Ocean acidification impact on the uptake of trace elements by mussels and their biochemical effects.

This study delves into the intricate interplay between ocean acidification (OA), metal bioaccumulation, and cellular responses using mussels (Mytilus galloprovincialis) as bioindicators. For this purpose, environmentally realistic concentrations of isotopically labelled metals (Cd, Cu, Ag, Ce) were added to investigate whether the OA increase would modify metal bioaccumulation and induce adverse effects at the cellular level. The study reveals that while certain elements like Cd and Ag might remain unaffected by OA, the bioavailability of Cu and Ce could potentially escalate, leading to amplified accumulation in marine organisms. The present findings highlight a significant rise in Ce concentrations within different mussel organs under elevated pCO2 conditions, accompanied by an increased isotopic fractionation of Ce (140/142Ce), suggesting a heightened potential for metal accumulation under OA. The results suggested that OA influenced metal accumulation in the gills of mussels. Conversely, metal accumulation in the digestive gland was unaffected by OA. The exposure to both trace metals and OA affects the biochemical responses of M. galloprovincialis, leading to increased metabolic capacity, changes in energy reserves, and alterations in oxidative stress markers, but the specific effects on other biomarkers (e.g., lipid peroxidation, some enzymatic responses or acetylcholinesterase activity) were not uniform, suggesting complex interactions between the stressors and the biochemical pathways in the mussels.

Will extreme weather events influence the toxic impacts of caffeine in coastal systems? Comparison between two widely used bioindicator species.

In the recent years, marine heatwaves (MHWs) have caused devastating impacts on marine life. The understanding of the combined effects of these extreme events and anthropogenic pollution is a vital challenge. In particular, the combined effect of MHWs on the toxicity of pharmaceuticals to aquatic life remains unclear. To contribute to these issues, the main goal of the present investigation was to evaluate how MHWs may increase caffeine (CAF) toxicity on the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Bioaccumulation levels and changes on oxidative stress, metabolic capacity and neurotoxic status related biomarkers were investigated. The obtained results revealed the absence of CAF accumulation in both species. However, the used contaminant generated in both bivalve species alteration on neurotransmission, detoxification mechanisms induction as well as cellular damage. The increase of antioxidant defence mechanisms was complemented by an increase of metabolic activity and decrease of energy reserves. The obtained results seemed magnified under a simulated MHWs, suggesting to a climate-induced toxicant sensitivities' response. On this perspective, understanding of how toxicological mechanisms interact with climate-induced stressors will provide a solid platform to improve effect assessments for both humans and wildlife.

Alpha-1 antitrypsin deficiency is significantly associated with atopy in asthmatic patients.

Background: Although the etiology and disease mechanisms of asthma and alpha-1 antitrypsin deficiency (AATD) are distinct, several reports indicate that asthma is common in AATD patients, however the relationships between asthma and AATD are poorly described in the literature.Objectives: The aim of the study was to investigate in a cohort of outpatients affected by mild to moderate asthma the clinical features that may differentiate asthmatic patients with and without mutation on SERPINA1 gene.Methods: Seven hundred thirty-five asthmatic outpatients underwent quantitative analysis of the serum level of alpha-1antitrypsin. According to the literature only sixty-seven out of seven hundred thirty-five asthmatic patients were submitted to genetic analysis to identify AATD and non-AATD subjects. Fifty-eight patients were studied. Clinical and functional data, including lung function, atopy and bronchial hyperactivity, were recorded.Results: The fifty-eight asthmatic patients were divided in AATD patients (n = 22) and non AATD patients (n = 36), according to genotype. The presence of atopy was significantly higher in patients with AATD than in those without AATD (91% vs. 64%; p = 0.031). AATD patients reported allergic manifestations more than non AATD patients (77% vs. 47%; p = 0.030).Conclusion: Our study shows that the presence of atopy in asthmatic patients with AATD is significantly higher than in asthmatic patients without gene mutation. In addition, a higher percentage of AATD patients self-reported allergic manifestations. No significant differences in respiratory symptoms, physical examination, disease severity or inflammation markers were found between AATD patients and non AATD patients.

Ovarian tissue cryopreservation and transplantation in menopause: new perspective of therapy in postmenopausal women and the importance of ethical and legal frameworks.

Menopausal transition entails a progressive decrease in hormone production by the ovaries that lead to important physical and psychological changes that could significantly affect quality of life. Hormone replacement therapy (HRT) administered from the onset of menopausal symptoms usually improves quality of life and life expectancy. Nevertheless, it is not risk-free. Ovarian tissue cryopreservation (OTC) has been investigated as a potential new strategy for delaying menopause and/or to avoid HRT. This review analyzes the critical points of HRT to assess whether OTC and subsequent reimplantation can affect postmenopausal management. We assessed available randomized clinical trials in PubMed, Cochrane Library, ISI web of science, and Scopus from August 2021 to November 2022, including studies and trials evaluating the efficacy of OTC in both cancer and menopausal patients, the efficacy of freezing techniques and the possible clinical scenarios that OTC can open, even from the standpoint of legal and ethical issues arising as such innovative techniques become mainstream. Lower duration of the graft and efficacy on estrogen secretions at a physiological and safer concentration of estrogen than conventional HRT based on hormonal supplements. OTC can reportedly trigger estrogen secretions at a lower and safer physiological concentration than conventional HRT. OTC and subsequent reimplantation remain a valid fertility-sparing approach in patients undergoing gonadotoxic treatments. Further studies are needed to better evaluate its safety and efficacy within postmenopausal therapy management and in order to lay out widely shared and evidence-based guidelines and best practices and perform such novel and innovative techniques in a legally and ethically safe fashion, in the best interest of patients and healthcare professionals.

Bioassay-based ecotoxicological investigation on marine and freshwater impact of cigarette butt littering.

Despite representing an extremely relevant portion (20-40%) of worldwide coastal litter, cigarette butts are still an underestimate environmental issue of limited scientific interest. Public authorities of different countries promote active removal of cigarette butts, but the issue remains problematic in terms of aesthetic, environmental and health-related impacts. There are few studies on the environmental side-effects of smoked cigarette butt litter despite being a worldwide issue. In this work, two ecotoxicological bioassay batteries were adopted to evaluate the environmental consequences of cigarette butt water-soluble ingredient release in both marine water and freshwater. Marine assays were generally more affected compared to freshwater. Interesting outcomes were observed with crustacean tests, showing a lower effect of smoked cigarette butt leachate when tested at maximum concentration. This finding were supported by heartbeat measures of Daphnia magna, which were accelerated at 100% of smoked cigarette butt leachate.

How to easily detect plant NADH-glutamate dehydrogenase (GDH) activity? A simple and reliable in planta procedure suitable for tissues, extracts and heterologous microbial systems.

Plant NADH glutamate dehydrogenase (GDH) is an intriguing enzyme, since it is involved in different metabolic processes owing to its reversible (anabolic/catabolic) activity and due to the oligomeric nature of the enzyme, that gives rise to several isoforms. The complexity of GDH isoenzymes pattern and the variability of the spatial and temporal localization of the different isoforms have limited our comprehension of the physiological role of GDH in plants. Genetics, immunological, and biochemical approaches have been used until now in order to shed light on the regulatory mechanism that control GDH expression in different plant systems and environmental conditions. We describe here the validation of a simple in planta GDH activity staining procedure, providing evidence that it might be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems.

Atlantic and Mediterranean populations of the widespread serpulid Ficopomatus enigmaticus: Developmental responses to carbon nanotubes.

Ficopomatus enigmaticus was adopted as model species for ecotoxicological bioassay, with its larval development as endpoint. Two different populations of the same species, collected in areas far from each other (Mediterranean Sea and Atlantic Ocean), were exposed to multi-walled carbon nanotubes, a class of emerging pollutants with a constantly increasing relevance in the landscape of nanomaterials production. Moreover, a molecular analysis based on Cyt b amplification and sequencing, was carried out to confirm that both populations belong to the same species. The aim of the present work was to strengthen existing results about F. enigmaticus relevance in ecotoxicological bioassays, adding the variable of population effect. For both populations the concentration-response curve of effect at different toxicant concentrations was similar and, at certain concentrations, overlapping, confirming the ecological relevance of the assay. These results posed an interesting acceptance on the introduction of this species as model in ecotoxicological bioassay scenery, underlining the relevance of a widespread wild species to compare effects of chemicals and environmental samples over large distances using the same bioassay.

Effects of copper on larvae of the marbled crab Pachygrapsus marmoratus (Decapoda, Grapsidae): Toxicity test and biochemical marker responses.

The importance of trace elements in ecotoxicological investigations is a well-known issue when monitoring polluted areas such as commercial harbors. Copper represents one of the most common metal contaminants, often detected in these areas as it is widely employed in various fields and has many sources of inflow in the marine environment. Pachygrapsus marmoratus is a widespread intertidal crab species that has been extensively studied in ecology, ethology and population genetics. Ecotoxicological studies have also been performed, exclusively on the adult stage. In the present study we investigated the mortality and biochemical (oxidative stress and neurotoxicity) responses of P. marmoratus larvae exposure to environmental relevant concentration of copper. Results showed dose-dependent responses in terms of larval mortality, with a calculated LC50 value of 0.5 mg/L of Cu2+. The LC50 concentration was used as the starting point for subsequent biochemical response evaluation. Results also demonstrated dose-dependent activation of antioxidant systems assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. Moreover, a significant enhancement of neurotransmitter activities was observed, assuming a possible direct interaction of copper with the enzymes or an increase of free copper ion aliquot into the cells.

Ficopomatus enigmaticus larval development assay: An application for toxicity assessment of marine sediments.

Ficopomatus enigmaticus is a serpulid polychaete, reef-forming, dioecious gamete spawner throughout different seasons and in different salinity conditions. Due to these characteristics, its candidacy as a model organism to monitor both marine and brackish matrices was assessed. Marine sediments collected in a polluted area near the port of Ancona (Italy) were used as case study. Besides the chemical characterization of sediments, the larval development of F. enigmaticus was adopted as an assay for ecotoxicological assessment of sediments in addition to a regulatory bioassay battery with different organisms and endpoints. The quality of sediments was classified according to the Italian Ministerial Decree for sediment management (Ministerial Decree 173, 2016) by using the SEDI-QUAL-SOFT® (ver. 109.0) software developed by the Italian Environmental Protection Agency (ISPRA). The F. enigmaticus larval development assay showed a quite similar sensitivity to C. gigas larval development assays, indicating its potential use in a regulatory battery of bioassays.

Use of administrative health databases to estimate incidence and prevalence of acromegaly in Piedmont Region, Italy.

PURPOSE: Recent studies from national registries have described changing patterns in epidemiology of acromegaly. Our retrospective study used administrative databases to estimate prevalence and incidence of acromegaly in the Piedmont Region, Italy. METHODS: This study was conducted in Piedmont between 2012 and 2016 on administrative health databases for inpatients and outpatients of any age. Enrollees were included if claims suggestive of acromegaly were identified in at least two of the following databases: Drug Claims Registry, Hospital Information System, Co-payment Exemption Registry and Outpatient Specialist Service Information System. RESULTS: 369 individuals (M = 146, F = 223) met our criteria. Overall incidence was 5.3 per million person years (95% CI 4.2-6.7), and prevalence was 83 cases per million inhabitants (95% CI 75-92). Mean age was 50.9 years. Both incidence and prevalence were slightly higher among women (rate ratio 1.08, prevalence ratio 1.43). Age-specific incidence was similar between sexes up to 39 years and diverged thereafter, with an increasing trend recorded among men. Prevalence was higher in women aged 40-79 years, and increased continuously up to 79 years in both sexes. CONCLUSIONS: This is the first population-based study conducted in Italy to estimate incidence and prevalence of acromegaly and results show a higher prevalence than previously reported. Although our algorithm requires proper validation, it constitutes a promising tool to describe the epidemiology of acromegaly.

Minimally Invasive Adhesive Rehabilitation for a Patient With Tooth Erosion: Seven-year Follow-up.

Tooth wear is a multifactorial condition of growing concern. In clinical practice, it is often a challenge for prevention and treatment since many etiological factors may be involved. This case report describes an esthetic rehabilitation of a young patient presenting tooth wear due to erosion. The etiological factor of this case was the patient sucking on lemons, an acidic fruit. The main complaint of the patient was the appearance of short maxillary incisors. The treatment involved orthodontic and restorative dentistry techniques. First of all, an orthodontic device was used to increase the vertical dimension of occlusion and create an adequate space for the direct restorations. The posterior teeth were restored with two direct composite resin techniques; the anterior teeth were then restored using a balanced occlusion. The seven-year follow-up of the case is presented. Replicas of the restorations were made and visualized under scanning electron microscopy up to the 12-month evaluation. In the clinical follow-up at seven years, maintenance of the results and restorations can be clearly seen.

Basin-scale analysis of the geomorphic effectiveness of flash floods: A study in the northern Apennines (Italy).

Large floods may produce remarkable channel changes, which determine damages and casualties in inhabited areas. However, our knowledge of such processes remains poor, as is our capability to predict them. This study analyses the geomorphic response of the Nure River (northern Italy) and nine tributaries to a high-magnitude flood that occurred in September 2015. The adopted multi-disciplinary approach encompassed: (i) hydrological and hydraulic analysis; (ii) analysis of sediment delivery to the stream network by means of landslides mapping; (iii) assessment of morphological modifications of the channels, including both channel width and bed elevation changes. The spatial distribution of rainfall showed that the largest rainfall amounts occur in the upper portions of the catchment, with cumulative rainfall reaching 300 mm in 12 h, and recurrence intervals exceeding 100-150 years. The unit peak discharge ranged between 5.2 and 25 m3 s-1 km-2. Channel widening was the most evident effect. In the tributaries, the ratio between post-flood and pre-flood channel width averaged 3.3, with a maximum approaching 20. Widening was associated with channel aggradation up to 1.5 m and removal of riparian vegetation. New islands formed due to the fragmentation of the former floodplain. In the Nure River, the average width ratio was 1.7, and here widening occurred mainly at the expenses of islands. Bed level dynamics in the Nure were varied, including aggradation, incision, and overall stability. The flood geomorphic effectiveness was more pronounced in the middle-higher portions of the basin. Planimetric and elevation changes were well correlated. Regression analysis of the relationship between widening and morphological/ hydraulic controlling factors indicated that unit stream power and confinement index were the most relevant variables. The study provides useful insights for river management, especially with regard to the proportion of the valley floor subject to erosion and/or deposition during large events.

Effect of acute and chronic excesses of dietary nitrogen on blood neutrophil functions in cattle.

Excess dietary nitrogen (EDN) is commonly expected in dairy herds, but no data are available regarding its consequences on cattle immunity. In this study neutrophil functions were assessed during EDN in steers. In experiment 1, 4 one-month periods, 4 diets [16% crude protein (CP; DM basis), 20% CP based on soybean meal, 20% CP based on urea, and 24% CP based on urea and soybean meal], and 4 steers were included in a crossover design to determine the effects of a chronic excess. In experiment 2, the repercussions of an acute excess were assessed with 2 periods of 10 d, the same 4 steers, and 2 diets containing 14 and 20% CP. Sampling was done during the fourth week of each period in experiment 1, and on d 0, 1, 2, 3, 7, and 9 of each period in experiment 2. Individual blood biochemistry parameters were measured and neutrophil factors, such as counts, recovery after isolation, surface expression of CD11b and CD62L, phagocytosis, diapedesis, reactive oxygen species (ROS) production, and bacteria killing, were determined. Data were analyzed by general linear models of R, with period, diet or biochemical component, and animal as explanatory variables. The outcome variables were biochemical or immune variables. The variables diet, period, and animal were forced as fixed effects. Data collected over the entire period of experiment 2 were pooled. Several multiples linear regressions or ANOVA were performed and a Bonferroni correction was applied. In experiment 2 (acute EDN), neutrophil counts were negatively associated with nitrogen intake, conversely to CD62L expression. The observed relative neutropenia may be due to neutrophil margination because CD62L-expressing neutrophils are more likely to stick to endothelium. Interestingly, ROS production was changed by EDN: chronic EDN (experiment 1) was negatively associated with opsonized zymozan (OZ)-induced ROS production and acute EDN (experiment 2) with spontaneous ROS production. For chronic EDN, ROS production upon phorbol 12-myristate 13-acetate was not modified, in contrast to OZ stimulation. Decreased ROS production during chronic EDN probably involves the early events leading to ROS production, as OZ acts through membrane receptors and phorbol 12-myristate 13-acetate directly activates protein kinase C. This is the first study to provide evidence that the modifications of neutrophil functions produced by excess nitrogen depend on the intensity and duration of the excess. Further studies, including epidemiological studies during risk periods, are needed to resolve the issues linked to EDN.

Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression.

In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients.

In vitro activation of mouse neutrophils by recombinant human interferon-gamma: increased phagocytosis and release of reactive oxygen species and pro-inflammatory cytokines.

Here, we investigated the ability of IFN-γ to modulate the functions of mouse neutrophils in vitro. Neutrophils incubated in the presence of IFN-γ showed enhanced phagocytosis in response to zymosan, opsonized zymosan or precipitated immune complexes of IgG and ovalbumin. The effect of IFN-γ was dose-dependent with an initial response at 10 U/ml and a maximal response at 150 U/ml; 2 h of incubation were required to reach the optimal response level. These stimuli can also induce IFN-γ-pretreated neutrophils to release reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and hypochlorous acid, as well as granule lysosomal enzymes and the pro-inflammatory cytokines TNF-α and IL-6. We found that increased expression of FcγR, dectin-1 and complement receptors (CRs) correlated with these effects in these cells. The enhancing effect of IFN-γ on the respiratory burst was found to be associated with up-regulation of the gp91(phox) and p47(phox) subunits of NADPH oxidase, as measured by their mRNA levels. The enhancing effect of IFN-γ on phagocytosis and ROS release may not only be relevant for the efficient killing of invading microorganisms, but may also produce oxidative stress on adjacent cells, resulting in a possible inflammatory role that could also be favored by the liberation of the pro-inflammatory cytokines TNF-α and IL-6.

Chronic cold stress in mice induces a regulatory phenotype in macrophages: correlation with increased 11ß-hydroxysteroid dehydrogenase expression.

Susceptibility to infections, autoimmune disorders and tumor progression is strongly influenced by the activity of the endocrine and nervous systems in response to a stressful stimulus. When the adaptive system is switched on and off efficiently, the body is able to recover from the stress imposed. However, when the system is activated repeatedly or the activity is sustained, as during chronic or excessive stress, an allostatic load is generated, which can lead to disease over long periods of time. We investigated the effects of chronic cold stress in BALB/c mice (4°C/4 h daily for 7 days) on functions of macrophages. We found that chronic cold stress induced a regulatory phenotype in macrophages, characterized by diminished phagocytic ability, decreased TNF-α and IL-6 and increased IL-10 production. In addition, resting macrophages from mice exposed to cold stress stimulated spleen cells to produce regulatory cytokines, and an immunosuppressive state that impaired stressed mice to control Trypanosoma cruzi proliferation. These regulatory effects correlated with an increase in macrophage expression of 11ß-hydroxysteroid dehydrogenase, an enzyme that converts inactive glucocorticoid into its active form. As stress is a common aspect of modern life and plays a role in the etiology of many diseases, the results of this study are important for improving knowledge regarding the neuro-immune-endocrine interactions that occur during stress and to highlight the role of macrophages in the immunosuppression induced by chronic stress.

Effect of academic psychological stress in post-graduate students: the modulatory role of cortisol on superoxide release by neutrophils.

Experimental and clinical evidence shows that neutrophils play an important role in the mechanism of tissue injury in immune complex diseases through the generation of reactive oxygen species. In this study, we examined the influence of academic psychological stress in post-graduate students on the capacity of their blood neutrophils to release superoxide when stimulated by immune complexes bound to nonphagocytosable surfaces and investigated the modulatory effect of cortisol on this immune function. The tests were performed on the day before the final examination. The state-trait anxiety inventory questionnaire was used to examine whether this stressful event caused emotional distress. In our study, the psychological stress not only increased plasma cortisol concentration, but it also provoked a reduction in superoxide release by neutrophils. This decrease in superoxide release was accompanied by diminished mRNA expression for subunit p47(phox) of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase. These inhibitory effects were also observed by in vitro exposure of neutrophils from control volunteers to 10(- 7) M hydrocortisone, and could be prevented by the glucocorticoid receptor antagonist RU-486. These results show that in a situation of psychological stress, the increased levels of cortisol could inhibit superoxide release by neutrophils stimulated by IgG immune complexes bound to nonphagocytosable surfaces, which could attenuate the inflammatory state.

Gene therapy with interleukin-10 receptor and interleukin-12 induces a protective interferon-γ-dependent response against B16F10-Nex2 melanoma.

Antitumor immune responses are associated with proinflammatory cytokines, whereas tumor-developing animals generally have increased the production of immunosuppressive cytokines. Here, we show that splenocytes from C57Bl/6 mice resistant to low doses of B16F10-Nex2 melanoma cells produced twofold or higher interferon-γ (IFN-γ)/interleukin-10 (IL-10) ratios, whereas cells from tumor-bearing animals produced predominantly IL-10. IL-10-knockout (IL-10KO) mice were significantly more resistant to B16F10-Nex2 development, producing increased amounts of IL-12 and IFN-γ. To neutralize IL-10 in vivo, aiming at cancer therapy, recombinant eukaryotic plasmid expressing the soluble extracellular region of the murine IL-10 receptor α-chain was constructed (pcDNA3-sIL-10R). Plasmid-treated melanoma-challenged animals showed extended survival time, the protective response was IFN-γ dependent and enhanced by co-immunization with a plasmid expressing IL-12. Dendritic cells (DCs) from IL-10KO mice, primed with B16F10-Nex2 antigens (TAg), secreted increased amounts of T-helper 1-type cytokines and increased the expression of surface activation markers. Vaccination of C57Bl/6 mice with TAg-activated IL-10KO DCs, as well as with TAg-primed DCs from C57Bl/6 mice transfected with pcDNA3-sIL10R plasmid, significantly increased animal survival. In conclusion, an IFN-γ-dependent protective response was induced against B16F10-Nex2 cells by neutralization of IL-10 with pcDNA3-sIL10R plasmid. This effect was enhanced by association with IL-12 gene therapy (80% protection), and could be mediated by TAg-primed DCs.

New approaches for the prevention of airway infection in ventilated patients. Lessons learned from laboratory animal studies at the National Institutes of Health.

Despite early diagnosis and appropriate antibiotic therapy, ventilator-associated pneumonia (VAP) remains the leading cause of death from hospital-acquired infection in ventilator-dependent patients. Strategies to prevent bacterial colonization of the trachea and lungs are the key to decrease mortality, hospital length of stay, and cost. It is well established that the VAP can result from entry of infected oropharyngeal/gastric secretions into the lower airways. Aspiration may occur during 1) intubation, 2) mechanical ventilation through leakage around the tracheal tube cuff, 3) suctioning of the tracheal tube when bacteria can detach from the biofilm within the tube, or 4) areosolization of bacterial biofilm during mechanical ventilation through the tracheal tube or the ventilator circuit biofilm. From experimental studies in sheep, we drew 3 relevant conclusions: 1) The tracheal tube and neck should be oriented horizontal/below horizontal to prevent aspiration of colonized secretions and subsequent bacterial colonization of the lower respiratory tract. 2) Continuous aspiration of subglottic secretions (CASS) can lower bacterial colonization of the respiratory tract, but at the price of severe tracheal mucosal damage at the level of the suction port. 3) Coating the interior of the tracheal tube with bactericidal agents can prevent bacterial colonization of the tube surface and of the entire respiratory circuit, during 24 hours of mechanical ventilation.