identify combinations of clinical markers and biomarkers, genetic markers, and phenotypes that can guide the personalized therapy of COPD patients. This critical review will therefore focus not only on currently established markers and biomarkers in COPD but also on possible future approaches toward precision medicine.

Prevalence of late-onset pompe disease in Portuguese patients with diaphragmatic paralysis - DIPPER study.

Pompe disease is a rare autosomal recessive neuromuscular disorder caused by acid α-glucosidase enzyme (GAA) deficiency and divided into two distinct variants, infantile- and late-onset. The late-onset variant is characterized by a spectrum of phenotypic variation that may range from asymptomatic, to reduced muscle strength and/or diaphragmatic paralysis. Since muscle strength loss is characteristic of several different conditions, which may also cause diaphragmatic paralysis, a protocol was created to search for the diagnosis of Pompe disease and exclude other possible causes. METHODS: We collected a sample size of 18 patients (10 females, 8 males) with a median age of 60 years and diagnosis of diaphragmatic paralysis of unknown etiology, followed in the Pulmonology outpatient consultation of 9 centers in Portugal, over a 24-month study period. We evaluated data from patient's clinical and demographic characteristics as well as complementary diagnostic tests including blood tests, imaging, neurophysiologic and respiratory function evaluation. All patients were evaluated for GAA activity with DBS (dried blood test) or serum quantification and positive results confirmed by serum quantification and sequencing. RESULTS: Three patients were diagnosed with Pompe's disease and recommended for enzyme replacement therapy. The prevalence of Pompe, a rare disease, in our diaphragmatic paralysis patient sample was 16.8%. CONCLUSION: We conclude that DBS test for GAA activity should be recommended for all patients with diaphragmatic paralysis which, despite looking at all the most common causes, remains of unknown etiology; this would improve both the timing and accuracy of diagnosis for Pompe disease in this patient population. Accurate diagnosis will lead to improved care for this rare, progressively debilitating but treatable neuromuscular disease.

Concurrent expression of cyclo-oxygenase-2 and epidermal growth factor receptor in canine malignant mammary tumours.

Canine mammary tumours (CMTs) are reported to express cyclo-oxygenase (COX)-2 and epidermal growth factor receptor (EGFR); however, no studies have evaluated concurrent expression of these proteins. In this study, 43 malignant CMTs were evaluated immunohistochemically for concurrent expression of COX-2 and EGFR and expression was correlated with malignancy. High COX-2 expression was associated with tumour size (P = 0.033), mitotic index (P = 0.040), nuclear grade (P = 0.021), histological grade of malignancy (P = 0.020) and lymph node metastasis (P = 0.029). High EGFR immunoreactivity was associated with tumour size (P = 0.001), necrosis (P = 0.001), mitotic index (P = 0.022), histological grade of malignancy (P = 0.041) and lymph node metastasis (P = 0.005). Simultaneous high-expression of COX-2 and EGFR was associated with high-nuclear grade (P = 0.049), high-histological grade of malignancy (P = 0.031) and the presence of lymph node metastasis (P = 0.025). A positive correlation between COX-2 and EGFR expression (r = 0.474; P = 0.001) was also observed. These results suggest that combined use of selective inhibitors of COX-2 and EGFR may be a useful approach to the treatment of malignant CMTs.

The socio-demographic, environmental and reservoir factors associated with leptospirosis in an urban area of north-eastern Brazil.

In an ecological study based on the 18 microregions that form the city of Recife, the capital of the Brazilian state of Pernambuco, associations between socio-demographic, environmental and reservoir factors and the incidence of leptospirosis in the city were investigated. Incidence over a 5-year period (2001-2005) and 14 variables were analysed, using central trend and dispersion measurements, Pearson's correlation and multiple linear regression. Variables relating to education, income, housing type, sewage system, rubbish collection and hydrographic factors were found to be significantly correlated with leptospirosis incidence (P<0.05 for each). Just two variables - the proportion of heads of households with incomes less than or equal to the legal minimum (U.S.$83.55/month), and the proportion of households from which rubbish was dumped in skips, lakes, rivers or the sea or on vacant land - explained 60% (P=0.017) of the differences in disease risk observed between the various areas of the city.

[Infant mortality rate and social disparity at Recife, the metropolis of the North-East of Brazil]. / Taux de mortalité infantile et disparités sociales à Recife, métropole du Nord-Est du Brésil.

We analyzed differences in infant mortality between areas of Recife, a city in the North East of Brazil, analyzing the relationship between living conditions and the risk of death. We compared infant mortality coefficients for 1995 with indicators of living conditions and collected data for the 770 infant deaths and the 27,965 live births. Neighborhoods were ranked according to the quality of living conditions and were grouped into four clusters. The infant, neonatal and postneonatal mortality coefficients were 27.53, 18.84 and 8.69 per 1,000 live births respectively. Lower quality living conditions were associated with higher coefficients. The main causes of infant deaths were perinatal disorders, the coefficient of which was 14.95 per 1,000 live births, followed by congenital malformations, gastroenteritis and bronchopneumonia. With the exception of congenital malformations, the coefficients of all these causes of death increased as living conditions worsened. These inequalities are generally obscured by the presentation of means for the city as a whole.

Mammalian genomes contain active recombinase recognition sites.

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.

Fetal mouse selenophosphate synthetase 2 (SPS2): characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system.

A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene. Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E. coli enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect cell system. Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme. The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide-dependent AMP formation from ATP. Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.

Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism?

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme's putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.

Biochemical and genetic characterization of multiple splice variants of the Flt3 ligand.

We have performed a comprehensive analysis of cell lines and tissues to compare and contrast the expression patterns of Flt3 ligand (FL), c-Kit ligand (KL), and macrophage colony-stimulating factor as well as their receptors, Flt3, c-Kit, and c-Fms. The message for FL is unusually ubiquitous, whereas that of its receptor is quite restricted, apparently limiting the function of the ligand to fetal development and early hematopoiesis. We have also sequenced a mouse FL genomic clone, revealing how the three splice variant FL mRNAs that we have isolated arise. The chromosomal location of the FL gene has been mapped, by in situ hybridization, to chromosome 7 in mouse and chromosome 19 in human. Natural FL protein has been purified from a stromal cell line and shown to be a 65 kD nondisulfide-linked homodimeric glycoprotein comprised of 30 kD subunits, each containing 12 kD of N- and O-linked sugars. Pulse-chase experiments show that one of the splice variants (T110) is responsible for producing the bulk of soluble FL, but only after it has first been expressed at the cell surface as a membrane-bound form. The other splice-variant forms produce molecules that are either obligatorily soluble (T169) or membrane-bound but released only very slowly (T118). Finally, even though most cell lines express some amount of FL mRNA, we found that very little FL protein is actually made, with T cells and stromal cells being the major producers. The data suggests that FL plays its roles over very short distances, perhaps requiring cell-cell contact.

Molecular cloning and characterization of lysosomal sialic acid O-acetylesterase.

O-Acetylation and de-O-acetylation of sialic acids have been implicated in the regulation of a variety of biological phenomena, including endogenous lectin recognition, tumor antigenicity, virus binding, and complement activation. Applying a strategy designed to identify genes preferentially expressed in active sites of embryonic hematopoiesis, we isolated a novel cDNA from the pluripotent hematopoietic cell line FDCPmixA4 whose open reading frame contained sequences homologous to peptide fragments of a lysosomal sialic acid O-acetylesterase (Lse) previously purified from rat liver, but with no evident similarity to endoplasmic reticulum-derived acetylesterases. The expressed Lse protein exhibits sialic-acid O-acetylesterase activity that is not attributable to a typical serine esterase active site. lse expression is spatially and temporally restricted during embryogenesis, and its mRNA levels correlate with differences in O-acetylesterase activity described in adult tissues and blood cell types. Using interspecific backcross analysis, we further mapped the lse gene to the central region of mouse chromosome 9. This constitutes the first report on the molecular cloning of a sialic acid-specific O-acetylesterase in vertebrates and suggests novel roles for the 9-O-acetyl modification of sialic acids during the development and differentiation of mammalian organisms.

A new approach to the study of haematopoietic development in the yolk sac and embryoid bodies.

To understand the mechanisms that control the differentiation of uncommitted mesoderm precursors into haematopoietic stem cells (HSCs) and the activation of haematopoiesis, we conducted a study to identify genes expressed at the earliest stages of both in vivo and in vitro haematopoietic development. Our strategy was to utilize Differential Display by means of the Polymerase Chain Reaction (DD-PCR) to compare patterns of gene expression between mRNA populations representing different levels of haematopoietic activity obtained from the mouse embryo, embryoid bodies (EBs) and mouse cell lines. We report the molecular cloning of two groups of genes expressed in the yolk sac: a group of genes expressed in the day-8.5 yolk sac at higher levels than in the day-8.5 embryo proper and up-regulated during EB development, and another group of day-8.5 yolk sac genes not expressed in the day-8.5 embryo proper or in EBs. Specifically, we describe the molecular cloning of the first nucleobase permease gene to be found in vertebrates, yolk sac permease-like molecule 1 (Ysp11). The Ysp11 gene has the unique property of encoding both intracellular, transmembrane and extracellular protein forms, revealing novel aspects of nucleotide metabolism that may be relevant during mammalian development.