Treatment of soft tissue recessions at titanium implants using a resorbable collagen matrix: a pilot study

OBJECTIVES: To histologically assess the effectiveness of a porcine-derived collagen matrix (CM) and a subepithelial connective tissue graft (CTG) for the coverage of single mucosal recessions at osseointegrated dental implants. MATERIALS AND METHODS: Chronic-type mucosal Miller Class I-like recessions (mean clinical defect height: 0.67 ± 0.33-1.16 ± 0.19 mm) were established at the buccal aspect of titanium implants with platform switch in six beagle dogs. The defects were randomly allocated to either (1) coronally advanced flap surgery (CAF) + CM, (2) CAF + CTG or (3) CAF alone. At 12 weeks, histomorphometrical measurements were made (e.g.) between the implant shoulder (IS) and the mucosal margin (PM) and IS and the outer contour of the adjacent soft tissue (mucosal thickness [MT]). RESULTS: All treatment procedures investigated were associated with an almost complete soft tissue coverage of the defect area (i.e. coronal positioning of PM relative to IS). Mean IS-PM and MT values tended to be increased in both CAF + CM (1.04 ± 0.74 mm/0.71 ± 0.55 mm) and CAF + CTG (0.88 ± 1.23 mm/0.62 ± 0.66 mm) groups when compared with CAF (0.16 ± 0.28 mm/0.34 ± 0.23 mm) alone. These differences, however, did not reach statistical significance. CONCLUSIONS: Within the limits of this pilot study, it was concluded that all treatment procedures investigated were effective in covering soft tissue recessions at titanium implants.

Preparation of intact bovine tail intervertebral discs for organ culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals

Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp.

Free-Ranging Wild Boar: A Disease Threat to Domestic Pigs in Switzerland?

The risk of transmission of pathogens from free-ranging wild boars (Sus scrofa scrofa) to outdoor domestic pigs (S. scrofa domesticus) is of increasing concern in many European countries. We assess this risk, using Switzerland as an example. We estimated 1) the prevalence of important pathogens in wild boars and 2) the risk of interactions between wild boars and outdoor pigs. First, we tested 252 wild boars from selected areas between 2008 and 2010 for infection with Brucella spp. Bacterial prevalence was estimated to 28.8% (confidence interval [CI] 23.0-34.0) when using bacterial culture (B. suis Biovar 2) and real-time polymerase chain reaction. Antibody prevalence was 35.8% (CI 30.0-42.0), which was significantly higher than in previous studies in Switzerland. We also tested 233 wild boars for porcine reproductive and respiratory syndrome virus (PRRSV). Antibody prevalence was 0.43% (CI 0.01-2.4) for EU-PRRSV and real-time reverse transcription polymerase chain reaction results were negative. These findings suggest that B. suis is increasingly widespread in wild boars and PRRSV is currently not of concern. Second, we documented the spatial overlap between free-ranging wild boars and outdoor piggeries by mapping data on their respective occurrence. Wild boars are most widespread in the mountain range along the western and northern Swiss borders, while most piggeries are located in central lowlands. A risk of interaction is mainly expected at the junction between these two bioregions. This risk may increase if wild boars expand eastward and southward beyond anthropogenic barriers believed to limit their range. Therefore, we evaluated the potential of expansion of the wild boar population. Population trends suggest a continuous increase of wild boars for the past 15 yr. Surveillance of selected wildlife passages using cameras on highways and main roads indicates that these barriers are permeable (average of up to 13 wild boar crossings per 100 days). Thus an increase of wild boar range should be considered. There may be a risk of B. suis spillover from wild boars in Switzerland, which could increase in the future. Data on the occurrence of interactions between pigs and wild boars are needed to assess this risk.

The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae

RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding beta-subunit (CD18) of beta2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the Pasteurellaceae family, Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and Actinobacillus equuli subsp. haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of Pasteurellaceae.

Fecal carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae in swine and cattle at slaughter in Switzerland

During the past decade, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae have become a matter of great concern in human medicine. ESBL-producing strains are found in the community, not just in hospital-associated patients, which raises a question about possible reservoirs. Recent studies describe the occurrence of ESBL-producing Enterobacteriaceae in meat, fish, and raw milk; therefore, the impact of food animals as reservoirs for and disseminators of such strains into the food production chain must be assessed. In this pilot study, fecal samples of 59 pigs and 64 cattle were investigated to determine the occurrence of ESBL-producing Enterobacteriaceae in farm animals at slaughter in Switzerland. Presumptive-positive colonies on Brilliance ESBL agar were subjected to identification and antibiotic susceptibility testing including the disc diffusion method and E-test ESBL strips. As many as 15.2% of the porcine and 17.1% of the bovine samples, predominantly from calves, yielded ESBL producers. Of the 21 isolated strains, 20 were Escherichia coli, and one was Citrobacter youngae. PCR analysis revealed that 18 strains including C. youngae produced CTX-M group 1 ESBLs, and three strains carried genes encoding for CTX-M group 9 enzymes. In addition, eight isolates were PCR positive for TEM beta-lactamase, but no bla(SHV) genes were detected. Pulsed-field gel electrophoresis showed a high genetic diversity within the strains. The relatively high rates of occurrence of ESBLproducing strains in food animals and the high genetic diversity among these strains indicate that there is an established reservoir of these organisms in farm animals. Further studies are necessary to assess future trends.

New Transposon Tn6133 in Methicillin-Resistant Staphylococcus aureus ST398 Contains vga(E), a Novel Streptogramin A, Pleuromutilin, and Lincosamide Resistance Gene

A novel streptogramin A, pleuromutilin, and lincosamide resistance determinant, Vga(E), was identified in porcine methicillin-resistant Staphylococcus aureus (MRSA) ST398. The vga(E) gene encoded a 524-amino-acid protein belonging to the ABC transporter family. It was found on a multidrug resistance-conferring transposon, Tn6133, which was comprised of Tn554 with a stably integrated 4,787-bp DNA sequence harboring vga(E). Detection of Tn6133 in several porcine MRSA ST398 isolates and its ability to circularize suggest a potential for dissemination.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro

Background Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (−NH2) and carboxyl (−COOH) surface modifications. Results Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. Conclusions The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies. Keywords: Multi-walled carbon nanotubes (MWCNTs); Pulmonary surfactant (Curosurf); Macrophages; Epithelial cells; Dendritic cells; Triple cell co-culture; Pro-inflammatory and oxidative reactions

Anti-Müllerian hormone levels in girls and adolescents with Turner syndrome are related to karyotype, pubertal development and growth hormone treatment

In girls and adolescents with Turner syndrome (TS), is there a correlation between serum AMH levels and karyotype, spontaneous puberty and other biochemical markers of ovarian function, or growth hormone (GH) therapy? SUMMARY ANSWER: Serum anti-Müllerian hormone (AMH) correlates with karyotype, pubertal development, LH, FSH and are measurable in a higher percentage of TS patients under GH therapy. WHAT IS KNOWN ALREADY: Most girls with TS suffer from incomplete sexual development, premature ovarian failure and infertility due to abnormal ovarian folliculogenesis. Serum AMH levels reflect the ovarian reserve in females, even in childhood. STUDY DESIGN, SIZE, DURATION: Cross-sectional study investigating 270 karyotype proven TS patients aged 0-20 years between 2009 and 2010. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Studies were conducted at three University Children's hospitals in Europe. Main outcome measures were clinical data concerning pubertal development as well as laboratory data including karyotype, serum AMH, LH, FSH, estradiol (E2), inhibin B and IGF. RESULTS AND THE ROLE OF CHANCE: Serum AMH was detectable in 21.9% of all TS girls and correlated strongly with karyotypes. A measurable serum AMH was found in 77% of TS girls with karyotype 45,X/46,XX, in 25% with 'other' karyotypes and in only 10% of 45,X TS girls. A strong relationship was also observed for measurable serum AMH and signs of spontaneous puberty such as breast development [adjusted odds ratio (OR) 19.3; 95% CI 2.1-175.6; P = 0.009] and menarche (crude OR 47.6; 95% CI 4.8-472.9; P = 0.001). Serum AMH correlated negatively with FSH and LH, but did not correlate with E2 and inhibin B. GH therapy increased the odds of having measurable AMH in TS (adjusted OR 4.1; 95% CI 1.9-8.8; P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The cross-sectional design of the study does not allow longitudinal interpretation of the data; for that further studies are needed. High percentage of non-measurable AMH levels in the cohort of TS require categorized analysis. WIDER IMPLICATIONS OF THE FINDINGS: Serum AMH levels are a useful marker of the follicle pool and thus ovarian function in pediatric patients with TS. These findings are in line with the published literature. The finding that GH therapy may affect AMH levels is novel, but must be confirmed by future longitudinal studies.

Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma

Continuous changes in the length of smooth muscles require a highly organized sarcolemmal structure. Yet, smooth muscle cells also adapt rapidly to altered environmental cues. Their sarcolemmal plasticity must lead to profound changes which affect transmembrane signal transduction as well as contractility. We have established porcine vascular and human visceral smooth muscle cultures of epithelioid and spindle-shaped morphology and determined their plasma membrane properties. Epithelioid cells from both sources contain a higher ratio of cholesterol to glycerophospholipids, and express a less diverse range of lipid-associated annexins. These findings point to a reduction in efficiency of membrane segregation in epithelioid cells. Moreover, compared to spindle-shaped cells, cholesterol is more readily extracted from epithelioid cells with methyl-beta-cyclodextrin and its synthesis is more susceptible to inhibition with lovastatin. The inability of epithelioid cells to process vasoactive metabolites, such as angiotensin or nucleotides further indicates that contractile properties are impaired. Phenotypic plasticity extends beyond the loss of smooth muscle cell marker genes. The plasma membrane has undergone profound functional changes which are incompatible with cyclic foreshortening, but might be important in the development of vascular disease.

Morphological alterations of exogenous surfactant inhibited by meconium can be prevented by dextran

BACKGROUND: Surfactant dysfunction due to inhibition is involved in the pathophysiology of meconium aspiration syndrome. Dextran addition has been shown to reverse exogenous surfactant inactivation by meconium, but the precise mechanisms and the morphological correlate of this effect are yet unknown. Morphological surfactant analysis by transmission electron microscopy (TEM) and stereology allows the differentiation of active (large aggregates = LA) and inactive (small aggregates = SA) subtypes. METHODS: To determine the in vitro effects of meconium and dextran addition on the morphology of a modified porcine natural surfactant (Curosurf), Curosurf samples were either incubated alone or together with meconium or with meconium and dextran, fixed and processed for TEM. Volume fractions of surfactant subtypes [lamellar body-like forms (LBL), multilamellar vesicles (MV), unilamellar vesicles (UV)] were determined stereologically. RESULTS: All preparations contained LBL and MV (corresponding to LA) as well as UV (corresponding to SA). The volume fraction of UV increased with addition of meconium and decreased with further addition of dextran. Correspondingly, the UV/(LBL+MV) ratio (resembling the SA/LA ratio) increased when meconium was added and decreased when dextran was added to the surfactant-meconium mixture. CONCLUSION: Meconium causes alterations in the ultrastructural composition of Curosurf that can be visualized and analyzed by TEM and stereology. These alterations resemble an increase in the SA/LA ratio and are paralleled by an increase in minimum surface tension. Dextran prevents these effects and may therefore be a useful additive to exogenous surfactant preparations to preserve their structural and functional integrity, thereby improving their resistance to inactivation.

Polymer stent coating for prevention of neointimal hyperplasia

AIMS: Restenosis has been the principal limitation of bare metal stents. Based upon the presumption that platelet and inflammatory cell recruitment initiate neointimal proliferation, we explored a novel polymer coating that reduces cell-stent interactions. The purpose of the present study was to investigate the effect of poly(L-lysine)-graft-poly(ethyleneglycol) (PLL-g-PEG) adsorbed to stent surfaces to reduce neointimal hyperplasia in the porcine restenosis model. METHODS AND RESULTS: Seven animals were instrumented each with 2 stainless steel stents (15 mm length, 2.5-3.5 mm diameter), randomly implanted in 1 major epicardial coronary artery. One stent was dip-coated with PLL-g-PEG, whereas the other stent served as the uncoated control stent. All animals were sacrificed after 6 weeks for histological examination. Neointimal hyperplasia was significantly less (-51%) in the PLL-g-PEG-coated stents (1.15 +/- 0.59 mm2) than in the uncoated control stents (2.33 +/- 1.01 mm2; p < 0.001). Conversely, lumen size was larger in the PLL-g-PEG-coated stents (2.91 +/- 1.17 mm2) than in the uncoated stents (2.04 +/- 0.64 mm2; p < 0.001). High magnification histomorphologic examination revealed no signs of inflammation or thrombus formation in either stent group. CONCLUSIONS: Polymeric steric stabilization of stents with PLL-g-PEG significantly reduces neointimal hyperplasia in the porcine restenosis model. Reduction of cell-stent interactions mediated by PLL-g-PEG appear to improve biocompatibility of stainless steel stents without evidence of adverse inflammatory or prothrombotic effects.

Reduction of postmortem angiography-induced tissue edema by using polyethylene glycol as a contrast agent dissolver

Postmortem investigation is increasingly supported by computed tomography (CT) and magnetic resonance imaging, in which postmortem minimal invasive angiography has become important. The newly introduced approach using an aqueous contrast agent solution provided excellent vessel visualization but was suspected to possibly cause tissue edema artifacts in histological investigations. The aim of this study was to investigate on a porcine heart model whether it is possible to influence the contrast agent distribution within the soft tissue by changing its viscosity by dissolving the contrast agent in polyethylene glycol (PEG) as a matrix medium. High-resolution CT scans after injection showed that viscosities above c. 15 mPa s (65% PEG) prevented a contrast agent distribution within the capillary bed of the left ventricular myocardium. Thereby, the precondition of edema artifacts could be reduced. Its minimal invasive application on human corpses needs to be further adapted as the flow resistance is expected to differ between different tissues.

Comparison of lactated Ringer's, gelatine and blood resuscitation on intestinal oxygen supply and mucosal tissue oxygen tension in haemorrhagic shock

OBJECTIVES: To evaluate the effects on intestinal oxygen supply, and mucosal tissue oxygen tension during haemorrhage and after fluid resuscitation with either blood (B; n=7), gelatine (G; n=8), or lactated Ringer's solution (R; n=8) in an autoperfused, innervated jejunal segment in anaesthetized pigs. METHODS: To induce haemorrhagic shock, 50% of calculated blood volume was withdrawn. Systemic haemodynamics, mesenteric venous and systemic acid-base and blood gas variables, and lactate measurements were recorded. A flowmeter was used for measuring mesenteric arterial blood flow. Mucosal tissue oxygen tension (PO(2)muc), jejunal microvascular haemoglobin oxygen saturation (HbO(2)) and microvascular blood flow were measured. Measurements were performed at baseline, after haemorrhage and at four 20 min intervals after fluid resuscitation. After haemorrhage, animals were retransfused with blood, gelatine or lactated Ringer's solution until baseline pulmonary capillary wedge pressure was reached. RESULTS: After resuscitation, no significant differences in macrohaemodynamic parameters were observed between groups. Systemic and intestinal lactate concentration was significantly increased in animals receiving lactated Ringer's solution [5.6 (1.1) vs 3.3 (1.1) mmol litre(-1); 5.6 (1.1) vs 3.3 (1.2) mmol litre(-1)]. Oxygen supply to the intestine was impaired in animals receiving lactated Ringer's solution when compared with animals receiving blood. Blood and gelatine resuscitation resulted in higher HbO(2) than with lactated Ringer's resuscitation after haemorrhagic shock [B, 43.8 (10.4)%; G, 34.6 (9.4)%; R, 28.0 (9.3)%]. PO(2)muc was better preserved with gelatine resuscitation when compared with lactated Ringer's or blood resuscitation [20.0 (8.8) vs 13.8 (7.1) mm Hg, 15.2 (7.2) mm Hg, respectively]. CONCLUSION: Blood or gelatine infusion improves mucosal tissue oxygenation of the porcine jejunum after severe haemorrhage when compared with lactated Ringer's solution.

Alternative protocol to initiate high-frequency oscillatory ventilation: an experimental study

INTRODUCTION: The objective was to study the effects of a novel lung volume optimization procedure (LVOP) using high-frequency oscillatory ventilation (HFOV) upon gas exchange, the transpulmonary pressure (TPP), and hemodynamics in a porcine model of surfactant depletion. METHODS: With institutional review board approval, the hemodynamics, blood gas analysis, TPP, and pulmonary shunt fraction were obtained in six anesthetized pigs before and after saline lung lavage. Measurements were acquired during pressure-controlled ventilation (PCV) prior to and after lung damage, and during a LVOP with HFOV. The LVOP comprised a recruitment maneuver with a continuous distending pressure (CDP) of 45 mbar for 2.5 minutes, and a stepwise decrease of the CDP (5 mbar every 5 minute) from 45 to 20 mbar. The TPP level was identified during the decrease in CDP, which assured a change of the PaO2/FIO2 ratio < 25% compared with maximum lung recruitment at CDP of 45 mbar (CDP45). Data are presented as the median (25th-75th percentile); differences between measurements are determined by Friedman repeated-measures analysis on ranks and multiple comparisons (Tukey's test). The level of significance was set at P < 0.05. RESULTS: The PaO2/FiO2 ratio increased from 99.1 (56.2-128) Torr at PCV post-lavage to 621 (619.4-660.3) Torr at CDP45 (CDP45) (P < 0.031). The pulmonary shunt fraction decreased from 51.8% (49-55%) at PCV post-lavage to 1.03% (0.4-3%) at CDP45 (P < 0.05). The cardiac output and stroke volume decreased at CDP45 (P < 0.05) compared with PCV, whereas the heart rate, mean arterial pressure, and intrathoracic blood volume remained unchanged. A TPP of 25.5 (17-32) mbar was required to preserve a difference in PaO2/FIO2 ratio < 25% related to CDP45; this TPP was achieved at a CDP of 35 (25-40) mbar. CONCLUSION: This HFOV protocol is easy to perform, and allows a fast determination of an adequate TPP level that preserves oxygenation. Systemic hemodynamics, as a measure of safety, showed no relevant deterioration throughout the procedure.