P < 0.01). VEGF mRNA and protein levels were lower in the KBV200 xenograft group treated with the combined regime (P < 0.01) than those in the VCR alone group. EGCG sensitises multidrug‐resistant OSCC to VCR, and this may occur through the inhibition of angiogenesis via VEGF down‐regulation.Oral squamous cell carcinoma (OSCC) is a very aggressive malignancy, and 50% of patients who receive curative treatment die from the disease or related complications within 5 years. Epigallocatechin‐3‐gallate (EGCG) is the most abundant bioactive ingredient of tea polyphenols in green tea and has anticancer properties. Here, we evaluated the preclinical efficacy of EGCG combined with vincristine sulfate (VCR) on the growth, angiogenic activity and vascular endothelial growth factor (VEGF) expression in xenograft nude mice inoculated with KBV200 cells. Compared with VCR alone, the combined use of EGCG and VCR strongly inhibited tumour growth and angiogenesis . Epigallocatechin‐3‐gallate (EGCG) may be a promising candidate in the quest for overcoming multidrug‐resistant OSCC. EGCG combined with vincristine sulfate (VCR) strongly inhibited tumour growth and angiogenesis  CAMchick embryo chorioallantoic membraneDAB3,3‐diaminobenzidineEGCGepigallocatechin‐3‐gallateMDRmultidrug resistanceMVDmicrovessel densityOSCCoral squamous cell carcinomaPCRpolymerase chain reactionPgpP‐glycoproteinRTVrelative tumour volumeVCRvincristine sulfateVEGFvascular endothelial growth factorOral squamous cell carcinoma (OSCC) is a very aggressive malignancy of head and neck cancer and has a dismal outcome, that is, more than 50% of patients who received curative treatment would die from the disease or related complications within 5 years , 3, 4, 5Multidrug resistance (MDR) is a phenomenon whereby human tumours acquire resistance to multiple anticancer drugs with diverse structures and mechanisms of action; MDR is the main cause of chemotherapy failure and high mortality rate of cancer . One of et al.  in vitro by using RPMI‐1640 supplemented with 10% (v/v) FBS, 100 U·mL−1 penicillin G and 100 µg·mL−1 streptomycin. The culture medium was added with 200 nmol·L−1 VCR to maintain VCR resistance. For subculturing, the cells were dissociated using 0.25% trypsin with 0.02% EDTA.KBV200 cells were cultured at 37 °C [5% (v/v) CO7 cells per 0.2 mL of PBS were injected into the right axilla of each nude mouse. When the average tumour size reached 125 ± 56 mm3 in 8 days, the mice were randomly divided into six groups  as follows: control (PBS only), VCR , EGCG , VCR combined with low‐dose EGCG , VCR combined with medium‐dose EGCG  and VCR combined with high‐dose EGCG . Drug doses were determined according to the preliminary experiment in the absence of cytotoxic effect. Tumour dimensions were measured with calipres every 2 days. Mean tumour volume was calculated using the following formula: V (cm3) = width2 × length × 0.52. The mice were sacrificed on day 13 after drug administration. The tumours were resected to measure the weight and calculate tumour inhibition (%) by using the formula (mean weightcontrol − mean weightdrug administration) × 100%/mean weightcontrol. Relative tumour volume (RTV) was determined using Vsacrificed/Vbefore drug administration, and relative tumour proliferation ratio (%) was measured using RTVdrug administration/RTVcontrol.KBV200 cells were cultured and isolated by typsinisation. Cell number was counted using a haemacytometre. The cells were resuspended in PBS, and 5 × 10After the mice were sacrificed, tumours were fixed immediately in 10% buffered formalin phosphate and embedded in paraffin. The sections were stained with SP kit in accordance with the manufacturer's instructions. The paraffin sections were routinely dewaxed with xylene and hydrated with gradient alcohol. EDTA buffer antigen retrieval was used for VEGF antigens, and CD34 antigens were citrate buffer retrieved. Primary antibodies (1 : 50) were applied at 4 °C overnight. The sections were washed with PBS and incubated with secondary antibodies at room temperature. The sections were coloured with 3,3‐diaminobenzidine and stained with haematoxylin. The negative control was prepared with the same steps, except that the primary antibody was used instead of PBS.Any cytoplasmic yellow brown‐stained tumour or endothelial cells were considered as VEGF‐positive cells. Positive rate was evaluated by calculating the percentage of positive cells with a minimum of 500 cells.et al. [Angiogenic activity was assessed by microvessel density (MVD) analysis using immunohistochemistry with antibodies to endothelial marker CD34 and determined according to the method of Foote et al. . In brieg for 10 min. Serum was collected and frozen at −80 °C. VEGF concentration was measured using ELISA kits in accordance with the manufacturer's instructions.Mouse blood was obtained and centrifuged at   1500 quantity one analytic software .The tumour tissue was collected, lysed and processed for total RNA isolation at 4 °C by using an RNA extraction and purification kit. Total RNA concentration in each sample was determined using a spectrophotometer. The integrity of the extracted RNA was confirmed by electrophoresis under denaturing conditions. Total RNA 1 µg) was reverse‐transcribed to cDNA by using the first cDNA synthesis, and primers were chemically synthesised with a DNA synthesiser. The primers used for VEGF and β‐actin gene amplification are shown in Table  µg was rspss 17.0 software package (IBM Corp.). Data are expressed as mean ± SD. Differences between groups were examined using ANOVA for repeated measures, followed by Duncan's multiple range test. P values < 0.05 were considered statistically significant.All statistical analyses were performed using P < 0.01, Table Chick embryo chorioallantoic membrane was used to analyse the effect of EGCG on angiogenesis. Newborn vessels in the PBS group were rich, clear and have intact branching structure. Despite no significant morphological difference in large vessels between EGCG and PBS groups, the results indicated that EGCG exhibited substantial growth inhibitory effect on medium and small vessels, especially on the latter . In addition, VCR possessed toxicity to mice, showing a significant loss of body weight (P < 0.01). However, EGCG can reduce the toxicity of VCR, showing a significant gain of body weight (P < 0.01). These results suggest that EGCG could sensitise multidrug‐resistant OSCC to VCR in vivo.All animals survived after the experimental model procedures. The evolution of body weights of the six study groups is shown in Table P < 0.01). However, the MVD in the combined treatment group was lower than that in the VCR group (P < 0.05).As shown in Fig. P < 0.01). The VEGF expression in the VE low group did not significantly differ from that in the VCR group. However, the VEGF expression levels in the mid and high VE groups were reduced compared with that in the VCR group (P < 0.01).As shown in Fig. P < 0.01). The serum concentration levels of VEGF were 3.52 ± 0.75 and 2.93 ± 1.49 pg·mL−1 in the mid and high VE groups, respectively. These values were obviously decreased (P < 0.01) compared with those in the VCR group.The serum levels of VEGF were measured using ELISA. As shown in Fig. P < 0.05).The mRNA of VEGF in the tumour tissues of different treatment groups was measured by RT‐PCR to further examine the involvement of EGCG in the regulation of VEGF at the mRNA level. EGCG demonstrated similar effects on VEGF mRNA and serum levels. As shown in Fig. et al. [−1; this finding indicates the huge effect of VEGF on MDR [Multidrug resistance is a critical cellular defence mechanism that protects tumour cells from chemotherapy agents; this phenomenon is the major cause of treatment failure in patients with cancer . Drug efet al.  found thF on MDR .Brucea javanica oil emulsion, could reverse the MDR of leukaemia, breast cancer, liver cancer, gastric cancer and oral epithelial carcinoma [Researchers have been searching for resistance reversal agents that have high efficiency, low toxicity and a wide range of targets. Although compounds, such as verapamil and tamoxifen, demonstrate resistance reversal activity in clinical applications, they still have limitations owing to the narrow spectrum of targets and considerable side effects , 44, 45.arcinoma , 47. The−1. EGCG can also inhibit the secretion of VEGF in tumour cells, resulting in suppressed tumour angiogenesis and restrained tumour growth [Epigallocatechin‐3‐gallate, the main component of tea polyphenols, exhibits powerful biological effects, such as antimutation, antitumour formation, MDR reversal and anti‐angiogenesis , 49. Ther growth , 51.in vivo possibly by partly diminishing blood vessels. This phenomenon blocks nutritional and paracrinal effects on tumour cells and thus induces apoptosis.Sustained angiogenesis is a critical factor during the growth and metastasis of solid tumours. After a solid tumour is formed, nutrients for tumour cells are mainly supplied by diffusion in the stage called avascular early stage of invasion. The diameter of tumour nodule is generally less than 2–3 mm during this period. Once neovascularisation occurred inside the tumour, the tumour growth is accelerated immediately . The tumHowever, the effect of EGCG on tumour growth differed from that on VEGF expression. We speculated that the difference could be due to two reasons. Firstly, VEGF detection was performed using immunohistochemistry, which is not sensitive to VEGF. Secondly, the effects of EGCG on tumour growth and VEGF possibly vary, that is, the inhibition of EGCG on tumour growth might be through an alternative pathway in addition to VEGF inhibition. However, the exact reason remains unknown and must be validated in future studies.Epigallocatechin‐3‐gallate exhibits potential as a new target for cancer therapy. Inhibiting tumour angiogenesis could be an effective therapeutic option to treat refractory chemotherapeutic drug‐resistant tumours.The authors declare no conflict of interest.LC, XG and YH wrote the manuscript. LC, GL and GZ made substantial contributions to the conception, design and intellectual content of the studies. LC, XG, YH, LL and GZ made key contributions to the analysis and interpretation of data. All authors read and approved the final manuscript and agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is appropriately investigated and resolved.


Therapeutic cancer vaccines are an attractive approach for treating malignant tumours, and successful tumour eradication depends primarily on controlling tumour immunosuppression status as well as heterogeneity of tumour cells driven by epigenetic alterations.Peptide-loaded dendritic cell (DC) prime and non-infectious peptide booster heterologous immunisations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides. Heterologous vaccination regimen targeting multiple shared tumour antigens simultaneously with PD-L1 blockade was assessed against murine myeloid leukaemia.122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumours. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukaemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumour eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukaemia.A synthetic PLK1Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimisation of cancer vaccines for the treatment of numerous viral diseases and malignant tumours. Nevertheless, the insufficient outcomes of current cancer immunotherapy can be largely attributed to the existence of immunosuppressive status such as elevated levels of T-cell inhibitory ligands, and to tumour heterogeneity that is driven by intrinsic factors such as genetic and/or epigenetic instability and alteration or loss of antigen expression eliciting reduced immunogenicity.7 Of these, blockade of T-cell inhibitory signals, especially PD-1/PD-L1 signal has achieved significant objective response rates correlated with intratumoural CD8 T-cell infiltration grade in the clinic.9 In contrast, tumour heterogeneity, mainly varied antigenicity remains a major challenge in the development of effective immunotherapies.Substantial progress in cancer immunology has provided clear evidence that antigen-specific cytotoxic CD8 T-cells are key immune effectors that eliminate malignant tumour cells. However, in many instances, tumour-reactive CD8 T-cells are present in low frequency and/or lack the critical functionality to eradicate established tumours.12 Particularly, as targeting a single antigen may be inadequate to eradicate malignant tumour cells due to tumour escape driven by low and/or loss of target antigen, numerous reports have shown that targeting multiple epitopes from a single antigen or simultaneously targeting different antigens elicited a promising anti-tumour immunity in vivo.15 However, a significant long-term clinical benefit has been hardly achievable with these vaccines, indicating that their immunogenicity is suboptimal. Thus, there is a clear need for further optimising strategies to potentiate peptide-based vaccine, and for identifying and defining immunogenic novel T-cell epitopes to achieve the desired therapeutic benefit.Several investigators including ours have attempted to utilise synthetic peptides representing CD8 T-cell epitopes as vaccines.16 Considering this, PLK1 has been an attractive target for cancer drug development,18 and demonstrated to be a shared antigen useful for inducing T-cell responses.19 In this study, we evaluated the therapeutic benefits of a synthetic peptide PLK1122, corresponding to a CD8 T-cell epitope from PLK1 against myeloid leukaemia that exhibit a high level of tumour heterogeneity evolving upon disease progression and/or relapse.22 Moreover, we demonstrate that targeting multiple shared antigens,23 survivin (Sur) and PLK1 simultaneously in conjunction with prolonged blockade of checkpoint inhibitory signal could be used as an effective strategy to overcome antigenic heterogeneity, resulting in complete eradication of clonally heterogeneous C1498 myeloid leukaemia. Collectively, the present findings should facilitate the development of more effective immunotherapeutic strategies for tumours that may circumvent current limitations associated with cancer vaccines.Since tumour cells are capable of altering targetable antigen expression to evade tumour immunity, we hypothesised that targeting survival-associated proteins such as shared antigens could be a way to overcome antigenic heterogeneity of tumours. Polo-like kinase-1 (PLK1), which is essential in cell-cycle regulation, is overexpressed in virtually all malignancies and its overexpression correlates with prognosis of cancer in patients.2 inhalation. Murine cell lines including C1498 leukaemia were obtained from the American Type Culture Collection and cultured as recommended by the provider. Luciferase-expressing C1498 transfectants (C1498-luc) were prepared using recombinant lentiviruses encoding firefly-luciferase-P2A-CD90.1 cDNA. After transduction, stable CD90.1-positive cells were isolated by flow cytometric sorting, and in some instances, C1498-luc clone was further isolated with cell cloning at limiting dilutions (referred as C1498Homo-luc).C57BL/6  mice were purchased from Orient-Bio and maintained in our animal facilities under specific-pathogen-free conditions. Animal care and experiments were conducted according to our institutional animal-care and use committee guide-lines. Mice were anaesthetised with inhalation of 1.5–2% isoflurane and euthanised humanely by COThe peptide sequences used in this study are as listed in supplementary Table 11 For vaccination, mice were immunised intravenously with 2 × 106 DCs pulsed with 10 µg/ml peptide(s) for 18 h. DCs were mixed with 30 µg poly-IC prior to injection, and 7-days later mice received an intravenous TriVax-boost immunisation consisting of 150 µg synthetic peptide(s), 50 µg poly-IC, and 100 µg anti-CD40 Abs. For PD-L1 blockade, anti-PD-L1 or anti-PD-1 Abs were administered intraperitoneally (200 µg/dose) after prime and boost immunisations twice on days +1 and +3, and for three-times on days +1, +3 and +5.Dendritic cells (DCs) were prepared as described previously.10 using freshly isolated splenic CD8 T-cells . For peptide dose curve responses, serial peptide dilutions were incubated with RMA-S cells overnight and freshly purified CD8 T-cells were added for an additional 40 h before harvesting supernatants to measure IFNγ-production by ELISA (eBioscience).For measuring antigen-specific CD8 T-cell responses, peripheral blood samples or splenocytes were incubated with 1 μg/ml peptide and 1 μg/ml GolgiPlug  at 37 °C. After 6 h, cells were stained for intracellular IFNγ following the directions provided by vendor (BD-Bioscience). For CD107a/b mobilisation assay, 2 μg/mL of fluorescence-conjugated anti-CD107a and CD107b Abs were added at the beginning of the stimulation period. To evaluate the in vitro T-cell tumour recognition, IFNγ-EliSpot assay were performed as described previously,6 C1498-luc cells, and 7-days later when tumours measured >3–5 mm in diameter the first immunisation was administered. For orthotopic myeloid leukaemia settings, mice were inoculated intravenously with 2 × 106 C1498-luc cells, and 7-days later, were given their first immunisation with peptide-loaded DCs. Seven-days later, the mice received a TriVax-booster immunisation. For in vivo depletion, each mouse received intraperitoneal administrations of 200 µg Abs against NK1.1, CD4, CD8, and CD25 on days −3 and −1 before the prime-boost immunisations. Depletions were confirmed with blood samples using flow cytometry (data not shown). The intravenous leukaemic tumour growth was assessed weekly by recording luciferase-bioluminescence from tumour tissue using Xenogen in vivo imaging system , and presented the time course of in vivo bioluminescence of average radiance in individual mice.To assess the therapeutic effects, mice were inoculated subcutaneously with 2 × 10Livers and Lungs were dissected and chopped into 2–5 mm sizes and dissociated using the gentleMACS Dissociator following the directions provided by vendor (Miltenyi-Biotec). Intracellular co-staining of Foxp3 was conducted as per the instructions provided by vendor (eBioscience). The relative PLK1 expression levels in freshly isolate C1498 cells were measured using the TaqMan™ Gene Expression Master Mix  according to the manufacturer’s instruction. Sequences of primers and probes are as follows: PLK1 forward, 5′‐GCCTGAGGCCCGCTACTAC‐3′, and reverse, 5′‐TGATTGCGGTGCAGGTACTG‐3′; probe, 5′‐FAM‐TGCGACAGATAGTCCTG‐BHQ-1‐3′; β‐actin forward, 5′‐CGATGCCCTGAGGCTCTTT‐3′ and reverse, 5′‐TGGATGCCACAGGATTCCA‐3′; probe, 5′‐FAM‐CCAGCCTTCCTTCTT‐BHQ-1‐3′. The levels of PLK1 gene expression divided by those of β‐actin gene expression were defined as relative PLK1 expression.t-tests, and survival analysis was established by Kaplan–Meier curves using log-rank tests. All analyses were performed using Prism 5.01 software (GraphPad).Results are representative of data obtained from at least two independent experiments. Statistical significance to assess the numbers of antigen-specific CD8 T-cells was determined by unpaired Student’s b-binding motifs. Eight peptide sequences and a mutated peptide PLK1345/9M, which has a substitution at position 9M for D to improve MHC-I binding affinity, were selected . PLK1345/9M had lower MHC-I dissociation rates in comparison to H-2Db-binder hgp10025 peptide. PLK1122 had fast dissociation rates at early time points but higher amounts of PLK1122 remained bound to H-2Kb in comparison to the H-2Kb-bound Trp2180 . Then, peptides PLK1121, PLK1122, PLK1345, and PLK1345/9M were evaluated for their capacity to induce CD8 T-cell responses capable of recognising PLK1-expressing tumour cells. PLK1122 and PLK1345/9M generated higher number of antigen-specific IFNγ-producing CD8 T-cells with lytic functionality (CD107a/b mobilisation) in comparison to that generated by PLK1121 and PLK1345 .First, we sought to identify at least one PLK1-derived CD8 T-cell epitope that could be naturally processed and presented on tumour cells, and thus be used for a peptide-based cancer therapy. Using three web-based MHC-peptide prediction algorithms, PLK1 was analysed for the presence of H-2ed Table . To asse345 Fig. . However345/9M and Ova55DC_TriVax had moderate therapeutic effects when compared to unvaccinated mice, mice that received PLK1122DC_TriVax vaccination had substantially reduced tumour progression as compared to those with PKL1345/9M and Ova55 peptides, marked by significantly lower bioluminescent signals (by day42), which correlated with the observed tumour growth . Likewise, we verified that PLK1122DC_TriVax vaccination could extend to another 7-day-established subcutaneous Hepa1-6 hepatoma, where tumour was completely eradicated . Notably, when the mice that rejected Hepa1-6 tumours were re-challenged on day48 with C1498-luc cells, the mice could reject the secondary tumour challenge . So far, these results imply that PLK1 protein can be used as a shared tumour antigen and PLK1122 is the most effective peptide in generating CD8 T-cells capable of recognising numerous PLK1-expressing tumour cells, resulting in potent therapeutic anti-tumour effects.Next, we evaluated whether PLK1-based peptide vaccination would offer a therapeutic benefit in vivo against established syngeneic tumours. Mice were first challenged subcutaneously with C1498-luc, and 7-days later, when tumours reached a size of 3–5 mm diameter the mice received PLK1-derived peptide-loaded DC prime_TriVax booster immunisation with a 7-day interval. Although PLK1122DC_TriVax vaccination regime in orthotopic C1498 myeloid leukaemia, which offer more clinically relevant tissue site-specific tumour setting. Mice were intravenously engrafted with C1498-luc cells, and 7-days later, the PLK1122DC_TriVax vaccinations were initiated. As shown in Fig. 55DC_TriVax had moderate therapeutic effects as compared to unvaccinated mice, whereas mice receiving PLK1122DC_TriVax had substantially reduced tumour progression in comparison to those with Ova55 peptides, resulted in significantly increased median survival of the mice by more than at least 1-week  to the PLK1lls Fig. . A peptiade Fig. . Next, w122DC_TriVax in myeloid leukaemia setting, we initially assessed for the presence of antigen-specific CD8 T-cells after euthanatising three mice in each group after 23-days of tumour infusion. As shown in Fig. S4A, B, mice that received the PLK1122DC_TriVax with PD-L1 blockade had comparably higher numbers of antigen-specific CD8 T-cells capable of effectively recognising C1498 tumour cells, whereas mice that received the Ova55DC_TriVax did not have any detectable PLK1122-specific CD8 T-cell responses. In this circumstance, we further investigated the presence of C1498 tumour cells, CD8 T-cell expansion, and the frequency of myeloid-derived suppressor cells (MDSC) in different tissues. The ratios of CD8 T-cells to regulatory T-cell and the frequency of CD90.1+C1498 cells were inversely correlated in several tissues . The combined treatment of PD-L1 blockade with PLK1122DC_TriVax led to decreased levels of Ly6GlowLy6Chigh monocytic MDSC subsets, but not Ly6GhighLy6Clow granulocytic polymorphonuclear MDSC, which were presented as percentage of CD11b+CD115high myeloid cells in the livers and lungs . Likewise, histochemical analysis also evidently displayed cancerous blast suppression in mice that received PLK1122 vaccinations in all tissues, except lungs .To further study any issues that impeded the efficacy of the combination of PD-L1 blockade with PLK1122DC_TriVax vaccination in same leukaemia settings. Surprisingly. the prolonged period of PD-L1 blockade dramatically enhanced the therapeutic efficacy of PLK1122DC_TriVax, in which complete tumour regressions were attained . In contrast, although moderate therapeutic effects were observed with anti-PD-1 Abs as compared to those with Ova55 peptides, no significant improvement of tumour regression was observed in comparison to that observed with anti–PD-L1 Abs. Particularly, at day 72, freshly isolated CD8 T-cells from survivor mice exhibited effective recognition not only to PLK1122-pulsed targets but also towards C1498 tumour cells , suggesting that the sufficient endurance of PD-L1 blockade could potentiate the therapeutic effectiveness of PLK1122DC_TriVax to C1498 myeloid leukaemia.In our further studies involving PD-L1 blockade, we evaluated the duration period of the combinatorial Abs delivery and the alternative efficacy of anti-PD-1 Abs since its blocking effect could also be achieved by hindering PD-1 receptors that are expressed on T-cells. We compared the efficacy of three doses of anti–PD-1 and anti–PD-L1 Abs  in combination with PLK124 we examined the expression levels of PLK1 protein, surface MHC-I, and surface PD-L1 on freshly isolated CD90.1+C1498 cells from liver tissue (referred to as C1498Liver) of mice that exhibited progressive tumour growth after receiving PLK1122DC_TriVax with PD-L1 blockade post 52-days of tumour infusion. As shown in Fig. Liver. However, when investigating the surface expression of MHC-I (H-2Db and H-2Kb) and PD-L1, the maternal C1498-luc revealed heterogeneous levels of MHC-I and relatively high level of homogenous PD-L1 expression, and these molecules were upregulated in response to inflammatory stimuli IFNγ that could be considered as inflammatory conditions of tumour sites in vivo  when administered in combination with anti-PD-L1 Abs. To verify the long-term anti-tumour immunity, the survivor mice were re-challenged (on day 105) with maternal heterogeneous C1498-luc cells without any further treatment. None of these mice developed tumours, in contrast to a naïve control group challenged with the same tumour  peptide, known as naturally processed H-2Db epitope25 was incorporated with PLK1122 because we sought to demonstrate our hypothesis by targeting clinically relevant shared antigens. As shown in Fig. 20 generated quite low numbers of antigen-specific lytic-functional CD8 T-cells in comparison to those generated by PLK1122, whereas multi-peptides PLK1122/Sur20DC_TriVax triggered simultaneous multivalent CD8 T-cells at levels comparable to that triggered by the administration of individual peptides. Correspondingly, freshly isolated splenic CD8 T-cells from PLK1122/Sur20-immunised mice recognised C1498 tumour cells at significantly better efficiency in comparison to their recognition by CD8 T-cells from individual peptide-immunised mice . The considerably improved effectiveness of PLK1122/Sur20DC_TriVax was also obtained even in the absence of PD-L1 blockade.Next, to validate the second possibility, Surice Fig. . Subsequ122/Sur20DC_TriVax in same leukaemia settings. The results presented in Fig. 122/Sur20DC_TriVax immunisation was effective in completely eradicating clonally heterogeneous C1498-luc leukaemia in the majority of the mice when administered in combination with prolonged period of PD-L1 blockade (6 out of 10 mice survived). Remarkably, PLK1122/Sur20DC_TriVax with PD-L1 blockade revealed higher numbers of PLK1122-specific CD8 T-cells as compared to using PLK1122DC_TriVax  C1498 myeloid leukaemia. Under these circumstances, the sustained PD1 blockade with PLK1122/Sur20DC_TriVax reduced the median tumour growth rate significantly, but no tumour rejections were observed . These overall results suggest that the induction of multiple antigen-targeting CD8 T-cells and sufficient endurance of PD-L1 blockade amplified intensely the therapeutic effectiveness of DC_TriVax regimen towards clonally heterogeneous C1498 myeloid leukaemia.Lastly, we assessed the therapeutic efficacy of sustained PD-L1 blockade combined with PLK1Vax Fig. . Moreove28 However, these studies use highly immunogenic foreign antigen-expressing C1498 cells to validate antigen-specific anti-tumour CD8 T-cell responses and thus have potential limitations of using artificial systems. In recent years, progress on identification of neoantigens with individual specificity, which are generated by non-synonymous mutations in the tumour genome, have revealed promising preliminary clinical outcomes.31 However, though neoantigen could be a theoretically ideal target as non-self-proteins, it is frequently hard to translate a genomic mutation to a neoantigen. Moreover, single neoantigen-based personalised vaccines could only eradicate a small number of tumour cells due to the heterogeneity in tumours. Unlike neoantigens, targeting most natural tumour antigens could broaden to a wide range of cancer coverage, but they are self-proteins which are over-expressed and/or mutated in the cancerous cells while being expressed at lower levels in normal cells attributing to potential T-cell tolerance toward self-proteins. Thus, identifying targetable self-antigens (raised in tumours) and defining novel T-cell epitopes are crucial for accomplishing T-cell-mediated cancer therapy in the clinical realm, in which the same target can be used in all patients with particular malignant tumours. In this aspect, our findings provide a promising preclinical strategy in which using PLK1-derived CD8 T-cell epitope peptides one could achieve high levels of tumour-reactive T-cell responses eliciting potent therapeutic effects.Over the past decades, the identification of tumour antigens for T-cells and their corresponding T-cell epitopes have expedited development of peptide-based vaccines against malignant tumours. In this study, our original intention was to evaluate the therapeutic efficacy of the combined immunotherapeutic strategy of peptide-based DCs prime_TriVax booster regimen with PD-L1 blockade in orthotopically established myeloid leukaemia, which offer more clinically relevant tissue site-specific tumour setting. Previous studies have shown that the in vivo engagement of PD-L1 blockade, CD40 ligation, and STING agonist are capable of enhancing leukaemic antigen-specific T-cell stimulatory capacity and inducing anti-leukaemic therapeutic effects in C1498 leukaemia models.122 (DSDFVFVVL) was the most effective of the predicted H-2b-binding candidates eliciting tumour-reactive CD8 T-cell responses in C57BL/6 mice. Interestingly, the high-scoring 8-mer PLK1123 (SDFVFVVL) and 10-mer PLK1121 (EDSDFVFVVL) in the computer-based algorithms were not as effective at inducing CD8 T-cell responses when compared to the PLK1122 peptide  to make a heteroclitic peptide vaccine, which could overcome the pre-existing immune dysfunction of cancer patients.32 PLK1345/9M revealed slower MHC-I dissociation rates in comparison to PLK1122 peptide, and PLK1345/9M vaccination could also evoke higher numbers of CD8 T-cells directed towards peptide-pulsed target but not effective against PLK1-expressing tumour cells  may not be expressed in sufficient amounts on tumour cells to allow for T-cell recognition, whereas PLK1122 is a naturally processed immunodominant epitope present on the surface of various malignant tumours.To our knowledge, the present work could be the first attempt to evaluate a peptide vaccine representing a PLK1-derived CD8 T-cell epitope. The 9-mer peptide PLK1ide Fig. . Additiolls Fig. . These r122-based vaccination could achieve higher numbers of functional and long-lasting cytotoxic CD8 T-cells capable of translating into significant therapeutic benefits against C1498 myeloid leukaemia. For these studies, we incorporated a novel vaccination regimen in which one could achieve high levels of CD8 T-cell responses using antigen-loaded DCs followed by peptide-based vaccine (TriVax) booster immunisation.11 Notably, PLK1122DC_TriVax regimen could be applied to various tumour-types to provoke tumour regression and prolonged survival, and tumour re-challenges in mice that rejected their initial tumours verified the presence of long-term antigen-specific CD8 T-cell memory immunity capable of rejecting even other types of tumour rechallenge . These results suggest that PLK1 could be a shared tumour antigen that is expressed at high levels in nearly all malignant tumours and PLK1122-based vaccination could enable effective control over numerous tumour growths. Interestingly, PLK1122DC_TriVax vaccination had a superior therapeutic efficacy toward localised implanted leukaemic tumours (like a solid mass), where complete tumour rejections were observed, in comparison to its efficacy against intravenously engrafted systemic haematological leukaemia. In agreement with this, a previous report has also shown that systemic introduction of C1498 cells induced a potent T-cell tolerance caused by the abortive expansion and deletion of leukaemia-reactive CD8 T-cells, while leukaemia-specific CD8 T-cell responses were effectively induced in localised leukaemic tumour-bearing mice.27 These results indicate that the characteristics of tumour-reactive CD8 T-cell responses induced by PLK1122DC_TriVax were quite diverse depending on whether the C1498 tumour challenge was local or systemic, and more essentially, further manipulations to PLK1122DC_TriVax regimen are needed to circumvent T-cell tolerance and dysfunctionality that could be induced by systemic C1498 leukaemia.Furthermore, PLK133 There is evidence that combining therapeutic cancer vaccines with additional modalities such as chemotherapy and/or checkpoint inhibitors have shown synergistic therapeutic outcomes in clinic,34 which is not just in killing the tumour cells but in inducing a range of alterations upon cancer and/or immune cells. So far, significant advances in potentiating anti-tumour efficacy by reversing immune tolerance in tumour site have been achieved through obstruction of immune checkpoints including PD-1/PD-L1 interactions. Thus, combined therapeutic strategies with PD-1/PD-L1 checkpoint inhibitors could strengthen tumour-specific immune responses though they may not work in all individuals and in all cancers. We and other have shown that PD-1/PD-L1 blockade prevents exhaustion of tumour-reactive T-cells even in haematological malignancies, leading to augmented effector function and persistence of antigen-specific T-cells at the tumour site.36 Indeed, we observed that the systemic administration of anti-PD-L1 Abs with PLK1122DC_TriVax led to an increase in the frequency of tumour-reactive CD8 T-cells that had higher antigen-recognition functionality, resulting in enhanced anti-tumour efficacy with an increase of overall T-cell numbers in systemic C1498 leukaemia . Although our results are in line with previous reports that demonstrate the synergistic effects of immune checkpoint inhibitors with vaccines,37 we believe that there is a clear need for additional strategies to optimise PLK1122DC_TriVax vaccination regimen to achieve the desired therapeutic benefits.It has become clear that multiple immune evasion mechanisms are present in tumour sites which potently inhibit tumour-reactive T-cell responses, leading to immune exhaustion and dysfunction eliciting tumour progression and poor clinical prognosis.40 Particularly, previous studies demonstrated that myeloid disorders including acute leukaemia and myelodysplastic syndromes exhibit clonal heterogeneity that evolves upon disease progression and/or relapse.22 In our studies, remarkable therapeutic efficacy  was accomplished with PLK1122DC_TriVax vaccination under PD-L1 blockade in clonally homogenous C1498Homo-engrafted leukaemic mice , which are overexpressed in the vast majority of human cancers with minimal expression in normal tissue, has proven effective in inducing antigen-specific T cell precursors and safe indicating no severe adverse events.48 Moreover, even in studies using TCR-engineered redirected T cells with high-avidity TCR specific for survivin and TERT, severe auto-reactive toxicity was not reported.50 As PLK1 protein is essential for cell-cycle regulation, they could be detected in highly proliferating normal tissues, targeting PLK1 antigen may occur potential adverse events. Nonetheless, we observed that the freshly isolated CD8 T cells from PLK1122DC_TriVax-vaccinated mice do not recognise normal liver cells, and the mice did not show common autoimmune pathophysiological symptoms such as weight change and abnormal behaviours. These observations imply that targeting PLK1 protein in immunotherapeutic strategies has a potential anti-tumour application in the future though it needs to be further investigated.The clinical outcomes of cancer vaccine therapies notwithstanding, potential concerns have been raised over targeting the overexpressed tumour antigens because they also show a low level of expression in some healthy tissues, which may induce unknown and possibly hazardous self-reactive adverse effect.Collectively, we present data demonstrating that combined immunotherapeutic strategy of multi-antigenic cancer vaccines with sustained PD-L1 blockade are able to induce potent tumour-reactive T-cells capable of overcoming the potential immune escaping of clonally heterogeneous myeloid leukaemia, resulting in remarkable therapeutic anti-tumour benefits. Additional studies with a focus on optimising the peptide vaccination regime that affect the specificity and avidity of tumour-reactive T-cells towards tumour escaping variants causing clonal heterogeneity could facilitate clinical applications in the treatment of various tumours, including haematologic malignancies.Supplementary data


Intravenous fluid administration should be considered as any other pharmacological prescription. There are three main indications: resuscitation, replacement, and maintenance. Moreover, the impact of fluid administration as drug diluent or to preserve catheter patency, i.e., fluid creep, should also be considered. As for antibiotics, intravenous fluid administration should follow the four Ds: drug, dosing, duration, de-escalation. Among crystalloids, balanced solutions limit acid–base alterations and chloride load and should be preferred, as this likely prevents renal dysfunction. Among colloids, albumin, the only available natural colloid, may have beneficial effects. The last decade has seen growing interest in the potential harms related to fluid overloading. In the perioperative setting, appropriate fluid management that maintains adequate organ perfusion while limiting fluid administration should represent the standard of care. Protocols including a restrictive continuous fluid administration alongside bolus administration to achieve hemodynamic targets have been proposed. A similar approach should be considered also for critically ill patients, in whom increased endothelial permeability makes this strategy more relevant. Active de-escalation protocols may be necessary in a later phase. The R.O.S.E. conceptual model  summarizes accurately a dynamic approach to fluid therapy, maximizing benefits and minimizing harms. Even in specific categories of critically ill patients, i.e., with trauma or burns, fluid therapy should be carefully applied, considering the importance of their specific aims; maintaining peripheral oxygen delivery, while avoiding the consequences of fluid overload. Intravenous fluids have been in clinical use for over a century, yet the medical and scientific community have only recently begun to appreciate the importance of judicious fluid administration, the necessity to handle them as any other drug we prescribe –4, and tResuscitation fluids are used to correct an intravascular volume deficit or acute hypovolemia; replacement solutions are prescribed to correct existing or developing deficits that cannot be compensated by oral intake alone [maintenance solutions are indicated in hemodynamically stable patients that are not able/allowed to drink water in order to cover their daily requirements of water and electrolytes [fluid creep, has been recently underlined [Three major indications exist for intravenous fluid administration , 4, 7–9:ke alone ; maintentrolytes , 11. In derlined , 13.https://www.fluidacademy.org).Although the use of intravenous fluids is one of the most common interventions in medicine, the ideal fluid does not exist. In light of recent evidence, a reappraisal of how intravenous fluids should be used in the perioperative and critical care setting is warranted. Here, we present the executive summary on this area of the International Fluid Academy . Therefore, there are two main categories of balanced solutions (Table − content (Cl− ≤ 110 mEq/L).Intravenous “balanced” solutions include crystalloids and colloids with minimal effect on the homeostasis of the trations . In addins Table : (1) flu2); (2) the concentration of non-volatile weak acids (ATOT); (3) the strong ion difference (SID), defined as the difference between the sum of all strong cations and the sum of all strong anions [ATOT [2, should balance these variations. Recent studies clearly showed that, in this regard, the ideal balanced solution should have an in vivo SID equal to the baseline concentration of HCO3− [3−, plasma pH will tend toward alkalosis; if the SID of the infused fluid is lower than plasma HCO3−, plasma pH will tend toward acidosis, as it is always the case for NaCl 0.9%, the so-called “normal” saline [According to the quantitative approach to acid–base equilibrium , 18, theg anions . These pns [ATOT –22. Ideaof HCO3− . If the ” saline .− content , as compared to NaCl 0.9%. Nonetheless, the final composition of such a fluid, especially when considering crystalloids, will depend on (1) tonicity; (2) electrical neutrality and (3) SID. Indeed, an isotonic balanced solution leaving unaltered acid–base equilibrium  will necessarily have a Cl− content > 110 mEq/L (as in Sterofundin-ISO). In contrast, a fluid with an SID of 24 mEq/L and a lower Cl− content will necessarily be slightly hypotonic (as with Lactated Ringer’s). Finally, an isotonic fluid with a low Cl− content will necessarily have a higher SID (as with PlasmaLyte), with a consequent alkalizing effect.As stated above, the definition of “balanced” solution includes also a category of iso- and near-isotonic fluids with a low ClBalanced and unbalanced (NaCl 0.9%) solutions might have slightly different effects on blood volume expansion, according to the clinical condition. Indeed, different kinetics showing approximately a 10% decrease in plasma volume expansion of balanced solutions as compared to NaCl 0.9% have been described in normovolemic healthy volunteers , 26. On Despite these controversies, which need further clarification, several definitive differences exist between these two categories of drugs. First, chloride-rich NaCl 0.9% causes a higher dose-dependent degree of acidosis and hyperchloremia, which possibly favors the contraction of vascular smooth muscles , 29, potWhen healthy volunteers received 2 L of either saline or Plasma-Lyte over 1 h, saline significantly decreased renal artery blood velocity, decreased renal cortical tissue perfusion, decreased urine output, and increased extravascular fluid accumulation compared with Plasma-Lyte . These f® versus NaCl 0.9% on the first day of major abdominal surgery led to significantly less renal failure requiring dialysis [Indeed, a large-scale propensity-matched observational analysis of U.S. insurance data showed that the use of PlasmaLytedialysis . In addidialysis , 34. Inddialysis , 36. Howdialysis  and a redialysis .p = 0.08) nor the incidence of persistent renal dysfunction  was statistically significant. A similar study performed by the same authors and published in the same issue of the New England Journal of Medicine, the SALT-ED trial, found a similar difference in the incidence of major adverse kidney events in non-critically ill adults [Two important and large randomized controlled trials comparing the use of balanced solutions and normal saline have been published in the last years. The SPLIT study was the first multi-center double-blind randomized controlled trial performed on 2092 patients, comparing balanced and unbalanced fluids in intensive care units. It showed no significant difference in the main outcome, i.e., incidence of acute kidney injury . While pl adults .In summary, we can avoid fluid-induced metabolic acidosis and excessive chloride loading simply using balanced solutions. There is increasing evidence that an excessive chloride administration may have a detrimental effect on renal function, even at low doses. Therefore, the use of balanced solutions, particularly in patients that potentially need a significant amount of intravenous fluids, seems to be a reasonable pragmatic choice . On the Albumin accounts for approximately 50% of the plasma protein content  and is tThe ALBIOS study, a large Italian randomized controlled trial, gave some suggestions on whether or not albumin administration improves outcomes in severe sepsis and septic shock . PatientColloids should remain in the intravascular space longer than crystalloids, provided that the endothelial barrier is intact, which is often not the case in critically ill patients . Given tThe significant cost and the availability of equally effective low-cost alternatives do not play in favor of albumin, although a subgroup analysis of the ALBIOS dataset may suggest that albumin infusion is likely cost-effective in patients’ septic shock . Up to dThe aim of perioperative fluid therapy, in parallel with the maintenance of the effective circulating blood volume, is to avoid both fluid overload and under-hydration, while maintaining patients’ fluid balance as close as possible to zero. Despite this rationale, it is not unusual for surgical patients to receive 5–10 L of fluid and 600–1000 mmol of sodium, leading to edema and adverse outcomes , which iFluid therapy is not only meant to compensate intraoperative losses but should also take into account those occurring prior to surgery, induced by poor water intake, bowel preparation, major inflammation associated with a stress response, and possibly, hemorrhage. Dehydration, however, is difficult to detect through clinical methods.Many studies examined whether a fluid load is capable of reducing hypotension caused by the induction of general/regional anesthesia. However, results regarding a preload strategy have been discouraging , 66.restrictive compared with the standard of care that was excessive  [In response to the ongoing administration of large volumes of crystalloid to patients undergoing major surgery, a ‘fluid restrictive’ strategy has been proposed. For example, Brandstrup et al. demonstrated in a multi-center randomized controlled trial that a more restrictive regimen was associated with better outcomes following colorectal surgery . Howevervolumes) . It is tvolumes) . What isvolumes) –72. ReceIt is common in Enhanced Recovery after Surgery (ERAS) protocols to find the term “intraoperative fluid restriction” . HoweverI2  =  0%), need for renal replacement therapy , and major infectious complications . It is worth mentioning that eligible trials were too small to draw firm conclusions on this issue.Fluid management in postoperative patients is a key determinant of their outcomes. While restoring effective volume is critical for these patients, fluid management should not compromise healing processes. Optimal fluid management should thus target efficient central hemodynamics and tissue perfusion while avoiding positive net fluid balance. In theory, colloids offer the advantages over crystalloids of higher plasma expansion capacity and longer plasma half-life. They have the theoretical disadvantage of delaying clotting time and increasing the risk of kidney injury. In randomized trials, the ratio of the cumulative dose of colloids over the cumulative dose of crystalloids ranged roughly from 0.41 to 1 . In patiIt should also be stated that there are opposing views regarding the use of starches . For exaAmong patients undergoing major abdominal surgery, the recent results of the FLASH trial, showed no significant difference in a composite outcome of death or major postoperative complications within 14 days after surgery .Pending the results of ongoing trials, there are currently insufficient data to ban the use of colloids in the surgical intensive care unit.Many patients undergoing surgery are not able to ingest food or fluids for some time following surgery and will require maintenance fluids. Recently, a debate emerged on the tonicity of these solutions: although guidelines traditionally recommended the use of hypotonic maintenance fluids, in pediatric literature, these were shown to be associated with an increased incidence of symptomatic hyponatremia , 84. TheA certain degree of hypervolemia is necessary to maintain organ perfusion during anesthesia and surgery. However, fluid given after the induction of anesthesia mainly increases “unstressed” blood volume, because vasodilatation occurs as a consequence of anesthesia. At this point, additional fluid administration is needed to optimize stroke volume, i.e., to add to the “stressed” intravascular volume . Many clFluid administration is one of the cornerstones of hemodynamic resuscitation in critically ill patients. How much fluid to give has been the subject of lively debate over the years. Too much fluid can have harmful consequences on multiple organ systems, e.g., worsening gas exchange, renal function and wound healing. Fluid overload is particularly likely to arise in conditions when capillary permeability is altered due to an inflammatory response, such as during sepsis. A positive fluid balance has been associated with worse outcomes in several studies in various groups of intensive care unit (ICU) patients , 93–95. It has to be acknowledged that a positive fluid balance could be a marker of disease rather than a pure iatrogenic or preventable problem and it would be erroneous to assume the default position of under-resuscitation. Indeed, inadequate resuscitation due to insufficient fluid administration may result in poorer tissue perfusion and hence organ dysfunction and failure, particularly in the early phase of treatment. A balance needs to be achieved, such that each patient receives sufficient, but not excessive, fluid for her/his needs. Crucially, different patients will have different needs and baseline fluid status depending on multiple factors including age, co-morbid disease and current diagnosis. In addition, it is mandatory to consider indices of fluid tolerance, such as CVP, lung water, oxygenation and hemoglobin levels. Fluid requirements vary during the course of illness. As such, fluids must be prescribed on an individual patient basis; the prescription should be regularly reviewed and tailored to the evolving clinical stage. The answer to the question of whether fluid overload is a problem in the ICU will thus depend on when it is asked. In the acute resuscitation/salvage phase, fluid administration is generous. While fluid overload should always be a concern, a positive fluid balance is a specific target of this phase.The term deresuscitation/de-escalation was first suggested in 2012  and finaEbb phase of shock [Flow phase [Ebb to Flow phase and will remain in a state of unresolved shock with positive cumulative fluid balance, and this is where active deresuscitation/de-escalation might have an important role.Fluid overload and a positive cumulative fluid balance are associated with increased morbidity and worse outcomes, as previously discussed. The natural course of events after a first insult  is a systemic inflammatory response with increased capillary permeability and organ dysfunction . The preof shock . In the ow phase . HoweverIt is unclear which is the best therapeutic option for deresuscitation/de-escalation. The administration of albumin in combination with diuretics  and the association of this strategy with the sequential application of PEEP set to counteract intra-abdominal pressure (IAP) have been proposed . In addi5 steps of Deresuscitation/De-Escalation need to be kept in mind: (1) define a clinical endpoint ; (2) set a fluid balance goal ; (3) set perfusion and renal safety precautions ; (4) re-evaluate after 24 h unless safety limits reached; (5) adjust the plan accordingly.In conclusion, it is crucial to ensure that the indication for fluid resuscitation no longer exists  before starting with deresuscitation. Furthermore, the four dynamic phases. The Acute Dialysis Quality Initiative (ADQI) group proposed S.O.S.D.  as acronym [International Fluid Academy Day (IFAD) meetings there was a clear preference for the R.O.S.E. acronym  as summarized below, in Fig. Two articles were published recently, almost simultaneously, referring to the dynamics of fluid therapy , 101. Thpositive. We do not support blind adherence to the surviving sepsis campaign guidelines adagio to administer 30 ml/kg of fluids within the first hour for all patients, as explained previously [In the first, salvage/resuscitation phase, when a patient presents with hemodynamic shock, the aim of the treatment is resuscitation and correction of shock with the achievement of an adequate perfusion pressure. A rapid fluid bolus should be given , normally in association with vasopressor administration. In parallel, emergency procedures to resolve any obvious underlying cause should be performed, with hemodynamic monitoring initiated. In this phase, the goal is early adequate goal-directed fluid management: fluid balance must be eviously . This maThe optimization phase starts when the patient is no longer in overt absolute/relative hypovolemia, but remains hemodynamically unstable. Some form of monitoring will by now be in place. Fluids should be administered according to individual needs, reassessed on a regular basis, e.g., using fluid challenge techniques , 104. FlAlthough a resuscitation based on microcirculatory endpoints is expected to result in analogous amelioration in the microcirculation, a lack of coherence may exist between macro- and microcirculation. Thus, markers of hypoperfusion should include also lactate, prolonged capillary refill time and mottling score .Once the patient is stable, the stabilization phase begins and evolves over days. In this phase, the aim of fluid management is to ensure water and electrolytes to replace ongoing losses and provide organ support. The target should be a zero or slightly negative fluid balance. It might be of interest, in this context, to underline the fact that in the major trials suggesting a harmful effect of starches , 107, thThe final phase is evacuation or de-escalation, with the purpose of removing excessive fluid. This will be frequently achieved by spontaneous diuresis as the patient recovers, although ultrafiltration or diuretics might be necessary. Of note, it was recently shown that diuretics might favor the recruitment of microcirculation, thus decreasing diffusion distances and improving oxygen extraction .Although traumatic brain injury remains the commonest cause of death following severe blunt injury, concomitant major hemorrhage will result in cerebral hypoperfusion, which undoubtedly contributes to secondary brain injury and death. As such, hemorrhage remains the most preventable cause of trauma mortality.An adequate intravascular volume, hemoglobin concentration and oxygen saturation are essential to maintain aerobic metabolism. Humans do not tolerate anaerobic metabolism and 90% of oxygen consumption is used in the formation of adenosine triphosphate (ATP), the major energy source for cell function. Rapid reversal of anaerobic metabolism is imperative to restore ATP and prevent irreversible cellular apoptosis and death .Recognizing that hypovolemia is the consequence of hemorrhagic shock, past strategies utilized crystalloids to restore intravascular volume, followed by blood transfusion. Crystalloids, however, do not carry oxygen, and oxygen delivery may only be enhanced by an adequate hemoglobin concentration. Furthermore, major hemorrhage is accompanied by a unique coagulation disorder, the Acute Coagulopathy of Trauma and Shock (ACoTS) , leadingWhile the above is a general recommendation, not all patients will require such an aggressive approach . Indeed,The standard approach has been to use conventional laboratory coagulation testing to determine the need for component therapy. These, however, are performed at room temperature and do not reflect individual steps in coagulation. Thromboelastometry has now been recognized as an essential tool to monitor coagulopathy in trauma . This deFollowing the CRASH-2 trial indicating the benefit of tranexamic acid given within 3 h from injury, such treatment has been included in many protocols for major hemorrhage . In the The understanding of burn shock pathophysiology and subsequent development of fluid resuscitation strategies resulted in dramatic outcome improvements in burn care during the last decades . HoweverHowever, over the last 15 years, multiple centers have reported excess fluid administration , 117. ThThis discrepancy between the predicted and the administered fluid is known as “fluid creep”, a term brought to life by Basil Pruitt .Recommendations for fluid resuscitation in burns are listed in Table The use of colloids in the first 24 h has been controversial since it was thought that the existing capillary leak would allow large molecules to leak out into the extravascular space and exert an osmotic pull increasing the formation of edema . In the Albumin is a natural plasma protein that contributes most to intravascular oncotic pressure in humans (see above). The most common solutions are 4%, 5% or 20% albumin. It is a relatively expensive solution and its availability may be limited in some countries. Although albumin resuscitation has been used with some reservations, especially in the acute phase of burn resuscitation, trials provide promising data regarding the use of albumin as an adjunctive therapy in burn resuscitation , 125. SiDrug—which fluid.Dose—calculate how much to give.Duration—duration of the IV fluid therapy.De-escalation—stop it as soon as possible.Consider the 5 Ps of fluid prescription as shown in Fig. “Give the right fluid in the right dose to the right patient at the right time”The bottom line is The prescription of fluid therapy is one of the most common medical acts in hospitalized patients but many of the aspects of this practice are surprisingly complex. It is time to introduce fluid stewardship in your ICU. To avoid fluid-induced harm, we recommend a careful evaluation of the chosen solution and a phase-wise approach to its administration, taking into account the clinical course of the disease or surgical procedure. Fluids should be prescribed with the same care as any other drug and every effort should be made to avoid their unnecessary administration.


Coles®Magazine and Woolworths Fresh® were analyzed and compared against a traffic-light system for classifying nutrients of concern in chronic disease. Nutrient content was compared across recipe type  and between recipes with and without health or nutrient claims. Overall compliance with the traffic-light criteria was low, with less than half of recipes meeting the target. Advertorial recipes had a higher energy (p = 0.001), saturated fat (p = 0.045) and sodium (p ≤ 0.001) content per serve, and per 100 g for sodium (p ≤ 0.001) compared to standard and celebrity recipes. Recipes with claims had greater compliance to the nutrient criteria (p < 0.001) compared to those without. These findings support previous research highlighting the poor nutritional quality of published recipes from a variety of sources.Recipe use impacts eating habits, yet there is limited research investigating the nutritional quality of recipes. Supermarket recipe magazines command large readerships, with over 4 million readers for each of the two major Australian supermarket publications. Assessing the nutrient content of featured recipes is therefore of public health interest. The nutrient content of 312 main-meal recipes from  A higher frequency of home cooking has been associated with improved diet quality ,2. ReseaAn Australian study comparing the nutrient content of celebrity-chef cookbook recipes against the Victorian Healthy Choices Traffic-Light System  found th® and Woolworths®. Coles®Magazine reaches an audience of 4.8 million people (19.2% of the population) and Woolworths Fresh® magazine reaches 4.2 million people (16.7% of the population) [There is currently limited research in an Australian context, with only two studies identified. These have focused on celebrity-chef cookbook recipes  and foodulation) ,12. The ulation) . To our ulation) .The huge reach and accessibility of supermarket recipe magazines, in addition to the documented influence of recipes on cooking and eating behaviors, indicates the potential for significant public health influence. This study aimed to assess the nutritional quality of recipes from supermarket magazines against a set of traffic-light criteria used and developed for a Government mass media healthy lifestyle campaign. A secondary objective was to compare the nutrient content of standard supermarket-produced recipes with advertorial and celebrity-chef recipes, as well as comparing the nutrient content of recipes with and without health or nutrient claims.® is a Western Australian social marketing campaign funded by the Western Australian Department of Health that encourages adults to eat well, be physically active, and maintain a healthy weight. The campaign commenced in June 2012 and is currently active as of May 2020. The campaign has run at various time points in other jurisdictions within Australia including Victoria, Northern Territory, South Australia, and Tasmania. In addition to elements such as mass media, research, and advocacy, evidence-based information and resources are developed and distributed to the population to facilitate behavior change. During the development of the campaign, a set of recipe guidelines, including nutrient criteria for nutrients of concern in chronic disease, were established [® website must meet the nutrient criteria and recipe guidelines. In addition, the nutrient criteria are promoted to the public as a way to assess the nutritional quality of everyday foods consumed.LiveLighterablished . These wablished ,17, the ablished  and Foodablished . RecipesThis project was a retrospective, cross-sectional analysis of the nutrient content and nutritional quality of recipes from Australian supermarket magazines.Coles®Magazine and Woolworths Fresh® published between July 2018 and June 2019. Each magazine produces a monthly issue, except for a combined January/February issue for Woolworths Fresh®. The sampling frame was drawn from the index page and included all recipes defined as a main-meal by the magazine, which included recipes by celebrity-chefs. The two magazines differed in the inclusion of advertorial recipes in the index list; Coles®Magazine included these in the index list while Woolworths Fresh® did not. Hence, Woolworths Fresh® was manually searched for additional advertorial main-meal recipes. The total number of main-meal recipes available across the time period for the two magazine types was 641, with the number of recipes in any one issue varying from 13 to 41. A sample of 13 recipes per issue was selected as this was the minimum number of recipes within a single issue. A simple random sampling technique was used, using an online random number generator (researchrandomizer.org), to select 13 main-meal recipes from each magazine. A double sample of 26 recipes was taken from the January/February issue of Woolworths Fresh®, giving a total sample size of 312 recipes to be included in the analysis.A complete list of all main-meal recipes was obtained from the online issues of The nutrient content of 312 main-meal recipes was analyzed using Foodworks 9 , using the Australian food composition databases AUSFOODS2017, AUSBRANDS2017 and NUTTAB2010 [When entering recipes into Foodworks, ingredients were entered in their cooked state, where appropriate, with conversion factors applied to adjust for differences in cooked and raw weight . The cooRecipes were entered into Foodworks by one author (JW), a student dietitian. A second author (AF), a dietitian experienced in the use of the Foodworks software, analyzed a random sub-sample of 20 recipes to determine inter-rater reliability. An acceptable percentage similarity was set at 85% . CompariAdditional information was collected on the types of foods being promoted in sampled advertorial recipes. Promoted foods were classified into groups as outlined in Charlton et al.’s method; core foods , unsaturated fats and oils, discretionary foods (energy-dense and nutrient-poor) and non-food items . The def® traffic-light criteria [® criteria was assessed, with recipes being compliant if they had a minimum of two nutrients classified as green and had no nutrients classified as red, as per the LiveLighter® recipe guidelines [® traffic-light criteria  [To assess nutritional quality, recipes were assigned a traffic-light color for total fat, saturated fat, sodium and dietary fiber based on the LiveLightercriteria . Table 1idelines . The ove rating) .® criteria. The level of statistical significance was set at p < 0.05 for all tests.Statistical analysis was conducted usingStatistical Package for the Social Sciences SPSS, Version 25 . Descriptive analysis was conducted for all data. All continuous data were assessed for normality using skewness, kurtosis, and normality plots. Independent t-tests and Mann-Whitney U tests were used to compare the nutrient content of recipes with and without health or nutrition claims. One-way analysis of variance ANOVA and Kruskal-Wallis tests were used to compare the nutrient content between recipe types, with least significant difference LSD used for post-hoc analysis. Chi-square and Fisher’s exact tests were used to compare differences in the traffic-light distribution of nutrients as per the LiveLighterA total of 312 main-meal recipes were included in this study (156 from each of the included supermarket publications). With respect to recipe type, there were a total of 234 standard recipes (75% of the sample), 42 advertorial recipes (13% of the sample), and 36 celebrity-chef recipes (12% of the sample). Health or nutrition claims were present on 75 of the sampled recipes (24% of the sample), while a further 237 recipes did not have claims (76% of the sample). Claims included health and nutrient claims such as “low fat” and food preference or dietary requirement claims such as “gluten-free”.p = 0.002) and energy (p = 0.001), saturated fat (p = 0.045) and sodium (p < 0.001) content per serve when compared to standard and celebrity recipes (p < 0.001), with advertorial recipes having the highest mean content (272 mg/100 g) (p = 0.001) and were significantly lower in energy (p < 0.001), fat (p = 0.001), saturated fat (p < 0.001) and sodium (p < 0.001) content per serve, compared to recipes without claims (p = 0.002), saturated fat (p < 0.001), and sodium (p < 0.001) content per 100 g compared to those without claims (The mean nutrient content and serve size of recipes is presented in  recipes . A signig/100 g) . Recipest claims . Similart claims .® criteria (see p = 0.047) and across all recipe types for sodium (p = 0.001). Celebrity recipes were more likely to be classified as red for total fat (36.1%), compared to standard recipes  and advertorial recipes . Advertorial recipes were more likely to be in the red category and less likely to be in the green category for sodium when compared to other recipe types (p = 0.003) and sodium (p < 0.001) (eria see ). There pe types . Compari< 0.001) , with re< 0.001) . An alte< 0.001) .p < 0.001)  .Sixty-four food products and two non-food products were promoted in the 42 advertorial recipes. Of the promoted food items, 33.3% were classified as core foods, 12.1% as unsaturated spreads and oils and 51.5% as discretionary items .® traffic-light criteria. To our knowledge, this is the first study investigating supermarket recipe magazines and the first Australian study to analyze a range of nutrients in magazine recipes.The purpose of this study was to assess the nutrient content of main-meal recipes included in Australian supermarket recipe magazines and compare this against a measure of nutritional quality, the LiveLighterThis study found that advertorial recipes contained more energy, saturated fat, and sodium per serve when compared to standard and celebrity recipes. This result is likely primarily due to the significantly greater serving size of advertorial recipes, as only sodium differed significantly when comparing per 100 g. The larger serving size may be explained by the financial motive of such recipes. Advertorial recipes are designed to sell a food product and advertising larger portions may be a strategy for increased sales, as is seen in fast food marketing strategies . However® and Woolworths® catalogues revealed a ratio of core to discretionary foods of 0.8:7 [The analysis of products advertised within advertorial recipes revealed that the majority were unhealthy “discretionary” food items, while a smaller proportion were healthy “core” foods. These findings are in line with content analysis studies of supermarket catalogues, identifying that the foods advertised by supermarkets are predominantly ultra-processed, discretionary items . A previof 0.8:7 . The fin® nutrient criteria for total fat, while advertorial recipes were less compliant with sodium guidelines. Furthermore, advertorial recipes were less compliant with the overall LiveLighter® recommendation to have a minimum of two nutrients in the “green” range and no nutrients in the “red” range. The increased portion size, significantly greater sodium content, high proportion of discretionary foods advertised and lower compliance with LiveLighter® nutrient criteria highlights the need to clearly mark advertorial recipes to provide consumers with adequate information to make informed dietary choices.Celebrity recipes were found to be less compliant with the LiveLighter® nutrient criteria than those without. These findings suggest that recipes making a claim are a better choice than those without a claim. It should be noted, however, that over one third of recipes with claims were still found to be non-compliant with LiveLighter® nutrient criteria. Interestingly, these results are in contradiction with other research and may not be transferable to other recipe sources [The present study found that recipes with a health or nutrient claim were significantly lower in energy, saturated fat, and sodium content per serving and per 100 g. Recipes making a claim were also found to be significantly more compliant with the LiveLighter sources ,32. Prev sources ,9,31,32.® nutrient criteria for nutrients of interest in chronic disease development . Similarly, most recipes did not comply with the overall LiveLighter® nutrient guidelines. This is consistent with other research on publicly available recipes that has found the majority to be of poor nutritional quality [® criteria for analysis, other studies have found similarly low compliance with other nutrient criteria. Trattner et al. found that only 0.14% of online recipes were fully compliant with the WHO criteria [Overall a small proportion of the recipes included in this study met LiveLighter quality ,6,7,8,9.criteria ,6,7,8. O® criteria, with most recipes classified as green. However, the LiveLighter® criteria for fiber is relatively low with less than 3 g per serve considered low fiber and more than 3 g per serve considered acceptable. Considering that the recipes included in this study were all main meals, a contribution of greater than 3 g of dietary fiber would likely be required to meet the daily adequate fiber intake of 25 g for women and 30 g for men [® criterion for dietary fiber may be too low for the purpose of assessing acceptable levels in main meals.Dietary fiber was the only nutrient to obtain a favorable rating with the LiveLighter for men . Similar® criteria for these nutrients. Secondly, only the main-meal recipes were included in this analysis and so it is unclear the extent to which other recipes included in supermarket recipe magazines, such as desserts and light meals, adhere to the LiveLighter® nutrient criteria. Lastly, a decision was made not to include sugar in the assessment of overall compliance with the LiveLighter® nutrient criteria as all recipes analyzed in this study were main-meal recipes and therefore were not high in sugar. As the recipes were still required to have a minimum of two nutrients in the green category the number of recipes conforming to the overall traffic-light criteria may have been lower than if sugar was included in the assessment, as the majority of recipes would then have had one nutrient classified in the green category automatically. Finally, although the proportion of the Australian population accessing supermarket magazines is high, there is no known data on how people are using these magazines i.e., how many readers are actually cooking recipes from these magazines. Hence, further research is required to properly assess the public health influence of these magazines.The present study has several limitations. First, data collection did not include ingredients such as seasoning and oil that were mentioned in the method section but not the ingredient list of the recipe. This means that the results may have underestimated the fat and sodium content of the recipes and overestimated the number of recipes meeting the LiveLighter® nutrient criteria. Future research could build on this by including additional nutrients or using broader nutrient criteria such as the WHO criteria used in other studies, making results more comparable to the body of research. Assessment of a wider range of recipe types, such as appetizer and dessert recipes, would also be beneficial, as well as a content analysis of supermarket magazines, e.g., the ratio of advertorial to non-advertorial recipes. Future research could also compare supermarket recipe magazines with other recipe sources such as subscription magazines and online recipe websites to identify the best choice when recommending recipes to the public.This study focused on the key nutrients of interest in chronic disease risk, in line with the LiveLighterThis research indicates that recipes promoted by Australian supermarkets are of poor nutritional quality when assessed against a set of traffic-light nutrient criteria for total fat, saturated fat, and sodium. The serving size of advertorial recipes was significantly larger and the sodium, fat, and energy content per serve was significantly higher when compared to standard and celebrity recipes. Advertorial recipes also promoted the use of unhealthy discretionary foods and were less likely to be compliant with the traffic-light criteria compared to other recipe types. Recipe magazines should therefore be used with caution and advertorial recipes should be clearly marked to allow the public to make informed choices about the meals they prepare and consume.


Persistent hyperlactatemia has been considered as a signal of tissue hypoperfusion in septic shock patients, but multiple non-hypoperfusion-related pathogenic mechanisms could be involved. Therefore, pursuing lactate normalization may lead to the risk of fluid overload. Peripheral perfusion, assessed by the capillary refill time (CRT), could be an effective alternative resuscitation target as recently demonstrated by the ANDROMEDA-SHOCK trial. We designed the present randomized controlled trial to address the impact of a CRT-targeted (CRT-T) vs. a lactate-targeted (LAC-T) fluid resuscitation strategy on fluid balances within 24 h of septic shock diagnosis. In addition, we compared the effects of both strategies on organ dysfunction, regional and microcirculatory flow, and tissue hypoxia surrogates.2 gradient/ arterial-venous O2 content difference ratio; and lactate/pyruvate ratio. There was no difference between CRT-T vs. LAC-T in 6 h-fluid boluses , or balances . CRT-T was associated with a higher achievement of the predefined perfusion target . No significant differences in perfusion-related variables or hypoxia surrogates were observed.Forty-two fluid-responsive septic shock patients were randomized into CRT-T or LAC-T groups. Fluids were administered until target achievement during the 6 h intervention period, or until safety criteria were met. CRT-T was aimed at CRT normalization (≤ 3 s), whereas in LAC-T the goal was lactate normalization (≤ 2 mmol/L) or a 20% decrease every 2 h. Multimodal perfusion monitoring included sublingual microcirculatory assessment; plasma-disappearance rate of indocyanine green; muscle oxygen saturation; central venous-arterial pCOCRT-targeted fluid resuscitation was not superior to a lactate-targeted one on fluid administration or balances. However, it was associated with comparable effects on regional and microcirculatory flow parameters and hypoxia surrogates, and a faster achievement of the predefined resuscitation target. Our data suggest that stopping fluids in patients with CRT ≤ 3 s appears as safe in terms of tissue perfusion.Clinical Trials: ClinicalTrials.gov Identifier: NCT03762005 (Retrospectively registered on December 3rd 2018) Persistent hyperlactatemia has been traditionally considered as a signal of tissue hypoperfusion or hypoxia in septic shock patients . Therefop = 0.06), required less fluid resuscitation during the intervention period, and presented less organ dysfunction at 72 h. A posterior Bayesian analysis showed a very high probability that CRT-targeted resuscitation may result in lower mortality and faster resolution of organ dysfunction compared to a lactate-targeted one  or percentage. Mann–Whitney U Test, chi-square or Fisher’s exact test were used when appropriate. Two-tailed p value < 0.05 was considered significant. Data were analyzed with Minitab v17  and Graphpad Prism  softwares.This study was conducted between June 2018 and October 2019, where it was stopped before completing the programmed sample size of 46 patients. The decision was made because of nil further recruitment after the start of a severe Chilean social outburst in October. During the study period, 149 patients were screened for potential protocol inclusion. Patient flow and causes for exclusion are represented in Fig. p = 0.9). The most common tests for fluid responsiveness assessment were pulse pressure variation , inferior vena cava variation , and passive leg raising with velocity–time integral .Baseline demographic, severity scoring, hemodynamic and perfusion characteristics are shown in Table p = 0.8), 28- day mortality , ICU  and hospital length of stay . Concerning specific resuscitation targets at 6 h, a higher proportion of patients in the CRT-T group achieved their objective , as shown in Fig. Table When assessing regional, microcirculatory and hypoxia-related parameters, no difference between groups was observed at the end of the intervention period, as shown in Table CRT-targeted fluid resuscitation was not superior to a lactate-targeted one in the primary outcome of fluid administration during the 6 h intervention period, neither in fluid balances nor organ dysfunction at 24 h. However, CRT-targeted resuscitation was associated with higher achievement of resuscitation targets during the intervention period and exhibited comparable effects to LAC-T on regional/microcirculatory flow parameters and hypoxia-surrogates.Our results could be viewed as in contradiction with the findings of the ANDROMEDA-SHOCK trial , particuA possible explanation of our findings is that since CRT exhibits a rapid response to flow increasing maneuvers it could be assessed in periods of 30 min thus allowing clinicians to stop resuscitation in a timely fashion. In contrast, lactate exhibits a relatively slow and biphasic recovery kinetics even after successful resuscitation .2 saturation and plasma disappearance rate for indocyanine green [Concerning the impact of both strategies on regional and microcirculatory flow, to the best of our knowledge this is the first study assessing this issue with a multimodal approach. In a previous study, Brunauer et al. found a significant correlation between changes in CRT with the pulsatility index of various hepatosplanchnic arteries during septic shock resuscitation . This isne green . AlthougAnother peripheral perfusion assessment method is the mottling score . Its pro2, leading to an exclusive anaerobic metabolism in affected territories [2 consumption and aerobic CO2 production are decreased and a critical amount of anaerobic CO2 is generated due to massive ATP degradation, with the resulting buffering of free H+ with plasma HCO3−. Once tissue hypoxia is established, the reduction of cell redox potential shifts the production of ATP to the anaerobic pathways, elevating the L/P ratio [2 production. This shift could rise the respiratory quotient and consequently the P(cv-a)CO2/ Da-vO2 ratio [None of the classic perfusion-related parameters reliably reflect the presence or absence of tissue hypoxia. Tissue hypoxia should englobe the idea of an impaired critical oxygen delivery, and/or the inability of the mitochondria to utilize Oritories . Consequ/P ratio  and furtO2 ratio . Both inO2 ratio , althougO2 ratio . In our 2 is flow-sensitive and was described in hemorrhagic shock, where it improves after successful resuscitation [2 decreases rapidly after a vascular occlusion test and recovers very fast after releasing compression [Our study has several limitations. First, there are inherent technical drawbacks and interpretation issues for each of the flow-related variables assessed in the study. PDR-ICG depends basically on liver blood flow but also on liver metabolic function . Thus, pcitation . StO2 depression , 24. Howpression . In this2/ Da-vO2, the use of central venous instead of mixed venous pCO2, and the use of differences in pressures and not CO2 contents could be criticized [Second, in the case of hypoxia surrogates, the background literature is scarce and both ratios are not universally accepted. Additional problems are the technical difficulties for assessing pyruvate, and the lack of clear cut-offs for abnormality. In the case of P(cv-a)COiticized . Howeveriticized , 26.Third, the small sample size may be a problem in physiologically focused studies. Therefore, we consider these results only as hypothesis-generating, but the data obtained may aid in bringing insights into the mechanisms behind the positive prognostic value of a normal CRT after initial fluid resuscitation. Fourth, CRT assessment might be subjected to inter-observer variability, but we used a standardized technique that decreases the likelihood of bias. Fifth, the premature stop of the study may have introduced bias but an independent statistician analyzed the results and concluded that recruitment of 4 more patients would not have changed the p values of the main findings. Finally, we could perform SDF and PDR-ICG techniques in only one of the two involved centers for logistic reasons, but we think that this does not invalidate our conclusions.CRT-targeted fluid resuscitation in septic shock was not superior to a lactate-targeted one on early fluid administration or fluid balances. However, it was associated with comparable effects on regional and microcirculatory flow parameters and hypoxia surrogates. In addition, achievement of allocated targets was higher for CRT-guided resuscitation during the 6 h intervention period. These data, although only hypothesis generating, expand the results of ANDROMEDA-SHOCK suggesting that potential benefits of CRT-targeted resuscitation should be tested in future studies beyond the limits of very early septic shock.Additional file 1: Methods used in the and cut-offs for fluid responsiveness assessment techniques in the present studyAdditional file 2: Proportion of target achievers at 6h with normal values of perfusion-related variables in both study arms