Efficacy and safety of high-flow nasal cannula therapy in elderly patients with acute respiratory failure.

INTRODUCTION: To assess the efficacy and safety of high-flow nasal cannula (HFNC) in elderly patients with acute respiratory failure (ARF) not due to COVID-19, refractory to treatment with conventional oxygen therapy and/or intolerant to noninvasive ventilation (NIV) or continuous positive airway pressure (CPAP) and without criteria for admission to intensive care units (ICU). METHODS: Prospective observational study of patients with ARF treated with HFNC who presented clinical and arterial blood gas deterioration after 24 h of medical treatment and oxygenation by conventional systems. The degree of dyspnoea, gas exchange parameters (arterial O2 pressure/inspired O2 fraction ratio (PaO2/FiO2); oxygen saturation measured by oximetry/ inspired fraction of oxygen (Sp02/Fi02), ROX index), degree of patient tolerance and mortality were evaluated. These were measured at discharge from the emergency department (ED), 24 h after treatment with conventional oxygenation and 60, 120 min and 24 h after initiation of HFNC. The results were analyzed for all patients as a whole and for patients with hypercapnia (arterial carbon dioxide tension (PaCO2) < 45 mmHg) separately. RESULTS: 200 patients were included in the study between November 2019 and November 2020, with a mean age of 83 years, predominantly women (61.9%), obese (Body Mass Index (BMI) 31.1), with high comorbidity (Charlson index 4) and mild-moderate degree of dependence (Barthel 60). A number of 128 patients (64%) were hypercapnic. None had respiratory acidosis (pH 7.39). Evaluation at 60 min, 120 min and 24 h showed significant improvement in all patients and in the subgroup of hypercapnic patients with respect to baseline parameters in respiratory rate (RR), dyspnoea, ROX index, PaO2/FiO2, SpO2/FiO2 and patient comfort. No changes in PaCO2 or level of consciousness were observed. HFNC was well tolerated. Ten patients (5%) died due to progression of the disease causing ARF. CONCLUSIONS: HFNC is an effective and safe alternative in elderly patients with ARF not due to COVID-19, refractory to treatment with conventional oxygen therapy and/or intolerant to NIV or CPAP and without criteria for admission to ICU.

The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.

Short communication: Linear discriminant analysis and type of oil added to dairy goat diets.

Gas chromatography fatty acid (FA) analysis of 112 milk fat samples from dairy goats fed a basal diet with no added oil or the same diet with 1 of 3 vegetable oils added [high oleic sunflower oil (HOSFO), regular sunflower oil (RSFO), or linseed oil (LO)] was used to identify the type of diet consumed through linear discriminant analysis. Twenty variables (19 FA and 1 FA ratio) were selected as valid predictors out of 84 variables tested. The Mahalanobis squared distance was minimal between HOSFO and RSFO groups and maximal between control and LO groups. Cross-validation showed that only one observation from RSFO group was misclassified into the HOSFO group. We concluded that linear discriminant analysis is a useful method to classify milk fat samples from dairy goats according to the particular vegetable oil (of the 3 oils tested here) added to the basal diet.

Study of the immunologic response of marine-derived collagen and gelatin extracts for tissue engineering applications.

The host immunologic response to a specific material is a critical aspect when considering it for clinical implementation. Collagen and gelatin extracted from marine sources have been proposed as biomaterials for tissue engineering applications, but there is a lack of information in the literature about their immunogenicity. In this work, we evaluated the immune response to collagen and/or gelatin from blue shark and codfish, previously extracted and characterized. After endotoxin evaluation, bone marrow-derived macrophages were exposed to the materials and a panel of pro- and anti-inflammatory cytokines were evaluated both for protein quantification and gene expression. Then, the impact of those materials in the host was evaluated through peritoneal injection in C57BL/6 mice. The results suggested shark collagen as the less immunogenic material, inducing low expression of pro-inflammatory cytokines as well as inducible nitric oxide synthase (encoded by Nos2) and high expression of Arginase 1 (encoded by Arg1). Although shark gelatin appeared to be the material with higher pro-inflammatory expression, it also presents a high expression of IL-10 (anti-inflammatory cytokine) and Arginase (both markers for M2-like macrophages). When injected in the peritoneal cavity of mice, our materials demonstrated a transient recruitment of neutrophil, being almost non-existent after 24 hours of injection. Based on these findings, the studied collagenous materials can be considered interesting biomaterial candidates for regenerative medicine as they may induce an activation of the M2-like macrophage population, which is involved in suppressing the inflammatory processes promoting tissue remodeling. STATEMENT OF SIGNIFICANCE: Marine-origin biomaterials are emerging in the biomedical arena, namely the ones based in marine-derived collagen/gelatin proposed as cell templates for tissue regeneration. Nevertheless, although the major cause of implant rejection in clinical practice is the host's negative immune response, there is a lack of information in the literature about the immunological impact of these marine collagenous materials. This work aims to contribute with knowledge about the immunologic response to collagen/gelatin extracted from blue shark and codfish skins. The results demonstrated that despite some differences observed, all the materials can induce a macrophage phenotype related with anti-inflammation resolution and then act as immuno-modulators and anti-inflammatory inducible materials.

Animal performance and milk fatty acid profile of dairy goats fed diets with different unsaturated plant oils.

The effect of supplementing a basal diet with 1 of 3 plant oils on productive efficiency and milk fatty acid composition was studied in dairy goats. Sixteen Malagueña goats were used in a 4×4 Latin square experiment with 21-d periods and 4 goats per treatment. The basal diet comprised 30% alfalfa hay and 70% pelleted concentrate. Experimental treatments were control (basal diet without added oil) and the basal diet supplemented with 48g/d of high oleic sunflower oil (HOSFO), regular sunflower oil (RSFO), or linseed oil (LO). Dry matter intake and body weight were not affected by treatments. Milk production was higher in HOSFO treatment and milk fat content was higher in RSFO and LO treatments, although no differences in milk energy production or milk renneting properties were found. The RSFO and LO treatments increased the proportion of vaccenic acid in milk fat more so than the HOSFO diet, and rumenic acid followed the same pattern. The content of trans10-18:1 remained low in all experimental diets (<0.7% of total fatty acid methyl esters) although HOSFO and RSFO diets increased it. The variations in the fatty acid profiles observed with the 4 diets, mainly the unsaturated fatty acid isomer contents, are extensively discussed. Compared with that in the control diet, the n-6:n-3 fatty acid ratio in milk fat substantially decreased with the LO, increased with RSFO, and did not change with HOSFO. The addition of moderate amounts of LO to the diets of dairy goats has favorable effects on milk fatty acid composition from the point of view of the human consumer, without negative effects on animal performance.

Anti-interleukin 10 receptor monoclonal antibody is an adjuvant for T helper cell type 1 responses to soluble antigen only in the presence of lipopolysaccharide.

Soluble foreign antigen usually leads to a transient clonal expansion of antigen-specific T cells followed by the deletion and/or functional inactivation of the cells. As interleukin (IL)-10 is a key immunoregulatory cytokine, we questioned whether neutralization of IL-10 during priming with soluble antigen could prime for a subsequent T helper cell type 1 (Th1) effector recall response. By using an adoptive transfer model to track the fate of antigen-specific T cell receptor (TCR)-transgenic CD4(+) T cells, we show that administration of soluble ovalbumin (OVA) protein, but not OVA(323-339) peptide antigen, together with an anti-IL-10 receptor (R) mAb led to the enhancement of a Th1 response upon rechallenge. Lipopolysaccharide (LPS) present in the protein was necessary for priming for Th1 recall responses in the presence of anti-IL-10R mAb, as removal of LPS abrogated this effect. Moreover, addition of LPS to the peptide did not itself allow priming for recall Th1 effector responses unless endogenous levels of IL-10 were neutralized with an anti-IL-10R mAb. A significant increase in OVA-specific IgG1 and IgG2a isotypes was observed when the protein antigen was administered with anti-IL-10R mAb; however, this was not the case with peptide antigen administered together with anti-IL-10R and LPS. Our data, showing that LPS receptor signaling and neutralization of endogenous immunosuppressive cytokines is essential for Th1 priming, has important implications for the design of relevant vaccines for effective in vivo immunotherapy.

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

Predictors and outcomes of disseminated tuberculosis in an intermediate burden setting.

SETTING: University-affiliated hospital located in Porto, North Portugal, an area with a low to intermediate incidence of tuberculosis (TB). OBJECTIVE: To identify predictors and outcomes of disseminated TB (dTB). DESIGN: A cohort of patients diagnosed with TB between 2007 and 2013 was retrospectively analysed. Patients with dTB criteria were characterized and compared to single organ TB cases. Factors independently associated with dTB were determined by multivariate logistic regression analysis. RESULTS: A total of 744 patients were analysed, including 145 with dTB. Independent risk factors for dTB were pharmacological immunosuppression (OR 5.6, 95% CI 2.8-11.3), HIV infection (OR 5.1, 95% CI 3.1-8.3), chronic liver failure or cirrhosis (OR 2.3, 95% CI 1.4-4.1) and duration of symptoms (OR 2.3, 95% CI 1.4-3.8). Compared to single organ TB, the clinical presentation of dTB patients differed by the absence of haemoptysis (OR 3.2, 95% CI 1.3-8.4) and of dyspnoea (OR 1.9, 95% CI 1.2-3.1), presence of weight loss (OR 1.8, 95% CI 1.1-2.9), night sweats (OR 1.7, 95% CI 1.1-2.7) and bilateral lung involvement (OR 4.4, 95% CI 2.8-7.1). Mortality and time until culture conversion were higher for dTB patients, although not reaching statistical significance. CONCLUSION: Immunosuppressive conditions and chronic liver failure or cirrhosis were associated with increased risk of dTB. The haematogenous spread may be dependent on longer symptomatic disease and usually progresses with bilateral lung involvement.

Human amniotic membrane intra-articular injection prevents cartilage damage in an osteoarthritis model.

Osteoarthritis (OA) is a degenerative joint disease that affects the soft tissues and bones of involved articulations as a result of deregulation between synthesis and extracellular matrix degradation in articular cartilage. The present study evaluated the effect of intra-articular injection of human amniotic membrane (AM) as a treatment in an OA animal model in the knee. Chemical OA was developed in the knees of New Zealand rabbits. Once OA was established, the right knees only were treated with an intra-articular injection of human AM, with the left knees considered as a negative control group. The evaluation was performed at 3 and 6 weeks post-treatment. At 3 weeks post-injection, the cartilage exhibited fibrillation, erosion, cracks and cell clusters in the negative control group, but not in the treated group (P=0.028). At 6 weeks post-injection, the left knees exhibited hypertrophy, cracks, cell clusters, decreased matrix staining and structure loss. However, the right knees exhibited cell clusters without evidence of disruption in cartilage integrity (P=0.015). These results suggested that the intra-articular injection of human AM delays histological changes of cartilage in OA.

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes.

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.

Osteogenic differentiation of two distinct subpopulations of human adipose-derived stem cells: an in vitro and in vivo study.

The first stem cells considered for the reconstruction of bone were bone marrow mesenchymal stem cells (BMSCs). Subsequently, cells with similar marker expression panel and differentiation potential were found in new sources of cells, such as adipose tissue. This source of stem cells has a promising future in tissue-engineering applications, considering the abundance of this tissue in the human body, the easy harvesting and the high number of stem cells that are available from such a small amount of tissue. The isolation of the adipose stem cells is generally performed by means of enzymatic digestion of the tissues, followed by a natural selection of the stem cells based on their capacity to adhere to the culture flasks, leading to a quite heterogeneous population. This constitutes a major drawback for the use of these cells, since the heterogeneity of the cell culture obtained can compromise their proliferation and differentiation potential. In the present study we have analysed the in vitro and in vivo behaviour of two selected subpopulations with high osteogenic potential. For this purpose, ASCs(CD29+) and ASCs (STRO-1+)subpopulations were isolated and in vitro cultured onto a biodegradable polymeric scaffold, using osteogenic medium, before implantation in a nude mice model. The biodegradable polymeric scaffold used is a fibre-mesh structure based on a blend of starch and polycaprolatone (SPCL) that has been successfully used in several bone tissue-engineering studies. The implanted ASCs-scaffold constructs promoted the formation of new bone tissue in nude mice. However, the results obtained show differences in the behaviour of the two ASCs subpopulations under study, particularly regarding their potential to differentiate into the osteogenic lineage, and allowed the indentification of ASCs (STRO-1+) as the best subpopulation for bone tissue-engineering applications.

Comparative Study of Serological Tests for Mycoplasma synoviae Diagnosis in Commercial Poultry Breeders.

Avian mycoplasmosis causes great economic losses to the poultry industry, and one of the major agents involved is Mycoplasma synovie (MS). Serum from commercial poultry breeders (n = 2781) was tested for MS by serum plate agglutination (SPA), hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay (ELISA). From 2,781 samples tested, 736 (26.46%) were positive in SPA. From 712 SPA-positive sera, 30 samples (4.21%) were positive in HI, and 150 samples (21.06%) were positive in ELISA. Copositivity between ELISA and HI was 90%, and conegativity was 82.0%. Agreement between HI and ELISA was rejected by McNemar's test (P ≤ .001), and Kappa coefficient showed a weak correlation between the two techniques (k = 0.25; 0.21 ≤ k < 0.40). Weak statistical correlation was observed between all serological tests (SPA, HI, and ELISA), and they should only be used for initial screening for MS.

In vivo short-term and long-term host reaction to starch-based scaffolds.

The implantation of biomaterials may elicit a host response to this foreign body, and the magnitude of that reaction depends on the host and on the implanted material. The aim of this study was to compare the inflammatory response induced by the implantation of starch-based (SPCL) scaffolds in two implantation rat models: subcutaneous (SC) and intramuscular (IM). Moreover, two methodologies, wet spinning (WS) and fibre-bonding (FB), were used to prepare the scaffolds. The short-term inflammatory/immune host reaction was assessed by SC and IM implantations in rats after 1 and 2 weeks, and the long-term host response was addressed after 8 and 12 weeks of SC implantation of both types of SPCL scaffolds in rats. After each time period, the scaffolds, surrounding tissue and nearby lymph nodes were explanted, and used for histological analysis and molecular biology evaluation. The results showed that SPCL-WS scaffolds seem to induce a slight lower inflammatory/immune reaction in both types of implantation models. Nonetheless, comparing the two models, the IM implantation resulted in a slightly higher inflammatory response than the SC implantation with early activation of the lymph nodes. The overall data suggests a good integration of the materials in the host, independently of the tissue location with a normal progress of the reaction for all the conditions.

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice.

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue-engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real-time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round-shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real-time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice.

Developments on drug delivery systems for the treatment of mycobacterial infections.

The clinical management of tuberculosis and other mycobacterial diseases with antimycobacterial chemotherapy remains a difficult task. The classical treatment protocols are long-lasting; the drugs reach mycobacteria-infected macrophages in low amounts and/or do not persist long enough to develop the desired antimycobacterial effect; and the available agents induce severe toxic effects. Nanotechnology has provided a huge improvement to pharmacology through the designing of drug delivery systems able to target phagocytic cells infected by intracellular pathogens, such as mycobacteria. Liposomes and nanoparticles of polymeric nature represent two of the most efficient drug carrier systems that after in vivo administration are endocytosed by phagocytic cells and then release the carried agents into these cells. This article reviews the relevant publications describing the effectiveness of the association of antimycobacterial agents with liposomes or nanoparticles for the treatment of mycobacterioses, particularly for Mycobacterium tuberculosis and M. avium infections. The increased therapeutic index of antimycobacterial drugs; the reduction of dosing frequency; and the improvement of solubility of hydrophobic agents, allowing the administration of higher doses, have been demonstrated in experimental infections. These advantages may lead to new therapeutic protocols that will improve patient compliance and, consequently, lead to a more successful control of mycobacterial infections. The potential therapeutic advantages resulting from the use of non-invasive administration routes for nanoparticulate systems are also discussed.

Neospora caninum: high susceptibility to the parasite in C57BL/10ScCr mice.

C57BL/10ScCr mice, lack Toll-like receptor 4 and a functional Interleukin-12 receptor. Taking this into account, susceptibility of these mice to Neospora caninum infection was assessed comparatively to that of immunocompetent C57BL/10ScSn mice. C57BL/10ScCr mice inoculated intraperitoneally with 5x10(5)N. caninum tachyzoites showed a high susceptibility to this parasite. All infected C57BL/10ScCr mice were dead by day 8 post-infection whereas all control C57BL/10ScSn mice survived this parasitic challenge. Immunohistochemical analysis of infected C57BL/10ScCr mice showed N. caninum tachyzoites spread in the pancreas, liver, lung, intestine, heart and brain whereas no parasites were detected in similarly infected C57BL/10ScSn controls. The higher susceptibility of C57BL/10ScCr mice to neosporosis correlates with reduced interferon-gamma mRNA expression and increased IL-4 mRNA expression, comparatively to C57BL/10ScSn controls, detected in the spleen after the parasitic challenge. C57BL/10ScCr mice could thus be used as a new experimental model where to study immunobiological mechanisms associated with host susceptibility to neosporosis.

Analysis of the immune response to Neospora caninum in a model of intragastric infection in mice.

To study experimental Neospora caninum infection initiated at the gastrointestinal tract, Toll-like Receptor 4- and functional IL-12Rbeta2 chain-deficient C57BL/10 ScCr mice were challenged intragastrically with 5 x 10(6) N. caninum tachyzoites. All parasite-inoculated mice eventually died with disseminated infection. In contrast, immunocompetent BALB/c mice challenged with 1 x 10(7) N. caninum tachyzoites by the intragastric (i.g.) or the intraperitoneal (i.p.) route remained alive for at least 6 months. Expansion of splenic B- and T-cells, the latter displaying both activated and regulatory phenotypes, and increased levels of IFN-gamma and IL-10 mRNA were detected in both groups of infected BALB/c mice compared with non-infected controls, whereas in the Peyer's patches only IFN-gamma mRNA levels were found to be increased. Parasite-specific IgG1, IgG2a and IgA antibody levels were elevated in the sera of all infected mice, whereas increased N. caninum-specific IgA levels were detected in intestinal lavage fluids of i.g. challenged mice only. These results show that N. caninum infection can be successfully established in mice by i.g. administration of tachyzoites. They also show that the immune response elicited in i.g. or i.p. infected BALB/c mice, although conferring some degree of protection, was not sufficient for complete parasite clearance.

The relative impact of bacterial virulence and host genetic background on cytokine expression during Mycobacterium avium infection of mice.

Resistance to Mycobacterium avium depends on both genetically encoded macrophage functions and acquired T-cell immunity. Cytokines may play a role in either type of resistance. We studied the expression of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) in naturally susceptible BALB/c (Bcgs) and naturally resistant C.D2 (Bcgr) congenic mice infected with two strains of M. avium (one highly virulent and another of low virulence). We observed that cytokine expression patterns correlated better with the virulence of the micro-organism than with the genetic background of the host. The control of the infection by the low virulence strain in either mouse strain was associated with an increased expression of IFN-gamma and IL-2. Only Bcgs mice infected with a virulent strain of M. avium were unable to restrict bacterial growth. An increased expression of IL-4, early during infection, was detected in the course of the latter infection but played no role in determining the susceptibility to infection. Neutralization of IFN-gamma or IL-2 with specific monoclonal antibodies led to an exacerbation of the infection in Bcgr mice by the two strains of M. avium and in Bcgs mice infected with the low virulence strain of M. avium.