Diploid yeast strains used in this work.
\r\n\tThis book will be very useful for a wide variety of readers, such as Chemists, Pharmaceuticals, Biochemists, Biotechnologists, Industrialists, Engineers, Researchers, Teachers and Students.
",isbn:"978-1-78984-072-8",printIsbn:"978-1-78984-071-1",doi:null,price:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"ff1acef627b277c575a10b3259dd331b",bookSignature:"Dr. Martin Alberto Masuelli",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8504.jpg",keywords:"Extraction, Purification, Characterization, Acid Hydrolysis, Basic Hydrolysis, Freezing/Thawing, Separative Processes, Clarification, Leaching, Drying, Lyophilized, Applications",numberOfDownloads:3,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"November 5th 2018",dateEndSecondStepPublish:"November 26th 2018",dateEndThirdStepPublish:"January 25th 2019",dateEndFourthStepPublish:"April 15th 2019",dateEndFifthStepPublish:"June 14th 2019",remainingDaysToSecondStep:"3 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,editors:[{id:"99994",title:"Dr.",name:"Martin",middleName:"Alberto",surname:"Masuelli",slug:"martin-masuelli",fullName:"Martin Masuelli",profilePictureURL:"https://mts.intechopen.com/storage/users/99994/images/system/99994.jpg",biography:"Martin Masuelli is a Inv. Adj. of Instituto de Física Aplicada – CONICET – UNSL and Associate Professor-UNSL, San Luis, Argentina. He holds a master’s degree and a PhD and Master thesis in Membrane Technology from National University of San Luis (UNSL). He is Director of Physics Chemistry Service Laboratory, UNSL. He is expert in polysaccharides and physics chemistry of macromolecules. He is author or co-author of more than 26 peer-reviewed international publications, six book chapters, 70 communications in national and international congresses, editor of five books. He is a member of the Sociedad Argentina de Ciencia y Tecnología Ambiental and Asociación Argentina de Fisicoquímica y Química Inorgánica. He is Editor in Chief and Founder, in July 2013, of Journal of Polymer and Biopolymers Physics Chemistry, Science and Education Publishing. He is editorial board of numerous Journals.",institutionString:"Universidad Nacional de San Luis",position:"Associate Professor",outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"2",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"Universidad Nacional de San Luis",institutionURL:"http://www.unsl.edu.ar/",country:{name:"Argentina"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"5",title:"Agricultural and Biological Sciences",slug:"agricultural-and-biological-sciences"}],chapters:[{id:"65638",title:"Pectins as Emulsifying Agent on the Preparation, Characterization, and Photocatalysis of Nano-LaCrO3",slug:"pectins-as-emulsifying-agent-on-the-preparation-characterization-and-photocatalysis-of-nano-lacro3",totalDownloads:3,totalCrossrefCites:0,authors:[null]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"270933",firstName:"Luka",lastName:"Cvjetkovic",middleName:null,title:"Mr.",imageUrl:"https://mts.intechopen.com/storage/users/270933/images/7797_n.jpg",email:"luka@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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It was said (but never proven) that those people could even protect themselves thereby against outbreaks of cholera.
\nEver since, there has been an increasing body of medical reports addressing the beneficial properties of Saccharomyces boulardii as a probiotic to treat cases of diarrhea, reconstituting the gut flora after antibiotic treatment and even in the treatment of patients suffering from Helicobacter pylori that can cause stomach ulcers (for a recent review, see [1]). High doses of lyophilized living cells (2–5 × 109) are administered in pills that dissolve in the gastrointestinal tract. It is assumed that S. boulardii cells do not establish in the gastrointestinal tract and are secreted with the feces. Therefore, several doses are recommended to be taken during several days. It has not been clearly shown if on their passage yeast cells interact with other microorganisms in the gastrointestinal tract. Despite several clinical studies indicating beneficial effects of S. boulardii, there have been also reports identifying it as the cause of fungemia in hospital patients (adults and children) that had received doses of this yeast to treat gastrointestinal disorders [2]. Though the beneficial effects of the baker’s yeast S. cerevisiae used for fermentation are unquestioned, certain wild S. cerevisiae isolates can have negative effects especially in people with a compromised immunosystem after undergoing surgery. Non-domesticated yeast strains as those isolated in hospitals can have invasive properties very different from those observed for domesticated yeast strains used in bakeries and breweries.
\nYeast strains used in the laboratory such as S288C are mostly derivatives of industrial yeast strains used for ages in breweries [3]. More recently, derivatives of diploid strain Σ1278b, which shows filamentous properties related to non-domesticated yeast strains [4], are used in molecular biology research. Surprisingly, the sequence identity of both yeast strains is only 46% [5] indicating considerable genetic variability due to adaptation to differing milieus and to human domestication of this eukaryotic species [6].
\nThanks to molecular genetic techniques, S. boulardii considered originally as a yeast species by itself [7] has been proven in recent years to be a variant of S. cerevisiae [8]. In this chapter, a diploid yeast strain similar to those strains used in most research laboratories, amenable to genetic manipulation when using conventional yeast protocols is shown.
\nTo further characterize this yeast, its growth properties at different temperatures are compared with other diploid yeast strains (all yeast strains used are summarized in Table 1). S. boulardii (I will keep this name in the text even though it is a S. cerevisiae strain) grows well on rich medium at 30°C as well as at 37°C but not at 40°C (Figure 1A). Growth at 37°C—though not ideal—is not uncommon to yeast strains such as the diploid strain BY4743 (a derivative of S288C) but as opposed to diploid strain RH2585/2586 (an Σ1278b derivative) which hardly grows at 37°C (Figure 1A). In that sense, growth at 37°C is not a particular and unique property of S. boulardii. It probably rather reflects its accommodation to hot climates such as those found often in Indochina.
\nName and properties of diploid yeast strain | \nAuxotrophic properties/antibiotic resistances | \n
---|---|
BY4743 | \nRequires histidine, leucine, methionine, and uracil | \n
RH2585/2586 | \nRequires histidine and uracil | \n
RH2585/2586 ∆flo8::kanX ∆flo8::NATR | \nNo requirements; G418- and NAT-resistant | \n
S. boulardii | \nNone | \n
S. boulardii ∆flo8::kanX ∆flo8::NATR | \nNo requirements; G418- and NAT-resistant | \n
S. boulardii ∆rem1::kanX ∆rem1::NATR | \nNo requirements; G418- and NAT-resistant | \n
S. boulardii ∆his3::kanX ∆his3::NATR | \nRequires histidine; G418- and NAT-resistant | \n
S. boulardii ∆ade2::kanX ∆ade2::NATR | \nRequires adenine; G418- and NAT-resistant | \n
Diploid yeast strains used in this work.
G418/geneticin and NAT/nourseothricin are selective antibiotics for yeast strains.
Properties of S. boulardii and other diploid yeast strains (all listed in Table 1). (A) Growth comparison of yeast strains at different temperatures. Indicated yeast strains were spread on YPD plates and incubated at indicated temperatures (30, 37, and 40°C) for 2 days; (B) filamentation properties of different yeast strains. Strains RH2585/2586, RH2585/2586 ∆flo8::kanX ∆flo8::NATR, and S. boulardii were incubated on SLAD plates (50 μM ammonium sulfate) for 1 day at 30°C and individual growth colony visualized under the microscope (upper panel: 20× magnification; lower panel: 100× magnification); (C) growth of S. boulardii on different media. S. boulardii (#1785) and derivatives ∆∆his3 (#1811) and ∆∆ade2 (#1812) were grown for 2 days at 30°C on minimal SD medium (left plate) or on YPD + G418 and nourseothricin (both at final 100 μg/ml) (right plate). Denote the characteristic pinkish color of strain #1812 due to the deletion of both ADE2 gene copies.
A further investigated property is S. boulardii’s potential capability to form filaments. For this purpose, it was grown on SLAD plates which carry only limiting concentrations of ammonium sulfate (50 μM, about 1000× less than conventional SD minimal medium). As observed under the microscope, the diploid strain RH2585/2586 clearly shows filamentous properties under such ammonium-limiting conditions (Figure 1B) [9]. Deletion of the FLO8 gene encoding a transcriptional factor required for filamentation and adhesion completely abolished its filamentous properties. Flo8 is required to express, for example, Flo11, a cell-surface glycoprotein [10]. As opposed to RH2585/2586, S. boulardii hardly showed any filamentation properties. Interestingly, sequencing of the PCR-amplified FLO8 gene of S. boulardii indicated that it does not carry a premature stop codon (not shown) found in non-filamentous yeast strains as those derived from S288C [10]. So, other up- or downstream genes required for filamentation are likely dysfunctional in S. boulardii. Its lack of filamentation probably explains its low toxicity and its lack of establishment capacity in the gastrointestinal tract that otherwise could make it more persistent and thereby more problematic for medical applications.
\nAnother interesting issue was to induce meiosis and sporulation in S. boulardii in order to obtain haploid progeny. For this purpose, diploid cells were incubated in a liquid medium with limiting nitrogen and very high potassium acetate concentration as a (poor) carbon source [11]. Despite several attempts, no tetrad formation was observed. In that respect, S. boulardii shows similar properties as RH2585/2586 (the filamentous diploid used in this work) that does not form tetrads upon treatment under the described conditions. In order to induce meiosis and sporulation, a double knockout of RME1 (Regulator of Meiosis 1) in S. boulardii was produced. RME1 is a negative regulator of meiosis that prevents the expression of meiosis-required proteins such as IME1 (Inducer of Meiosis 1) and promotes mitosis [12]. The S. boulardii ∆∆rme1 derivative did not show any phenotypical differences to the parental wild-type strain in terms of growth temperature (Figure 1A). Unfortunately, no tetrads were obtained from this knockout strain either. I conclude that S. boulardii does not undergo meiosis and haploid tetrad formation at least under laboratory conditions used here.
\nAs shown in Figure 1C, S. boulardii does not carry any auxotrophic markers as it grows well on minimal medium (SD) devoid of amino acids or nucleic acid components such as adenine or uracil. Auxotrophic marker genes could be easily obtained by deleting the ADE2 (adenine biosynthesis) or HIS3 (histidine biosynthesis) genes (Figure 1C). These deletions were obtained by introducing dominant auxotrophic marker genes that provide resistance to the antibiotics kanamycin/G418 or nourseothricin. Deletion of a single gene copy of ADE2 or HIS3 still allowed for growth on minimal medium plates (not shown) indicating the clear diploid character of this yeast. Only double deletion of both HIS3- or ADE2-gene copies (which made it resistant against both kanamycin/G418 and nourseothricin; Figure 1C) made this yeast strain auxotrophic for histidine or for adenine. Newly gained histidine auxotrophy was used in a subsequent experiment to transform it with yeast plasmids carrying HIS3 as a selectable marker gene (see subsequent text).
\nAs an auxotrophic histidine-deficient yeast strain was now available, I decided to transform it with conventional yeast plasmids that complement for the lack of HIS3. For this purpose, S. boulardii ∆∆his3 (#1811) was grown in minimal SD medium supplemented with histidine. Cells were made competent by treating them with Li-acetate following a well-established yeast transformation protocol [13].
\nAfter 2–3 days of incubation at 30°C, his+-transformants were nicely observed (Figure 2; left panel), indicating that a simple yeast transformation protocol was sufficient to transform this yeast strain and to recover its prototrophy. Plasmids used for transformation (p301HIS3 GAL-p20-HA from S. cerevisiae and Candida albicans) allow for the expression of the protein p20 (a modulator of the activity of eIF4E, the cap-binding protein; see subsequent text) when growing cells in a medium containing galactose. As shown on a Western Blot obtained from yeast extracts, p20 from different sources was expressed in galactose but not in glucose-containing media (Figure 2; right panel). This confirms that, in S. boulardii, the GAL1/10 promoter is regulated in an identical manner as in conventional yeast strains used in the laboratory [14].
\nTransformation of S. boulardii ∆∆his3 and expression of p20. (Left panel) Transformation of S. boulardii ∆∆his3. S. boulardii ∆∆his3 was transformed with plasmid p301HIS3 p20 (S. cerevisiae)-HA (segment 2) or with plasmid p301HIS3 p20 (C. albicans)-HA (segment 3) and grown on minimal medium plates (without histidine) for 3 days at 27°C. As a negative control, no plasmid DNA was added (segment 1); (right panel) expression of HA-tagged p20 after induction with galactose. S. boulardii ∆∆his3 extracts from cells transformed with p301HIS3 p20 (S. cerevisiae)-HA (lanes 1 and 3) or transformed with p301HIS3 p20 (C. albicans)-HA (lanes 2 and 4) grown on medium containing 2% glucose or 2% galactose as indicated are shown. Individual colonies from segments 2 and 3 were picked and grown in liquid SD medium for 24 h. Subsequently, half of the cells were collected, washed (2× with water), resuspended in SGal (minimal medium with 2% galactose), and incubated for 24 h at 27°C. Collected cells were boiled in 2× SDS-sample buffer, proteins separated on a 15% SDS-PAGE gel. Separated proteins were blotted onto nitrocellulose, subsequently incubated with monoclonal antibodies against the HA-tag (1:2000 Dilution in 2% skim milk). For visualization of the Western Blot, WesternBright ECL kit (advansta) was used.
p20, a small acidic protein of 161 amino acids, is encoded by the non-essential gene CAF20 which only exists in a variety of yeast species (such as S. glabrata, Kluyveromyces lactis, C. albicans, S. cerevisiae). Its function is related to regulating the activity of the cap-binding protein eIF4E during translation in a yet not-well-understood manner [15]. A sequence alignment of different yeast species (see Figure 3; upper panel) shows a clear homology but not identity of the corresponding p20 proteins. Especially conserved are peptide motifs at the amino terminus (which are required for binding to eIF4E; the canonical motif YxxxxLL/I/F highlighted) and at the carboxy terminus (where precise function has still to be determined).
\nSequence comparison of p20 from different yeast species. Multiple sequence alignment of p20 from different yeast sources (CANGA, Candida glabrata; KLUYV, Kluyveromyces lactis; CANAL, Candida albicans; YEAST, Saccharomyces cerevisiae) was done with the help of Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Highlighted amino acids; see text. For sequencing of S. boulardii p20 gene, the complete ORF and adjacent sequences were PCR-amplified with oligonucleotides used for S. cerevisiae p20 gene amplification and the obtained PCR product was sequenced in both directions (shown is the part encoding the p20 open reading frame of S. boulardii).
The CAF20 gene from S. boulardii was PCR-amplified using genomic DNA and oligonucleotides hybridizing at the 5′ and 3′ region of the S. cerevisiae CAF20 gene. Subsequent sequencing showed that CAF20 from S. cerevisiae and S. boulardii is nearly identical. The only difference detected is a conserved amino acid substitution (leucine to valine; highlighted) at position 16 (Figure 3; lower panel). Among those yeast species that carry the p20 gene conservation varies between 30 and 90% (not shown). The almost identity of both sequences shown here clearly confirms that S. boulardii is a variant of the species S. cerevisiae.
\nIn this work, I present data indicating that S. boulardii is a diploid S. cerevisiae strain. It thrives well under laboratory conditions at different temperatures (up to 37°C) which is not unusual for different laboratory yeast strains. S. boulardii does not show filamentous properties even though its FLO8 gene does not carry the typical premature stop codon identified in many laboratory (and industrial) yeast strains. S. boulardii does not undergo meiosis or form haploid progeny when incubated in sporulation-inducing media. S. boulardii does not carry any identifiable auxotrophic gene markers. It can be easily manipulated to obtain knockout derivatives by inserting genes conferring antibiotic resistance and obtain thereby auxotrophic progeny. Additionally, S. boulardii can be easily transformed with conventional yeast plasmids allowing also for the expression of proteins regulated by the galactose-inducible GAL 1/10 yeast promoter.
\nIn accordance with those properties, it is probably not detrimental for human health (at least not for immunocompetent individuals) as it will not easily establish in the gut or penetrate the intestinal blood barrier. All this does not mean that it has beneficial physiological properties and I would like to ask the question: is this not just a further conventional yeast strain?
\nS. boulardii was obtained from I. M. Castro (Belo Horizonte/Brazil) [16]. I would like to thank Daniela Ross-Kaschitza from my laboratory for carefully reading this manuscript. This work was supported by the Swiss National Foundation grant 31003A_146722/1.
\nAcute respiratory distress syndrome is characterized by an increase of the permeability of the lungs alveolar-capillary membranes leading to the extravasation of the intravascular plasma of the lungs capillary network surrounding the alveoli to the alveolar spaces that were previously filled by air. This accumulation of liquid rich in proteins inside the alveolar spaces turns an air-filled lungs into a heavy high-osmotic pressure liquid-filled lungs and the consequent collapse of the lowermost lung regions, shunt, refractory hypoxemia, decrease in lungs compliance and increase in dead space that are more pronounced the more severe the permeability changes of the pulmonary alveoli-capillary membrane. Regarding the physiopathology of ARDS, the hallmark mechanism of injury is inflammation leading to increased endothelial and epithelial permeability and liberation of receptors for angiopoietin-2 and advanced glycation end products (RAGE) [1, 2, 3, 4].
\nAccording to the recent Berlin definition, severe acute respiratory distress syndrome is defined by bilateral pulmonary infiltrates of recent onset (less than 1 week) in a patient that have a PaO2/FIO2 equal or less than 100 with a positive end-expiratory pressure equal or more 5 cm H2O with no evidence of cardiac failure or hypervolemia. The patient also needs to present a risk factor for ARDS development as respiratory infection, gastric content aspiration, lungs contusion, blood products transfusion, sepsis, high-risk trauma, high-risk surgery, shock, and pancreatitis [1, 2, 3, 4].
\nThe extravascular lung water index (EVLWi) is calculated as the intra-thoracic total volume minus the intra-thoracic blood volume indexed by predicted body weight measured using a transpulmonary thermodilution method. The pulmonary vascular permeability index (PVPI) was calculated as extravascular lung water divided by the pulmonary blood volume [5]. Theoretically, the greater the EVLWi and PVPi, the greater the severity of ARDS. Recently, Kushimoto and colleagues [5] evaluated the relationship among the severity categories of ARDS as defined by the Berlin definition, EVLWi and PVPi to confirm their predictive validity for severity of ARDS. They measured EVLWi and PVPi in 195 patients with an EVLWi of ≥10 mL/kg, which fulfilled the Berlin definition of ARDS in 23 intensive care units for three consecutive days. Patients with moderate and severe ARDS had higher acute physiology and chronic health evaluation II (APACHE II) and sequential organ failure assessment scores (SOFA) on the day of enrollment compared to patients with mild ARDS. Patients with severe ARDS had higher EVLWi (severe, 19.1; moderate, 17.2; mild, 16.1; P < 0.05) and PVPI (3.2; 3.0; 2.7; P < 0.05). When the authors evaluated 495 independent measurements over three consecutive days, they observed a moderative and negative correlation between PaO2/FIO2 ratio and EVLWi (r = −0.355, P < 0.001) and PaO2/FIO2 and PVPi (r = −0.345, P < 0.001 The authors observed an association between ARDS severity according to Berlin definition and 28-day mortality rate: severe, the odds ratio 4.167 relative to mild.
\nHemodynamic data from the ARDSnet Fluids and Catheter Therapy Trial (FACCT) [6] that analyzed 475 patients randomized to receive a pulmonary artery catheter for ARDS management, none of the baseline measures of cardiopulmonary dysfunction distinguished survivors from nonsurvivors. When the authors measured the transpulmonary gradient (TGP), they observed that 73% of the ARDS patients monitored with the Swan-Ganz had an elevated TGP (>12 mm Hg). Patients with a TPG > 12 mm Hg had a significantly greater mortality rate than patients with a TPG < 12 mm Hg (30 vs. 19%; P = 0.02). In multivariate analysis, an elevated TPG and a high PVRi remained an independent predictor of an adverse outcome in this ARDS population. In a recent, prospective and observational study in an academic medical intensive care unit in France [7], 226 consecutive patients with moderate to severe ARDS ventilated who received a protective ventilation (plateau pressure less than 30 cm H2O and mean PEEP of 8.8 ± 3.6 cm H2O, underwent transesophageal echocardiography (TEE) within the first 3 days after the diagnosis of ARDS. Cor pulmonale (dilated right ventricle associated with septal dyskinesia), was detected in 49 patients (prevalence of 22%; 95% confidence interval, 16–27%). Patients who had cor pulmonale presented a significantly higher 28-day mortality rate (60 vs. 36%, P < 0.01) compared with the ARDS patients without cor pulmonale. Sepsis and higher values of driving pressure were associated with the presence of cor pulmonale that was an independent risk factor for 28-day mortality in their population. Taking these results into consideration, a subgroup of ARDS severity stratification: ARDS with right ventricular dysfunction should be proposed especially because different ventilatory strategies (prone position, low driving pressures, titrated PEEP levels), distinct pharmacologic therapy (pulmonary artery vasodilators) should be tested in order to improve prognosis of this subgroup of ARDS patients [4].
\nOther factors that are associated with severe ARDS are respiratory system compliance of less than 20 mL/cm H2O, pulmonary dead space fraction greater than 0.60, as well as high APACHE II and SAPS II score as well as multiple organ failures (the higher the organ failures, the higher the patient mortality) [1, 2, 3, 4].
\nIchikado [8] showed that fibroproliferation signs in high-resolution CT evaluation of early ARDS patients were correlated with higher mortality and ventilator dependency. The lung SAFE study [3], an international, multicenter, prospective cohort study of patients undergoing invasive or noninvasive ventilation, conducted in 459 ICUs from 50 countries across five continents showed that 2.377 out of 29.144 patients developed ARDS in the first 48 h and whose respiratory failure was managed with invasive mechanical ventilation. The period prevalence of mild ARDS was 30.0% (95% CI, 28.2–31.9%); of moderate ARDS, 46.6% (95% CI, 44.5–48.6%); and of severe ARDS, 23.4% (95% CI, 21.7–25.2%). The cumulative frequency distribution of tidal volume was similar in patients in each severity category, with 65% of patients with acute respiratory distress syndrome (ARDS) receiving a tidal volume of 8 mL/kg of predicted body weight or less. In contrast, a right shift of the cumulative frequency distribution curves of plateau pressures was seen for increasing ARDS severity category, with plateau pressure of more than 30 cm H2O in 8.5% of patients for which these data were available. There was a lower likelihood of survival to day 28 with increasing severity of acute respiratory distress syndrome (ARDS) at day 1. Patients with a driving pressure of greater than 14 cm H2O on day 1 of ARDS criteria had a higher mortality. Taking into consideration, these data that tidal volume ventilation was similar across the ARDS severity but inspiratory driving pressure less than 14 cm H2O was associated with decreased mortality of those patients one could argue that in the future tidal volume should be titrated according to the derived driving inspiratory pressure.
\nAmato and colleagues [9] analyzed individual data from 3562 patients with ARDS enrolled in nine previously published randomized clinical trials of mechanical ventilation using a multilevel mediation analysis. They observed a strong association between driving pressure and ARDS survival even though all the ventilator settings that were used were lung protective (RR of death: 1.36, 95% CI 1.17–1.58, P < 0.001). These observations suggest that tidal volume might be adjusted to the resultant airway driving pressure in addition to the adjustment to the predicted body weight. They also observed that airway driving pressures higher than 15 cm H2O were associated with increasing rates of mortality in ARDS patients. Recently, Villar and colleagues [10] analyzed the data from two observational studies enclosing 778 patients with moderate and severe ARDS. They assessed the risk of hospital death based on quantiles of tidal volume, positive end-expiratory pressure, plateau inspiratory pressure and airway driving pressure evaluated 24 h after ARDS diagnosis while the patients were ventilated with lung protective ventilation. The authors verified that positive end expiratory pressure and tidal volume that were set according to a protective lung ventilation strategy had no impact on mortality while a plateau pressure higher than 29 cm H2O and a driving pressure higher than 19 cm H2O were associated with a higher hospital mortality.
\nAs respiratory system driving pressure does not account for variable chest wall compliance or different degrees of intra-abdominal pressures or even more to the presence of inspiratory efforts or asynchrony, esophageal manometry can be used to measure transpulmonary pressure that represents the lungs parenchyma stress during tidal volume ventilation. Recently, Baedorf and colleagues examined the relationships between respiratory system and transpulmonary driving pressure measured at baseline, 5 min and 24 h after PEEP titration and 28-day mortality in 56 ARDS patients. They observed that PEEP titration to target positive end-expiratory transpulmonary pressures resulted in both improved elastance and driving pressures and was associated with improved 28-day mortality.
\nHowever, future studies regarding the evaluation of respiratory system and transpulmonary driving pressure in ARDS patients with normal and increased abdominal pressure and various degrees of respiratory system compliance is still needed in order to establish the value of both as a bedside ventilator target as well as a prognosticator of evolution and mortality of those patients.
\nIncreased dead space in the first day of mechanical ventilation, increased PaCO2 levels with protective ventilation and multiple organ failure are all associated with higher mortality in severe ARDS. However, specific therapeutic aiming to decrease dead space fraction, decrease PaCO2 levels or even multimodal therapeutic approach to treat multiple organ failure still need to be defined and tested [4].
\nRecently, Villar and colleagues [11] showed in a cohort of 778 patients that severe ARDS occurred in about 37.5% at ARDS diagnosis and after 24 h of ARDS onset 20.8% and moderate to severe ARDS had an overall mortality of 38.8%. They also showed that the underlying cause of ARDS influence in the mortality ratio (the mortality ratio was higher in pancreatitis and progressively lower in sepsis, pneumonia and trauma).
\nLow-tidal ventilation (≤6 mL/kg of predicted body weight) must be initiated as soon as the ARDS patient is intubated and mechanically ventilated. The predicted body weight (PBW) can be calculated as follows: for women, PBW = 45.5 + 0.91 (height in centimeters—152.4) and for men, PBW = 50.0 + 0.91 (height in centimeters—152.4). It is well documented that lower tidal volumes (6 mL/kg of predicted body weight) compared to higher tidal volumes (12 mL/kg of predicted body weight) associated with PEEP levels titrated by a PEEP/FIO2 table reduced mortality in a randomized, clinical trial that analyzed 861 ARDS patients (ARMA trial) [12].
\nIt is crucial to adjust tidal volume to lung size that depends of the height and sex, but more importantly is to adjust the tidal volume to functional lung size that depends on the ARDS severity (lung compliance), sex, height, and chest wall compliance. The patient with severe ARDS and a low compliance will be ventilated with high airway pressures if ventilated with 6 mL/predicted body weight. Amato and colleagues [9], in 2015, reported that driving pressure (ΔP), that can be also be represented by tidal volume (VT)/respiratory system compliance (CRS), in which VT is intrinsically normalized to functional lung size, was a ventilatory variable more strongly associated with survival than VT or PEEP in patients who are not actively breathing. Using a statistical tool known as multilevel mediation analysis to study individual data from 3562 patients with ARDS enrolled in nine previously reported randomized trials, they examined ΔP as an independent variable associated with survival. In the mediation analysis, they estimated the isolated effects of changes in ΔP resulting from randomized ventilator settings while minimizing confounding due to the baseline severity of lung disease. The authors observed that among ventilation variables, ΔP was most strongly associated with survival. A 1-SD increment in ΔP (approximately 7 cm of water) was associated with increased mortality (relative risk, 1.41; 95% confidence interval [CI], 1.31–1.51; P < 0.001), even in patients receiving “protective” plateau pressures and VT (relative risk, 1.36; 95% CI, 1.17–1.58; P < 0.001). Individual changes in VT or PEEP after randomization were not independently associated with survival; they were associated only if they were among the changes that led to reductions in ΔP (mediation effects of ΔP, P = 0.004 and P = 0.001, respectively). They concluded that ΔP was the ventilation variable that best stratified risk. Decreases in ΔP owing to changes in ventilator settings were strongly associated with increased survival.
\nMoreover, recent evidences [13] showed that in severe ARDS patients, inspiratory efforts during assisted ventilation could worsen ventilator lung injury induced by the mechanical ventilation during the ventilatory support of the ARDS patients. This associated and added injury could explain the results of a phase IV randomized controlled trial in moderate-severe ARDS patients (PaO2/FIO2 < 150), comparing cisatracurium to placebo for 48 h showed an improved adjusted 90-day survival rate and increased ventilator-free in the cisatracurium group without a significant increase in muscle weakness. Short-term paralysis may facilitate patient-ventilator synchrony in the setting of lung protective ventilation. Short-term paralysis would eliminate patient triggering and expiratory muscle activity. In combination, these effects may serve to limit regional overdistention and cyclic alveolar collapse. Paralysis may also act to lower metabolism and overall ventilatory demand [14]. Recently, Sottile and colleagues [15] showed that the use of neuromuscular blockade in ARDS patients receiving low tidal volume ventilation and a PaO2/FIO2 ratio less than 120 were associated with decreased biomarkers of epithelial (serum surfactant protein-D) and endothelial (serum Von Willebrand factor) lung injury and systemic inflammation (serum interleukin 8).
\nAt the same time, that an adequate sedation, an adequate short-term paralysis and low tidal volume were set in severe ARDS patients [16], an adequate respiratory rate must be concurrently set in order to keep a minute ventilation around 7–8 L/min and a PaCO2 around 40–60 mm Hg and a pH above 7.2. In the more severe ARDS patients, sometimes after the adjustment of a minute ventilation around 7–8 L/min with tidal volumes lesser than 6 mL/kg of predicted body weight, the PaCO2 levels stay above 80 mm Hg and pH less than 7.2 (specially patients with septic shock and metabolic acidosis). In these cases, the VCO2 must be assessed and be kept as least as possible (fever control, low carbohydrate intake) and hemodialysis can be initiate (especially in ARDS patients with concomitant acute renal failure) in order to help control the metabolic acidosis. Efforts must be taken to decrease the pulmonary dead space by means of recruitment maneuvers and PEEP titration, tidal volume and respiratory rate adjustments or even the initiation of prone ventilation. In the most difficult cases, tracheal gas insufflation or extracorporeal CO2 removal or extracorporeal oxygenation should be started in order to keep the protective low tidal volume ventilation [17].
\nPermissive hypercapnia can carry potential harmful consequences including pulmonary vasoconstriction and pulmonary hypertension, proarrhythmic effects of increased discharge of catecholamines and cerebral vasodilation yielding increased intracranial pressure. When applying protective ventilation in patients with severe ARDS, special attention should be given to patients with pulmonary hypertension and right ventricular dysfunction that could not tolerate high PaCO2 and low pH levels. Nonetheless, permissive hypercapnia should probably be used with caution in patients with heart disease and is relatively contraindicated in those with elevated intracranial pressure. In ARDS cases with pulmonary hypertension and right ventricular dysfunction, prone position ventilation should be preferred [17].
\nRecently, three large clinical trials [18, 19, 20], including acute lung injury/ARDS patients ventilated with low tidal-volume, have compared different PEEP strategies (high vs. low), but none of them could show a significant difference in mortality. Moreover, a recent meta-analysis [21] has pooled those trials, revealing some combined benefits of the high PEEP strategy; still, the survival benefit was modest and limited to the subgroup of ARDS patients with PaO2/FIO2 < 200 (moderate and severe ARDS according to Berlin definition). Conceptually, one could argue that none of the “high-PEEP” strategies was designed to test the “open-lung hypothesis” postulated by Lachmann, that is, the hypothesis that most of the collapsed lung tissue observed in early ARDS can be reversed at an acceptable clinical cost, potentially resulting in better lung protection [22, 23, 24]. According to a recent study by Borges and colleagues [25], a straight test of the “open-lung hypothesis” would certainly require more aggressive recruiting maneuvers in association with individualized, decremental PEEP titration. Recently, de Matos and colleagues [26] reported the experience with maximal recruitment strategy (MRS) in 51 patients with ARDS. MRS consisted of 2-min steps of pressure-controlled ventilation, fixed driving pressure of 15 cm H2O, respiratory rate of 10 breaths/min, inspiratory/expiratory ratio of 1:1, and stepwise increments in PEEP levels from 10 to 45 cm H2O (recruitment phase). After that, PEEP was decreased to 25 cm H2O and, then, from 25 to 10 cm H2O (PEEP titration phase) in steps of 5 cm H2O, each one lasting 4 min monitored by thoracic tomography images. At each of the steps, computer tomography image sequences from the carina to the diaphragm were acquired during an expiratory pause of 6–10 s. Visual inspection of the images was performed during the tomographic examination in order to assess the lung collapse in the lungs bases for immediate clinical decision, and after an offline quantitative analysis was realized. Nonaerated parenchyma decreased significantly from 53.6% (interquartile range (IQR): 42.5–62.4) to 12.7% (IQR: 4.9–24.2) (P < 0.0001) after MRS. The opening plateau pressure observed during the recruitment protocol was 59.6 (±5.9 cm H2O), and the mean PEEP titrated after MRS was 24.6 (±2.9 cm H2O). The mean PaO2/FiO2 ratio increased from 125 (±43) to 300 (±103; P < 0.0001) after MRS and was sustained above 300 throughout 7 days. MRS showed a statistically significant decrease in nonaerated areas of the ARDS lungs that was accompanied by a significant increment in oxygenation. The potentially recruitable lung was estimated at 45% (IQR: 25–53). ICU mortality was 28% and hospital mortality was 32%. The independent risk factors associated with mortality were older age and higher driving pressures. There were no significant clinical complications with MRS or barotrauma. A better evolution of these ARDS patients with less necessity of oxygen supplementation in the recovery phase of the disease and a better quality of life were observed in these patients [26].
\nA recent systematic review and meta-analysis [27] that analyzed the effects of recruitment maneuvers for adult patients with acute respiratory distress syndrome showed an overall pool effect of a significant decrease on mortality in these patients and no associated increase in barotrauma. However, soon after, a large prospective, multicenter and controlled trial (ART trial) [28] that compared recruitment maneuver and best-compliance PEEP titration in 501 ARDS patients with 509 ARDS patients ventilated with low PEEP showed an increased 6-month mortality in both groups, but higher in the recruitment and PEEP titration group (65.3 vs. 59.9%, respectively, P = 0.04). However, in our opinion, the recruitment maneuver tested in ART trial was abrupt and short (started at 25 cm H2O PEEP, duration of 1 s and not imaging monitored) what could have contributed to the higher levels of observed barotrauma and mortality [29]. When we combined the results of the systematic review and meta-analysis with the ART results, we observed a total of 1144 patients undergoing recruitment maneuvers and PEEP titration and 1179 controls with standard ventilation with a final result of no significant differences in mortality relatite risk of 0.91 [95% CI, 0.74–1.13]. The most effective recruitment maneuver and PEEP titration in ARDS remain to be determined [29].
\nOur group is used to apply maximal recruitment strategy maneuvers in our severe ARDS patients with PEEP titration with thoracic tomography with good results. Here, we present a case of a 47-year-old man, previously asymptomatic, that started with cough, dyspnea, and an acute hypoxemic respiratory failure. The chest-X-ray showed a bilateral pulmonary infiltrate that predominates in the lower lungs fields (Figure 1). SpO2 in ambient air was 77% and after oxygen mask of 100% SpO2 was 88% and he needed intubation and mechanical ventilation. Arterial pressure is of 11 × 7 cm Hg and HR of 110 rpm. Hemocultures and tracheal secretion were collected as well as a nasopharyngeal swab for respiratory viruses. Oseltamivir, clarithromycin and ceftriaxone were initiated. A transthoracic echocardiogram showed normal right and left ventricular function and a normal arterial pulmonary pressure. A protective mechanical ventilation was initiated with tidal volume of 6 mL/kg/predicted body weight (420 mL), RR of 20 rpm, PEEP of 15 cm H2O, FIO2 of 100%, SpO2 de 90% and an arterial blood gas analysis showed a pH of 7,35, PaCO2 of 50 mm Hg, PaO2 of 60 mm Hg, sodium bicarbonate of 23 and base excess of −1, lactate of 10 mg/dL, SvO2 of 70 mm Hg, Hb of 13 g/dL, 12,000 leucocytes, platelets of 250,000, reactive C-protein of 120 mg/L, BNP of 40 pg/mL. He was submitted to maximal recruitment strategy maneuvers and PEEP titration of 25 cm H2O in the tomography room (Figure 2). The PaO2/FIO2 ratio increased from 60 to 200, and after 3 days, he was with PEEP of 15 cm H2O and a PaO2/FIO2 of 300. After 10 days, he was extubated and presented in ambient air a SpO2 of 95% and an expressive improvement in chest X-ray (Figure 3).All the collect cultures and the nasopharyngeal swab for respiratory viruses were negative. The thoracic tomography performed 3 days after extubation showed an important improvement of the lungs infiltrates (Figure 4).
\nChest X-ray showing bilateral pulmonary infiltrates that predominate in lower lungs fields.
Thoracic tomography before and after maximal recruitment maneuvers and PEEP titration. Pre-recruitment PEEP of 10 cm H2O. Pos-recruitment PEEP of 25 cm H2O.
Chest-X-ray day 1, day 3, and day 10 after admission (day of extubation).
Thoracic tomography at first day after admission and thoracic tomography 3 days after extubation.
Recent evidence showed that prolonged prone position ventilation (16 h) must be used in early ARDS with PaO2/FIO2 < than 150 with PEEP levels of or more than 5 cm H2O in order to significantly improved 90-day mortality compared to supine ventilation (PROSEVA trial) [30].
\nRecent meta-analysis also showed that in the era of low tidal ventilation, the prone position use improved mortality of moderate/severe ARDS patients that needed invasive mechanical ventilatory support [31]. If PEEP titration during prone position, ventilation should improve survival of ARDS patients, which is still a matter of debate.
\nFor patients in whom gas exchange is refractory to conventional ventilation and other advanced therapies, extracorporeal membrane oxygenation (ECMO) may be appropriate as salvage therapy. Venovenous ECMO may be able to support refractory hypoxemia in the setting of severe ARDS. It may also be used for carbon dioxide removal when respiratory system compliance is severely compromised and efforts to maintain plateau airway pressures within acceptable parameters lead to unsustainable levels of hypercapnia and respiratory acidosis.
\nProspective randomized controlled trial of ECMO in severe ARDS, reported in 2009, was the Conventional Ventilation or ECMO for Severe Adult Respiratory Failure (CESAR) trial [32], in which 180 subjects with severe ARDS were randomized to conventional mechanical ventilation or referral to a specialized center for consideration of ECMO. The United Kingdom randomized and prospective clinical trial (CESAR) revealed a survival advantage in the ECMO group; the ECMO group had a 63% survival after 6 months, while the control group had a 47% survival rate. The study was criticized because there was no standardized protocol management for the control group and some patients in the ECMO group did not receive the proposed treatment. The authors demonstrated that this strategy is also likely to be cost-effective in settings with similar services to those in the United Kingdom. Patients should be considered for weaning from venovenous ECMO once the underlying disease process for which ECMO was initiated has sufficiently resolved so that they can be safely and adequately supported by protective ventilatory strategy and oxygenation support without evidence of excessive respiratory work of breathing. Markers of sufficient native lung function recovery include adequate gas exchange reserve, acceptable respiratory system compliance, and improvement in chest images [32].
\nAnother recent approach for application of extracorporeal carbon dioxide removal new devices (ECMO-R) in ARDS patients is the observation in thoracic tomography of ARDS patients that in severe ARDS, even the low tidal volume ventilation with 6 mL/kg of predicted body weight can cause tidal hyperdistension in the nondependent regions of the lungs accompanied by plateau airway pressures greater than 28 cm H2O and elevated plasma markers of inflammation. Application of ECMO-R in these severe ARDS patients could allow the authors to decrease the tidal volume to less than 6 mL/kg with a consequent plateau pressure less than 25 cm H2O that was associated with lower levels of lung-derived inflammatory cytokines and a lower radiographic index of lung injury [33], but prognostic implications of ECMO-R devices application in clinical practice are still under investigation [34]. Pumpless interventional lung assist (iLA) is also used in patients with ARDS and is aimed at improving extracorporeal gas exchange with a membrane integrated in a passive arteriovenous shunt. iLA can be used in severe ARDS patients as an extracorporeal device to remove CO2 enabling low tidal volume and a reduced inspiratory plateau pressure in the mechanical ventilator in extremely severe ARDS patients. iLA device was used in 51 severe ARDS patients by Zimmermann and colleagues with a decrease in PaCO2 allowing the ultraprotective ventilation (lower tidal volume and plateau inspiratory pressures) with a hospital mortality of 49% [35]. More recently, the use of an ultraprotective strategy using 4 mL/kg of predicted body weight associated with low flow extracorporeal carbon removal in 15 moderate ARDS patients was described by Faneli and coworkers [36]. Additional data to the use of ECMO in ARDS patients will be added with the publication of EOLIA trial (ClinicalTrials.gov Identifier: NCT01470703), a prospective and randomized trial that evaluated the role of ECMO in severe ARDS that has finished but not published yet.
\nIn conclusion, the severe ARDS is defined as an acute bilateral pulmonary infiltrate onset, in a patient with a PaO2/FIO2 equal or less than 100 with a positive end expiratory pressure equal or more than 5 cm H2O that have an ARDS risk factor with no signs of cardiac failure or hypervolemia. Thus, when an intensivist evaluates an ARDS patient severity, he/her has to take into consideration the patient’s age, cause of ARDS, the PaO2/FIO2 ratio, response to PEEP, prone position, PaCO2 with protective ventilation (6 mL/predicted body weight), right ventricular function and level of pulmonary artery pressure, presence of shock (and necessity of vasoactive drugs), APACHE II score, number of organ failures (specially renal failure). The severe ARDS patients present a higher mortality ratio and required an extremely careful and specialized treatment. The cause of ARDS initiation should be addressed and promptly treated. These patients present a more difficult mechanical ventilatory support (higher airway pressures with low tidal ventilation and higher PaCO2 levels). They should be adequately monitored (airway and esophageal pressure measurements, bedside echocardiography and lung ultrasound, if possible). Protective ventilatory strategy must be offered and monitored (low tidal volume (less than 6 mL/kg of predicted body weight) and low distending inspiratory driving pressures (less than 15 cm H2O) with adequate PEEP levels, and early prone position applied for more than 16 h. The possible benefits for adjunctive ventilatory support therapy (higher PEEP, recruitment maneuvers, inhalatory nitric oxide, ECMO and continuous hemodialysis) in the refractory cases should be offered, observing and monitoring the cross-talking among the multiple organ dysfunctions and guiding and changing the treatment according to the patients’ responses. The more difficult cases must be treated in specialized centers with expertise supervision [4, 17, 26, 37, 38].
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. 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After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"Vienna University of Technology",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:null},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:"Assist. Prof.",profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). He is the member of many Pharmaceutical Associations and acts as a reviewer of scientific journals and European projects under different research areas such as: drug delivery systems, nanotechnology and pharmaceutical biotechnology. Dr. Sezer is the author of many scientific publications in peer-reviewed journals and poster communications. 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I received a B.Eng. degree in Computer Engineering with First Class Honors in 2008 from Prince of Songkla University, Songkhla, Thailand, where I received a Ph.D. degree in Electrical Engineering. My research interests are primarily in the area of biomedical signal processing and classification notably EMG (electromyography signal), EOG (electrooculography signal), and EEG (electroencephalography signal), image analysis notably breast cancer analysis and optical coherence tomography, and rehabilitation engineering. I became a student member of IEEE in 2008. During October 2011-March 2012, I had worked at School of Computer Science and Electronic Engineering, University of Essex, Colchester, Essex, United Kingdom. In addition, during a B.Eng. I had been a visiting research student at Faculty of Computer Science, University of Murcia, Murcia, Spain for three months.\n\nI have published over 40 papers during 5 years in refereed journals, books, and conference proceedings in the areas of electro-physiological signals processing and classification, notably EMG and EOG signals, fractal analysis, wavelet analysis, texture analysis, feature extraction and machine learning algorithms, and assistive and rehabilitative devices. I have several computer programming language certificates, i.e. Sun Certified Programmer for the Java 2 Platform 1.4 (SCJP), Microsoft Certified Professional Developer, Web Developer (MCPD), Microsoft Certified Technology Specialist, .NET Framework 2.0 Web (MCTS). 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