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Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Resumo Os processos administrativos de reconhecimento de terras indígenas (TIs) no Brasil podem levar décadas para chegar a termo. Povos indígenas que habitam em TIs não reconhecidas em caráter final são mais vulneráveis a uma série de violações de direitos, o que torna premente sua conclusão. O presente artigo pretende oferecer uma resposta à seguinte pergunta: por que alguns processos demarcatórios demoram mais que outros? Primeiramente, arrolamos cinco condições constantes da literatura sobre demarcação de terras indígenas que podem postergá-las. Na sequência, aplicamos o método qualitative comparative analysis (QCA), em sua modalidade crisp set, a um conjunto de quarenta casos de demarcações de TIs. Apresentamos duas conclusões: que a presença de interesses econômicos é uma condição importante para explicar o longo tempo de alguns processos demarcatórios, mas não é necessária nem suficiente, e que a conjunção desse fator com a judicialização do processo demarcatório explica a maioria dos casos de demarcações de longa duração.

Hyperthyroidism is an endocrine disorder whose treatment of choice is methimazole, due to the low risk of adverse reactions. Drug-induced liver injury by methimazole is rare, especially in pediatric age, but when it occurs it is potentially serious. We present a case report of drug-induced liver injury due to methimazole in a 12-year-old boy diagnosed with hyperthyroidism due to Graves’ disease. The patient had signs and symptoms of thyrotoxicosis, but not clinical findings related to the liver condition. After the discontinuation of treatment and ablative therapy with Iodine131, there was an excellent clinical and analytical evolution, with recovery of cytocholestasis to almost normal values. Thus, early recognition of this serious adverse effect through frequent monitoring of liver enzymes along with thyroid function is essential to avoid unfavorable outcomes.

Microbiological diagnosis of osteoarticular infections (OI) is crucial for a successful treatment. A prospective multicenter study including 262 synovial fluids with suspicion of acute OI was performed between July 2021 and October of 2022. BioFire Joint Infection Panel multiplex-PCR test was performed and results were compared with conventional cultures of synovial fluid specimens. In total, 136 microorganisms were detected, and fourteen samples were positive for more than one microorganism. In monomicrobial infections (n = 87) agreement with culture was 69%. In 26 samples, the multiplex PCR yield an additional positive result when culture result was negative. It helped in the detection of fastidious microorganisms as K. kingae and N. gonorrhoeae. This multiplex PCR has proven to be a useful technique that can be used for patients with high suspicion of acute OI in a rapid and automated manner.

Background: Infections by multidrug-resistant (MDR) microorganisms are associated with increased morbidity and mortality in burn patients. This study aimed to analyze the evolution of MDR bacteria over a five-year period at Coimbra Burns Unit (CBU) in Portugal, seeking to assess the possible associations of specific bacteria with presumed risk factors. Methods: The data obtained consisted of identified bacteria present in any microbiological sample from each patient (including blood, central venous catheter, urine, tracheal aspirate and/or wound exudate). Univariate models and a multivariate model were constructed for each of the MDR bacteria species that infected at least 50 patients or that had five or more MDR strains. Statistical hypothesis tests with a p-value less than 0.05 were considered significant. Results: Of a total of 341 samples obtained, 107 were MDR, corresponding to 10 species. Globally, there was no significant variation in MDR bacteria frequency over the period under analysis. Some risk factors and/or trends were identified for some species, but none was linked to all of them. Conclusions: The risks for the development of MDR in bacteria in burn patients are multifactorial, mainly linked to longer hospital stays, the use of invasive devices and inadequate antimicrobial treatment. However, the influence of these risks regarding specific bacterial species is not straightforward and may rely on individual characteristics, type of treatment and/or local prevalent flora. Due to the severity of multidrug-resistant infections, continued microbiological surveillance with the aid of rapid diagnostic tests and prompt institution of appropriate antimicrobial therapy are crucial to improving outcomes for burn patients.

ABSTRACT Imipenem-relebactam is a novel β-lactam-β-lactamase inhibitor combination. We evaluated the in vitro activity of imipenem-relebactam and comparators against Enterobacterales clinical isolates recovered in 8 Spanish and 11 Portuguese intensive care units (ICUs) (SUPERIOR, 2016–2017; STEP, 2017–2018). Overall, 747 Enterobacterales isolates (378 Escherichia coli, 252 Klebsiella spp., 64 Enterobacter spp., and 53 other species) were prospectively collected from ICU patients with complicated intraabdominal (cIAI), complicated urinary tract (cUTI), and lower respiratory tract (LRTI) infections. MICs were determined (ISO-broth microdilution), and whole-genome sequencing (WGS) was performed in a subset of isolates displaying susceptible and resistant imipenem-relebactam MICs. Imipenem-relebactam (98.7% susceptible) showed similar activity to ceftazidime-avibactam (99.5% susceptible) and higher than ceftolozane-tazobactam (86.9% susceptible). Imipenem-relebactam was inactive against 1.3% (10/747) isolates, all of them due to carbapenemase production (9 K. pneumoniae and 1 E. cloacae). Imipenem-relebactam was active against 100% of extended-spectrum β-lactamase (ESBL)-E. coli and ESBL-Klebsiella spp. isolates and 80.4% of carbapenemase-Klebsiella spp. producers. Carbapenemase genes were confirmed by WGS in 41 Klebsiella spp.: OXA-48 (20/41), KPC-3 (14/41), OXA-181 (4/41), NDM-1 (1/41), OXA-48 + VIM-2 (1/41), and KPC-3 + VIM-2 (1/41). In Klebsiella spp. isolates, relebactam restored imipenem susceptibility in all KPC-3 producers, and resistant isolates (7/41) were mostly OXA-48 + CTX-M-15-K. pneumoniae high-risk clones (7/9). Intercountry differences were detected as follows: OXA-48 (17/21) was dominant in Spain, unlike KPC-3 (14/15) in Portugal. Imipenem-relebactam was 100% active against CTX-M-15-ST131-H30Rx-E. coli high-risk clone, predominant in both countries. Our results depict the potential role of imipenem-relebactam in ICU patients with cIAIs, cUTIs, and LRTIs due to wild-type ESBL- and carbapenemase-producing Enterobacterales, particularly KPC producers. IMPORTANCE We comparatively evaluate the in vitro activity of a drug combination consisting of a carbapenem (imipenem) and a novel inhibitor of beta-lactamases (relebactam), a mechanism that destroys beta-lactam antibiotics. We assess the activity against a collection of Enterobacterales clinical isolates recovered from difficult-to-treat infections in patients admitted to different intensive care units in Portugal and Spain. Imipenem-relebactam shows excellent activity in avoiding common resistance mechanisms in this setting, such as extended-spectrum beta-lactamases and carbapenemases widely distributed, including KPCs. We show few resistant isolates (

Most antibodies display very low brain exposure due to the blood-brain barrier (BBB) preventing their entry into brain parenchyma. Transferrin receptor (TfR) has been used previously to ferry antibodies to the brain by using different formats of bispecific constructs. Tetravalent bispecific tandem immunoglobulin Gs (IgGs) (TBTIs) containing two paratopes for both TfR and protofibrillar forms of amyloid-beta (Aβ) peptide were constructed and shown to display higher brain penetration than the parent anti-Aβ antibody. Additional structure-based mutations on the TfR paratopes further increased brain exposure, with maximal enhancement up to 13-fold in wild-type mice and an additional 4–5-fold in transgenic (Tg) mice harboring amyloid plaques, the main target of our amyloid antibody. Parenchymal target engagement of extracellular amyloid plaques was demonstrated using in vivo and ex vivo fluorescence imaging as well as histological methods. The best candidates were selected for a chronic study in an amyloid precursor protein (APP) Tg mouse model showing efficacy at reducing brain amyloid load at a lower dose than the corresponding monospecific antibody. TBTIs represent a promising format for enhancing IgG brain penetration using a symmetrical construct and keeping bivalency of the payload antibody.

Background Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier. Method We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry. Results 12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized β1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnβ1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues. Conclusions This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.

Multicultural societies are marked by the coexistence of ethnic, sexual, religious, racial, and cultural minorities and mainstream groups. This coexistence can either be tense or collaborative. How to bridge the gap between the political demands of majority and minority groups? What are the obstacles to meaningful participation? What are the main challenges faced by such societies? And finally, how do we encourage large-scale debates around issues of minorities? In order to provide answers to these questions, this review examines Intercultural Deliberation and the Politics of Minority Rights by R. E. Lowe-Walker (2018), Deliberative Democracy Now: LGBT Equality and the Emergence of Large-Scale Deliberative Systems by Edwina Barvosa (2018), and Deliberative Democracy, Political Legitimacy, and Self-determination in Multicultural Societies by Jorge M. Valadez (2018).

Objetivo: A infeção no pós-operatório é considerada uma das mais importantes causas de morbi-mortalidade após transplante hepático. Poucos estudos analisam a incidência de culturas positivas do líquido de preservação do enxerto e o outcome dos respetivos recetores. Estudamos a incidência e o impacto clínico de infeção do líquido de preservação de orgão para transplante hepático. Métodos: Cultivamos o líquido de preservaãoo (Celsior®) em 225 transplantes de fígado durante quatro anos consecutivos; os recetores de alto risco foram submetidos a um protocolo de profilaxia antibiótica de infeção pós-transplante, durante 48 horas, que consistia numa cefalosporina de 3a geração e netilmicina. Resultados: Setenta líquidos de preservação foram considerados positivos com uma três patógenos identificados. Destes, 31% eram flora saprofítica da pele, porém, em 29 casos (41,1%), isolamos patógenos de alta virulência. Apenas oito doentes desenvolveram febre no pós-operatório devido ao microorganismo isolado no líquido de preservação. Conclusão: Foram identificadas culturas positivas em 31,1% dos casos, sendo que um terço corresponde a flora saprofítica da pele. Os nossos resultados não suportam a realização por rotina de cultura do líquido de preservação, desde que haja um regime profilático de antibióticos adequado.

Abstract Background Perceived risk of HIV plays an important role in the adoption of protective behaviours and HIV testing. However, few studies have used multiple-item measures to assess this construct. The Perceived Risk of HIV Scale (PRHS) is an 8-item measure that assesses how people think and feel about their risk of HIV infection. This cross-sectional study aimed to assess the psychometric properties (reliability and validity) of the European Portuguese version of the PRHS, including the ability of this scale to discriminate between individuals from the general population and HIV-uninfected partners from sero-different couples on their perceived risk of HIV infection (known-groups validity). Methods This study included 917 individuals from the general population (sample 1) to assess the psychometric properties of the PRHS. To assess the known-groups validity, the sample comprised 445 participants from the general population who were in an intimate relationship (sub-set of sample 1) and 42 HIV-uninfected partners from sero-different couples (sample 2). All participants filled out a set of questionnaires, which included a self-reported questionnaire on sociodemographic information, sexual behaviours, HIV testing and the PRHS. Sample 1 also completed the HIV Knowledge Questionnaire – 18-item version. Results The original unidimensional structure was reproduced both in exploratory and confirmatory factor analyses, and the PRHS demonstrated good reliability (α = .78; composite reliability = .82). The differential item functioning analyses indicated that the items of the PRHS, in general, did not function differently for men and women or according to HIV testing. Significant associations with sexual risk behaviours and HIV testing provided evidence for criterion validity. The known-groups validity was supported. Conclusions The PRHS is a suitable scale in the evaluation of the perceived risk of HIV, and its psychometric characteristics validate its use in the Portuguese population. Furthermore, the present study suggests that interventions improving individuals’ HIV risk perceptions may be important since they were associated with different sexual behaviours and the likelihood of HIV testing.

Transient receptor potential vanilloid 1-4 (TRPV1-4) expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood–brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells [rat brain microvessel endothelial cells (rPBMEC)]. A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (hPBMEC)], showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 > > TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 μM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 μM), a highly potent and specific TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 μM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.

Introdução: A emergência de bactérias produtoras de β-lactamases de espetro expandido é um problema mundial, com importância crescente nas infeções adquiridas na comunidade, nomeadamente nas infeções urinárias. Os dados pediátricos de utilização de antimicrobianos não carbapenemos nestas infeções são escassos. O objetivo do estudo foi analisar a terapêutica antibiótica instituída nas infeções urinárias causadas por estes agentes, assim como a evolução clínica e laboratorial. Material e Métodos: Estudo retrospetivo efetuado num hospital pediátrico entre junho de 2007 e dezembro de 2017. Foram incluídas todas as crianças com urocultura positiva para Enterobacteriaceae produtoras de β-lactamases. Resultados: Foram diagnosticadas 175 infeções urinárias causadas por Enterobacteriaceae produtoras de β-lactamases, das quais 34 (19%) foram adquiridas na comunidade: 25 Escherichia coli (74%), 4 Klebsiella pneumoniae (12%), 4 Proteus mirabilis (12%) e 1 Proteus vulgaris (3%). Em 30 (88%) episódios tratou-se da primeira infeção urinária. Após conhecimento do microrganismo e suas suscetibilidades, 33 (97%) crianças foram reavaliadas e 24 (71%) repetiram urocultura, que foi positiva em três (13%). Em seis (18%) casos foi alterado o antimicrobiano. No mês subsequente, quatro (12%) crianças tiveram nova infeção urinária e 30 (88%) crianças realizaram investigação imagiológica, sem deteção de malformações nefro-urológicas. Discussão: Na última década, cerca de 20% das infeções urinárias causadas por Enterobacteriaceae produtoras de β-lactamases foram adquiridas na comunidade, com um número relativamente estável ao longo dos anos. Estas crianças não apresentavam malformações nefro-urológicas. Conclusão: Embora o número de casos seja pequeno, a evolução clínica e microbiológica mostrou que a maioria foi tratada com sucesso com antimicrobianos não carbapenemos, com baixa ocorrência de novos episódios.

[This corrects the article DOI: 10.1371/journal.pone.0162797.].

Abstract Background Early sepsis diagnosis is crucial for the correct management of burn patients, and it clearly influences outcomes. The systemic inflammatory response triggered by burns mimics sepsis presentation and complicates early sepsis diagnosis. Biomarkers were advocated to aid the diagnosis of early sepsis. Serum procalcitonin (PCT) exhibits fair accuracy and good correlation with sepsis severity, being used in diverse clinical settings. However, few studies have evaluated perioperative changes in PCT levels in burn patients. The present study evaluated PCT kinetics during the first days after burn injury and subsequent surgical interventions to assess PCT utility in distinguishing septic from non-septic inflammatory responses. Methods This study was a retrospective observational study of all burn patients admitted to the Coimbra Burns Unit (Portugal) between January 2011 and December 2014 who presented with a total burn surface area ≥ 15% and who underwent subsequent surgery. PCT kinetics were investigated a) during the first five days after burn injury and b) preoperatively during the five days after surgery in three subsets of patients, including those with no preoperative and no postoperative sepsis (NN), no preoperative but postoperative sepsis (NS), and preoperative and postoperative sepsis (SS). A total of 145 patients met the selection criteria and were included in the analysis. Results PCT levels in the first five days after burn injury were significantly higher in patients who developed at least one sepsis episode (n = 85) compared with patients who did not develop sepsis (n = 60). PCT values > 1.00 ng/mL were clearly associated with sepsis. Study participants (n = 145) underwent a total of 283 surgical interventions. Their distribution by preoperative/postoperative sepsis status was 142 (50.2%) in NN; 62 (21.9%) in NS; and 79 (27.9%) in SS. PCT values exhibited a parallel course in the three groups that peaked on the second postoperative day and returned to preoperative levels on the third day or later. The lowest PCT values were found in NN, and the highest values were observed in SS; the NS values were intermediate. Conclusions PCT kinetics coupled with a clinical examination may be helpful for sepsis diagnosis during the first days after burn injury and burn surgery.

Abstract Background Due to greater infection susceptibility, sepsis is the main cause of death in burn patients. Quick diagnosis and patient stratification, early and appropriated antimicrobial therapy, and focus control are crucial for patients’ survival. On the other hand, superfluous extension of therapy is associated with adverse events and arousal of microbial resistance. The use of biomarkers, necessarily coupled with close clinical examination, may predict outcomes, stratifying patients who need more intensive care, and monitor the efficacy of antimicrobial therapy, allowing faster de-escalation or stop, reducing the development of resistance and possibly the financial burden, without increasing mortality. The aim of this work is to check the suitability of procalcitonin (PCT) to fulfill these goals in a large sample of septic burn patients. Methods One hundred and one patients, with 15% or more of total body surface area (TBSA) burned, admitted from January 2011 to December 2014 at Coimbra Burns Unit (CBU), in Portugal were included in the sample. All patients had a diagnosis of sepsis, according to the American Burn Association (ABA) criteria. The sample was factored by survival (68 survivors and 33 non-survivors). The maximum value of PCT in each day was used for statistical analysis. Data were summarized by location measures (mean, median, minimum, maximum, quartiles) and dispersion measures (standard error and range measures). Statistical analysis was performed with SPSS© 23.0 IBM© for Windows©. Results There were statistically significant differences between PCT levels of patients from the survivor and non-survivor groups during the first and the last weeks of hospitalization as well as during the first week after sepsis suspicion, being slightly higher during this period. During the first 7 days of antimicrobial therapy, PCT was always higher in the non-survivor, still without reaching statistical significance, but when the analysis was extended till the 15th day, PCT increased significantly, rapidly, and steadily, denouncing therapy failure. Conclusion Despite being not an ideal biomarker, PCT proved to have good prognostic power in septic burn patients, paralleling the evolution of the infectious process and reflecting the efficacy of antimicrobial therapy, and the inclusion of its serial dosing may be advised to reinforce antimicrobial stewardship programs at burn units; meanwhile, more accurate approaches are not available.

Ochrobactrum anthropi is a Gram-negative bacillus widely distributed in nature. It is a low virulence and low pathogenic microorganism and human infection by this agent is considered rare. This microorganism can cause bacteremia and in some cases can lead to osteomyelitis and endocarditis. Included in Brucellaceae family, this bacterium is phenotypically and genetically closely related to the Brucella genus and may be misidentified by rapid identification systems. The authors describe a patient admitted to the Infectious Diseases Department with vertebral osteomyelitis initially identified as Ochrobactrum anthropi. Despite appropriate antimicrobial therapy, the blood cultures remained positive and there were no signs of clinical improvement. This raised suspicion of a possible misidentification. It was decided to initiate antimicrobial therapy to include the Brucella genus, with slow but progressive clinical improvement. Samples were sent to Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) for genotyping, confirming the initial suspicion of misidentification and identifying Brucella melitensis as the causal agent. Timely diagnosis of brucellosis is essential for the correct management and prevention of its consequences for the patient and for safe handling of the laboratory samples, preventing laboratory-acquired infection

Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence.

The non-human primate (NHP)-brain endothelium constitutes an essential alternative to human in the prediction of molecule trafficking across the blood–brain barrier (BBB). This study presents a comparison between the NHP transcriptome of freshly isolated brain microcapillaries and in vitro-selected brain endothelial cells (BECs), focusing on important BBB features, namely tight junctions, receptors mediating transcytosis (RMT), ABC and SLC transporters, given its relevance as an alternative model for the molecule trafficking prediction across the BBB and identification of new brain-specific transport mechanisms. In vitro BECs conserved most of the BBB key elements for barrier integrity and control of molecular trafficking. The function of RMT via the transferrin receptor (TFRC) was characterized in this NHP-BBB model, where both human transferrin and anti-hTFRC antibody showed increased apical-to-basolateral passage in comparison to control molecules. In parallel, eventual BBB-related regional differences were Investig.igated in seven-day in vitro-selected BECs from five brain structures: brainstem, cerebellum, cortex, hippocampus, and striatum. Our analysis retrieved few differences in the brain endothelium across brain regions, suggesting a rather homogeneous BBB function across the brain parenchyma. The presently established NHP-derived BBB model closely mimics the physiological BBB, thus representing a ready-to-use tool for assessment of the penetration of biotherapeutics into the human CNS.

The blood–brain barrier (BBB) hinders the brain delivery of many anticancer drugs. In pediatric patients, diffuse intrinsic pontine glioma (DIPG) represents the main cause of brain cancer mortality lacking effective drug therapy. Using sham and DIPG-bearing rats, we analyzed (1) the brain distribution of 3-kDa-Texas red-dextran (TRD) or [14C]-sucrose as measures of BBB integrity, and (2) the role of major ATP-binding cassette (ABC) transporters at the BBB on the efflux of the irinotecan metabolite [3H]-SN-38. The unaffected [14C]-sucrose or TRD distribution in the cerebrum, cerebellum, and brainstem regions in DIPG-bearing animals suggests an intact BBB. Targeted proteomics retrieved no change in P-glycoprotein (P-gp), BCRP, MRP1, and MRP4 levels in the analyzed regions of DIPG rats. In vitro, DIPG cells express BCRP but not P-gp, MRP1, or MRP4. Dual inhibition of P-gp/Bcrp, or Mrp showed a significant increase on SN-38 BBB transport: Cerebrum (8.3-fold and 3-fold, respectively), cerebellum (4.2-fold and 2.8-fold), and brainstem (2.6-fold and 2.2-fold). Elacridar increased [3H]-SN-38 brain delivery beyond a P-gp/Bcrp inhibitor effect alone, emphasizing the role of another unidentified transporter in BBB efflux of SN-38. These results confirm a well-preserved BBB in DIPG-bearing rats, along with functional ABC-transporter expression. The development of chemotherapeutic strategies to circumvent ABC-mediated BBB efflux are needed to improve anticancer drug delivery against DIPG.

Organic cation transporters (OCTs) participate in the handling of compounds in kidneys and at the synaptic cleft. Their role at the blood-brain barrier (BBB) in brain drug delivery is still unclear. The presence of OCT1,2,3 (SLC22A1-3) in mouse, rat and human isolated brain microvessels was investigated by either qRT-PCR, quantitative proteomics and/or functional studies. BBB transport of the prototypical substrate [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+) was measured by in situ brain perfusion in six mouse strains and in Sprague Dawley rats, in primary human brain microvascular endothelial cells seeded on inserts, in the presence or absence of OCTs and a MATE1 (SLC49A1) inhibitor. The results show negligible OCT1 (SLC22A1) and OCT2 (SLC22A2) expression in either mice, rat or human brain microvessels, while OCT3 expression was identified in rat microvessels by qRT-PCR. The in vitro human cellular uptake of [3H]-MPP+ was not modified by OCTs/MATE-inhibitor. Brain transport of [3H]-MPP+ remains unchanged between 2- and 6-month old mice, and no alteration was observed in mice and rats with inhibitors. In conclusion, the evidenced lack of expression and/or functional OCTs and MATE at the BBB allows the maintenance of the brain homeostasis and function as it prevents an easy access of their neurotoxicant substrates to the brain parenchyma.

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB.

Tuberculosis imposes high human and economic tolls, including in Europe. This study was conducted to develop a severity assessment tool for stratifying mortality risk in pulmonary tuberculosis (PTB) patients. A derivation cohort of 681 PTB cases was retrospectively reviewed to generate a model based on multiple logistic regression analysis of prognostic variables with 6-month mortality as the outcome measure. A clinical scoring system was developed and tested against a validation cohort of 103 patients. Five risk features were selected for the prediction model: hypoxemic respiratory failure (OR 4.7, 95% CI 2.8-7.9), age ≥50 years (OR 2.9, 95% CI 1.7-4.8), bilateral lung involvement (OR 2.5, 95% CI 1.4-4.4), ≥1 significant comorbidity-HIV infection, diabetes mellitus, liver failure or cirrhosis, congestive heart failure and chronic respiratory disease-(OR 2.3, 95% CI 1.3-3.8), and hemoglobin