In this study, we determined whether the amniotic fluid of mice harbors a microbiota in late gestation. The profiles of the amniotic fluids of pups located proximally or distally to the cervix were characterized through quantitative real-time PCR, 16S rRNA gene sequencing, and culture (N = 21 dams). These profiles were compared to those of technical controls for bacterial and DNA contamination. The load of 16S rRNA genes in the amniotic fluid exceeded that in controls. Additionally, the 16S rRNA gene profiles of the amniotic fluid differed from those of controls, with Corynebacterium tuberculostearicum being differentially more abundant in amniotic fluid profiles; however, this bacterium was not cultured from amniotic fluid. Of the 42 attempted bacterial cultures of amniotic fluids, only one yielded bacterial growth – Lactobacillus murinus. The 16S rRNA gene of this common murine-associated bacterium was not detected in any amniotic fluid sample, suggesting it did not originate from the amniotic fluid. No differences in the 16S rRNA gene load, 16S rRNA gene profile, or bacterial culture were observed between the amniotic fluids located Proximally and distally to the cervix. Collectively, these data indicate that, although there is a modest DNA signal of bacteria in murine amniotic fluid, there is no evidence that this signal represents a viable microbiota. While this means that amniotic fluid is not a source of microorganisms for in utero colonization in mice, it may nevertheless contribute to fetal exposure to microbial components. The developmental consequences of this observation warrant further investigation.The existence of an amniotic fluid microbiota  in mammals is controversial. Its existence would require a fundamental reconsideration of fetal  Indeed, the amniotic fluid is essential for fetal development and maturation , 2. As sin utero , 89, 90.in utero , 91–95.Investigations of human amniotic fluid in normal pregnancy have yielded contradictory results. Multiple studies using DNA sequencing techniques , 96–98 aThe existence of an amniotic fluid microbiota would require a fundamental reconsideration of the role of intra-amniotic microorganisms in fetal development and pregnancy outcomes. Such reconsideration would require the implementation of animal models to perform mechanistic experimentation of host immune-microbe interactions. Yet, there have been only a limited number of studies investigating the presence of an amniotic fluid microbiota in animal models, specifically cattle, horses, sheep, goats, and rats , harborsad libitum. Females (8-12 weeks old) were mated with males of demonstrated fertility. Daily examination was performed to assess the appearance of a vaginal plug, which indicated 0.5 days post coitum (dpc). Dams were then housed separately from the males and their weights were checked daily. An increase in weight of ≥ 2 g by 12.5 dpc confirmed pregnancy. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) (Protocol No. 18-03-0584) of Wayne State University.C57BL/6 mice were obtained from The Jackson Laboratory  and bred at the C.S. Mott Center for Human Growth and Development at Wayne State University, Detroit, MI, USA in the specific-pathogen-free (SPF) animal care facility. Mice were housed under a 12 h:12 h light/dark cycle and had access to food , and water Twenty-one pregnant mice were included in this study Figure 1For all mice, proximal and distal amniotic fluid samples (~40 µL each) were cultured in 200 µL of Brain-Heart-Infusion (BHI) broth supplemented with 5 mg/L of hemin and 2 µg/L of vitamin K1 under oxic and anoxic conditions for 48 hours. For the first eight mice in the study, 40 µL of the BHI culture were then plated on supplemented BHI agar plates and cultured under the respective atmospheric condition for an additional 48 hours, and resultant bacterial isolates were taxonomically characterized. For the last 13 mice in the study, 40 µL of the BHI culture were subsequently plated on supplemented BHI agar plates and cultured under the respective atmospheric condition if turbidity of the broth culture was observed after 48 hours of incubation. Any potential growth of bacteria in BHI broth cultures of proximal and distal amniotic fluid samples was then assessed through qPCR and 16S rRNA gene sequencing. As each amniotic fluid sample was cultured under oxic and anoxic conditions, 125 µL each from the oxic and anoxic broth cultures were pooled and stored at −80°C. The 16S rRNA gene loads and profiles of these amniotic fluid broth cultures were compared to those of six uninoculated BHI broth negative controls using qPCR and 16S rRNA gene sequencing.Corynebacterium tuberculostearicum, in particular, was more relatively abundant in proximal and distal amniotic fluid samples than in background technical controls  per mL and CFU limits of detection were calculated. All cultures were performed in duplicate and were CFU counts were averaged between the duplicate plates. Colony counts of the stock cultures indicated the original stock contained approximately 976,250 CFU/mL. Following 48 hours of broth enrichment, plated cultures were countable for the 10-3 (630 CFUs) and 10-4 dilutions (11 CFUs); no colonies were observed for the 10-5 dilution. After backcalculating the dilution and original inoculum volume, approximately four CFUs were inoculated into the 10-4 broth dilution tube, indicating that the limit of detection for C. tuberculostearicum was four CFUs.Secondarily, after finding that DNA from the 16S rRNA gene of Genomic DNA was extracted within a biological safety cabinet from amniotic fluid and BHI broth samples, as well as positive  and negative  controls using the DNeasy PowerLyzer Powersoil kit , with minor modifications to the manufacturer’s protocols as previously described , 108. SpTo measure total 16S rRNA gene abundance within samples, amplification of the V1-V2 region of the 16S rRNA gene was performed according to the protocol of Dickson et al. , with mivia the dual indexing strategy developed by Kozich et al.  and the online MiSeq protocol  with minor modifications. These modifications included allowing truncation lengths of 250 bp and 150 bp and a maximum number of expected errors of 2 bp and 7 bp for forward and reverse reads, respectively. To allow for increased power to detect rare variants, sample inference allowed for the pooling of samples. Additionally, samples in the resulting sequence table were pooled prior to removal of chimeric sequences. Sequences were then classified by using the silva_nr_v132_train_set database with a minimum bootstrap value of 80%, and sequences derived from Archaea, chloroplast, or Eukaryota were removed.Raw sequence reads were processed by using DADA2 (v 1.12) . An analdecontam version 1.6.0  using phyloseq . The bacW = 6, p = 0.0003) and distal  amniotic fluid samples than in blank extraction controls. However, the 16S rRNA gene signal did not differ between paired proximal and distal samples    and distal  amniotic fluid samples than in controls , Pseudomonas (ASV 24), and Sphingobium (ASV 33) . Prior to removing potential background DNA contaminants, the 16S rRNA gene profiles of both the proximal and distal amniotic fluid samples differed from that of negative controls  or in evenness (Shannon-Wiener diversity) . The structure of these profiles did not differ either by mouse dam ID  or proximity to the cervix  .To address whether the 16S rRNA gene signals in amniotic fluid may represent potential background DNA contamination, the R package n medium , 120, whLactobacillus murinus NBRC 14221 (NR_112689). The distal amniotic fluid sample from Dam #3 did not have 16S rRNA gene concentrations outside the range of other amniotic fluid samples in the study  yielded bacterial growth   had at least 500 sequence reads remaining . The structures of the proximal and distal culture 16S rRNA gene profiles did not vary by mouse dam ID  or differ based on proximity to the cervix .Secondarily, for 13/21 dams , we further characterized the 16S rRNA gene concentration and profile of the amniotic fluid-inoculated BHI broths and compared these data to those of stock control broth. Overall, the 16S rRNA gene signal of inoculated broths did not exceed that of stock control broth  acnes, Staphylococcus epidermidis, Ralstonia, Streptococcus anginosus, and Peptoniphilus dominance. The second study , typical commensal skin bacteria  has been previously detected in human amniotic fluid using molecular techniques; however, this bacterium was not recovered using conventional culture methods (Streptococcus ASV detected in the current study (ASV 13) had an identical sequence match with multiple members of the Mitis group of the genus Streptococcus, which are common inhabitants of the oral cavity and upper respiratory tract in humans , but a species-level taxonomy could not be assigned, indicating that the V4 region of the 16S rRNA gene is not adequate for differentiation of the Pseudomonas species, at least those detected herein. Sphingobium is typically an environmental microorganism  can be found below: The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) (Protocol No. 18-03-0584).KT, NG-L, and RR co-conceived the study. AW, JMG, JG, VG-F, and DK performed the laboratory work and data processing. AW, ZS, and KT completed statistical analysis. AW, ZS, JG, NG-L, and KT co-wrote the manuscript. All authors read, edited, and approved the manuscript.Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U. S. Department of Health and Human Services (NICHD/NIH/DHHS), and, in part, with federal funds from the NICHD/NIH/DHHS under Contract No. HHSN275201300006C. This research was also supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dr. Romero has contributed to this work as part of his official duties as an employee of the United States Federal Government.This research was supported, in part, by the Perinatology Research Branch (PRB), Division of Intramural Research, 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.


We study the dynamics of a squirmer in a nematic liquid crystal using the multiparticle collision dynamics (MPCD) method. A recently developed nematic MPCD method  which employs a tensor order parameter to describe the spatial and temporal variations of the nematic order is used to simulate the suspending anisotropic fluid. Considering both nematodynamic effects (anisotropic viscosity and elasticity) and thermal fluctuations, in the present study, we couple the nematic MPCD algorithm with a molecular dynamics (MD) scheme for the squirmer. A unique feature of the proposed method is that the nematic order, the fluid, and the squirmer are all represented in a particle-based framework. To test the applicability of this nematic MPCD-MD method, we simulate the dynamics of a spherical squirmer with homeotropic surface anchoring conditions in a bulk domain. The importance of anisotropic viscosity and elasticity on the squirmer’s speed and orientation is studied for different values of self-propulsion strength and squirmer type . In sharp contrast to Newtonian fluids, the speed of the squirmer in a nematic fluid depends on the squirmer type. Interestingly, the speed of a strong pusher is smaller in the nematic fluid than for the Newtonian case. The orientational dynamics of the squirmer in the nematic fluid also shows a non-trivial dependence on the squirmer type. Our results compare well with existing experimental and numerical data. The full particle-based framework could be easily extended to model the dynamics of multiple squirmers in anisotropic fluids. Microswimmers are natural or artificial self-driven entities which are capable of converting stored or ambient energy into a systematic motion in a suspending fluid medium . RecentlE. coli, B. subtilis, and P. mirabilis) move parallel to the nematic director (anisotropy axis) in a biocompatible LC, namely a solution of water and biocompatible compound DSCG (disodium cromoglycate) [While the dynamics of microswimmers in Newtonian fluids are reasonably well studied, the dynamics of microswimmers in anisotropic and elastic environments such as LCs were given due consideration only recently –9. Unlikglycate) –15. The glycate)  have stuglycate) –18. Thisglycate) , 19, 20.glycate)  and defeglycate) –26 have A closer look into the theoretical literature shows that dynamics of microswimmers in LCs have been investigated using either the lattice Boltzmann method  or finitIn the following we provide a detail description of the nematic MPCD-MD algorithm which we use to study the dynamics of a squirmer moving in a nematic LC medium.The basic MPCD method employs pointlike particles to represent the fluid. These particles are not fluid molecules rather they represent a parcel of fluid. To describe a Newtonian fluid on a coarse-grained level, the fluid is represented by velocity . In the mentclass2pt{minimL is elastic constant (note that we have assumed the one-elastic-constant approximation) and The evolution of the particle-based tensor order parameter given by 1\documentipliers . The molheory as  \documenIn the basic MPCD method, streaming and collision steps are performed in such a way that the mass, linear momentum, angular momentum and temperature of the fluid remain constant. This scheme effectively combines the hydrodynamics (the Navier–Stokes behavior at a coarse-grained level) and thermal fluctuations. For Newtonian fluids, particles are moved ballistically in the streaming step over a time interval ing form 3\documenthe form 5\documenhat cell . To calchat cell .j in the cell relative to the center-of-mass of the cell, and To perform the collision step at fixed discrete time intervals, the simulation domain is divided into small cubic cells of length ach cell , 43. In ion step .The particle-based framework of MPCD makes it straightforward to model the dynamics of embedded particles  in a fluid medium by coupling the MPCD algorithm with molecular dynamics (MD) like scheme for embedded particles .R with prescribed surface velocity [C. reinhardtii cells), pusher for E. coli), and neutral for The squirmer is a rigid spherical particle of radius velocity  \documenIn the simulation domain, mentclass2pt{minimigned as 8\documenDuring the streaming step, the squirmer motion is updated by employing an MD-like step in which we integrate the equation of motion in the following formNote that the fluid particles interact with the squirmer via collision during the streaming step . To impotreaming . At the During the collision step, the squirmer is coupled to the fluid via the virtual particles . These vIn the MPCD framework, the collision cell length  numbers ). In theL). In the present study, we have taken 5CB as a model LC fluid [The nematic fluid is described by six viscosity coefficients  in lyotropic chromonic LC is about 2 times smaller than in a Newtonian medium. Figure The speed of a squirmer, v et al.  recently$\beta = $$\end{doz direction ; this is due to the fact the resistance to flow is smaller in the direction of the director. On the other hand, for the puller the streamlines are more compressed. Inspecting Fig. Figure eriments  and fulleriments . Figure We have proposed a particle-based mesoscopic simulation method for microswimmers in nematic LCs. The existing nematic MPCD method is extended by combining it with an MD scheme for squirmer. We have tested this nematic MPCD-MD method for a single squirmer in an unbounded nematic LC medium. We correctly reproduce the orientation dynamics of squirmer in nematic LCs. The proposed method could be easily extended to study the collective dynamics of multiple squirmers in LCs.We find that the speed of a squirmer moving within a nematic fluid depends nonlinearly on both


Background: Manual lymph drainage (MLD) is one of the common treatments for breast cancer-related lymphedema (BCRL). Although the primary goal of MLD is to drain the excessive fluid accumulated in the affected upper limb and trunk to an area of the body that drains usually, the use of MLD is decided based on swelling and subjective symptoms, without assessing whether there is fluid accumulated in the affected region. The purpose of this study was to examine truncal fluid distribution in a sample of BCRL patients and investigate any correlation between such fluid distribution and swelling or subjective symptoms.Methods and Results: An observational study was conducted with 13 women who had unilateral, upper extremity BCRL. Fluid distribution was evaluated by using two magnetic resonance imaging (MRI) sequences: half-Fourier acquisition single-shot turbo spin echo and three-dimensional double-echo steady-state. The presence of swelling was determined by lymphedema therapists, and subjective symptoms were measured by using a visual analog scale. On MRI, no participants had any free water signals in the trunk. However, seven had swelling and all 13 had some kind of subjective symptoms on the affected side of their trunk.Conclusions: These results suggest that swelling and subjective symptoms do not correlate with the presence of truncal fluid. For such cases, a different approach than MLD may be needed to address truncal swelling and related subjective symptoms. Checking for the presence of fluid in the truncal region may help MLD be used more appropriately. Axillary lymph nodes are junctions that drain lymph from ipsilateral arm, breast, and nearby areas into deep networks of lymphatic vessels. Accordingly, lymphedema often develops in the trunk and upper limb on the same side as the affected breast.3After breast cancer treatment, 8%–56% of patients suffer from breast cancer-related lymphedema (BCRL).5 However, to date, BCRL studies mainly have focused on the upper limb rather than the trunk, as lymphedema is more apparent in the arm than in the trunk.3 Therefore, truncal fluid accumulation in BCRL patients remains largely unexamined.Truncal lymphedema can cause some degree of physical and psychological sequelae .6 Although the primary goal of MLD is to drain the excessive fluid accumulated in the affected upper limb and trunk into a normally draining area, the decision to use MLD is based on the presence of swelling and subjective symptoms, without determining whether there is fluid accumulated in the region.Manual lymph drainage (MLD) is one of the most common treatments for edema in the trunk and upper limbs of BCRL patients.7 This finding suggested that swelling might not correlate with the presence of fluid in the upper limb. Though not yet studied, this finding might be true for the trunk, as well. Therefore, the purpose of this study was to use MRI to examine truncal fluid distribution in BCRL patients, and to investigate the correlations between fluid distribution, and swelling or subjective symptoms.Recently, Niwa et al. reported that magnetic resonance imaging (MRI) showed that some BCRL patients' affected upper limbs had no fluid signals despite being swollen.This study used an observational design and was conducted from February 2019 to February 2020.Patients with unilateral BCRL were recruited from among those referred for lymphedema care to the lymphedema outpatient unit of Nagoya University Hospital or the Japanese Red Cross Nagoya Daini Hospital.8 Second, they had received primary and adjuvant breast cancer treatment  that was completed at least 6 months before the study. Those receiving ongoing hormonal therapy were allowed to participate. Third, their physician had given permission for them to have the MRI examination.All participants were assessed for eligibility by using the following criteria. First, they had been diagnosed with BCRL by a physician. To diagnose lymphedema, the affected arm circumference must have exceeded the contralateral arm by >1 cm. This threshold level has been demonstrated to be adequate for the diagnosis of lymphedema in Japanese BCRL patients.This study was approved by the Ethics Committee of Nagoya University  and the Japanese Red Cross Nagoya Daini Hospital (no. 1323), and it conforms to all conventions governing ethical conduct as stated in the Declaration of Helsinki. All participants gave their informed consent before study inclusion, and their anonymity was preserved.10Fluid distribution was examined by using MRI. This type of imaging is appropriate for evaluating lymphedema status, because it provides excellent contrast for visualizing soft tissues and fluid accumulation without ionizing radiation.Images were obtained by using a 3-Tesla MRI scanner . Two sequences were used: half-Fourier acquisition single-shot turbo spin echo (HASTE) and three-dimensional double-echo steady-state (3D-DESS).7The 3D-DESS sequence is suitable for three-dimensional observation of fluid accumulation in lymphedematous tissue and was chosen to examine imaging differences between each participant's trunk and affected upper limb.The 3D-DESS imaging parameters were as follows: repetition time = 14.16 ms; echo time = 5.00 ms; flip angle = 28°; bandwidth = 250 Hz/pixel; field of view = 256 mm; number of slabs = 1; slices per slab = 160; and slice thickness = 1.00 mm. To reduce the physical burden on subjects, the acquisition time was shortened by using generalized auto-calibrating partial acquisition, a type of simultaneous acquisition of spatial harmonics. The parallel acquisition technique factor was set to 2. Moreover, to lessen image distortion, a two-dimensional distortion correction filter was used. The imaging range was limited to the affected side, from the midsagittal plane of the upper body. The imaging area was divided into three sections: the upper region (shoulder joint and chest), the middle region (elbow and upper abdomen), and the lower region (wrist and lower abdomen). The acquisition time was approximately 8 minutes per section, for a total of 24 minutes for one side.HASTE is a high-speed turbo-spin echo T2-weighted sequence. This sequence was selected to provide an image of the whole trunk, as it can be used with a short breath-holding time and was suitable for observing free water, which has a long transverse relaxation time.none, and the spectral attenuated inversion recovery was set at strong.The HASTE imaging parameters were as follows: repetition time = 700 ms; echo time = 69 ms; flip angle = 120°; bandwidth = 391 Hz/pixel; field of view = 360 mm; number of slabs = 24; and slice thickness = 6.00 mm. The breath-holding time was 20 seconds, the fat suppression was set at The presence of signal intensity areas was determined by the consensus of three experienced observers using three-dimensional maximum intensity projection analysis of the MRI images.Truncal swelling was evaluated by the inspection and palpation of the participants by the therapists-in-charge of lymphedema patients. The lymphedema therapists classified each participant into one of two groups: those with and those without truncal swelling.11 These particular symptoms were selected from previous studies conducted to verify the effectiveness of BCRL treatment.12Subjective symptoms  were measured by using a visual analog scale.Eleven of the 13 participants were observed by using both the 3D-DESS and HASTE sequences, and two were observed by using only 3D-DESS. For the 3D-DESS sequence, eight participants had hyper-intense signals in the upper, middle, and lower regions of their affected upper limbs, but not in the subcutaneous area of their trunks . Five paThe HASTE sequence showed that none of the participants had hyper-intense signals in the subcutaneous area, but all participants had such signals in either their stomach or spine .The lymphedema therapist determined that seven participants had truncal swelling. The number of participants with subjective symptoms is shown in 13 This study was the first to use MRI to assess fluid distribution in the trunks of BCRL patients. Our results suggest that none of the participants had free water in the truncal region, and eight had free water only in their upper limbs.In fluid-sensitive MRI sequences, hyper-intense signals indicate the presence of free water and the distinctive patterns found in the affected upper limbs of BCRL patients.14 In our study, the reason for the absence of the free water is not clear, as the relationship between lymphedema's progress or chronicity and fluid distribution remains unknown. However, it has been suggested that the rate of fluid discharge in the upper limbs may differ from that of the truncal region.According to Fumiere et al. and Tassenoy et al., as lymphedema progresses and becomes chronic, hyper-intense MRI signal images tend to lessen due to the progression of fibrillization.Our MRI results suggest that none of the participants had free water in the trunk. In contrast, seven participants were assessed as having truncal swelling, and all had some subjective symptoms in the trunk. These results suggest that there is no correlation between swelling conditions or subjective symptoms and the presence of fluid in the trunk. Therefore, we posit that swelling and subjective symptoms are caused by something other than the presence of fluid. It is possible that the swelling and subjective symptoms are due to asymmetric development of muscle or fat tissue resulting from surgical wounds.This study is the first to assess BCRL participants for the presence of fluid in the truncal region by using MRI. However, because the results indicated that the participants did not have fluid accumulation in the trunk, it remains unclear how fluid is distributed in the truncal region of BCRL patients. Further studies with more patients are required to confirm this study's results.15 However, our results showed that BCRL patients did not have free water in the trunk despite exhibiting swelling or related subjective symptoms. Considering the absence of free water in the truncal region, MLD in this region may not be necessary for such patients. Instead, a different approach may be needed to address truncal swelling or subjective symptoms. Therefore, checking the truncal region for the presence of fluid by using MRI may allow MLD to be used more appropriately.To ensure the process of draining lymph from a lymphedematous arm by using MLD is effective, truncal fluid generally is drained first. Therefore, draining fluid from the trunk is important not only for reducing patient discomfort but also for ensuring the success of lymphedema treatment for the upper limb.Our findings suggest that swelling conditions or subjective symptoms in grade-2 BCRL patients do not necessarily correlate with the presence of fluid in the trunk.


Patients with inflammatory bowel disease (IBD) are at an increased risk of herpes zoster (HZ). HZ is caused by reactivation of the varicella zoster virus (VZV) and is prevented by strong VZV-specific cell-mediated immunity. The aim of our study was to evaluate whether patients with IBD had lower or equivalent protection compared with healthy controls (HCs) at age 50 years and older.via ELISPOT, and humoral immunity was measured via a quantitative VZV antibody enzyme-linked immunosorbent assay assay.We performed a cross-sectional study at a single academic center and evaluated cellular and humoral immunity to VZV in patients with IBD at age 35–49 years vs HCs aged 50–59 years. All patients with IBD were on stable medication regimens for at least 3 months. VZV-specific cell-mediated immunity was measured P = 0.54). In addition, there was also no significant difference between ELISPOT counts in immunosuppressed patients with IBD (N = 45) and HCs (P = 0.32). We also found no correlations between ELISPOT counts and age . Patients with IBD had similar IgG VZV antibody levels  compared with HCs (median 23.5 mIU/mL (range 4–34); P = 0.54).Seventy-seven patients with IBD and 12 HCs were enrolled in the study. There was no significant difference in ELISPOT counts between patients with IBD and HCs (Young patients with IBD have equivalent cellular and humoral immunity to VZV as healthy older adults in whom HZ immunization is recommended. Patients with inflammatory bowel disease (IBD) are at a 2-fold increased risk of herpes zoster (HZ), independent of whether they are on immunosuppressive therapy, compared with the general population . Those oHZ is caused by reactivation of latent varicella zoster virus (VZV) due to age-related waning of VZV-specific cell-mediated immunity (CMI) . It is kIn the United States, licensure of the vaccine does not guarantee insurance coverage of the vaccine until the Advisory Committee on Immunization Practice (ACIP) makes a recommendation. The Affordable Care Act states that ACIP recommendations for immunization are required to be covered without cost sharing . Thus, uThis cross-sectional pilot study compared quantitative VZV antibody and ELISPOT counts in patients with IBD and HCs who presented for routine clinical care at the University of Wisconsin Digestive Health Center  between December 2017 and May 2019. The inclusion criteria for both cohorts required a history of chicken pox, no history of any zoster vaccine or HZ, and positive VZV qualitative antibody. Zoster vaccine history was verified in the Wisconsin Immunization Registry, an Internet immunization information system tracking immunization histories of Wisconsin residents, which is linked to the electronic health record .The inclusion criteria for the IBD cohort were age 35–49 years, having an established diagnosis of IBD, and on stable medical treatment for at least 3 months. The age of 35 years and older was chosen because we wanted to include only individuals who had varicella infection (chicken pox) and not those immunized with varicella vaccine, which started in 1995 and was routinely used for children through age 12 years . The varELISPOT is an enzyme-linked assay for detecting and enumerating lymphocytes that produce cytokines in response to an antigen. Cytokine ELISPOT sets for IFN-γ  were used according to the manufacturer's instructions. Two hundred thousand lymphocytes were incubated with VZV antigen  ,21. Cytovia ELISPOT between patients with IBD and HCs. The secondary outcomes were as follows: (i) evaluating VZV-specific CMI between types of IBD and immunosuppressed patients with IBD and those with IBD on nonimmunosuppressing regimens (ii) comparing VZV-specific antibodies between patients with IBD and HCs; (iii) evaluating correlation between age and VZV-specific CMI; and (iv) correlation between VZV-specific CMI and VZV-specific antibodies.The primary outcome of the study was to compare VZV-specific CMI via ELISPOT were compared among groups using the Kruskal-Wallis tests and between groups using the Mann-Whitney U tests. A Spearman correlation was used to determine whether relationships existed between responder cell frequency and antibody concentration or age. Our sample sizes conferred an 87% power to detect a difference of 20 responder cells in HCs to 10 responder cells in patients with IBD  among HCs and 76 (range 5–295) among patients with IBD (P = 0.36)   or between those who were treated with a nonimmunosuppressing regimen, those in Group A and vedolizumab monotherapy, , and those who were on an immunosuppressing regimen . Furthermore, there was no significant difference in the median number of IFN-γ spots among the IBD groups categorized by treatment regimen .All patients with IBD and HCs were able to respond to VZV stimulation. The median number of IFN-γ spot-forming cells per 2 × 10P = 0.59), as measured with the quantitative IgG ELISA and adjusted to the World Health Organization standard. Among immunosuppressed patients with IBD, IgG VZV antibody levels  did not differ significantly from IgG VZV antibody levels in nonimmunosuppressed patients . We also found no difference in VZV antibody concentrations between those with UC  and Crohn's disease . They also did not correlate with VZV-specific cell number .All patients with IBD and HCs had measurable antibodies per inclusion criteria. Patients with IBD had similar IgG VZV antibody levels (median 19 mIU/mL range 0.5–218) compared with HCs  program, through the NIH National Center for Advancing Translational Sciences (NCATS), grant UL1TR002373. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.Potential competing interest: F.C. has been a consultant for Takeda Pharmaceuticals, Celgene Pharmaceuticals, and Arena Pharmaceuticals and received research support from Takeda Pharmaceuticals and Sanofi for work unrelated to the topic of this manuscript.✓ Patients with inflammatory bowel disease (IBD) are at an increased risk of herpes zoster (HZ).✓ Many patients are not eligible to be vaccinated against HZ.✓ Varicella zoster virus (VZV)-specific cell-mediated immunity (CMI) plays a critical role in preventing HZ.✓ Patients with IBD with previous VZV infection had preserved CMI to VZV stimulation.✓ Young patients with IBD have equivalent VZV-specific CMI as healthy older adults in whom HZ immunization is recommended.


Following the publication of the original article , the autThe correct funding note is given below.Funding note:This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 116026 H2020 EU "HARMONY" project. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. Moreover the research leading to these results has received funding from the European Union’s Horizon 2020 research and innovation programme “VEO” Grant n. 874735, and from ETN-ITN “ImforFuture” Grant n. 721815. The “HARMONY” and “VEO funding bodies were partially used to pay staff salaries, while the “ImforFuture” funding body partially supported the purchase of the high performing machine server used for the experiment.The author group has been updated above and the original article  has been


Despite the frequent inclusion of fluid therapy in the treatment of many conditions in horses, there are limited studies available to provide evidenced-based, species-specific recommendations. Thus, equine fluid therapy is based on the application of physiology and extrapolation from evidence in other veterinary species and human medicine. The physiologic principles that underly the use of fluids in medicine are, at first glance, straightforward and simple to understand. However, in the past 20 years, multiple studies in human medicine have shown that creating recommendations based on theory in combination with experimental and/or small clinical studies does not consistently result in best practice. As a result, there are ongoing controversies in human medicine over fluid types, volumes, and routes of administration. For example, the use of 0.9% NaCl as the replacement fluid of choice is being questioned, and the theoretical benefits of colloids have not translated to clinical cases and negative effects are greater than predicted. In this review, the current body of equine research in fluid therapy will be reviewed, connections to the controversies in human medicine and other veterinary species will be explored and, where appropriate, recommendations for fluid therapy in the adult horse will be made based on the available evidence. This review is focused on the decisions surrounding developing a fluid plan involving crystalloids, synthetic colloids, and plasma. Fluid therapy is a key component in treatment and supportive care of horses with a variety of conditions, especially those with critical illness. Despite its frequent utilization, it remains challenging to make evidence-based treatment recommendations. This is due to the limited number and design of studies related to fluid therapy that have been performed in horses . The majAt the simplest level, fluids are drugs and, as such, have the potential for both life-saving benefits and serious, possibly life-threatening, adverse effects. In order to better align their utilization with their characterization as drugs, human medicine has begun a paradigm shift toward considering their use within the “four Ds” of drug therapy: drug, dosing, duration, de-escalation . TreatinRoute of Administration will discuss when and how to utilize the various routes of fluid administration available in the horse. As with all drugs, the administration route must be determined early on when developing a fluid therapy plan. In horses, the most commonly utilized routes are intravenous (IV) and intragastric (IG). Rectal (PR) fluid administration may be an alternative and has been a topic of recent interest. Once the route of fluid administration is decided, fluid type and dose must be selected. Information on fluid type and dose for IG and PR fluid administration are provided in Intragastric Fluids and Rectal Fluids, respectively. Following this, Intravenous Fluids - Crystalloids and Intravenous Fluids - Colloids will discuss the choice of type and dose of IV fluid therapy, with Intravenous Fluids - Crystalloids focused on the crystalloids and Intravenous Fluids - Colloids focused on colloids. Throughout this review, we aim to delve into the controversies surrounding choosing the most appropriate route, fluid type, and dose and report the most current literature, in order to help the equine practitioner navigate this territory. Where appropriate, recommendations for treatment of the adult horse will be made, based on the available evidence. It should be noted that the present review does not touch on transfusion medicine, nor parenteral nutrition, information for which has been well-reviewed elsewhere  and dose. lsewhere –9.Factors that must be taken into account when choosing route of administration include those related to the patient's medical conditions, such as the indications and contraindications for fluid therapy associated with the underlying disease and co-morbidities, as well as non-medical considerations, such as patient temperament, owner finances, and the environment. Together, these determine the likelihood that the treatment will be successful and the potential for adverse effects. via many routes.An important first question when choosing the route of administration should be: what is the purpose of fluid therapy? The indications for fluid administration are diverse, but can be generally categorized into emergency resuscitation, replacement of deficits, meeting daily requirements, and targeted treatment of a specific problem. Emergency fluid resuscitation is needed for shock due to decreased effective circulating volume (preload) and circulatory collapse associated with conditions such as severe acute hemorrhage or sepsis. Fluids are also indicated to replace existing deficits, or dehydration. Daily fluid requirements include both maintenance administration to cover expected sensible and insensible losses and to offset ongoing losses. Specific reasons for fluid therapy may include providing nutrients, correcting acid-base and electrolyte abnormalities, diuresis, and, particularly in horses, increasing water content in the ingesta and feces. Although for some indications the choice of route is clear, as is the case for most treatment decisions, more often it is not a black and white or right and wrong answer. For example, IV fluids are a clear choice for resuscitation, but correction of dehydration can be achieved One of the most common reasons for fluid administration is resuscitation of the critically ill patient with insufficient preload due to hypovolemic, distributive, or obstructive shock. These patients require rapid, significant intravascular volume expansion. It should not be surprising that this is best achieved by administering fluids directly into the vascular space (IV). IG and PR routes require absorption and the amount that can be administered as a bolus or over a short period of time is limited. Additionally, in the hypovolemic state, perfusion to the gastrointestinal tract is reduced which slows absorption, further restricting the use of enteral routes for stabilization. Thus, the IV route is the clearly superior method for initial therapy. Once the intravascular volume has been sufficiently expanded and perfusion to the gastrointestinal tract is restored, replacement of deficits with IG or PR may be effective.Intravenous Fluids - Crystalloids and Intravenous Fluids - Colloids.Commonly, fluid resuscitation in the equine patient is performed using large volumes of IV isotonic crystalloids, administered as a bolus. Recommendations regarding total dose have varied, although most focus on administration of boluses of approximately 20–25% of a total “shock dose” (80–90 ml/kg for horses) repeated (up to 4–5 times) until indices of perfusion have improved . In the via a variety of routes. In fact, IG and PR routes have been shown to be at least as effective as IV routes for correcting dehydration  as well as insensible (sweat and respiratory) losses. In the adult equid, this requirement is typically reported to be 2–4 ml/kg/h or 40–60 ml/kg/day, although recent work suggests a significant reduction in this requirement when horses are off feed , 25. As Rhodococcus equi, colitis, and coagulopathies); or IV administration of a hypertonic crystalloid or mannitol for cerebral edema.Specific problems that are treated with fluid therapy include hydration of intestinal contents; provision of nutrition; correction of anemia; treatment of failure of passive transfer; or reduction of cerebral edema, to name a few. Logically, the type and route of fluid therapy required will vary based on these underlying problems. For example, when considering the aforementioned conditions, the practitioner may utilize: IG fluid administration for improving ingesta/fecal water content; parenteral (IV) or enteral (IG) routes to meet nutritional needs; IV administration of whole blood; IV administration of hyperimmune plasma for failure of passive transfer and treatment of specific conditions  or dosing (rate of administration).Rate/Dose of Crystalloid Administration.While IV fluid administration is commonplace, it is perhaps the route posing the highest risk for complications – due to both the requirement for an indwelling IV catheter and the potential for medical errors when selecting and tailoring the fluids themselves. Catheter complications can vary in severity and consequence and may include phlebitis or thrombosis, septic thrombophlebitis, insertion site abscessation, or acquisition of an air embolus . The admvia an IG tube must not exceed the stomach capacity of the horse and, as noted previously, requires a functional proximal gastrointestinal tract. Mixing errors when preparing electrolyte solutions for administration can result in electrolyte and acid base abnormalities and exceeding recommended dosing for MgSO4 as a laxative can result signs of Mg toxicity. While little objective evaluation of the PR administration route exists in horses, some concern has been raised about the type of fluid administered. It has been suggested that hypotonic solutions, such as tap water, may be damaging to the rectal mucosa  has been an alternative to commercially available options. These fluids are generally formulated by adding a powdered electrolyte mixture to commercially available reverse osmosis or distilled water meant for human consumption . Non-stevia gravity flow requires fluids to be suspended at a significant height. It should be noted that, if facility limitations are likely to negatively impact the standard of care the patient receives, then hospitalization should be considered.In addition to the considerations discussed above, sometimes circumstances limit the options available to a patient. Patient temperament can affect the choice of fluid administration route. For example, horses have variable tolerance of indwelling nasogastric tubes and some have a propensity for removing their own catheters. While there are ways to mitigate these issues, if other factors are equal this should be a factor in planning. Where and how the horse is housed can also affect the choice of route of fluid administration. For example, constant rate fluid administration may not be an option in extreme cold due to freezing and rapid IV administration IG therapy has a long history of use in equine fluid therapy, with proposed benefits over IV administration that include reduced cost, ease of administration, and improved safety. In addition to these considerations, there is some evidence to suggest that IG administration may be preferable to IV under some circumstances.IG fluid therapy should be considered as a route of fluid administration for replacement of dehydration, providing maintenance requirements, and treating specific diseases. While IG administration is a becoming a more commonly utilized route for treating horses suffering from large intestinal impaction, for other equine cases it is frequently overlooked and has been the subject of little research. IG administration of either isotonic electrolyte solution or plain water has been shown to increase fecal water content more effectively than IV fluid therapy , 36. Trevia nasogastric tube . The extracellular fluids will then be relatively hypotonic and further redistribution into the intracellular fluid space will occur to balance tonicity between these compartments. By comparison, if an isotonic electrolyte solution is administered, the fluid will once again be initially taken up and equally divided between the extracellular fluid spaces. However, there will not be the osmotic drive to initiate further redistribution intracellularly, and therefore more of the absorbed fluid will remain in the extracellular  space. Although plain water and electrolyte solutions increase gastrointestinal water content and plain water has been shown to correct dehydration in horses, concerns have been raised regarding the hypotonicity of plain water resulting in the possibility of serious electrolyte abnormalities . HoweverThe purpose of the glucose/dextrose component in human rehydration solutions is to drive glucose cotransport of sodium within the intestinal tract, ultimately creating more solvent drag and fluid uptake. Interestingly, however, several studies in horses have not supported their use over a electrolyte solution not containing glucose , 44. EckThe buffering component of human rehydration solutions is included in an attempt to counteract metabolic acidosis, particularly in diarrheic patients. Many of the oral rehydration solutions used in the horses do include sodium bicarbonate in varying proportions , 0.37 g KCl , and 3.78 g NaHCO3 in 1 L tap water) . Howevervia nasogastric tube. Bolus dosing can be convenient, as it does not require a stall set-up that allows for a continuous administration. With gastric emptying times for fluid reported at around 15 min, frequent boluses of appropriate volumes should be possible and well-tolerated, so long as normal small intestinal function is adequate , but no effect on hydration was identified. A separate part of this study evaluated how far orally fluid migrated in horses and found it rarely reached the level of the pelvic flexure . Horses that were deemed to be mildly dehydrated, based on loss of body weight and evidence of hemoconcentration, were administered their calculated fluid deficit of a homemade polyionic solution PR in boluses of no more than 5 L at a time. The procedure was well-tolerated and resulted in a measurable improvement in hematocrit and total protein concentrations, with minimal changes in overall electrolyte balance .via continuous gravity flow over the course of a 6-h period. In the main randomized, crossover study, six horses were treated with each of 3 fluid therapy treatments  and a control treatment where no fluid was administered. Results indicated that horses administered tap water per rectum at these rates tolerated this well and exhibited evidence of hemodilution (based on decreased PCV/TS) similar to that achieved via the IV and IG routes  soft enema tube . High sodium, hypertonic solutions, are most frequently utilized to rapidly shift fluids into the intravascular space during resuscitation and can also be useful for treatment of cerebral edema. Correction of sodium imbalance is another indication for administration of hypo- or hyper-tonic (low or high sodium) fluids. The distinctions between most commonly available commercial crystalloid solutions are discussed below. In addition to these basic crystalloid options, some additional alternatives exist, such lactate-rich polyionic solution and bicarbonate solutions, which have specific purposes related to restoring circulating volume or normalizing acid-base disturbances , 56. ModReplacement fluids are designed to “replace” deficits, generally those associated with hypovolemia and dehydration. Maintenance fluids are designed to “maintain” homeostasis by providing requirements associated with maintenance needs and expected losses. Because the deficits being replaced in cases of hypovolemia (intravascular) and dehydration  involve the extracellular fluid space primarily, replacement fluids have a composition similar to extracellular fluid—high in sodium and chloride, low in potassium, calcium, and magnesium. In contrast, the losses being addressed with maintenance fluids are lost from both intra- and extra-cellular fluid spaces through fluids such as urine, which are generally comparatively low in sodium and chloride and high in potassium, calcium, and magnesium. Thus, maintenance fluid composition is closer to total body water electrolyte composition. The low sodium concentration of maintenance fluids makes them hypotonic unless dextrose is added.Commercially available replacement fluid options available include: 0.9% sodium chloride (0.9% NaCl)Such a significant sodium load results in natriuresis and thus diuresis, which is logically counterproductive and can result in depletion of additional electrolytes. In the study by Lester et al., kaliuresis and diuresis persisted in the recovery period following administration of IV polyionic replacement fluids. Horses in the IV treatment group were unable to completely restore hydration during the treatment period and illustrated a rebound dehydration during the 24 h following treatment . SimilarSodium that is not able to be excreted contributes to fluid retention and predisposes to fluid overload and development of edema. The general approach to fluid therapy in adult equids has been to consider them relatively tolerant of overzealous fluid therapy efforts in comparison to their small animal counterparts, due in large part to the comparatively fewer cases of cardiac dysfunction or renal insufficiency. However, in the diseased adult patient or neonate with less tolerance for large sodium shifts, the potential for sodium and fluid overload should be acknowledged and avoided.A recent controversy within crystalloid fluid therapy is the shift away from using 0.9% NaCl as the replacement fluid of choice in favor of a more balanced electrolyte solution. Historically, saline has been the mainstay fluid of choice in human medicine due to the perception that it is the most physiologically appropriate choice. However, this has been questioned due to concerns regarding the effects of increased chloride load from its administration –60.Physiologic 0.9% saline, or normal saline, contains sodium and chloride only, and each of these components exists in higher levels in this fluid than is present in normal equine plasma (154 vs. ~130–140 mmol/L for sodium and 154 vs. ~90–100 mmol/L for chloride) . Normal While a balanced electrolyte solution is favored for the majority of equine patients, there are certain disease states in which sodium chloride would be preferable. The first of these is logically patients with a hypochloremic metabolic alkalosis, as the propensity for a hyperchloremic metabolic acidosis may actually be of benefit in correcting this disturbance. Normal saline has also generally been selected preferentially over balanced electrolyte solutions in hyperkalemic patients. This is because all balanced electrolyte solutions contain some potassium, which may theoretically worsen the hyperkalemia. However, the acidosis induced by high chloride fluids induces a shift of potassium out of cells which may increase potassium to a greater degree than lactated ringer's solution , 64. LasMost commonly hypertonic saline is available as a 7.2% solutionThe rapid volume expansion achieved with hypertonic saline administration makes this an excellent choice in the resuscitation of the hypovolemic patient. In an experimental study in which anesthetized horses underwent controlled exsanguination to achieve induced hemorrhagic shock, administration of hypertonic saline resulted in rapid plasma volume expansion and urine output, along with sustained elevations in numerous cardiovascular parameters including cardiac output, stroke volume, mean arterial pressure, and contractility . ComparaThe rate of crystalloid administration is typically dictated by the indication for the fluid therapy. However, as briefly discussed above, to date there has often been an inclination to treat with very aggressive fluid therapy rates, especially for particular problems such as intestinal impactions. In addition to the concerns regarding high fluid rates resulting in counterproductive diuresis (particularly with high sodium content of replacement fluids commonly used in equine practice), other potential drawbacks of this approach include cost, lack of actual benefit, and the potential for detrimental fluid overload.Interestingly, studies evaluating the use of IV fluids to improve fecal water content have been conflicting. Lester et al.  identifivia diuretic therapy, when appropriate  solutions, dextrans, and gelatins. HetastarchColloids are relatively large, charged, osmotically active molecules. The amount of osmotic pressure created by colloids in a solution is measured as colloid osmotic pressure and also known as oncotic pressure. The number of molecules, rather than their size, is responsible for the osmotic pull. For this reason, in plasma, it is albumin, not globulin, that is the predominant molecule responsible for oncotic pressure, because there are more molecules of albumin. It is for a similar reason that smaller molecular weight HES solutions should provide more volume expansion that larger molecular weight HES. Due to the large size and charge of colloid molecules, normal vessels are not permeable to colloids, which is in contrast to other osmotically active molecules such as electrolytes and glucose. Whereas, electrolyte and glucose concentrations quickly equilibrate between the plasma and interstitial spaces, colloids remain within the vascular space, maintaining an osmotic gradient that encourages transcapillary movement from the interstitium to the intravascular space and keeps water in the vasculature, providing a more sustained intravascular volume expansion , 85.f). If applied to crystalloid administration, the addition of isotonic fluids within the vascular space results in an increased volume and, therefore, hydrostatic pressure along with dilution of colloid/protein and, therefore, a decrease in oncotic pressure within the vessel. Both changes in the capillary favor a net movement of fluid into the interstitium. If applied to colloid administration, the increase in volume still results in an increase in hydrostatic pressure, but the increase in colloid results in an increase in oncotic pressure within the vessel, pulling fluid into the vessel. The goal is to increase the oncotic pressure enough to offset and outweigh the increased hydrostatic pressure to favor net movement of fluid from the interstitium and into the intravascular space. Thus, theoretically colloid administration should result in a more significant volume expansion effect for a given volume administered, with less redistribution of the fluid to the interstitium, providing a more sustained expansion and minimizing the negative effects of accumulation of interstitial fluid.The underlying principle behind the use of colloid therapy to produce sustained volume expansion was the standard Starling equation of transcapillary movement. In summary, the equation states that the direction and amount of transcapillary fluid movement is related to the balance of hydrostatic and colloid osmotic, or oncotic, pressures between the capillary and the interstitium and a factor representing the permeability of the vessel wall (Kf) allow colloids to accumulate, along with the water they attract  or inducing more rapid and sustained volume expansion than crystalloids during fluid resuscitation of critically ill patients . RelatedDespite the theoretical benefits of colloid administration, results of clinical studies have failed to consistently show advantages over crystalloids in human medicine , 85. IniIn general, regardless of the type, colloid products are more costly than crystalloid alternatives, when compared on a volume basis . HoweverWith respect to improved COP, administration of synthetic colloids to healthy horses and ponies in an experimental setting has been shown to result in an increase in oncotic pressure for as long as 24 h post-administration , 96. WheWhile the colloid literature in human medicine has focused a great deal on outcomes , similar studies within the equine literature are sparse. Several studies have evaluated the effect of colloid administration on hematologic parameters that are considered indicative of hemodilution, supporting the premise that these products enhance the circulating fluid volume. Previous reports have indicated that both synthetic colloid and natural colloid options reduce hematocrit (HCT) or packed cell volume (PCV) and/or total protein (TP) or total solids (TS) concentrations in healthy horse , 104. A Recent reports in the human literature have brought to light concerns regarding the potential for complications following synthetic colloid use. In conjunction with the now limited reliable data supporting their use, this has led to a significant move away from their use in human medicine . The priin vitro in cats and in dogs with naturally occurring hemoperitoneum  are likely to be no more effective at increasing fecal water than lower rates, despite significantly increasing the cost and increasing the risk of electrolyte abnormalities and volume overload.Use of non-sterile isotonic fluid options may be ill-advised due to the potential for mixing errors, bacterial and endotoxin contamination, and increased risk for catheter site complications.Normal saline has been repeatedly reported to result in hyperchloremic metabolic acidosis, which in other species has been associated with complications and possibly poorer outcomes. As such, balanced electrolyte solutions are likely preferable in the majority of patients. Exceptions include situations in which these effects may be therapeutic . There are more debatable benefits in cases of hyperkalemia.While the proposed mechanism by which colloids exert their effects has been questioned, the equine literature to date does support their influence on colloid oncotic pressure and volume expansion.Synthetic colloid administration has been shown to exert some, albeit mild, effects on hemostatic parameters similar to those seen in people and small animal patients, particularly at higher doses. As such, it may be wise to limit their administration to lower doses. It is important to note that there is no data from which to evaluate the potential for additional side effects seen in other species.As can be seen throughout this review, with respect to evaluating fluids in the context of being a drug, the preponderance of the equine literature has focused on the first two “Ds” - drug and dosing - but very little has been evaluated with respect to duration or de-escalation. Work in other species has shifted to consideration of how these factors influence outcome, with particular emphasis on concerns such as fluid overload . This haThe authors contributed equally to the this manuscript through researching the topic and writing and editing the manuscript. 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.


Scarce drug penetration in solid tumours is one of the possible causes of the limited efficacy of chemotherapy and is related to the altered tumour microenvironment. The abnormal tumour extracellular matrix (ECM) together with abnormal blood and lymphatic vessels, reactive stroma and inflammation all affect the uptake, distribution and efficacy of anticancer drugs.We investigated the effect of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) pre-treatment in degrading hyaluronan , one of the main components of the ECM, to improve the delivery of antitumor drugs and increase their therapeutic efficacy. The antitumor activity of paclitaxel (PTX) in HA synthase 3-overexpressing and wild-type SKOV3 ovarian cancer model and in the BxPC3 pancreas xenograft tumour model, was evaluated by monitoring tumour growth with or without PEGPH20 pre-treatment. Pharmacokinetics and tumour penetration of PTX were assessed by HPLC and mass spectrometry imaging analysis in the same tumour models. Tumour tissue architecture and HA deposition were analysed by histochemistry.Pre-treatment with PEGPH20 modified tumour tissue architecture and improved the antitumor activity of paclitaxel in the SKOV3/HAS3 tumour model, favouring its accumulation and more homogeneous intra-tumour distribution, as assessed by quantitative and qualitative analysis. PEGPH20 also reduced HA content influencing, though less markedly, PTX distribution and antitumor activity in the BxPC3 tumour model.Remodelling the stroma of HA-rich tumours by depletion of HA with PEGPH20 pre-treatment, is a potentially successful strategy to improve the intra-tumour distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.The online version contains supplementary material available at 10.1186/s13046-021-02070-x. Many molecular mechanisms responsible for resistance to anticancer drugs have been elucidated over the years. The co-existence of heterogeneous populations of cancer cells with different sensitivity explains why, even after an initial response, most solid tumours relapse, becoming resistant to treatments.Growing evidence suggests that the resistance of many solid tumours can also be due to insufficient and heterogeneous tumour drug distribution , 2. StudThe penetration capacity of a drug depends on its physical-chemical properties and on the neoplastic cells’ characteristics, but the low delivery in tumour tissue is mainly related to the altered tumour microenvironment (TME) . AbnormaHA is a glycosaminoglycan and a major component of the normal ECM, whose accumulation is significantly increased in several solid malignancies, particularly in advanced stage disease . High HATo improve the delivery of antitumor drugs and increase their therapeutic efficacy, some strategies based on ECM modification have been tested. Lowering HA levels in TME with a hyaluronidase has been suggested as a “distribution enhancing approach”. By cleaving polymeric HA into substituent units, hyaluronidase can liberate trapped water molecules, thus rapidly lowering TIFP and enabling collapsed vessels in the tumour to open. Thus, it would improve tumour perfusion and drug permeation. rHuPH20, a recombinant human hyaluronidase approved for subcutaneous injection to facilitate the absorption of other injected drugs  or fluids, has been further developed for anticancer purposes. PEGPH20 is an investigational PEGylated form of rHuPH20 that has a longer plasma half-life (about 10.3 h for PEGPH20 compared to 3 min for rHuPH20 in mice) and it is suitable for intravenous administration . In precIn patients, PEGPH20 is tolerated, although prophylactic steroids and anticoagulant are required to prevent musculoskeletal and thromboembolic events , 32. TumA fuller description of the connections between drug distribution, TME characteristics and antitumor activity could help elucidate the true potential of this therapeutic approach. We investigated the distribution of paclitaxel (PTX) after PEGPH20 pre-treatment using mass spectrometry imaging (MSI) and correlated it with antitumor activity and tissue modifications in different preclinical models.Paclitaxel  and paclitaxel-D5  were dissolved in ethanol at a concentration of 1 mg/ml. Serial dilutions of drug were prepared in 50% ethanol from 0.5 to 100 pmol/μl for all MSI experiments.For treatment purposes, PTX was dissolved in 50% Cremophor EL (Sigma) and 50% ethanol and further diluted 1:5 in saline immediately before use.The PEGPH20 formulation  consisted of the active compound dissolved in histidine buffer  at 5 mg/ml. It was diluted in saline solution (NaCl 154 mM) just before the treatment.2 nanoparticles  were used as a matrix for MSI experiments, dissolved at the concentration of 1 mg/ml in ethanol 50%/KCl 0.5%. The TiO2 nanoparticle suspension was vortexed and sonicated for 3 min just before use, to reduce agglomeration and sedimentation.TiOParental SKOV3 (SKOV3) and HAS3-overexpressing SKOV3 (SKOV3/HAS3) cell lines were provided by Halozyme Therapeutics Inc. SKOV3/HAS3 were obtained by transducing SKOV3 cells with a retrovirus carrying human HA synthase 3 (HAS3) cDNA. These cells are characterized by production of high HA levels. Both cell lines were maintained in McCoy medium added with 10% FBS and glutamine. The BxPC3 pancreatic adenocarcinoma cell line was maintained in RPMI with 10% FBS and glutamine. All the cell lines were mycoplasma free and authenticated by short-tandem repeat (STR) profiling.-nu/nu mice were obtained from Envigo RMS. They were maintained under specific pathogen-free conditions, housed in isolated vented cages, and handled using aseptic procedures after an acclimatization period of one week.Six- to 7-week-old female NCrARRIVE guidelines 2.0 .All procedures involving animals and their care were conducted in conformity with the following laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law ; Mario Negri Institutional Regulations and Policies providing internal authorization for persons conducting animal experiments ; the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/UE) and in line with Guidelines for the welfare and use of animals in cancer research {Workman, 2010}. Animal experiments were reviewed and approved by the IRFMN Animal Care and Use Committee (IACUC) that included “ad hoc members for ethical issues. Animals were housed in the Institute’s Animal Care Facilities, which meet international standards; they are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, and experimental protocols and procedures revision. Experiments were reported following the 6 cells) were implanted subcutaneously in the flank of nude mice. Tumour growth was measured with a Vernier caliper two/three times a week, and tumour weights (1mm3 = 1 mg) were calculated as [length (mm) * width2 (mm2)]/2. When tumours reached approximately 150–200 mg, mice were assigned to the experimental group by stratified randomization based on tumour weight (8 to 9 mice/group) and treated with PEGPH20  alone or with PTX  or with PEGPH20 followed by PTX . Control groups received the same volume of the respective vehicles. Mice were weighted two/three times a week to evaluate general toxicity. Tumour volume was measured thrice a week up to the ethical endpoint (1500 mm3) then mice were euthanized. For the analysis of the tumour growth curves, each tumour weight (TW) measure was normalized to the tumour weight of the same mouse at the start of treatment . Antitumor efficacy was expressed as best T/C% were T and C were the mean of the RTWs of treated and control mice, respectively according to the standards of the National Cancer Institute (NCI) of the United States  were calculated as follows: k = [log TWt(i + 1)-log TWt]/ (t(i + 1)-ti). The experimental groups were compared two by two using a Wilcoxon Rank-Sum test, stratified by intervals, on the obtained k values . Student’s t-test for unpaired samples was used to evaluate differences in drug distribution experiments. Differences in the pharmacokinetic profiles were analysed by two-way ANOVA (Graph Pad Prism 8).The treatment effect on xenograft tumour growth curves was formally tested using a non-parametric approach. For each mouse, the partial tumour growth rates (k) in every interval [tp-value< 0.001 compared to control, p-value< 0.001 compared to single treatments). An in depth analysis of the tumour growth curves is shown in Additional file The effect of PEGPH20 pre-treatment on PTX antitumor activity was assessed in parental and HAS3-overexpressing SKOV3 tumours Fig. . PTX indTo verify the hypothesis that PEGPH20 enhances the PTX antitumor efficacy by improving its distribution in tumour tissue, we performed an in-depth analysis of spatial distribution of PTX within tumour tissue, using an in-house developed MSI technique. Measures of drug concentrations in tumour homogenates and plasma pharmacokinetics are not fully indicative of drug distribution in tumour tissue. Since heterogeneous penetration of pharmacological compounds in neoplastic tissue is a known mechanism of drug resistance, the determination of drug intratumor penetration adds valuable information. The MSI analysis of PTX is shown in Fig. MSI images were further processed to extract quantitative parameters describing drug distribution. In addition to the mean and CV% of the drug concentration calculated at pixel level, we examined a panel of GLSZM features selected for their significance in relation to the diffusion and homogeneity of the drug in the tissue. The complete panel of GLSZM features and an interpretation of their meaning in this context is reported in Additional file The complete pharmacokinetic analysis after a single dose of PTX 20 mg/kg, alone or after PEGPH20 pre-treatment in both tumour models is reported in the Additional Information. PEGPH20 pre-treatment minimally affect PTX pharmacokinetic behaviour. Indeed, PTX concentrations in tumour samples collected at different time points were comparable in the two different experimental groups (with or without PEGPH20 pre-treatment). Similar levels were also found for plasma in the SKOV3 model while circulating PTX was lower after PEGPH20 than vehicle pre-treatment in SKOV3/HAS3 model but only 1 h after PTX. Similarly, PTX levels in liver were lower in the combination in both experimental models 1 h after PTX.To determine whether the different PTX distribution in tumour was due to differences in tissue architecture, we did histological examinations on tumour sections adjacent to those analysed by MSI. H&E staining confirmed that SKOV3 tumours were composed of sheets/solid areas of neoplastic cells arranged in lobules, infiltrated by a moderate to large amount of thin fibrovascular stroma and occasionally thicker fibrous septa Fig. . VariablIn SKOV3/HAS3 tumours, a looser arrangement of the tissue structure was evident, with loss of defined lobules. HA ability to bind large amounts of water molecules might be responsible for the looser arrangement of the tumour tissue observed in SKOV/HAS3 samples: a large amount of water is frozen and released during sample preparation, producing the artefacts characterized by clear/empty spaces dissecting the tumour tissue. The PEGPH20 pre-treatment restored a more compact tumour architecture, similar to that of parental SKOV3 tumours Fig. . No diffp-value = 0.025).Specific HA depletion in SKOV3/HAS3 tumours following PEGPH20 treatment was quantified in tissue sections stained for HA using the immuno-adhesin HTI-601 with DAB used as the chromogen Fig. . A high MSI analysis was done following the same therapeutic regimen as PEGPH20 and PTX used for antitumor activity experiments . The results confirmed that PEGPH20 pre-treatment improved drug distribution in SKOV3/HAS3 but there were no differences between the two treatments in the parental SKOV3 model Fig. . The comThe clinical relevance of our results was verified by testing the effect of PEGPH20 on PTX distribution in a xenograft model of pancreatic cancer, a tumour known for its dense, desmoplastic stroma with abundant ECM. The MSI analysis of PTX results are shown in Fig. Quantitative analysis of the MSI images indicated that PEGPH20 in BxPC3 induces a pattern of modifications in our panel of features similar to that of the SKOV3/HAS3 model: an increase of the mean pixel drug concentration, with lower LZE, higher HGZE and lower IV Fig. . These ap-value 0.019; Fig. No difference was seen between the experimental groups in PTX total concentrations measured by HPLC in tumour homogenates Fig. . The BxPHeterogeneous spatial distribution of drugs in solid tumours represents a sort of pseudo-chemoresistance, because tumour cells can survive therapy simply because of lack of exposure . This isHA is a TME component that is often overexpressed in various tumours , 22. HA PEGPH20 was developed for its ability to disrupt HA in tumour tissue, inhibiting HA’s protumor properties, reducing TIFP and solid stress. Indeed, PEGPH20 can be exploited in a combination strategy, to improve the penetration of concomitant drugs through tumour tissue. Previous preclinical studies have shown that PEGPH20 increases the delivery of doxorubicin in pancreatic cancer  and favoIn our study, we selected a HA-overexpressing ovarian cancer cell line and its parental counterpart as tumour models with different HA contents. Cells were implanted ectopically in nude mice. PEGPH20 pre-treatment increased PTX antitumor activity in the ovarian cancer model with high HA levels. In the parental model expressing lower HA levels, PEPGH20 did not affect PTX efficacy. Consistently with clinical data, we also found that the PTX monotherapy was less efficacious in ovarian cancer with high HA levels than in the parental cell line, supporting the correlation between HA content and tumour aggressiveness, chemoresistance and worse prognosis .A single pre-treatment with PEGPH20 did not significantly increase PTX levels in the tumour, although there was a positive tendency. Plasma and liver levels of PTX were also similar, or even lower, in PEGPH20-pretreated mice than control ones. These pharmacokinetic results were really consistent with the reported data on effect of PEGPH20 pre-treatment on systemic and tissue distribution PEGylated polystyrene nanoparticles for drug delivery in HA expressing tumours . InsteadThe effect of PEGPH20 on tumour PTX distribution and antitumor activity was at least partly confirmed in the BxPC3 xenograft, even though the results were less striking. Also in this model, PEGPH20 reduced HA tumour content and slightly improved PTX distribution and antitumor efficacy.The data obtained in the pancreatic model may help to interpret the contradictory results of clinical trials. A phase II trial in patients with HA-high metastatic PDA (HALO 202)  showed sNonetheless, on the basis of our preclinical data, further clinical development of PEGPH20 should not be excluded, as certain tumours (e.g. ovarian cancer) and therapeutic settings could benefit. Further studies are still required to understand the interplay between TME, tumour cells and drug distribution better.This study demonstrated that PEGPH20 pre-treatment can improve PTX distribution in tumour tissue in two models with high HA levels, and ultimately increase PTX antitumor efficacy. Thus remodelling the extracellular matrix of HA-rich tumours is a promising new strategy to improve the intratumor distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.Additional file 1. Additional methods and results sections.


Social media activity on a research article is considered to be an altmetric, a new measure to estimate research impact. Demonstrating software on Twitter is a powerful way to attract attention from a larger audience. Twitter integration of software can also lower the barriers to trying the tools and make it easier to save and share the output. We present three case studies of Twitter bots for cheminformatics: retrosynthetic analysis, 3D molecule viewer, and 2D chemical structure editor. These bots make software research more accessible to a broader range of people and facilitate the sharing of chemical knowledge, concepts, and ideas. Twitter is a social networking service on which users post short messages called tweet, interact with each other by sending a reply and share other users’ tweets by retweet. Scientists are using hashtags such as #chemtwitter, #RealTimeChem, #ScienceTwitter, #compchem and #OpenScience to create communities on Twitter for networking and information exchange [Social media such as Twitter has become an important communication tool for scientists. exchange . Twitterexchange , which iexchange –5. AttraA Twitter bot is an automated program on Twitter controlled through Twitter API. Bots are common on Twitter, and they usually deliver tweets like news and blog updates . TwitterIn this paper, we report case studies of cheminformatics tools demonstrated on Twitter by introducing the functionality of our Twitter bots and Twitter cards. The Twitter account @souyakuchan  was origIn this section, we introduce a Twitter bot @retrosynthchan , which cThe workflow of the bot program is as follows: (1) retrieve a reply to the bot via Twitter API, (2) check whether the given string is valid as SMILES using RDKit  and sendA reply in Twitter is a message toward a specific account, and it usually starts with the screen name of the receiver . At the time of this writing, the Twitter API provides a method to retrieve tweets that contain a specific keyword in real-time (POST statuses/filter ). The boAfter receiving a reply, the string is separated into parts by a space. The second part is assumed to be a SMILES string since the first part is the bot’s screen name. The SMILES string is considered to be valid if it can be successfully parsed by RDKit  with theRetrosynthetic analysis of the molecule represented by the given SMILES is conducted by AiZynthFinder , open-soThe generated synthetic route images are sent to the user as a reply with images. Twitter allows users to attach only up to four images in one post, so three images are connected into one image using Pillow  to send A screenshot of the bot in action is shown in Fig. The bot attracted attention from Twitter users interested in chemistry, and it gained more than one hundred followers in the first 2 weeks. The followers include chemists in academia, researchers in the drug industry, undergraduate or graduate chemistry students, etc. The Twitter bot helped the retrosynthesis software outreach to a broader range of people.Next, we introduce a 3D molecule viewer which can be played on a Twitter timeline inlinely via our web server chemical.space . This feAs a 3D molecule viewer, we adopted LiteMol , which imention tweet, the bot immediately replies with an URL in which the 3D viewer Player Card can be expanded. Users can view the 3D structure interactively as it is in the Twitter timeline.A screenshot of the viewer played on a Twitter timeline is shown in Fig. https://chemical.space/editor) is one of the easiest ways to share one’s own idea of chemical structures on Twitter. Users can share their manually edited structural formula as a SMILES or Chemical Markup Language (CML) [Finally, we introduce a Twitter Player Card which offers an easy way to share a molecule inside a tweet. The 2D chemical structural formula editor integrated with tweet posting function (available at ge (CML)  format. https://chemical.space/editor compresses CML strings by gzip using pako [Among the existing well-designed traditional browser-based 2D molecule editors such as JSME  and Marving pako  library,ing pako .https://chemical.space/editor is shown in Fig. The 2D molecule editor hosted on As presented in these case studies, it is technically possible to make any cheminformatics tool accessible and noticeable from social networking services in the form of interfacing with Twitter bots. However, to make effective impressions on the scientific community and stimulate discussion, it is necessary to use appropriate hashtags that are widely prevailing and be tweeted by accounts with a large number of followers and strong influence. It would be helpful for scientists to follow accounts of mutual interest regularly and grow their follow/follower count appropriately. Tweets with high-impact and sensational content are likely to generate more impressions, but the information described in the tweet must have a certain level of accuracy. As with the sanitization of structural formulas , it is aWe presented three examples of Twitter integrated cheminformatics tools in this study. Twitter integration can offer a convenient interface to run cheminformatics software without a laborious environment setup, and users can easily share the result by tweet and retweet. Providing a demonstration on social media will be a new approach to present one’s research and stimulate discussion in a more accessible manner. If you would like to add your own tool to our bot functions, please contact the authors for support.Project name: Twitter Retrosynthesis Bothttps://github.com/n-yoshikawa/retrosynthesis_botProject home page: Operating system(s): Platform independentProgramming languages: PythonOther requirements: AiZynthFinder, RDKit, Tweepy, PillowLicense: MIT LicenseCase study 2 and 3Project name: chemical.spacehttps://github.com/ddquest/chemical.spaceProject home page: Operating system(s): Platform independentProgramming languages: PythonOther requirements: LiteMol, RDKit, TweepyLicense: MIT LicenseCase study 1


Escherichia Coli 055:B5 endotoxin) in 7 pigs, and 3 pigs were included in the control group. The endotoxin was infused until the mean arterial pressure (MAP) dropped below 50 mmHg (TH0); then, the model animal was reanimated with fluids and vasopressors. We performed fluid challenges every hour for 6 h. ROC curve analysis and a linear mixed model were performed. The area under the curve of PPV decreased from 0.95 (0.81–1.00) to 0.60 (0.17–1.00) at TH0. Its cutoff increased from 10.5 to 22.00% at TH0. PPV showed an inverse relationship with stroke volume, mean systemic filling pressure, MAP, and systemic vascular resistance (SVR) . The PPV operative performance as a predictor of fluid responsiveness decreased with the progression of shock. This could lead to an inverse association between PPV and the following variables: MAP and SVR.Several limitations regarding pulse pressure variation (PPV) use have been reported. Our aim was to describe changes in the PPV operative performance as a predictor of fluid responsiveness during the development of a swine endotoxin shock model and to assess hemodynamic variables associated with PPV changes. A swine porcine endotoxin shock model was established ( The use of intravenous fluid therapy based on physiological variables related to fluid responsiveness has been beneficial in these patients4, whereas its use without defined hemodynamic goals has been associated with an increased medical complication rate5.Appropriate fluid therapy is key for the management of critically ill patients in intensive care units or the operating room6. Among these indices, pulse pressure variation (PPV) has been used as a predictor of fluid responsiveness in mechanically ventilated patients in several clinical settings. However, some limitations have been described8.Dynamic indices related to cardiac preload have been generally used as predictors of fluid responsiveness. A positive response to fluid challenge is usually defined as a 10–15% increase in cardiac output (CO) or stroke volume (SV)9, a variable that summarizes the features of arterial vascular load in humans10. Therefore, its operative performance and its cutoff as a predictor of fluid responsiveness could change in hypotensive states.Although this hemodynamic variable has been studied for 20 years, little is known about its operative performance as a predictor of fluid responsiveness during hypotensive states, such as septic shock. Moreover, Monge et al. showed that PPV is related to effective arterial elastanceThis study aimed to describe changes in the PPV operative performance and cutoff of PPV as a predictor of fluid responsiveness during the induction of endotoxic shock in a swine model. We also used a statistical model to explore the possible relationship between PPV and other hemodynamic variables that could explicate this decrease in the PPV operative performance.11, Resolution 008430 of 1993 issued by the Colombian Ministry of Health, and Law 84 of 1989 issued by the Congress of Colombia, the “Estatuto Nacional de Protección de Animales” . Additionally, this research was conducted according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines12.The study was carried out in accordance with the principles for the care and use of animals in research established by the Guide for the Care and Use of Laboratory Animals (NIH Guide)This study was approved by the Bioethics Committee of the Faculty of Veterinary Medicine of Universidad Nacional de Colombia (CB-FMVZ-UN-031-19). The study was conducted at the simulation laboratory of Instituto de Simulacion Medica (INSIMED) from January to December 2019. Ten female pigs  were premedicated using tiletamine-zolazepam  at 4.4 mg/kg doses through intramuscular injection. Then, each pig was cannulated through the marginal ear vein, and anesthesia was induced through the inhalation of isoflurane at a 1.5 minimum alveolar concentration (MAC) using an anesthesia mask. Once under general anesthesia, the pig was intubated and mechanically ventilated with a tidal volume of 10 ml/kg and a respiratory rate of 20 breaths/minute. For the maintenance of general anesthesia, isoflurane at doses of 1.5 MAC was used. Additionally, in all animals, the internal jugular vein and the femoral artery were cannulated using a central venous catheter (ARROW CV-17702) and an artery catheter (PiCCO PV2015L20-A). The quality of blood pressure signals was tested using a rapid washout test. All measurements were performed with the animal in a supine position, considering the phlebostatic axis as the zero reference.Animals were placed on a stationary operating table and thermoregulated using medical blanket warmers, keeping their body temperature at a minimum level of 38 °C. During the development of the model, intravenous fluid was administered using normal saline solution (SS) at an infusion rate of 3 ml/kg/hour.13: MSFP = 0.96 (CVP) + 0.04 (MAP) + 0.5 (CO).A monitor with the pulse contour cardiac output system (TFT Mindray BeneVision N22 Patient Monitor) was used. CO was calculated as the average of three thermodilution boluses (20 ml of < 8 °C SS through the jugular venous catheter). SV was calculated as CO/heart rate (HR). Systemic vascular resistance (SVR) and PPV were automatically calculated by the PiCCO system. Finally, MSFP was estimated using the method of ParkinAfter completion of the surgical procedures, animals were allowed to stabilize MAP (variation < 10%) at least for 10 min. After hemodynamic stabilization, animals were assigned to the control group  or the endotoxin group .E. Coli 055: B5, Sigma, St. Louis, MO) through the central venous catheter at an infusion rate of 7 μg/kg/h that was increased every 10 min  until reaching 20 μg/kg/h 14. The infusion of endotoxin ended when an MAP < 50 mm Hg for at least 10 min was achieved. Pigs in the control group were not administered endotoxin.Animals in the endotoxin group received a continuous infusion of endotoxin , in accordance with the international criteria for this procedure established by the American Veterinary Medical Association (AVMA).Time zero (T0) was defined in the endotoxin group and the control group as the time immediately after hemodynamic stabilization. TH0 was defined in the endotoxin group as the time when an MAP < 50 mm Hg was reached. In the control group, this time was set at 3 h of observation. This time was selected in the control group because it was the median time required to reach an MAP < 50 mm Hg for at least 10 min during the pre-experimental standardization phase of the endotoxin models.A total of 6 fluid challenges were performed in each group. The first three fluid challenges were performed every hour starting at T0 . Afterward, three fluid challenges were performed every hour starting at TH0 . Hemodynamic measurements were performed each time. This approach allowed us to assure that both groups had the same amount of fluid administered and that the variables could be compared.15. Animals in which a fluid challenge induced an increase in CO > 10% were defined as fluid responders6 6. CO was measured by transpulmonary thermodilution in all cases. The study protocol is depicted in Fig. All fluid challenges consisted of 4 ml/kg SS IV infused for 5 min through a central venous catheter. This is a standardized approach to perform a fluid challenge in humans16.Data are presented using medians and interquartile ranges. Receiver operating characteristic (ROC) curve analysis was conducted for each fluid challenge to assess changes in the PPV operative performance over time in the endotoxin group. Its 95% confidence intervals (95% CI) were calculated by the bootstrap method. The cutoff was calculated by maximizing the sum of sensitivity and specificityTwo-way analysis of variance (ANOVA) was conducted to assess differences between both groups. Additionally, one-way ANOVA was performed to determine changes in the variables over time in the endotoxin group. Post hoc analysis was carried out using the Bonferroni correction for multiple comparisons. Categorical variables were evaluated by the Chi-square test in each fluid challenge.A linear mixed model was performed to determine the associations between PPV and SVR, MAP, SV, baseline PPV, MSFP, and the endotoxin or the control group. Time was used as the random effect, the variables in the model were assessed by the restricted maximum likelihood (REML), and the contribution on each variable was quantified using the estimated value and its standard deviation. Models were compared using the Akaike information criterion (AIC), the Bayesian information criterion (BIC), and the REML. The models with the lowest AIC and BIC were considered the best models.18. A p value < 0.05 was considered statistically significant for all analyses.Data were analyzed using R statistical softwarep < 0.05) (Table p < 0.05). SVR showed similar behavior when measured at TH0 and TH1 (p < 0.05)  at T0 and increased to 23.5% at TH0 (18.0–26.5%) in the endotoxin group  19 and SVR. In clinical conditions when SVR is low, MSFP decreases20, and the relationship between the stressed/unstressed volume changes, leading to a clinical condition of relative hypovolemia. PPV will be affected by MSFP; a decrease in MSFP would decrease the driving pressure for venous return, leading to a decrease in preload and SV. This clinical condition may not show a fluid response despite a high PPV value because the fluid load leads to an increase in the unstressed volume preventing an increase in MCFP, and hence an increase in venous return and SV21. Therefore, a high PPV does not mean that there is a high likelihood of fluid response in this setting. Moreover, when MAP increased due to noradrenaline infusion, the operative performance improved, hence confirming the relationship between the PPV operative performance and SVR described above.These findings should be considered when a fluid challenge will be performed in critically ill patients with hypotensive states, and PPV will be used as a predictor of fluid responsiveness. Some studies have reported several limitations of PPV as a predictor of fluid responsiveness9. Meanwhile, PPV is related to load arterial variables. Therefore, SVV should be a better predictor of fluid challenge in this setting. Since SVV is a central parameter and PPV is a peripheric parameter. Indeed, their PPV/SVV ratio  could describe the relationship between the ventricular system and arterial system 9.We believe that these changes do not happen with stroke volume variation (SVV); since SVV is related to ventricular elastanceOur findings also showed an increase in the cutoff when an MAP < 50 mmHg was reached. Several cutoffs have been reported in the literature in different clinical settings; however, we are not aware of studies that reported an increase in the cutoff in patients with low arterial pressure. This parameter should be taken into account since it increases the amount of fluid used for reanimation, increasing the risk of fluid overload.9, a variable that summarizes the features of arterial vascular load in humans10. Other studies described a decrease in PPV after alpha-agonist infusion in experimental models of hemorrhagic shock, which could suggest a relationship between PPV and load arterial variables23. We suggest that this relationship could explain the low PPV operative performance when low arterial pressure was achieved.Other findings from our study included an inverse relationship between PPV and load arterial variables, such as SVR and MAP. Some studies support this relationship. Monge et al. showed an association between PPV and effective arterial elastance (Ea)Finally, we suggest that these findings should not be considered a limitation of PPV; instead, PPV should be considered a variable of preload dependency or a variable that allows us to recognize changes in the relationship of the stressed/unstressed volume, and the absence of changes related to a fluid challenge should alert us of the need for increased SVR. Thus, the risk of fluid overload will decrease.27. Second, we used the MSFP formula proposed by Parkin and Leaning13 instead of using an invasive measure of this variable. Nevertheless, this formula has yielded good results over time in swine models28. Moreover, the MSFP values found in the present study are similar to those reported in studies conducted in humans29. Third, our findings should not be extrapolated to all clinical settings since our model of endotoxin shock was characterized by low SVR with a compensatory increase in CO, such as early septic shock. Finally, Ea was not included in our regression model since this is calculated as MAP/SV or PP/SV; and, could have collinearity among Ea and MAP or SV. New studies are needed that allow the determination of the causes of the worsening of the PPV operative performance in this setting, assessing stressed/unstressed volume, MSFP, and venous return.The present study had some limitations. First, the evolution of PPV observed in our study may have been caused by increased vascular permeability, resulting in a decreased stressed blood volume, or by splenic red cell sequestration, as has been widely reportedIn conclusion, the PPV operative performance decreased, and the cutoff increased when MAP was < 50 mmHg. We also found an inverse association between PPV and load arterial variables, such MAP and SVR, that could be related to changes in the PPV operative performance.Supplementary Information 1.Supplementary Information 2.