Resuscitative fluid therapy aims to increase stroke volume (SV) and cardiac output (CO) and restore/improve tissue oxygen delivery in patients with circulatory failure. In individualized goal-directed fluid therapy (GDFT), fluids are titrated based on the assessment of responsiveness status . Fluid administration may increase venous return, SV and CO, but these effects may not be predictable in the clinical setting. The fluid challenge (FC) approach, which consists on the intravenous administration of small aliquots of fluids, over a relatively short period of time, to test if a patient has a preload reserve , has been used to guide fluid administration in critically ill humans. In responders to volume expansion , FC administration is repeated until the individual no longer presents a preload reserve  or until other signs of shock are resolved . Even with the most recent technological developments, reliable and practical measurement of the response variable (SV or CO changes induced by a FC) has posed a challenge in GDFT. Among the methods used to evaluate fluid responsiveness in the human medical field, measurement of aortic flow velocity time integral by point-of-care echocardiography has been implemented as a surrogate of SV changes induced by a FC and seems a promising non-invasive tool to guide FC administration in animals with signs of circulatory failure. This narrative review discusses the development of GDFT based on the FC approach and the response variables used to assess fluid responsiveness status in humans and animals, aiming to open new perspectives on the application of this concept to the veterinary field. Iatrogenic hypervolemia due to a liberal fluid administration strategy causes tissue/lung edema and worsens patient outcome , while monitoring the patient's the response   and cardiac output (CO) in response to volume expansion] appears to reduce patient mortality, duration of stay in the intensive care unit (ICU), and duration of mechanical ventilation . The useReferences from PubMed database since January 2000 were included in this narrative review. Primary search terms were fluid challenge, GDFT, and fluid responsiveness. Human and animal studies  involving the above mentioned terms were selected. To approach the target variables used to assess the response to a fluid challenge, studies comparing newer CO measurement technologies with reference methods of CO measurement in humans, dogs, and cats were selected. Studies published before 2000 considered seminal for understanding the current state of knowledge were also included in the review.via an effective increase Vs  which contain ~70% of blood volume , 5. Baserease Vs . Anotherrease Vs . Lower drease Vs , 7. Otherease Vs . The negIn conscious patients with signs of acute circulatory failure , intravascular volume expansion is beneficial only if it significantly increases SV and CO. Titration of intravenous fluids to maximize SV and CO, while avoiding hyper- or hypovolemia is an ongoing paradigm in the medical field. The Frank-Starling curve provides the physiological basis for understanding the concept of fluid responsiveness. To test the fluid responsiveness status of an individual, a change in preload must be provoked and the observed response in SV or CO recorded . Responda priori to fluid administration (peak), pulse pressure variation (PPV), systolic pressure variation (SPV). plethysmographic variability index (PVI) and stroke volume variation derived from pulse contour analysis (SVVPCA) , or that reflect end-diastolic volume, are not reliable to predict fluid responsiveness and have limited usefulness to guide fluid administration –32. Accuted dogs , 21, 22.ted dogs , 21, 22.a priori to volume expansion  , 37. In xpansion . The CVCxpansion –41.A fluid challenge is the rapid administration of a relatively small volume of fluid to test if an individual has a cardiac preload reserve. Inasmuch as small amounts of fluids are titrated “to effect,” based on pre-established goals, the fluid challenge also provides volume replacement with lower risk of volume overload , 10. PerIn individuals with signs of poor tissue perfusion that are responders to the first fluid challenge (SV or CO increase ≥10–15% above baseline), maximization of SV and CO can be achieved by administering additional fluid challenges , 10. BefOne question that remains opened is the end-point of fluid resuscitation in individuals that show preload dependence. Unless there is evidence of significant hypovolemia, it has been the suggested in humans admitted for emergency care that the volume of intravenous fluids should be initially limited to 20–30 ml/kg before vasopressors are considered to treat hypotension . The updvia a venoconstrictive action  and/or CO and does not imply that there is a circulating volume deficit . Althouge action , 49.According to a systematic review of literature, the fluid challenge most commonly used in adult human patients with signs of poor tissue perfusion consisted of 500 ml of crystalloids or colloids (~7 ml/kg to an adult patient weighing 70 kg), administered during 20–30 min . The tarvia a fluid challenge approach in healthy/normovolemic dogs under anesthesia , precise , and provide good ability to track changes in the target variable , 63. Accvia minimally invasive or non-invasive procedures  (PATD) was used to evaluate the response to a fluid challenge in mechanically ventilated human individuals with acute circulatory failure related to sepsis  technique has been the clinical gold standard method for CO monitoring since its introduction in the 1970's . Pulmonaer (PAC) . Cardiaco sepsis , 26, 30 o sepsis . Howevero sepsis , 68. Foro sepsis –69. In vIntroduced during the 1990's in the medical field, transpulmonary thermodilution (TPTD) is a less invasive indicator dilution technique than PATD because changes in blood temperature induced by the thermal indicator are measured by a thermistor tipped catheter inserted into a central artery of the systemic circulation  , 69. ExpTPTD) to track changes in CO measured by the reference standard (COPATD) using polar plot analysis is presented in TPTD can be used in replacement of COPATD because of an acceptable precision of agreement and good trending ability between these two techniques  to evaluate fluid responsiveness. The minimum percent change in CO that can be trusted as significant and not related to the imprecision of the method [i.e. the least significant change (LSC)] was reported decrease from 20% with single COTPTD measurements to 12% with triplicate COTPTD measurements in humans, which is adequate to detect responders to volume expansion (CO increases ≥ 15%)  than previously reported in humans (LSC = 12%), which could allow reliable detection of changes in CO ≥ 10%  . AveragiCO ≥ 10% .TPTD monitoring during the fluid resuscitation phase is limited by the fact that it requires not only a central venous catheter for injection of the thermal indicator , but also a thermistor tipped catheter placed in a central artery   and hematoma formation should be considered as potential complications  , 69. Alty)  can be performed through a peripheral venous catheter in humans and dogs , 83. Theand cats –87. Howeand cats . The litvia injection of additional amounts of thermal  or chemical (lithium chloride) indicators, the pulse contour analysis (PCA) method provides continuous, real time estimation of CO and SV.While indicator dilution techniques provide intermittent CO values that need to be updated by the generation of new indicator dilution curves PATD in small animals are summarized in Real time estimation of CO and SV could be particularly convenient to evaluate the response to a fluid challenge during volume resuscitation . HoweverTPTD (PiCCO2 system and VolumeView/EV1000 system) or with CO measured by lithium dilution (LIDCOplus and PulseCO systems). Studies have suggested that the calibrated PCA method could be useful to evaluate the response to a fluid challenge in humans (2) showed good correlation with changes in COPATD and COTPTD induced by volume expansion (correlation coefficient ≥ 0.70) (The same monitors that measure CO intermittently by indicator dilution techniques (TPTD and lithium dilution) also provide real time CO and SV estimations by the calibrated PCA method. This technique is based on analysis of the arterial pressure waveform to estimate real time CO and SV using proprietary algorithms, after calibration of the system with COn humans , 79, 92. ≥ 0.70) , 92.2 system) could discriminate responders (COTPTD increases ≥ 15%) from non-responders (COTPTD increases <15%) to volume expansion , ProAQT/Pulsioflex]. Real time SV monitoring with an uncalibrated PCA device (ProAQT/Pulsioflex) was used to evaluate the ability of a mini fluid challenge  to predict the response to 250 ml of physiological saline in mechanically ventilated human patients undergoing neurosurgery . HoweverIn spite of continuous changes in the algorithm of one uncalibrated PCA device (FloTrac/Vigileo) over the recent years, studies have shown a poor agreement and limited ability to track phenylephrine-induced changes in CO measured by echocardiography  and by PPATD , there are conflicting results regarding its ability to track changes in CO measured by a reference method. Earlier studies in pigs, where a range of CO values were induced by hemorrhage and inotropic administration (dobutamine), reported an acceptable agreement (PE <30%) between PRAM and CO measured by an aortic flow probe and between PRAM and PATD . HoweverPATD . HoweverEcho and SVEcho, respectively) can be used to evaluate the response to a fluid challenge. While transthoracic echocardiography is useful in conscious individuals with signs of circulatory failure, transesophageal echocardiography is a practical means of monitoring the response to a fluid challenge in anesthetized patients during surgery or in unconscious/sedated individuals under mechanical ventilation. According to this technique SVEcho is firstly measured by multiplying the area of the left ventricular outflow tract (LVOT) during mid systole  by the velocity time integral (VTI) of the LVOT. Because the LVOT area during mid systole does not to change over the respiratory and cardiac cycle, one single measurement of the LVOT area is usually performed for a patient as SV will vary exclusively as function of changes in aortic flow VTI  with COPATD  in dogs that were normotensive and hypotensive during anesthesia . It must be borne in mind that the reliability of VTI measurements is highly operator dependent. To account for operator related errors, studies evaluating fluid responsiveness with echocardiography should ideally report intra and inter-operator agreement . The relWhen VTI is used to evaluate fluid responsiveness, one important question that arises is the level of precision  of aortic flow measurements obtained during stable hemodynamic conditions. If the precision of the method is poor, it may not be able to correctly identify responders to volume expansion . In humaThe ability of a method to detect changes in SV induced by a fluid challenge can be inadequate during conditions of hemodynamic instability/irregular heart rhythm because the method's variance is composed not only by the precision of the method but also by the method's general variability about the true values (defined as “trueness”) . TherefoMeasuring the VTI demands several keystrokes and could be time-consuming, which may be less practical in the emergency setting. However, with the recent development of algorithms incorporated into ultrasound machines, it has become possible to perform automated VTI measurements. Use of tools that allow automated VTI measurements has the potential for optimizing evaluation of fluid responsiveness in the emergency settings .Velocity time integral measurements are overestimated in the presence of moderate-to-severe aortic regurgitation and sub-aortic obstruction (aortic stenosis) . Dynamicvia a Doppler transducer inserted into the thoracic esophagus. The velocity of blood flow in the descending aorta plotted over time is used to determine the stroke distance (distance of the envelope that contains blood flow velocity vs. time curve measured in centimeters) (EDM) is calculated by stroke distance times the cross sectional area of the aorta. A nomogram developed for human patients  estimates the cross sectional area of the aorta, assuming a 45° angle between the aorta and the esophageal probe (EDM) is calculated by the multiplying SV/stroke distance by the heart rate  imeters) –116. Theal probe –116. Theart rate –116.EDM cannot be considered interchangeable with COPATD, this device can show an acceptable/marginal trending ability with COPATD, suggesting that this method could be used to guide volume expansion in goal directed fluid therapy  in pediatric patients with congenital heart defects and in patients undergoing coronary artery bypass surgery  , 115. Al therapy .EDM does not present an acceptable agreement with COPATD in dogs (PE = 39%)  . OtherwiE = 39%) . AdditioPATD  is a non-invasive method of continuous CO monitoring based on impedance cardiography. Measurement of left ventricular SV and CO is based on a transcutaneous electrical AC voltage applied to the chest to calculate changes in resistance during the cardiac cycle –125. StuPATD , which cPATD .PATD in dogs (PE = 61–63%) and humans (PE = 69%) (TPTD (PATD in dogs (Pulse wave transit time (PWTT) is another recently developed technique that provides continuous, non-invasive CO estimations. This method measures the time elapsed between the R wave of the electrocardiogram, which corresponds to the beginning of ventricular systole, and the appearance of the corresponding pulse wave on a pulse oximeter. Measurement of CO by PWTT is based on the principle that the transit time of pulsatile blood flow, detected by a pulse oximeter, is inversely proportional to CO , 128. HoE = 69%) , 127. Ca%)  and pulmonary vascular permeability index (PVPI) in patients monitored with TPTD technology , 134. InBased on experimental studies healthy anesthetized dogs, it appears that reference values of EVLWI in humans cannot be extrapolated to canine species, as mean EVLWI values of 12 ml/kg have been reported in healthy anesthetized dogs receiving a standard fluid rate of 2 ml/kg/hour of LRS . In normHydrostatic pulmonary edema may occur if a fluid challenge is administered to individuals presenting a restrictive left ventricular filling pattern. Features of diastolic dysfunction leading to a restrictive filling pattern include an excessive acceleration of early transmitral flow (E wave), and a decreased longitudinal myocardial fiber lengthening at the level of the mitral annulus during early diastole (e' wave), measured by pulse wave Doppler and by tissue Doppler imaging, respectively . The E/eMyxomatous mitral valve disease and dilated cardiomyopathy are among the heart diseases that lead to elevated LVFP and increased risk of pulmonary edema from bolus fluid administration in canine species . In catsAlthough, data are lacking in veterinary medicine, left ventricular diastolic dysfunction has been recognized as a common feature in humans with sepsis or septic shock . SystoliIn the presence of echocardiographic changes suggestive of elevated LVFP a restrictive fluid therapy strategy should be considered . OtherwiAn ideal fluid challenge should reliably discriminate responders from non-responders to volume expansion by means of the smallest volume possible to minimize this risk of fluid overload. An excessively large volume of IV fluids used to test the preload dependency increases the risk of tissue edema. Otherwise, if the volume of fluid used as a “challenge” is excessively small, the resultant increase preload might not be enough to identify responders to volume expansion, yielding false negative results. In hemodynamically stable post-cardiac surgery human patients the administration of progressively increasing volumes of isotonic crystalloids (1 to 4 ml/kg over 5 min) increased the proportion of responders from 20% (1 ml/kg) to 65% (4 ml/kg) . TherefoNot only the volume of fluid, but also the duration of the infusion influences the recognition of responders to volume expansion. Meta-analysis studies in humans have shown that more prolonged infusion times (>30 min) decreases the proportion of responders to volume expansion . A thirdExperimental evidence suggests that administration of 25% of the “shock dose” of crystalloids in dogs 20 ml/kg) can increase the risk of tissue edema because a larger cumulative volume of fluids may be administered until animals are recognized as non-responders to volume expansion . Lower v ml/kg cavia fluid administration in high-risk small animal patients.Knowledge on the assessment of fluid responsiveness has experienced a significant development during the past two decades in the human medical field. In patients admitted with signs of circulatory failure, fluid resuscitation guided by a fluid challenge approach has the potential to improve patient outcome. Considering the principle of “less is more,” to minimize the risk of fluid overload, it has been recognized that smaller volumes of fluids, or mini-fluid challenges, may allow identification of responders to volume expansion induced by a standard fluid challenge. To reduce the risk of edema from fluid overload, recommendations for the use of 25% of the “shock dose” should be revised in favor of smaller volumes of crystalloids as standard fluid challenge (up to 10 ml/kg over 5–10 min in dogs and <10 ml/kg over 10–15 min in cats). When using the fluid challenge approach, it should be recognized that the response variable measured before and after the fluid challenge  will significantly impact the identification of responders to volume expansion. In veterinary medicine, goal-directed fluid therapy aiming to maximize CO and SV is in its infancy. Among the CO and SV measurement technologies that could be used to assess the response to a fluid challenge, aortic flow VTI obtained by transesophageal and transthoracic echocardiography appears to be a promising tool to evaluate fluid responsiveness in animals. Esophageal Doppler monitoring might be particularly useful to guide fluid administration in anesthetized dogs and cats or in animals under mechanical ventilation in the ICU. Future studies are needed to assess fluid responsiveness status in dogs and cats with signs of circulatory failure and the potential benefits that could be achieved by maximizing CO and SV FT-N: manuscript conception and writing (~70% of manuscript text). AV: Manuscript preparation, writing (~30% of manuscript text), and critical review. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Tumour blood vessels, characterised by abnormal morphology and function, create an immunosuppressive tumour microenvironment via restricting the appropriate leucocyte subsets trafficking. Strategies to trigger phenotypic alteration in tumour vascular system to resemble normal vascular system, named vascular normalisation, promote effective trafficking of leucocytes into tumours through enhancing the interactions between leucocytes and endothelial cells. This review specifically demonstrates how targeting tumour blood vessels modulates the critical steps of leucocyte trafficking. Furthermore, selective regulation of leucocyte subsets trafficking in tumours can be achieved by vasculature-targeting strategies, contributing to improved immunotherapy and thereby delayed tumour progression.The effectiveness of immunotherapy against solid tumours is dependent on the appropriate leucocyte subsets trafficking and accumulating in the tumour microenvironment (TME) with recruitment occurring at the endothelium. Such recruitment involves interactions between the leucocytes and the endothelial cells (ECs) of the vessel and occurs through a series of steps including leucocyte capture, their rolling, adhesion, and intraluminal crawling, and finally leucocyte transendothelial migration across the endothelium. The tumour vasculature can curb the trafficking of leucocytes through influencing each step of the leucocyte recruitment process, ultimately producing an immunoresistant microenvironment. Modulation of the tumour vasculature by strategies such as vascular normalisation have proven to be efficient in facilitating leucocyte trafficking into tumours and enhancing immunotherapy. In this review, we discuss the underlying mechanisms of abnormal tumour vasculature and its impact on leucocyte trafficking, and potential strategies for overcoming the tumour vascular abnormalities to boost immunotherapy via increasing leucocyte recruitment. The presence of specific subtypes of leucocytes in tumours is closely related to improved prognosis for cancer patients . The abiThe imbalance of angiogenic mediators in tumours is prone to strengthen tumour angiogenesis and elicit the tumour blood vessels to develop abnormally, in terms of structure and function. The aberrant tumour vasculature plays an important role in generating an immune suppressive microenvironment, preventing the trafficking of leucocytes from infiltrating into the tumour bed. Anti-angiogenesis therapy can result in altered leucocyte trafficking to enhance immunotherapy. However, the overall clinical achievement of anti-angiogenic agents has been less than what was expected. This lack of benefit for cancer patients appears to be due to “intrinsic resistance” (resistance to anti-angiogenic therapies observed at the beginning of the treatment) and “acquired resistance”  to the drugs . In receIn this review, we summarise the current knowledge on the role of tumour blood vessels in controlling leucocyte trafficking, importantly on the selective trafficking of subpopulations of leucocytes. We discuss potential therapeutic strategies for modulating tumour vasculature to influence leucocyte recruitment.Normal blood vessels are composed of evenly spaced and well-differentiated arterioles, capillaries, and venules . In contThe abnormalities in tumour vascular structure contribute to an immature and dysfunctional tumour vasculature. Increased expression of angiogenic factors, in particular vascular endothelial growth factor (VEGF) results in disrupted junctions and as such, tumour blood vessels are extremely permeable to intravascular fluids with resulting increased interstitial fluid pressure (IFP) . In addiA growing body of evidence has revealed that unproductive and aberrant tumour-associated blood vessels emerge as an important contributor to the tumour immunoresistant microenvironment. More precisely, tumour blood vessels not only influence how cancer cells escape the anticancer immunosurveillance and respond to immunotherapy, but they also determine the number and activity of leucocytes that invade into tumours ,18,19. SVascular normalisation has been regarded as an effective vascular-targeting therapeutic strategy since it was first described by Rakesh Jain and colleagues ,14. InstIn the process of vascular normalisation, disorganised and highly proliferating tumour ECs become more quiescent and less active. The normalised endothelium tends to form tighter interendothelial junctions between neighbouring cells involving adherens junction molecules such as vascular endothelial (VE)-cadherin and tight junction -1, ZO-2, and Claudin-5) . PericytVascular normalisation, however, appears to be dose-dependent. For example, high doses of anti-angiogenic drugs  are prone to destroy the tumour blood vessels with impaired drug delivery whilst too low a dose fails to work on tumour blood vessels ,39. FurtLeucocytes use an array of cell adhesion molecules and chemokines to attach and transmigrate across ECs in order to penetrate into connective tissue stroma at an inflammatory site  3]. The. The3]. Tumours demonstrate specific tumour microenvironments characterised by differential chemokines and chemotactic factors that affect leucocyte recruitment . LeucocyIn a normal inflammatory response, the initial capture of leucocytes from the blood circulation is regarded as a prerequisite for leucocyte rolling along the adhesive endothelial surface  and can An essential mechanism of tumour escape from immunity is the fact that tumour ECs fail to capture leucocytes from the blood circulation, thereby limiting the number of immune cells within the tumour mass ,54,55. TIn general, the rolling of leucocytes on the luminal surface of ECs is predominantly governed by a cascade of carbohydrate binding proteins called E-, P-, and L-selectins, which are expressed on the surface of both ECs (E- and P-selectins) and leucocytes (L-selectin). E- and P-selectins can bind to glycosylated ligands on the leucocytes, most notably P-selectin glycoprotein ligand 1 (PSGL1) ,60. BothThe leucocyte–endothelial cell adhesion in tumour blood vessels is diminished under both basal and cytokine-stimulated circumstances . OverproThe majority of adherent leucocytes tend to relocate from the initial site of adhesion to the nearest junctional extravasation site in the endothelium before diapedesis, a distinct process termed intraluminal crawling or “locomotion” . This cr+ neutrophils in the venules while CD8+ T cells crawled in both collecting and post-capillary venules, indicating different leucocyte subpopulations may crawl along distinct vascular structures or components within the TME  [In tumours, leucocytes exhibit an effective amoeboid movement that is characterised by pseudopod protrusion at the leading edge of the cell, followed by directional locomotion toward a chemotactic source derived from the tumour tissue . Within n, etc.) . Furthern, etc.) . To bettn, etc.) ,78.The infiltration of leucocytes across the vascular wall into tissues (namely leucocyte TEM or diapedesis) is an indispensable physiological behaviour that happens during both the adaptive and innate immune response and in the process of immune surveillance and homing . Only acParacellular transmigration requires the adhesion of the leucocyte to the endothelium, but also needs the clustering of ICAM-1 and VCAM-1 which result in the formation of docking structures or transmigratory cups on the endothelial apical surface membrane . The cluAs leucocytes transmigrate through the EC barrier, they are encapsulated by the lateral border recycling compartment (LBRC), a recently described peri-junctional network of interconnected tubulovesicular membrane in ECs ,99. Targ2-integrin occupancy, appropriate shear stress, and the existence of a range of key chemokines on the ECs [Leucocytes are also able to transmigrate via a transcellular mode, whereby the leucocytes are taken up by ECs and are transported in a vesicle-like component from the luminal to the abluminal side . The pri the ECs ,109,110.In the process of transcellular migration, LBRC membrane surrounds the leucocyte and ICAM-1 expressed on the apical surface of ECs is enriched around the leucocyte adhesion sites . This en2 mAb 2E6 [Tumour blood vessels, in contrast to normal blood vessels, present aberrant vascular structures and functions that inhibit leucocyte transmigration . VEGF se mAb 2E6 .Indeed, neutrophil transmigration was markedly enhanced within the tumour blood vessels, which has been thought to be linked to the increased stiffness of ECs . The tum+ T cells, NKs, and DCs, and poor survival associated with neutrophils, Tregs, etc. Thus, efficient admission to these well-defined leucocyte subpopulations presents conspicuous value in research as well as in clinical applications [Leucocytes, or white blood cells (WBC), exert fundamental effects on the human immune system. They can be divided into three main subpopulations: lymphocytes, monocytes, and granulocytes. The overall survival of cancer patients is correlated with type and level of leucocyte subpopulations in the tumour parenchyma, with good survival associated with CD8ications ,123. In ications . Althougications . Therefo+ T cell infiltrate, and impressive clinical responses to a plethora of immunotherapies that reinvigorate them [+ T lymphocytes [Lymphocytes are regarded as an essential subset of leucocytes that are the mediators of immunity. Lymphocytes consist of three major subpopulations: T cells, B cells, and natural killer (NK) cells . Among tate them . Of notephocytes . Given tphocytes .+ T cells [+ T cells in the presence of tumour-derived VEGF, prostaglandin E2 and IL-10 stimulation [A growing body of evidence has shown that tumour blood vessels set up difficult hurdles for the trafficking of T lymphocytes, in particular effector CD8 T cells ,144. Abn T cells ,18. The  T cells . This nomulation . Hence, Although normalisation of tumour blood vessels was perceived as a mechanism to limit vessel expansion and decrease the hypoxic TEM, one of the unexpected outcomes is the return to a more controlled regulation of leucocyte trafficking. Normalisation strategies where such effects have been studied are summarised as below.+ T cells [+ T cells into the TME. In particular, boosted pericyte coverage coincided with the time of peak CD8+ T cell infiltration [Pericytes: In tumours, pericytes appear to be absent or loosely attached onto the ECs, which results in the tumour blood vessels becoming more permeable than normal vessels. The high permeability of the tumour blood vessels triggers irregular blood flow, contributing to insufficient T cell trafficking within the tumour parenchyma . An eleg T cells . In addiltration .+ T cells transmigrating into the tumour bed as well as enhanced CD8+ T cell activity [+ T cells into tumours was mainly through the transcellular route, which was mediated via AKT/GSK3β/β-catenin signalling pathway in ECs [+ T cells preferentially pursued transcellular migration when EC junctions became tight [EC junctions: We have reported that specific upregulation of VE-cadherin by a novel microRNA-based agent, CD5-2, could transform the tumour vasculature to a more normalised phenotype with improved endothelial junctions, and reduced hypoxic TEM. The normalisation resulted in increased CD8activity . Interesy in ECs . This wame tight .Hypoxia: Hypoxia has been widely held to be the driving force that leads to dysfunctional vascularisation . To this+ T cells [+ T cells into tumours [+ T cell infiltration into tumours [+ cytotoxic T cells into tumours, improving outcomes when in combination with anti-PD-1 in various mouse tumour models [Anti-angiogenic therapy: Pro-angiogenic growth factors produced by tumour cells can downregulate the expression of a collection of adhesion molecules , inhibiting extravasation of T lymphocytes across the tumour endothelium ,157. Dis T cells . Low-dos tumours . Moreove tumours . Additior models .+ T cell infiltration have been gaining attention. For instance, bone morphogenetic protein 9 (BMP9) was reported to promote the normalisation of tumour blood vessels via activating activin receptor-like kinase 1 (Alk1) signalling, yielding increased influx of T cells [+ T cell infiltration into tumours [Other strategies: Recently, a variety of new strategies to bridge tumour vascular normalization and CD8 T cells . Althoug T cells ,165. Ind T cells . Further tumours .HEVs are anatomically diverse post-capillary venules that emerge as main portals of entry for lymphocytes into lymph nodes and other secondary lymphoid organs . The end+ and CD8+ cytotoxic T cells [The density of HEVs is tightly associated with the number of tumour-infiltrating CD3 T cells . High de T cells . Allen e T cells . Further T cells . Notably T cells ,171.high tumours compared with HEVlow tumours, gene profile of HEVs has shown upregulation of genes encoding lymphoid chemokines including chemokine (C-C motif) ligand (CCL) 19 (CCL19), CCL21, and CXCL13 as well as T-cell homing receptors (CCR7 and LSEL) [+ DCs were shown to be required for the maintenance of HEVs in an LTα1β2-dependent manner [In HEVnd LSEL) . Geneticnd LSEL) ,133. Addnd LSEL) . Surprist manner . In contt manner . Thus, mAngiogenesis is able to trigger EC anergy, a failure to respond to a large amount of proinflammatory cytokine stimulation such as TNF-α, IL-1, and IFN-γ. This anergy at least partially results from continuous stimulation by angiogenic factors such as VEGF and FGF, which inhibit TNF-α-mediated upregulation of VCAM-1, ICAM-1, and chemokines ,174,175.Monocytes are heterogeneous circulating white blood cells that play a fundamental role in tissue homeostasis, protective immunity, and both promotion and resolution of inflammation ,177. Simhi macrophages led to disruption or loss of endothelial junctions, transient vascular leak, and tumour cell intravasation, providing insight into the mechanism of tumour distant metastasis [Tumour-associated macrophages (TAMs) predominantly derive from circulating monocytes that stem from the bone marrow . TAMs artastasis .+ T cell infiltration in various HCC mouse models [+ T cell trafficking into the TME via the repression of CXCL9 and CXCL10 expression [TAM trafficking into tumours is dependent on CCL2 expression. The CCL2 inhibitor, bindarit inhibited M2-like macrophage trafficking and reduced human melanoma xenografts . CCL2 ane models . Indeed,e models . Hedgehopression . Zhu andpression .There are a number of approaches being tested for conversion of a M2-rich into an M1-rich TAM microenvironment. For instance, Zoledronic acid, a potent drug for suppression of spontaneous tumour growth, exerted striking anti-angiogenic effect in part through repolarisation of pro-angiogenic M2-like TAMs to suppressive M1-like TAMs . MoreoveDCs act as key orchestrators of the immune response. Monocyte-derived DCs (mo-DCs), also referred to as inflammatory DCs (inf-DCs), can be recruited into tissues and appear to be the most abundant DC population in the process of inflammation ,195. TheModulation of DC trafficking has been achieved through a number of different routes. These include (1) activation of the endothelium by exposure to hypoxia, oxidized low density lipoprotein or TNF-α , depleti+ DCs into the TME and promote the anti-tumour immune response [+ DC infiltration [(2) Targeting the extracellular matrix around the tumour blood vessels, which is high in collagen, also can modulate DC infiltration. A fusion protein (CBD-CCL4) of chemokine CCL4 and the collagen-binding domain (CBD) of von Willebrand factor  was able to recruit CD103response . Furtherltration .(3) Targeting tumour vasculature associated signalling pathways, including blockade of VEGF-A and ANG2 reinforced the antigen-presenting ability of DCs, and the combined therapy potentiated DCs to present an activated phenotype characterised by increased expression of MHC-II and CD86 . VEGF biGranulocytes or polymorphonucleocytes are characterised by the presence of granules and include neutrophils, eosinophils, and basophils , and are2 integrin family that are predominantly expressed on neutrophils, binding to ICAM-1 expressed on ECs [Neutrophils are the most enriched cell population in the immune system  and haved on ECs , and find on ECs . High ned on ECs  and CD17d on ECs .The transmigration of neutrophils into the tumour parenchyma can occur by enhanced expression of adhesion molecules and chemokines which include CXCL1, CXCL8 and CCL15 ,217,218.Normalising tumour vasculature via improving the endothelial junctions (mainly through increasing VE-cadherin expression) resulted in decreased neutrophil infiltration into tumour parenchyma ,146. Fur+ T cells, through action to alter the TME with increased IFN-γ and TNF and to induce tumour vascular normalisation [Eosinophil accumulation in the peripheral blood and tissues is a typic hallmark of multiple common diseases such as atopic disorders, parasitic infections, and cancer . Similarlisation . Furtherlisation . Notablylisation . HoweverThe goal of immunotherapy is to harness the immune response to deliver tumour killing potential. However, the immune suppressive nature of the tumour microenvironment hinders the infiltration of leucocytes into tumour parenchyma. Thus, strategies for converting “cold tumours” into “hot tumours” as evidenced by increased and appropriate leucocyte infiltration into tumour parenchyma, have gained attention. Since blood vessels, and in particular the endothelium, are central players in controlling the delivery of leucocytes into tissue, it has become a new target for anti-cancer therapies. Indeed, anti-angiogenic treatment can promote leucocyte trafficking and reduce the number of newly formed blood vessels. Further, vascular normalisation as a novel vasculature-targeting therapy facilitates the trafficking of leucocytes via improving tumour vascular structure and function. Of particular note now are strategies that will enhance selective immune subsets and will aid their activation while inhibiting access of detrimental subsets into the tumour to improve cancer immunotherapy and bring great benefit for cancer patients.


We evaluated the BSI results in 2018-2020.The IDEA Program monitors the 8,643 organisms were consecutively collected (1/patient) from 74 medical centers located in the US , western EU , and eastern EU . Organisms were susceptibility tested by reference broth microdilution methods in a central laboratory. S. aureus , E. faecalis , S. epidermidis , β-hemolytic streptococci , and E. faecium , but rank order varied markedly by geographic region. The top 3 GP organisms were SA, EF, and BHS in the US; SA, EF, and EFM in W-EU; and SA, S. pneumoniae, and BHS in E-EU. Dalbavancin was highly active against methicillin-susceptible (MSSA) and -resistant (MRSA) SA, with an MIC90 of 0.03 mg/L in all 3 regions (Table). Among SA, MRSA rates were higher in the US (41.3%) than W-EU (21.5%) or E-EU (27.3%), and ceftaroline susceptibility ranged from 95.4% (W-EU) to 96.6% (US). Vancomycin (VAN) susceptibility varied from 97.3% (E-EU) to 98.3% (W-EU) among EF (97.5% in US), and dalbavancin was active against all VAN-S EF . Among SEP, all isolates were inhibited at ≤0.25 mg/L of dalbavancin  and oxacillin resistance ranged from 66.9% in W-EU to 86.5% in E-EU (73.2% in US). BHS exhibited low dalbavancin MIC values  and high S rates for its comparators. Among EFM, VAN-S rates varied from 36.6% in the US to 61.6% in E-EU and 76.1% in W-EU, and dalbavancin inhibited all VAN-S EFM at ≤0.25 mg/L .Overall, the most common GP organisms were Dalbavancin exhibited potent activity and broad spectrum against a large collection of contemporary GP bacteria recovered from patients with BSI in US and EU. Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  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Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Michael D. Huband, BS, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support) Dee Shortridge, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support) Cecilia G. Carvalhaes, MD, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. 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Polygalae Radix, and Kampō, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock.The mammalian circadian clock system regulates physiological function. Crude drugs, containing We examine effective crude drugs, which cover at least one or two of Kampō, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects.in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice  to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 μg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments.We prepared 40 crude drugs. In the Polygalae Radix (ED50: 24.01 μg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm.We found that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.Our results indicate that  The following day, MEFs were stimulated with 100 nM dexamethasone  for 2 h to synchronize the clock gene expression rhythm before being placed in D-MEM  supplemented with 0.1 mM d-luciferin sodium salt  and 10% heat-inactivated FBS. The MEFs were incubated at 37 °C, and bioluminescence was monitored for 1 min at 10 min intervals for six days using a dish-type luminometer .The rhythmic expression of PER2::LUC was measured using a real-time LUC assay in MEFs derived from PER2::LUC knock-in mice  . The cell count was determined using a TC10TM automated cell counter .In experiment 5, a CaMKII inhibitor , an inactive analogue of KN93 , or an ERK 1/2 inhibitor  was added to the culture medium before bioluminescence measurements were taken.ad libitum before the experiments. Experimental animal care was conducted with the authorization of the Animal Welfare Committee of Waseda University (2015-A37).Eight-week-old male C57BL/6J mice were housed in an animal room maintained at a temperature of 22 ± 2 °C, humidity of 60 ± 5%, and a 12 h light/dark cycle  condition). Zeitgeber time (ZT) 0 was defined as the lights-on time, and ZT12 was defined as the lights-off time. The mice were provided a regular diet  and water in vivo experiment. We fed mice with AIN-93M supplemented with or without crude drug, which had the most shortening effect on the period length in the in vitro experiments. This crude drug was given for 2 weeks under LD condition and then for 4 weeks under constant dark (DD) condition. We evaluated the locomotor activity levels under LD and DD conditions, and calculated the free-running period length, which is regarded as an endogenous rhythm in the absence of environmental light signals, under DD condition. In the previous experiments, in vivo administration dose of herbal medicines widely ranged from 0.5 g/kg  and analysed using the CLOCKLAB software , as previously described . Peaks were defined as points at which the bioluminescence was higher than both sides and were confirmed from the waveform. Bioluminescence was measured for 6–10 consecutive days. We numbered them in order starting from the front  were smoothed using an adjusting-averaging method with 2 h running means, as previously described  with Tukey’s or Dunnett’s multiple comparisons test, or two-way ANOVA with Tukey’s multiple comparisons test, while non-parametric analysis was conducted using the Mann–Whitney test, the Kruskal–Wallis test with Dunn’s multiple comparisons test, or the Mann–Whitney test with false discovery rate (FDR) multiple testing correction. In addition, GraphPad Prism  calculated the ED50 and 95% confidence intervals.All values are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism version 6.03 . We verified whether data were normally or non-normally distributed using the D’Agostino–Pearson normality test/one-sample Supplemental Figure 1. Since the PER2::LUC expression rhythm period and the amplitude of peak 1 in each group’s control were slightly different due to differences in cultured conditions and/or the sensitivity of the LumiCycle equipment, the results were recalculated with 100% of the PER2::LUC expression rhythm period and the amplitude of peak 1 in each group’s control , Cnidii Rhizoma (no. 7) and Polyporus (no. 32) significantly shortened PER2::LUC expression rhythm period, while Gardeniae Fructus (no. 5) and Schisandrae Fruits (no. 26) significantly prolonged it  or each crude drug (100 μg/mL), and bioluminescence was monitored for several days. In this experiment, we used distilled water as a control, because we had confirmed in previous experiments that mixing 20 μL of distilled water as vehicle with 2 mL of culture medium/35-mm dishes had no effect on the PER2::LUC expression rhythm. In addition, some previous studies also used distilled water as control   . These rPolygalae Radix had the most significant shortening effect on period length. However, it also decreased PER2::LUC amplitude due to cytotoxicity or desynchronization of cellular clocks. Therefore, we conducted two separate experiments; first, we replaced the medium containing 0 or 100 μg/mL Polygalae Radix with a fresh medium containing 0 μg/mL Polygalae Radix on day 5 (arrow head), and we measured the amplitude of peak 6 ; however, after the change of medium, the amplitude of each peak was recovered to the similar levels as those of peaks in the control group . The number of living cells in both groups  was similar . At concentrations above 50 μg/mL, Polygalae Radix significantly decreased the amplitude . These results indicated that Polygalae Radix elicited its shortening effect in a dose-dependent manner.To evaluate the dose-dependency of me lower  in respot manner . Moreovemplitude . In addiPolygalae Radix, activated the CaMKII and ERK1/2 pathways .In addition, to identify what component of Polygalae Radix induced shortening of PER2::LUC expression rhythm period length through the activation of CaMKII.These results indicated that Polygalae Radix on the free-running period of mice under DD condition as a switch from in vitro experiments to in vivo experiments. Mice were acclimated to control food (AIN-93M) for the first week, divided into the ‘control food’ and ‘Polygalae Radix’ groups, and fed with control food and control food supplemented with 1% Polygalae Radix, respectively (Polygalae Radix was 14.02 g/kg for LD50 (Guan et al. Polygalae Radix added to the control diet was set at 1% in the present experiment, which was the equivalent of approximately 1 g/kg. Mice were fed thus for 2 weeks under LD condition, and then released under DD condition for the following 4 weeks. Locomotor activity rhythms in the ‘1% Polygalae Radix’ group under LD or DD conditions were higher around ZT12 and CT12 than in the control group (Polygalae Radix had no effect on locomotor activity rhythm period length under LD condition (Polygalae Radix shortened free-running period length in mice.We examined the effects of ectively . Consideol group . Polygalondition . Howeverondition . Collectin vitro and/or in vivo (Isojima et al. Polygalae Radix was found to show the most significant effect. In addition, we found that the effects of Polygalae Radix were suppressed by the inhibition of CaMKII activation. Moreover, the period shortening effects of Polygalae Radix were confirmed in vivo, and included the evaluation of the free-running period of locomotor activity rhythm in mice.Several studies have identified pharmacologically active and bioactive compounds that affect clock gene expression rhythm period length in vitro experiments showed that Polygalae Radix decreased the amplitude of PER2::LUC and Bmal1-Eluc expression rhythms to the same proportion as it shortened period length. Other in vitro experiments revealed that Polygalae Radix was not cytotoxic, as Polygalae Radix had no effect on living cell count after six days of culturing and MEFs cultured in media containing 0 or 100 μg/mL Polygalae Radix showed similar bioluminescence levels after medium change. As well known, during long-term cell culturing, the circadian rhythm is attenuated when each cell is out of phase. However, medium change resets the rhythm and restored higher amplitude rhythms. These results indicate that Polygalae Radix is not cytotoxic at this concentration. The ED50 for period shorting effect was 24.01 μg/mL, and this concentration may be quite low, because Polygalae Radix is complex of crude components.The Polygalae Radix shortened not only PER2::LUC rhythm period length in MEFs but also the free-running period length in mice. Several signalling pathways, especially CaMKII pathways, are involved in the determination of biological rhythm period length (Kon et al. Bmal1-luc expression rhythm period length (Kon et al. Polygalae Radix activated the ERK1/2 and CaMKII pathways (Hu et al. Polygalae Radix may activate CaMKII and shorten PER2::LUC expression rhythm period length. Moreover, a previous study showed that CamK2aK42R mutant mice, which has no kinase activity of CaMKII, showed a longer free-running period length as compared to wild type mice (Kon et al. We found that Polygalae Radix activated the CaMKII signalling pathway and shortened clock gene expression rhythm and locomotor activity rhythm period lengths. However, an important limitation of this study is that we did not identify what component of Polygalae Radix has the shortening effect on biological rhythm. It is possible that Polygalae Radix also contains other substances that have already been reported to affect biological rhythms, such as catechin and nobiletin (Li et al. This study showed that in vitro experiments showed that low Polygalae Radix concentrations had no effect on PER2::LUC amplitude but slightly shortened PER2::LUC expression rhythm period. In vivo experiments showed that control food supplemented with 1% Polygalae Radix increased locomotor activity levels around ZT12 and CT12, and slightly shortened the free-running period. In the previous experiments, in vivo administration dose of herbal medicines widely ranged from 0.5 g/kg (Motohashi et al. Polygalae Radix added to the control diet was set at 1%, and this concentration was the equivalent of approximately 1 g/kg per day. Based on present results along with previous observations, 1% is a low Polygalae Radix concentration and its shortening effects would have been better observed with increase in concentration to the 3–5% range, for example. However, increasing the concentration of Polygalae Radix may have decreased the amount of food consumed by mice, and decrease in food consumption leads to advance the locomotor activity rhythms (Mendoza et al. The Polygalae Radix, Polyporus and Bupleuri Radix were good candidates for the effective acute manipulation of the peripheral circadian clock phase, with stimulation time-of-day dependency in vitro and in vivo (Motohashi et al. Polyporus had a shortening effect and that Bupleuri Radix had no effect on circadian period length. These results indicate that the circadian period length pathway may be different from that of the circadian phase. Actually, previous studies have reported that nobiletin affects circadian period length and phase by stimulating different pathways (He et al. In previous studies, we found that as opposed to Polygalae Radix shortened clock gene expression rhythm period length via the CaMKII pathway and shortened the free-running period length. Compared to the effects of many medicinal drugs, crude drugs are generally better tolerated (Sardesai 2+-related pathways, including the CaMKII pathway, regulate sleep duration in mammals (Tatsuki et al. Polygalae Radix may be regarded as a new therapy and that the CaMKII pathway may be regarded as a target pathway, for disorders caused by delayed sleep phase syndrome, extreme eveningness and social jetlag. This is because these conditions require a phase advance and/or shortening of circadian rhythms.In conclusion, Sardesai . MoreoveClick here for additional data file.


Water retention capacity of HPMC varied linearly with Mv. The formation and curation of gels were monitored by rheology employing consecutive steps such as heating ramp (25–90°C), time sweep (90°C), cooling ramp (90–25°C), time sweep (25°C), and frequency sweep. Additionally, creep-recovery tests were performed. HPMC above 1.5% w/w delayed the range of gelatinization temperature of starch up to 2°C. Viscoelasticity and stiffness of corn starch gels with HPMC depend on both the amount of polymer added and Mv of the HPMC. Finally, to achieve corn gels with mimetic viscoelastic properties to wheat gel , HPMC with relatively low viscosity (low Mv) is necessary to be added at certain content.The objective of this study is to determine the effect of the addition of hydroxypropyl methylcellulose (HPMC)  with three different viscosities  to corn starch  gels. Average viscosimetric molecular weights (M Nowadays, gluten-free products are increasingly in demand due to more people are diagnosed as having celiac disease. Most bakery products are made with wheat flour that contains gluten, but there are flours (some of them underexploited) such as corn, rice, chestnut, and acorn, among others, useful to produce gluten-free starchy food materials . HoweverHydrocolloids are capable to control the swelling power, solubility, and rheology of starch aqueous systems, throughout the stabilization of emulsions, suspensions, and foams. In fact, they are widely used in the manufacturing of starchy foodstuffs improving the properties of starch gels .A common hydrocolloid used for cited purposes is the hydroxypropyl methylcellulose (HPMC) that belongs to the group of cellulose ethers and has a wide range of applications in food, cosmetics, adhesives, agriculture, and textiles . HPMC is5 g/mol) and, at constant guar gum content  the peak viscosity and setback increased with the lowest molecular weight guar gum and the addition of guar gum delayed the pasting temperature from 75.7°C (for CS) up to 79.6°C.In relation to the effect of the addition of hydrocolloids with different sizes to corn starch (CS), Funami et al.  studied In these previous studies, the effect of the addition of some hydrocolloids, such as xanthan gum, guar gum, and HPMC, among others, was investigated but without considering the effect of molecular size of HPMC. Likewise, considering the specific interaction depending on the starch source, it was hypothesized that the molecular size of HPMC added to CS, commonly used in gluten-free products, might affect starch interactions and in consequence viscoelasticity of CS gels. Therefore, the aim of this study is to determine the effect of the addition of three different HPMC with different molecular weights on CS gels by means of the evaluation of the viscoelastic behavior.2O at 20°C, . All the materials were provided by Sigma Aldrich.Corn starch  and wheat starch . HPMC, with constant methoxyl and hydroxypropyl content, 28.7 and 9.1%, respectively, of three different apparent viscosities, 40–60 (HPMC L), 80–120 (HPMC M), and 2,600–5,600 cP (HPMC H), at 2% in H0) and HPMC (μ) solutions, the relative viscosity (μμ = μ/0) and the specific viscosity (μsp = μr− 1) were calculated. Using the values of μr and μsp, the intrinsic viscosity (μ) was calculated by the Huggins (Eq. 1) and Kraemer (Eq. 2) Equations . Solutions were prepared with a concentration of 0.1%  in distilled water. For each HPMC, three dilutions were performed . All measurements (at least 5 replicates) were performed at 25°C (± 0.1°C). At these low hydrocolloid content, density was constant and equal to the density of solvent (water). With the absolute viscosities of solvent /C, and KH and KK are the Huggins and Kraemer constants, respectively. The values of viscosity average molecular weight, Mv, were determined by the Mark–Houwink Equation (Eq. 3):where μwhere (μ) (dl/g) is the intrinsic viscosity and K and α values depended on the solute–solvent system at a constant temperature.–1) was calculated by Eq. (4).Water retention capacity (WRC) values were determined to characterize the CS and HPMC-CS mixtures. WRC was calculated following the protocol established by Robertson et al. . BrieflyFRW is the fresh residue weight and DRW is the dry residue weight. WRC of HPMC was not directly determined due to water forms a viscous gel :where cous gel , but it WRCCS is the WRC of CS, wdHPMC and wdCS are the dry weights of HPMC and starch in the mixture, respectively.where Dispersions with constant solids content (30% w/w) were prepared by mixing CS and HPMC (by substitution) at different concentrations  and distilled water. First, the solids were gently blended and later distilled water was added. The slurry was mildly stirred (100 rpm) using a magnetic stirrer, for 10 min at room temperature. Aqueous HPMC solutions (2.0% w/w) were prepared by mixing HPMC and distilled water. These mixtures were kept in agitation until completely dissolved. The rheological characterization was performed with a stress-controlled rheometer  using a plate–plate geometry . Samples (1.2 ml) were loaded between the parallel plates and compressed up to obtain a gap of 0.5 mm. In the case of HPMC solutions, the same geometry was used with a smaller gap (0.3 mm) with a volume of sample of 0.9 ml. The measurements were performed at different temperatures [from 25 up to 90°C (± 0.1°C)], controlled by a Peltier system. All the samples were covered with light paraffin oil to prevent water evaporation. Tests were carried out at least in triplicate.–1, 1 Hz, 100 Pa), to accomplish starch gelatinization; (ii) time sweep ; (iii) temperature sweep ; (iv) time sweep ; and finally (v) frequency sweep  inside LVR of formed gels (preliminary tests were performed to determine the corresponding LVR). For HPMC solutions (2.0% w/w in water) a strain sweep from 0.1 to 100% at a constant frequency (1 Hz) was made to analyze the LVR. Then, a temperature sweep was carried out from 35 to 80°C at a constant strain of 10% and a constant frequency of 1 Hz (inside LVR). Strain sweep tests from 0.1 to 10% at a constant frequency (1 Hz) were made on dispersions and gels to define the corresponding linear viscoelastic regions (LVRs). Second, dispersions were subjected to the procedure previously reported  with min–1) = γ/σ, where γ is the strain experimentally measured. The Burgers model , of 100 or 400 Pa within the LVR during the creep phase. Before the measurements, the gels were rested for 15 min in the rheometer to allow the sample equilibrium. During the creep period, the selected constant stress was applied for 120 s and the recovery (stress zero) period lasted 120 s . The resrs model  was empl0J (Pa–1) is the instantaneous compliance, mJ (Pa–1) is the viscoelastic compliance, λ (s) is the mean retardation time, t (s) is the phase time, η0 (Pa⋅s) is the zero-shear viscosity, and Jmax (Pa–1) is the maximum creep compliance. The goodness of fitting was evaluated by means of the corresponding coefficients of determination [R2 and the root mean square error (RMSE)].where p-values ≤ 0.05 . All the experimental results were expressed as mean ± SD from at least triplicate experiments.Experimental data were analyzed through one-factor analysis of variance (ANOVA), followed by the Duncan test and considering significant H and KK constants, intrinsic viscosity (μ) values, and the average viscosimetric molecular weight (Mv) for each HPMC. Mv was calculated by the Mark–Houwink equation, Eq. (3), where the values of K and α parameters were 3.39 × 10–4 dl/g and 0.88, respectively, previously determined by Vázquez et al.  and obtained values from 3.78 to 14.98 dl/g and Mv values from 39.7 × 103 to 190.0 × 103 g/mol. Bustamante et al. z et al.  studied e et al.  studied H and KK obtained were from 0.21 to 0.63 and from –0.26 to –0.04, respectively. As the nominal viscosity of the HPMC increased, the KH, KK, (μ), and Mv significantly (p < 0.05) increased as well. HPMC solubility can be related to intrinsic viscosity (v) causes a decrease in the solubility of the biopolymer in water. The values of KH and KK could be analyzed to study the interactions between the polymers and solvents. In fact, KH < 0.5 and KK < 0 indicate good solvents (strong interactions polymer–solvent). An additional criterium given by KH – KK close to 0.5 based on the solvent goodness (solvent θ), defined a condition in which neither inter- or intramolecular polymer aggregation is produced and particulates behave as non-perturbed units and polymers adopt an extended conformation (flexible coil) in the solvent used (H = 0.63 > 0.5 and KH – KK close to 0.7) from theoretical considerations for good solvent–polymer interactions and this fact could be tested out in its low water solubility and slow solubilization.The values of Kiscosity . This beCS was 0.95 ± 0.01 (g water/g d.b.) whereas WRC values of HPMC L, M, and H, were 8.54 ± 0.28, 8.92 ± 0.22, and 12.97 ± 0.45 (g water/g d.b.), respectively. Yılmaz et al.  are more accessible for water and therefore, more water amount could be retained. Additionally, a linear correlation was found between intrinsic viscosity (μ), of HPMC and WRCHPMC (R2 > 0.99). As (μ) was proportional to Mv, WRCHPMC also varied linearly with Mv of HPMC (R2 > 0.99).In order to calculate the WRC of the different HPMC samples, mixtures of starch and HPMC were made. By means of the Eq. (4) the WRC of CS and mixtures were calculated. Then, using Eq. (5) the WRC of HPMC was evaluated. WRCz et al.  reported0) was evaluated from the first inflection point of elastic modulus (G′) and the final gelatinization temperature (Tf) was determined from the point in which the slope of G′ changes after the peak  to 62.8 ± 1.1°C (for 2.0%), while with HPMC M was 61.8 ± 1.1°C (for 0.5 and 1%) and 62.8 ± 1.1°C (for 1.5 and 2.0%). However, T0 value for HPMC H hardly changed, 62.8 ± 0.8°C, independently of its content. In addition, the Tf was delayed in a similar way, from 74.2 ± 0.8°C for CS gels to 75.3 and 76.4 ± 0.8°C with the presence of HPMC. This delay of the gelatinization process could be related to the water absorption of the added hydrocolloid (2O at 20°C) by substitution. Moreover, Zhang et al. (Dispersions were subjected to a temperature sweep (step i) where the onset gelatinization temperature   . For theerature) .As it can be observed in Analyzing in detail the results shown in 90′ = G′90f – G′90i, were evaluated where G′90f is the G′ after 30 min and G′90i is the initial value. For CS gel, without the addition of HPMC, ΔG90′ was −970 Pa, starch gels with HMPC L and M showed lower changes, but always G′90f < G′90i. These variations decreased with the amount of HPMC added. However, with the addition of HPMC H, two different trends were observed depending on the amount of polymer used. Negative values of ΔG90′ were also determined at low content (0.5% w/w), but increasing HPMC H content (>1.0% w/w) positive ΔG90′ values were determined achieving 1,560 Pa at 2.0% w/w. These results showed that Mv of HPMC clearly modifies the formation rate of gels and their final characteristics. In our tests, despite tested polymers had the same substitution degree, when they were added at the same content, the gel features were significantly different. The addition of HPMC, independently of Mv, dampened the decay of G′ observed in CS gels due to the presence of hydroxyl groups that are capable to form additional hydrogen bonds and therefore stronger structures   to evaluructures . The pos25′ = G′25f – G′25i. 25i and ΔG25′ values for tested gels. Globally, G′25i values decreased with increasing polymer addition of HPMC L and M. This result indicated that short-time starch retrogradation given by the rapid recrystallization of amylose molecules was slowed down by these HPMC additions due to the increase of G′ during cooling (step iii) was significantly lower. However, this decrease of G′25i only was observed at the lowest content of HPMC H (0.5% w/w) because beyond that level G′25i showed a steady increase with the level of HPMC addition (2.0% w/w) maintaining the trend observed at 90°C. In fact, the addition of hydrocolloids can increase, decrease, or have no effect on the extent of (short- and long-time) starch retrogradation, depending on the gel preparation method, temperature, time, added amount of hydrocolloid, and measurement techniques of starch retrogradation  decreased (up to 865 Pa with HPMC L at 2.0% w/w). No significant differences among ΔG25′ values of gels with different amounts of HPMC H added were observed, and these changes were close to that determined in CS gel (p > 0.05).In the next stage, the temperature sweep from 90 to 25°C (step iii), radation . The afoThe previous time sweep (step iv) showed that the gels have reached a stable state and are ready to analyze their structures. For this reason, frequency sweeps (step v)  were carried out to characterize the viscoelastic properties of gels . For allFor comparative purposes, the frequency sweep for a WS gel  formed under the same conditions that tested CS gels included in v) a positive linear trend was found (R2 > 0.98) with HPMC content. The firmness of gels with HPMC L and M decreased linearly with the amount of polymer added and no significant differences (p > 0.05) between both polymers were found. In samples with HPMC H, it was observed that the previous trend is maintained only at the low addition (0.5% w/w) level. At higher content (>1.0% w/w), the opposite effect is observed, and consequently, the change of G′ diminished  due to the increase of G′ with additional amounts of polymer added. This fact is given by the high viscosity of HPMC in the continuous phase . Parameters obtained using the Burgers model (Eqs 6 and 7) are shown in 0 values varied from 43.0 10–6 Pa–1 for CS gel up to 56.5, 57.7, and 40.2 10–6 Pa–1 for gels at maximum polymer addition (2.0% w/w) of HPMC L, M and H, respectively. For WS gel, J0 was 49.6 × 10–6 Pa–1. The Jm values obtained varied in a restricted range between 3.3 and 5.3 × 10–6 Pa–1, excepting for CS gels with HPMC L and M (at 2.0% w/w) which were noticeably higher, 13.92 and 44.70 × 10–6 Pa–1, respectively. In general, λ and μ0 varied between 4.1 and 5.7 s and 286 and 597 Pa⋅s, respectively. The Jmax values varied from 46.2 to 56.1 × 10–6 Pa–1, excepting for CS gels with HPMC L and M (at 2.0% w/w), 73.6 and 108.4 × 10–6 Pa–1, respectively. These results were also in agreement with the obtained results by oscillatory tests where G′ and tan δ values varied sharply showing lower G′ values and higher tan δ than remaining samples. J0 varied inversely with respect to G′ (from frequency sweep at 0.1 Hz and 25°C) and a successful linear correlation between both the parameters could be established (R2 > 0.96). Consequently, the trends found for G′ with HPMC amount in the oscillatory tests are also valid here. Similarly, Onyango et al.  values increased when the HPMC content increased in gluten-free doughs based on chestnut flour.The Burgers model satisfactorily fitted the experimental creep and recovery data  of HPMC. The temperature of gel point was invariant with Mv (when substitution degree is maintained constant), but HPMC with high Mv showed an aggregation step at temperatures near (below) gel point. The HPMC addition to CS gels causes significant changes in viscoelasticity and stiffness of samples. These effects depend on the amount of polymer added and the Mv of the HPMC.Water retention capacity of tested HPMC varied linearly with the average viscosimetric molecular weight  of HPMC of 27.2⋅103 g/mol or 1.5% (w/w) of HPMC of 32.7⋅103 g/mol must be added. Nevertheless, the use of HPMC with high Mv is not adequate for this purpose.Based on the viscoelastic response and creep-recovery tests, maintaining constant solid content (30% w/w), CS gels plus HPMC with mimetic features to WS gels can be only obtained under certain circumstances. HPMC with relatively low MThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LM: methodology, data curation, and writing—original draft preparation. CR: supervision, validation, and writing—reviewing, and editing. RM: conceptualization, data curation, supervision, validation, and writing—reviewing and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.