To evaluate the accuracy and precision for assessing fluid intake by examining the ability of nurses, patients and healthy people to visually estimate fluid volumes, thereby reflecting the fluid monitoring process in clinical practice.A cross‐sectional study.This study used the convenience sampling method and involved twenty‐five participants from three groups; nurses, patients and healthy people. The participants carried out a set of different visual volume assessments of two types of fluids using two fluid containers. The exact volumes were measured, and the results were compared with the target volumes.High variations were observed in the fluid volume assessments for patients, nurses and healthy persons and also were observed to be an effect of environmental factors (fluid container or fluid type) on volume perceptions. This highlights the importance of finding new and innovative ways of measuring fluids for oral intake in a hospital setting, to ensure accurate and reliable data on fluid balance and thereby increase patient safety. Inclusion criteria for all participants were that they should be above the age of 18 years, speak and understand Danish, be cognitively able to give consent and have an absence of tremors.An additional inclusion criterion for the nurse group participants was that they should have more than 6 months of professional experience as a nurse. For the patient group participants, an additional inclusion criterion was that they should not be diagnosed with severe heart or kidney disease since these patients are well‐trained in the assessment of fluid intake due to the fluid intake restrictions being a part of their medical treatment.2.3The study was conducted from August 2020 to September 2020. All participants carried out a set of different visual assessments of fluid volume as described in the framework showed in Figure All data were recorded in a spreadsheet . In addition to the fluid assessments, the participants were asked questions about their daily fluid intake, the type of fluid they preferred to drink, whether see‐through or opaque, the number of hospitalizations they previously went through and their experience with assessing fluid intake volumes.2.4SD) were included. An acceptable error margin of 10% was set for the accurate evaluation of data, as described in the study by Tattersall  using descriptive statistics (numbers and percent), and for continuous data, the mean and standard deviation .33.1SD) age was recorded for the participants in the patient group, which showed a mean of 54 (13) years, compared to the participants in the nurse group with a mean of 37 (9) years and the healthy group with a mean of 45 (13) years (Table SD) of 11 (3) (Table SD) of 9 (2) full glasses/cups of fluid consumed per day. The primary fluid consumed was recorded as ‘see‐through’ for all three groups.The characteristics for the three groups of participants, nurses, patients and healthy persons, are summarized in Table an of 54 3 years, an of 54 3 years, When asked about their experience in assessing fluid intake, the majority, 92%, of nurse group participants had previously assessed fluid intake. In contrast, only a few, 12%, of the patient group participants had previous experience, and the healthy group participants had no experience.3.2SD) in pouring accuracy ranging from 16.9–18.5 ml. The nurse group participants poured under the 100 ml target in the glass, with a mean difference of −17 ml, and poured slightly over the 100 ml target in the cup, with a mean difference of +4 ml.The participants were asked to repeatedly pour in the same volume of water to a target volume of 100 ml in the two different fluid containers, a glass and a cup. This assessment was repeated three times. The mean volumes of water poured into the fluid containers for each participant group are shown in Figure SD) of 27.1 ml was observed in the pouring accuracy for the nurse group participants when they used a cup. In contrast, the variation (SD) when using the glass was 17 ml. The healthy group participants poured to target in the glass and close to target in the cup, with a mean difference of −0.5 and +2.2 ml, respectively. Besides, a lower variation (SD) in pouring accuracy ranging from 10.5 and 8.2 ml was observed for the healthy group participants  were observed among the volumes poured in the fluid containers for all participants, especially for the patient and nurse groups. The nurse group participants poured in a mean volume of water in both fluid containers that was closest to the hospital's standard definition of full fluid containers  of 10.7–13.6 ml was found for this group of participants.For all three groups, high variations  of 10.2–21.4 ml. In contrast, the healthy group participants poured mean volumes under the standard volume of a full glass for both fluid types and poured mean volumes over the standard volume of a full cup for both fluid types. This group of participants showed the lowest variation (SD) of 6.6–11.3 ml. Except for the mean volume of coffee poured in the glass by the patient group participants, the mean volumes poured by the patient and healthy group participants were within the acceptable 10% error margin  than the two other participant groups. However, further studies are needed to determine whether this was related to the nurses having more experience in assessing volumes of different types of fluids and in different fluid containers, through the use of fluid balance charts, than the patient and healthy group participants.The clinical implications of unreliable fluid volume assessments might vary from patient to patient, ranging from having no effect to a more severe impact on the effect of treatment and the course of illness (Diacon & Bell, 4.1Despite the inclusion criteria used for including participants in this study, the participating patients might have been in a health‐related condition impacting their ability to assess fluid volumes. All were cognitively able to provide consent but might still have had pain, anxiety or other conditions that affected their overall ability for making such measurements. Most of the nurses had prior experience in fluid volume assessment, whereas most of the patients and healthy persons did not. It is unknown how this experience or the lack of it has impacted the participant's ability to assess volumes. Therefore, specific training might result in more accurate assessments, as seen in some of the results among nurses in this study. In addition, the use of standard hospital fluid containers in this study might have given nurses and patients the advantage of having seen these containers before, whereas the healthy persons might not have seen them before. Even so, both the glass and the cup used in this study did however reflect the size of other cups and glasses often used in a Danish context.5The results in this study point to several potential areas in which future solutions towards more accurate fluid volume measurements can be developed depending on the context and purpose of the monitoring process. In addition, more focus on accurate monitoring of fluid balance and oral fluid intake might include new ways of collaboration and sharing of data across healthcare sectors and medical‐technical equipment, thereby improving patient care, increasing patient safety and preventing illness. This will also enable nurses to link fluid intake to patient care outcomes and support them in their clinical decision‐making. Low technical solutions could include using glasses or cups with a printed measure of volume on the inside or outside. High technical solutions could include hydration monitoring apps (Steven et al., MBSS and MLB are affiliated with an organization with a non‐financial interest in the subject matter discussed in this manuscript.CFM, MBSS, MLB and HK designed and directed the project; HK collected the data; CFM analysed the data; CFM and HK wrote the article. All authors discussed the results and commented on the manuscript.


The acknowledgement omitted the following: “The research grant was awarded to Associate Professor Dr Subapriya Suppiah by the Malaysian Ministry of Education, that is, the Fundamental Research Grant Scheme (FRGS) with a grant number 5540244 and reference code number FRGS/1/2019/SKK03/ UPM/02/4, helped support this research. ”.In the article titled “Diagnostic power of resting‐state fMRI for detection of network connectivity in Alzheimer’s disease and mild cognitive impairment: A systematic review” by Buhari Ibrahim,Subapriya Suppiah, Normala Ibrahim, Mazlyfarina Mohamad, Hasyma Abu Hassan, Nisha Syed Nasser, M Iqbal Saripan  and project code: 04‐01‐19‐2119FR and project number 5540244 that was awarded to Associate Professor Dr. Subapriya Suppiah .”


This study further evaluated the Haemophilus influenzae , Haemophilus parainfluenzae , Moraxella catarrhalis (490 isolates), and Streptococcus pneumoniae . The isolates were collected primarily from CARTIs (90.8%) and pneumonia in hospitalized patients . Organisms were tested using reference broth microdilution methods in a central laboratory. The study included a total of 2,476 fastidious organisms: 90 values of 0.5 mg/L against H. influenzae and 1 mg/L against H. parainfluenzae isolates. All 18 BLNAR isolates from these two species were inhibited at ≤1 mg/L of tebipenem. The MIC90 values observed for ertapenem and meropenem was 0.25 mg/L for these organisms. Tebipenem displayed good activity against M. catarrhalis . Tebipenem inhibited 100% of S. pneumoniae isolates at ≤1 mg/L. Tebipenem activity  was 8-fold greater than ertapenem  against S. pneumoniae isolates.Tebipenem had MICHaemophilus isolates, including all BLNAR, were inhibited at ≤1 mg/L. All M. catarrhalis isolates were inhibited at ≤0.03 mg/L. Although tebipenem activity correlated with penicillin resistance, all S. pneumoniae isolates were inhibited at ≤1 mg/L. Tebipenem in vitro activity was greater than ertapenem when tested against S. pneumoniae isolates. This data supports the possible development of tebipenem as an oral option for combating CARTIs caused by these organisms.Tebipenem displayed potent activity against fastidious organisms causing respiratory tract infections. Greater than 99.7% of all TableS J Ryan Arends, PhD, AbbVie (formerly Allergan) (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Abby L. Klauer, n/a, Cidara Therapeutics, Inc. (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Nicole Cotroneo, Spero Therapeutics  Ian A. Critchley, Ph.D., Spero Therapeutics  Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)


The great diversity of the invertebrate community thriving in the deepest sections of the gypsum karst system of the Monte Conca sinkhole  suggests the existence of a complex food web associated with a sulfidic pool and chemoautotrophic microbial activity. To shed light on the peculiarity of this biological assemblage, we investigated the species composition of the invertebrate community and surveyed trophic interactions by stable isotope analysis. The faunal investigation conducted by visual censuses and hand sampling methods led to the discovery of a structured biological assemblage composed of both subterranean specialized and non-specialized species, encompassing all trophic levels. The community was remarkably diverse in the sulfidic habitat and differed from other non-sulfidic habitats within the cave in terms of stable isotope ratios. This pattern suggests the presence of a significant chemoautotrophic support by the microbial communities to the local food web, especially during the dry season when the organic input from the surface is minimal. However, when large volumes of water enter the cave due to local agricultural activities  or extreme precipitation events, the sulfidic habitat of the cave is flooded, inhibiting the local autotrophic production and threatening the conservation of the entire ecosystem. In terms of energy inputs, subterranean ecosystems are generally regarded as oligotrophic environments, especially in the inner and most isolated parts . NutrienThiobacillus thioparus, a bacterium capable of oxidizing hydrogen sulfide to sulfuric acid. The organic material produced in situ allows the development of a rich and diversified community composed of different species, 37 of which are endemic of this subterranean groundwater ecosystem  × 1000 and δ15N = [(15N/14N) − 1] × 1000, where δ13C or δ15N are reported in part per thousand (‰). Atmospheric N2 is the standard for nitrogen, while Vienna PeeDee belemnite (VPDB) is the standard for carbon.Isotopic composition was determined using an isotope mass spectrometer. Stable isotope data are presented in the delta (δ) notation as the relative difference between the ratios of the sample and the standards: δThe Stable Isotope Ratio Analysis was performed at the University of New Mexico, Department of Earth and Planetary Sciences, Albuquerque, NW, USA.13C values were statistically compared with the Student’s t-test. For this purpose, only the species shared by the two habitats were analyzed. Normality was at first tested using the ‘shapiro.test’ function from ‘stats’ package, ver. 4.2.0 [To test for the actual separation between the sulfidic and non-sulfidic habitats, δr. 4.2.0 . The F-t2S in the sulfidic habitat (Sulfide concentrations were negligible (0.03 ppm and 0.18 ppm) in the control plot b. Values habitat c were geAir and water temperatures in the control site were relatively stable in June, July and August . Temperature dropped in February, reaching 11.5 °C and 10.5 °C, respectively. d.Air and water temperature in the sulfidic habitats followed the same trend across the year, but values were generally higher than those measured in the non-sulfidic habitat, with values at least 0.6 °C higher (June). The highest deviation was +3.6 °C, recorded in February d. Water Nepa cinerea or the beetle Paranchus albipes were found in the dry and the wet season, hinting at their possible presence in the cave throughout the year. The general epigean origin of the assemblage is related to passive water transportation during the wet season, contributing to an overall and seasonal increase of the species diversity at the inner parts of the cave. Predators were highly diversified in the assemblage (24 species), including 18 species at the cave entrance, 16 in the non-sulfidic habitat and 20 in the sulfidic one. Detritivores were represented by 20 species, .The biological survey determined the presence of 54 species  and non-sulfidic habitats (7), as well as the number of detritivores (14 and 7 species respectively), that become the most diverse group in the sulfidic habitat .Pseudamnicola (Pseudamnicola) moussonii , Proasellus montalentii  and Tubifex blanchardi . Among predators, we detected Haemopis sanguisuga  and Nepa cinerea   a. The laMetellina merianae, Holocnemus pluchei), while others, such as the troglophile Kryptonesticus eremita, were also found dwelling on organic debris and on cave walls in the terminal section of the cave, in both sulfidic and non-sulfidic habitats.Among terrestrial species, spiders were usually found under rocks, dead wood or in crevices in the walls. Some were only present in the twilight zone of the cave , n = 1 larva and 1 adult of Meladema coriacea , n = 4 Kryptonesticus eremita , n = 3 Nepa cinerea , n = 3 Paranchus albipes , n = 1 sample of Proasellus montalentii , n = 1 sample of Tubifex blanchardi . For the non-sulfidic habitat, we analyzed n = 1 larva Dytiscidae , n = 1 Julidae (Diplopoda), n = 5 Kryptonesticus eremita, n = 1 Meladema coriacea, n = 2 Nepa cinerea, n = 1 Oxychilus lagrecai .Stable isotope analysis was performed on the most detectable biological species. For the sulfidic habitat we analyzed 13C value of −40.18‰ and a δ15N value of 11.77‰, indicating a chemoautotrophic food source. This differed significantly from the organic matter collected in the control habitat, where values of δ13C were −23.46‰  was isotopically light, showing a δ −23.46‰ a, indica13C = −41.08 to −25.26‰) but rather similar in nitrogen (δ15N = 10.29 to 15.66‰) than the one collected in the non-sulfidic habitat (13C = −10.64 to −25.34‰) but similar in nitrogen (δ15N = 8.75 to 16.08‰). For instance, the δ13C value for Tubifex blanchardi and Proasellus montalentii were −41.078‰ and −40.94‰, respectively. Both T. blanchardi and P. montalentii likely feed on microbial mats as their δ13C values differ considerably from the other invertebrates collected in the sulfidic habitat . As both are predators, they probably feed on organisms isotopically lighter such as the isopod Proasellus montalentii.The beetles Nepa cinerea, the spider Kryptonesticus eremita and the beetle Meladema coriacea, allowed a direct comparison of their isotopic values and C:N ratios. For these specimens, the δ13C values were also isotopically heavier in the control habitat  compared to the sulfidic one . The C:N ratio values were similar in both habitats. Predators in the non-sulfidic habitat Nepa cinerea, Kryptonesticus eremita and Meladema coriacea had a value of 4.2, 4.1 and 3.9, respectively, whereas in the sulfidic habitat, their values were 4.2, 4.3 and 4.3, respectively.Species present in both the sulfidic and the control habitat, such as the bug 13C values were statistically significant  confirming a clear separation of the two habitats, at least during the dry period  origin. Remarkably, the number of species of detritivores in the sulfidic habitat was twice as high as in the control habitat. It seems likely that such diversity is favored by the high quantity and quality of the white autotrophic microbial biofilm representing the base of the food web in the sulfidic habitat.T. blanchardi dwell in the H2S-rich water of the sulfidic pool. Here, they probably proliferate facilitated by their ability to survive for long periods in anoxic, sulfidic or heavily polluted areas  in the sulfidic pools also parallels the presence of a diversified assemblage of predators. Among others, the water-scorpion Nepa cinerea can tolerate moderate levels of pollution [During the dry season, dense clusters of  Neumann  and Mart Neumann ) as well Neumann . The greollution  and so tollution ,40,41,42Lessertia barbara does not appear to be at disadvantage by the extreme conditions characterizing the sulfidic habitat. The species was previously uniquely known in Italy from another gypsum cave in Sicily , [The spider rapani), . Remarkarapani), ), in sevrapani), , and onerapani), . InteresKryptonesticus spp. and Phanetta subterranea Emerton 1875) living close to the sulfidic pools and acid droplet on the spider webs were also observed in Movile, Frasassi and Parker caves [The occurrence of nesticid and linyphiid spiders .The isotopic values for invertebrates that live in the sulfidic habitat differed significantly from those collected in the non-sulfidic one. Similarly, Roach et al.  revealedHowever, besides the driest period, the flooding during the wet season prevents the establishment of trophic chains exclusively based on chemoautotrophic production. The wet season causes large volumes of water to runoff from the surface and enter the Monte Conca sinkhole. This input facilitates the contact between the sulfidic and non-sulfidic habitats and a strong dilution of the hydrogen sulfide content of the water. The sulfide concentration measured was in fact considerably reduced in the wet season, also in the sulfidic habitat.Escherichia and Lysinibacillus, likely derived from the agricultural regions around the Monte Conca sinkhole. As reported by several authors, changes in surface land use, such as agriculture activities or sewerage system in urban areas, might alter the amount and composition of water entering the cave, promoting changes in speleothem growth and in cave microbial diversity [Davis et al.  detectediversity ,54,55). iversity  revealedMonte Conca sinkhole is a karst system developed in gypsum. It represents one of the few spots in the world that includes a sulfidic pool located within the cave, where the subterranean invertebrate community relies seasonally on autochthonous food produced chemoautotrophically by sulfur-oxidizing bacteria. Accordingly, the Monte Conca sinkhole could be considered an intermediate semi-closed ecosystem, unlike fully closed systems such as the Movile Cave in Romania, and open ones, as found in most sulfidic caves across the world ,22.The land use overlying the cave, in particular agricultural activities and extremization of meteorological events pose major threats to this ecosystem, as changes in the natural hydrological regime might increase the amount of water flowing into the cave, favoring the input of anthropogenic pollutants and altering the structure of its unique chemotrophic based food chain. Consequently, the amount of organic matter produced by the sulfur oxidizers, crucial for the structuring of the local biological assemblage, could fade over time. Such alterations could lead to severe disturbance of this exceptional ecosystem, thus threatening its existence. Due to its considerable scientific importance, Monte Conca sinkhole needs immediate and unquestionable protection.


A pleural fluid adenosine deaminase (ADA) has been used globally to assist in the diagnosis of a tuberculous pleural effusion (TPE) with a notable negative predictive value.Mycobacterium tuberculosis.We report a case of a patient with a negative pleural fluid ADA who was found to have culture-positive and biopsy-proven This case shows the importance of pursuing gold standard diagnostic studies when clinical suspicion remains high despite negative preliminary testing. We further describe gaps in research to improve pleural fluid biomarkers for TPE. M. tuberculosis cases [M. tuberculosis but carries substantial morbidity. Pleural fluid molecular markers have been developed to forgo invasive testing. Pleural fluid adenosine deaminase (ADA) has a high sensitivity (0.92) and specificity (0.90) for tuberculous pleural effusions (TPE), with a positive likelihood ratio of 9 and a negative likelihood ratio of 0.10 summarized across studies [Tuberculosis (TB) is the world’s leading killer by a single infectious pathogen. Pleural effusions occur in nearly 5% of all is cases . In endeis cases . Pleuralivity 0.9 and spec studies . Some haivity 0.9 and specM. tuberculosis on the pleural sample was negative (Mayo MTBRP). Sputum AFB cultures revealed no growth. Sputum NAAT (Cepheid GeneXpert MTB/RIF) for TB was negative. A pleural fluid ADA (ARUP 3002978) returned at 6.5 IU/L by quantitative spectrophotometry. Given the very low ADA value, negative NAAT and that cultures of the pleural fluid were preliminarily without growth, alternative diagnoses were considered, and anti-TB therapy was withheld. The otherwise more exhaustive diagnostics included a multiplexed respiratory virus polymerase chain reaction panel, urine antigen studies for Histoplasmosis and Blastomyces species, and autoimmune serologies; all of which were unremarkable. While computed tomography imaging did not show enlarged lymph nodes to suggest lymphoma and pleural cytology did not demonstrate malignant cells, there were a few small calcified supraclavicular lymph nodes and thus given the very low ADA value and lack of alternate diagnosis, a thoracoscopic pleural biopsy was performed. On hospital day 21, his initial pleural fluid cultures grew M. tuberculosis, and later his pleural biopsy also grew M. tuberculosis. While awaiting the diagnostic work-up and without anti-TB therapy, he received numerous administrations of antipyretics and continued to lose weight. Once the diagnosis of TB was ultimately made, he was initiated on typical four-drug treatment for drug-susceptible TB. However, he subsequently developed hepatoxicity and required treatment interruption before continuing a triple-drug, pyrazinamide-sparing regimen necessitating an extended total duration. His total hospital course was 30 days, and now has completed treatment. He is clinically improved to near baseline health with a resolved pleural effusion by repeat imaging.We present a case of a human immunodeficiency virus-negative 37-year-old man with 4 months of an intermittent, non-productive cough with associated weight loss (body mass index of 19). He had recently spent 6 weeks in the Amazonian jungle and had traveled to Botswana and India within 2 years of presentation. His brother had been previously treated with active TB. His symptoms progressed with the onset of chest discomfort prompting medical evaluation. A chest x-ray showed a right pleural effusion without other pulmonary nodules  is effective in ruling out TPE, even in low prevalence areas –9. HowevM. tuberculosis specific antigens, have been studied in recent years as potential tests to rapidly rule in or rule out a TPE, however meta-analyses have shown variable diagnostic accuracy [Despite the incorporation of ADA into diagnostic algorithms for TPE, the diagnosis remains challenging Table . Pleuralaccuracy –16. Nuclaccuracy . A recenaccuracy . Xpert Maccuracy . Other naccuracy . Some reDiagnosing and confirming TPE can be difficult as pleural culture can take weeks and biopsy is invasive or may be unavailable in TB endemic settings. While pleural fluid ADA has been shown previously to have an excellent negative predictive value, we present a case of ADA-negative TPE without risk factors for a falsely low ADA value. In a patient with a high likelihood of TB, a pleural fluid ADA should not be used exclusively to rule-out TPE. Further research should focus on the predictive accuracy of multiple pleural fluid biomarkers and pragmatic trials of TB treatment initiation.


Radiation has been a pillar of cancer therapy for decades. The effects of radiation on the anti-tumour immune response are variable across studies and have not been explicitly defined in poorly immunogenic tumour types. Here, we employed combination checkpoint blockade immunotherapy with stereotactic body radiation therapy and examined the effect on tumour growth and immune infiltrates in subcutaneous and orthotopic mouse models of pancreatic cancer. Although immune checkpoint blockade and radiation were ineffective alone, their combination produced a modest growth delay in both irradiated and non-irradiated tumours that corresponded with significant increases in CD8+ T cells, CD4+ T cells and tumour-specific T cells as identified by IFNγ ELISpot. We conclude that radiation enhances priming of tumour-specific T cells in poorly immunogenic tumours and that the frequency of these T cells can be further increased by combination with immune checkpoint blockade. PancreaAlthough combination chemotherapy regimens are the mainstay of pancreatic cancer treatment, radiation is frequently included, particularly in resectable or locally advanced disease as a means of reducing tumour burden prior to surgery –12. DespImmunotherapies have transformed the state of cancer therapy ,21. WhilImmune checkpoint blockade reinvigorates existing T cell responses and is thus most effective when used in patients with robust endogenous T cell priming. In order to increase the effectiveness of immune checkpoint blockade against pancreatic cancer, various strategies have been employed to augment priming and infiltration of tumour-specific T cells ,28. NotaTo this end, a landmark study examined the immunologic effects of SBRT, anti-CTLA-4 and anti-PD-1 in mouse models of melanoma and pancreatic cancer, and found that radiation plays a non-redundant role in combination with CTLA-4 and PD-1 blockade . The autHere, we used a poorly immunogenic model of pancreatic cancer that is critically unresponsive to combination checkpoint blockade. We investigated the immunologic impact of combining dual CTLA-4 and PD-1 blockade with radiation and compared tumour immune infiltrates and therapeutic efficacy in the subcutaneous and orthotopic settings. We show only PDAC immune infiltrates in a distant, unirradiated tumour, indicating the combined systemic effects of radiation and immune checkpoint therapy. We find that addition of SBRT extended mouse survival and correlated with increased IFNγ-producing, tumour-specific T cells. These results support the use of radiation to induce immunogenic cell death as part of a combination immunotherapy regimen, even in cancers that are otherwise poorly immunogenic.. 2. 2.12 in RPMI media (Life Technologies) supplemented with 10% (v/v) FBS, 2 mmol l−1 L-glutamine (Gibco), 1% (v/v) penicillin/streptomycin (Gibco), 1% (v/v) MEM non-essential amino acids (Gibco), 1 mmol l−1 sodium pyruvate (Gibco) and 0.1 mmol l−1 β-mercaptoethanol (Sigma).The 6694c2 cell line was derived from a LSL-KrasG12D;p53+/floxed, Pdx-cre, YFP-floxed mouse and was a gift from Ben Stanger (University of Pennsylvania) . Cells w. 2.2C57BL/6 mice were purchased from Jackson Laboratories (stock no. 000664) and housed according to DFCI guidelines.. 2.32 when tumours reached 2000 mm3 or had gross ulceration. Other humane endpoint criteria included body condition score less than or equal to 2 or weight loss greater than 10% of initial body weight, although no mice in this study met these humane endpoints.A total of 200 000 6694c2 cells were subcutaneously injected in HBSS (Gibco). Tumour size was measured twice weekly, and tumour volume calculated by multiplying the three dimensions of the tumour. Mice reached study endpoint criteria and were euthanized with CO. 2.4Orthotopic tumours were inoculated as previously described . Briefly. 2.5−1 αPD-1 (BMS RMP1-14), and/or 10 mg kg−1 αCTLA-4 (BMS 9H10) weekly. Control mice were treated with 10 mg kg−1 of each relevant isotype control, also weekly. Treatment was begun 6 days after tumour implantation.Mice treated with immune checkpoint blockade were administered 10 mg kg. 2.6Mice were inoculated with bilateral subcutaneous flank tumours (described above), and one of the two tumours was irradiated with a single 5 Gy dose of SBRT. A small animal radiation research platform was used for radiation, allowing radiation to be delivered precisely to the tumour site.. 2.7Tumours were excised, and orthotopic tumours were weighed. Tumours were chopped and incubated at 37°C in RPMI with collagenase IV (Sigma) and trypsin inhibitor (Life Technologies). Tumour pieces were further manually digested before being filtered through a 40 μM cell strainer, washed with PBS and centrifuged. The resulting pellet was resuspended in PBS with 2% FBS and stained with two separate panels of flow cytometry antibodies. Samples were stained for 30 min at 4°C, washed with PBS and resuspended in 1% formalin before analysis on a Sony spectral flow cytometer (SP6800). Flow cytometry antibodies were purchased from BioLegend. Myeloid panel: CD11c FITC (no. 117305), CX3CR1 PE (no. 149006), Ly6G PE-Cy7 (no. 127617), SiglecF Brilliant Violet 421 (no. 155509), CD11b Pacific Blue (no. 101223), Ly6C Brilliant Violet 570 (no. 128030), I-A/I-E Brilliant Violet 510 (no. 107635), CD45 Brilliant Violet 711 (no. 103147), NK1.1 Brilliant Violet 785 (no. 108749), F4/80 APC (no. 123116).Lymphoid panel: CD45 Brilliant Violet 711 (no. 103147), CD11b Pacific Blue (no. 101223), CD25 Alexa Fluor 488 (no. 102017), CD103 PE (no. 121405), PD-1 PE-Cy7 (no. 109109), CD4 Brilliant Violet 510 (no. 100553), B220 Brilliant Violet 605 (no. 103243), CD8α Brilliant Violet 785 (no. 100749), CD11c APC (no. 117309).. 2.8ELISpot plate (BD Biosciences) was treated with sterile-filtered 70% ethanol before being washed three times with sterile PBS 1×. IFNγ capture antibody (BD Biosciences no. 551881) was plated and the plate was sealed and left to incubate at 4°C overnight. Positive control wells were also plated with anti-CD3ε (BioLegend no. 100340). The plate was washed three times with sterile PBS and blocked with 10% (v/v) FBS in PBS overnight at 4°C. IFNγ-stimulated 6694c2 or B16F10 cells were plated, except in the unstimulated and positive control wells. Pancreatic draining lymph nodes were excised from treated mice bearing orthotopic tumours, and lymph nodes were macerated to form a single-cell suspension. Lymph node cells (one-third of lymph node per well) were plated on top of pre-plated tumour cells with human IL-2 (PeproTech), and positive control wells also received anti-CD28 (BioLegend no. 102116). Plate was incubated at 37°C for 24 h before being washed with sterile water followed by PBST. IFNγ detection antibody (BD Biosciences no. 551881) was added, and plate was incubated for 2 h at room temperature. Wells were washed with PBST, and streptavidin-HRP (BD Biosciences) was added. Plate was incubated for 1 h at room temperature before being washed with PBST and PBS. AEC chromagen substrate (BD Biosciences) was added, and plate was developed. Plate was dried and analysed.. 3. 3.1a) . W. Wa) . W. Wf) [47. 3.3a–d) in order to understand any potential abscopal effect of the therapy. Orthotopic pancreatic tumours had fewer CD4+ and CD8+ T cell infiltrates than subcutaneous tumours and a larger fraction of SiglecF+ granulocytes. Combination radiation and immune checkpoint blockade modestly shifted the immune profile of orthotopic tumours but did not increase CD4+ or CD8+ T cells to the frequencies observed in subcutaneous tumours (a–d).To determine whether radiation and combination checkpoint blockade would affect the immune microenvironment of primary pancreatic tumours, we compared the immune composition of 6694c2 tumours implanted either subcutaneously or orthotopically in the pancreas. In both cases, mice were also inoculated with a subcutaneous tumour on the right flank to serve as the irradiated lesion. We compared the immune microenvironment of the non-irradiated orthotopic or subcutaneous tumours a–d in or tumours a–d.Figu. 3.4a). Combination radiation and immune checkpoint blockade induced a trending decrease in orthotopic tumour mass in treated mice that did not reach statistical significance, potentially due to limitations in sample size (b).To more closely determine the effect of radiation and combination checkpoint blockade on orthotopic tumour growth, rather than to compare subcutaneous and orthotopic tumours, we followed the same treatment scheme, whereby mice were inoculated with both a subcutaneous tumour (to serve as the irradiated lesion) and an orthotopic tumour, and mice were treated as shown a. Combinple size b.Figurec). Frequencies of tumour-infiltrating CD8+ T cells increased significantly, although the overall number was still low, at around 1% of CD45+ cells (d). Treg and immunosuppressive myeloid population frequencies were unchanged . Because anti-tumour T cell priming often occurs in the tumour-draining lymph node, we analysed T cells in the tumour-draining lymph nodes by IFNγ ELISpot to determine whether the modest increase in CD8+ T cells corresponded to an increase in tumour-specific T cell priming (figure 4f) [figure 4f).To determine whether combination treatment affected the T cell response to orthotopic PDAC tumours, we analysed immune infiltrates by flow cytometry. Tumour-infiltrating CD4+ T cells showed a trending increase in combination-treated mice c. Freque5+ cells d. Treg agure 4f) ,48. Afte. 4Radiation exerts both immune-promoting and immune-suppressing effects, which complicates its use in combination immunotherapy regimens. Here, we combined dual PD-1 and CTLA-4 blockade with radiation in a poorly immunogenic model of pancreatic cancer and found that the combination therapy augments tumour-specific T cell priming. These findings are consistent with a phase II trial that evaluated two dosing regimens of SBRT combined with either durvalumab (anti-PD-L1) or durvalumab plus tremelimumab (anti-CTLA-4) . In thisMouse studies in melanoma have revealed that, when added to checkpoint blockade therapy, radiation diversifies the clonality of the anti-tumour T cell response . We showBeyond increasing T cell priming, radiation-induced immunogenic cell death also induces negative feedback in the form of immunosuppressive myeloid cells that infiltrate the area to support wound healing and tissue homeostasis. Damaged tissue can not only recruit myeloid cells from circulation, but it can also affect hematopoiesis in the bone marrow, leading to the systemic release of immune-suppressive myeloid cells into circulation . PreviouPDAC is among the most treatment-refractory of all tumour types. Although induction of tumour-specific T cell responses is insufficient to provide long-term tumour control in this disease , we prop


The goal of burn resuscitation is to provide the least amount of fluid necessary to maintain end-organ perfusion and prevent burn shock. The objective of this analysis was to examine how the Burn Navigator (BN), a clinical decision support tool in burn resuscitation, was utilized across 5 major burn centers in the United States.A non-interventional, observational trial of 300 adult patients with embedded prospective and retrospective components was undertaken to examine the effectiveness of the BN in burn resuscitation. 5 ABA-verified burn centers enrolled patients. Data examining patient demographics, burn characteristics, fluid volumes, and resuscitation-related complications were examined. Statistical analysis compared the 5 sites in terms of these variables.+ 16.8 years), BMI (29.2 + 6.9), ISS (21.2 + 12.8), or median TBSA . Primary crystalloid infusion volumes at 24 hours differed significantly when measured in ml/kg/TBSA . Similarly, total fluids, which includes colloid adjuncts, drip medications and enteral fluids, differed between groups when measured in both ml/kg  and ml/kg/TBSA  at 24 hours. Post-hoc adjustment for pairwise comparisons resulted in a loss of significance between most of the sites. There was a total of 156 resuscitation-related complications reported across the 5 sites with an average incidence of 44.4 % incidence.A total of 285 patients were eligible for analysis. There was no difference among the centers in terms of average age (45.5 The Burn Navigator appeared to standardize fluid resuscitations across 5 major US burn centers. With primary fluid volumes near the Parkland formula, the device can be utilized effectively in burn centers, and further study should exam the utility of this device in facilities that do not commonly treat burn injuries, as well as the battlefield.


We and others have reported that methicillin-resistant or methicillin-susceptible Staphylococcus aureus  and other pathogens assemble a proteinaceous matrix in synovial fluid. This proteinaceous bacterial aggregate is coated by a polysaccharide matrix as is characteristic of biofilms. In this study, we identify proteins important for this aggregation and determine the concentration ranges of these proteins that can reproduce bacterial aggregation. We then test this protein combination for its ability to cause marked aggregation, antibacterial tolerance, preservation of morphology, and expression of the phenol-soluble modulin (PSM) virulence factors. In the process, we create a viscous fluid that models bacterial behavior in synovial fluid. We suggest that our findings and, by extension, use of this fluid can help to better model bacterial behavior of new antimicrobial therapies, as well as serve as a starting point to study host protein–bacteria interactions characteristic of physiological fluids.Biofilms are typically studied in bacterial media that allow the study of important properties such as bacterial growth. However, the results obtained in such media cannot take into account the bacterial localization/clustering caused by bacteria–protein interactions  Despite aggressive treatment and the use of local supratherapeutic concentration of antimicrobials, joint infections can persist , even inin vitro, where microscopic clusters are seen in serum and microscopic and macroscopic clusters are visible in synovial fluid  and other pathogens assemble a proteinaceous matrix that is coated by polysaccharides as is characteristic of biofilms   . In lighSynovial fluid is central to aggregation and is a protein-rich viscous fluid composed of proteins filtered from blood and secreted proteins/proteoglycans from cells (predominantly synovium and cartilage). In our studies, we have recognized that synovial fluid shows donor variability, perhaps where composition reflects disease state . To circin vivo biofilm characteristics.In this study, we set out to determine which proteins in synovial fluid were predominantly responsible for staphylococcal aggregation. To do that, the abundant proteins sequestered in synovial fluid bacterial aggregates were identified and their roles were investigated. Based on these determinations, we next evaluated if the presence of these proteins/proteoglycans could reproduce the phenotype of synovial fluid bacterial aggregates, where our criteria were (1) morphology, (2) composition, (3) antimicrobial tolerance, and (4) virulence factor production. As a consequence of these studies, we have developed a “pseudo” synovial fluid (pSynF) that embodies many of the characteristics of native synovial fluid and serves as a readily accessible means to study bacterial behavior in fluids that more closely approximate Human synovial fluid and other surgical waste were obtained from therapeutically necessary joint aspirations or operations. This material, designated as “waste” (no identifiers), was retrieved and designated as “not human research” by the Thomas Jefferson University Office of Human Research Protections in keeping with the revised Federal Policy for the Protection of Human Subjects .®25923TM and MRSA LAC USA300 .MSSA ATCC  strains ] were grExcept for the PJI samples, synovial fluid used in this manuscript was collected as discarded material from routine office aspirations from patients with sterile fluid excess. Eight of those were used without pooling for determination of MSSA aggregate characteristics by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Otherwise, 2–5 samples were pooled to allow volumes large enough to perform the various activity/expression assays throughout the rest of the manuscript. The infected samples were collected as waste from revision surgeries for treatment of confirmed PJI and represented implant-adherent biofilm and non-attached biofilm. A small sample was scraped from the implant (biofilm sample), and another sample was retrieved from the discarded fluid material that was flushed from the bone cavity (floating aggregate). Material was placed into labeled 50-ml conical tubes, fixed by incubation in formalin, and, after rinsing, prepared for imaging by scanning electron microscopy (SEM). The identity of the bacterial strain causing PJI was not supplied with the fixed samples.in vitro, MSSA (105 CFU/ml) were incubated in TSB or human synovial fluid for 24 h, 37°C, without agitation using 1 cm × 2 mm Ti6Al4V coupons (kind gift of Zimmer). Surfaces were gently rinsed 3 × with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde, ON, 4°C. Samples were then prepared for SEM. Clinical examples were composed of samples of infected reamings from a PJI patient as well as several scrapings from the implant removed during the patient’s treatment. From this formalin-fixed material, at least three samples were removed and rinsed and solid material was affixed to a slide followed by dehydration, sputter coating, and SEM analysis. Micrographs were compared for fibrous material, presence of blood cells, and apparent biofilm-like structures that could be attributed to bacteria. Images representative of these are shown.To form biofilms Streptococcus equi, 91% high molecular weight (MW); Alfar Aesar, Tewksbury, MA) was dissolved  to 4.05 mg/ml in sterile TSB. Fibrinogen  was dissolved to 60, 90, and 120 mg/ml in PBS, pH 8.5, by incubation at 37°C, 3 min, followed by vortexing. Albumin  was dissolved to 60, 120, and 180 mg/ml in PBS, pH 8.5. All stocks were sterile filtered (0.2 μm filter) and stored at 4°C for up to 2 days.Hyaluronic acid . Each individual condition was determined in triplicate, with three independent experiments to yield an n = 8 or 9 for each condition. The combination of 3.0 mg/ml HA, 9 mg/ml Fg, and 10 mg/ml Alb in TSB was referred to as pSynF.A concentration matrix of Fg, Alb, and HA was created in 96-well plates. TSB (140 μl) containing 0, 1.5, or 3.0 mg/ml HA was added to each well; 20 μl of Fg stocks were added to give 0, 6, 9, or 12 mg/ml and 20 μl of Alb stocks to yield 0, 6, 12, and 18 mg/ml. Lastly, 20 μl of 107 or 108 CFU/ml MSSA or MRSA and incubated for a minimum of 90 min, 37°C, 125 rpm. Either during or after aggregation, amikacin (AMK) was added (0–200 μg/ml) and incubated for 6 h. At 6 h, bacteria were pelleted and washed . All pellets were then washed with PBS, resuspended by pipetting/vortexing and, after serial dilution, plated on 3MTM PetrifilmTM aerobic count plates , followed by direct counting . This dispersal method was validated by the (1) disappearance of visible aggregates and (2) equivalence of CFU (after Trypsin) with CFU recovered from TSB using an equal bacterial inoculum.Pooled synovial fluid was formed by combining 2–5 samples from individual donors to better allow reproducibility. Here, 0.2, 1.0, or 3.0 ml of human synovial fluid , pSynF, human serum  or TSB in 6-, 24-, or 96-well tissue culture plates , respectively, were inoculated with 10Bacterial aggregates were fixed with 4% paraformaldehyde, room temperature (RT), 10 min, and dehydrated by sequential incubation  with 10, 30, 50, 70, 90, and 100% ethanol in PBS. Samples were dried ON, sputter-coated with gold, and imaged using a Hitachi TM-1000 SEM.n = 8), each separately labeled with a different number. Before fractionation, samples were diluted to 3% with sterile PBS. Aggregates formed as above were pelleted at 13,000 × g, 3 min, washed, and resuspended in 1 ml PBS by repeated vortexing and pipetting. Laemmli sample buffer with β-mercaptoethanol was added (1:1), samples heated at 70°C for 10 min, placed on ice, and fractionated  on an SDS-polyacrylamide gel (7.5%), using the discontinuous buffer system of TM Dual Color Standards, Bio-Rad, Hercules, CA, United States) were run on the same gel, as were serum, Fg (Alfa Aesar), and Alb (Thermo Fisher Scientific).Synovial fluid samples were obtained from individual donors . In all gels and blots, results were repeated using additional aggregates formed in the same or different synovial fluid sample with similar appearances. Densitometry was performed using three different exposures of each blot. The amount of Fg in samples was calculated by comparison to the band area in 100 μg Fg control for each of the exposures.Following fractionation, protein bands were transferred  to nitrocellulose membranes (Bio-Rad) using the Trans Blot Turbo Transfer System (BioRad) with Turbo transfer buffer. Membranes were blocked by incubation in 10% nonfat milk (BioRad) in Tris-Buffered Saline (1×) containing 0.5% Tween-20 (10% TTBS), 60 min, followed by incubation with mouse anti-Fg  or rabbit anti-Alb  antibody, in 5% TTBS, 4°C, ON, with shaking. The blot was then washed 3× with 10% TTBS, incubated in horseradish peroxidase (HRP)-linked goat anti-mouse  or HRP-linked goat anti-rabbit antibody , RT, 2 h, washed 3×, followed by visualization using PiercePooled synovial fluid was formed by combining 2–5 samples from individual donors to better allow reproducibility. Protein was precipitated from pooled human synovial fluid  by additThe viscosity of pooled synovial fluid and pSynF was measured by a homemade viscometer  using thT = time (s) for a 2-mm diameter (d) ball bearing to traverse 0.1 m (L) of liquid in a 2-ml serological pipette. η = viscosity, ρb = 5156.6 kg/m3 = ball density; ρl = 1,000, 1,002, or 1,034 kg/m3, which are the density of water, synovial fluid, or pSynF, respectively; g = 9.8 m/s2 = gravitational acceleration; θ = π/3 radians = angle of the pipette with respect to the horizontal. Finally, all values were adjusted by multiplied by the ratio of the established viscosity of water over the viscosity of water in this experiment , 10 min, RT. Stained aggregates were rinsed three times with sterile PBS and imaged at 488 nm using a confocal laser scanning microscope (Zeiss LSM800).Bacterial aggregates were rinsed three times with sterile PBS, fixed with 4% paraformaldehyde, 37°C, 15 min, rinsed three times with sterile PBS, and stained with 5 μg/ml AlexaFluor psmα-lux, LAC psmβ-lux, or LAC P3-lux strains of USA300 MRSA were inoculated (Cf ∼106 CFU/ml) in TSB, human serum, pooled synovial fluid, or pSynF to a final volume of 5.0 ml, followed by incubation for up to 24 h . Every 2 h, while maintaining temperature, samples were vortexed, 2 × 100 μl were removed and placed in clear bottom white plates, and luminescence was measured using a VeritasTM microplate luminometer . In parallel, absorbance (λ = 600 nm) was measured on the same samples to determine bacterial growth (Tecan Infinite M1000 plate reader). Luminescent values were normalized to the absorbance at 600 nm and expressed as relative luminescent units (RLUs). Each point was determined in duplicate in a minimum of three independent experiments.LAC t-test or the Mann–Whitney U-test for simple comparisons. More complex comparisons were analyzed by one-way or two-way ANOVA with a Tukey’s post hoc test. Alpha values were set to 0.05, and the statistical analyses were performed using SPSS IBM ® Statistics Version 26 or Prism GraphPad 9.0.1.Statistical significance was determined using the Student’s in vitro in human synovial fluid approximated the aggregates formed in an infected joint. By SEM (in vitro images are representative of at least three samples), the adherent biofilm retrieved from an implant in a case of clinically confirmed PJI was rich in proteins and even showed the presence of red blood cells. In vitro, MSSA biofilm formed in human synovial fluid on smooth titanium alloy (Ti6Al4V) was relatively sparse and much simpler than the PJI biofilm. The major similarity was that bacteria/biofilm showed clear fibers within and between biofilm clusters. In TSB, MSSA was largely clustered and covered by biofilm; any underlying fibrils were not as apparent as on Ti6Al4V. The non-adherent aggregate retrieved from the PJI fluid  showed large globular bacterial aggregates immobilized on a fibrous matrix and encased in biofilm. Like the PJI aggregate, large, globular, floating MSSA aggregates were formed in vitro in human synovial fluid from a single donor; individual bacteria were also visible and many of these were obscured by biofilm coating   . By Westn = 3) , the Fg n = 9 (TSB), 12–13 (SynF)]. MSSA that typically exists in small clusters within TSB showed no significant increase in CFU after trypsin treatment. In synovial fluid, CFU were ∼1 log lower than the measured inoculum; trypsin treatment resulted in an ∼1.5 log increase in CFU. Thus, (1) synovial fluid aggregation reduced the number of retrieved CFU by counting one large cluster as one colony, and (2) trypsin treatment dispersed the aggregates to release the bacteria from the aggregates. This trypsin dispersal resulted in numbers of CFU consistent with those measured for the same inoculum in TSB.To be able to determine the effect of altered Fg content on bacterial aggregation, we set up a trypsin dispersal assay. Specifically, we tested the ability of trypsin to digest surface proteins and other proteinaceous components that facilitate bacterial clustering in TSB and aggregation in pooled synovial fluid . In TSB, 30 μg/ml of AMK eradicated MSSA (∼9 log decrease). In 12 mg/ml Alb + TSB, or 3 mg/ml HA +TSB, AMK tolerance increased so that there was only an ∼5–5.5 log decrease. It is notable that MSSA in Fg + TSB exhibited full sensitivity to AMK. MSSA in either Alb + HA + TSB or Fg + Alb + TSB showed intermediate tolerance (∼3 log decrease). MSSA exhibited the greatest tolerance to AMK in Fg + HA + TSB and the complete pSynF (HA + Fg + Alb + TSB) where 30 μg/ml AMK could only decrease MSSA levels by ∼1.5 log.We then tested the effect of each of the different components of pSynF when added to TSB on MSSA tolerance to AMK, an aminoglycoside antibiotic important for severe infections ; n = 8–9p < 0.001).We then compared MSSA antibiotic responsiveness in pooled synovial fluid to that in pSynF . In TSB,To complete these analyses, we measured the viscosity of pSynF, an important property of the proteoglycan-rich synovial fluid. While the viscosity of the pSynF (∼7.5 cP) was much greater than that of water (∼1 cP), it was lower than the viscosity of human synovial fluid, which was measured as ∼40 cP in our system . CollectPreviously, we showed that both MSSA and MRSA aggregates, i.e., floating biofilms, formed in synovial fluid were covered with a polysaccharide intercellular adhesin (PIA) matrix . We thusS. aureus virulence factor expression, which is under the control of the agr operon, is also highly influenced by the microenvironment. We have previously shown that virulence factor production in S. aureus is downregulated in synovial fluid . P3 luc expression in TSB showed a rapid and marked rise over time, whereas the expression in serum and synovial fluid was only minimally detectable. However, P3 luc expression in pSynF fell between that of synovial fluid and TSB, despite the presence of aggregation. For both psmα and psmβ, luc expression in TSB showed a marked steep rise over time, similar to the expression patterns of P3 in TSB. As we had observed previously, both serum and synovial fluid showed very low levels of psmα and psmβ luc activity. Importantly, psmα and psmβ luc expression patterns in pSynF were very similar to those measured for serum and synovial fluid.al fluid . We thernd pSynF ; n = 6. We have suggested that the phenotype of bacteria in the joint directly impacts the ability of antimicrobials to combat PJI, a devastating complication of joint replacements. In previous work, we showed that synovial fluid, the lubricious fluid within the joint space, causes aggregation of many bacterial species, including MSSA and MRSA. Furthermore, we established that proteinase K and tissue plasminogen activator (tPA), presumably through activation of the serine protease plasmin, can inhibit aggregate formation, disperse the floating MSSA or MRSA aggregates formed in synovial fluid, and restore antibiotic sensitivity . These oin vitro in synovial fluid. While these aggregates show many normal components of a biofilm, based on our earlier work, the degradation of PIA or extracellular DNA is insufficient to disrupt the aggregates – a proteinase is required  . Based oInterestingly, Alb and HA were also important for aggregation, as demonstrated in the combinatorial addition experiments. Within the range of 1.5–3.0 mg/ml HA, 6–12 mg/ml Alb, and 6–12 mg/ml Fg, MSSA and MRSA aggregated, as evidenced by decreased CFU. Furthermore, if only one of the components was present, the ability to reproduce the characteristic antibiotic tolerance was attenuated. Interestingly, antibiotic tolerance measured in HA + Fg + TSB was similar to that in pSynF, despite the dependence on Alb that aggregation was shown to have. It is interesting that the Fg concentration showed an upper limit as the 12 mg/ml Fg concentrations started to become more gel-like (rather than distinct aggregates) in the presence of MSSA. Importantly, we chose the concentrations of the different components based on statistical tests using aggregation, antibiotic tolerance, and ultimately literature values for human synovial fluid (which fell within the ranges we had determined) .The proteins sequestered within the MSSA aggregates formed in pSynF approximated the protein composition of that in human synovial fluid, although individual and cross-linked chains of Fg were not always equivalent in the pSynF vs. synovial fluid aggregates, perhaps pointing to the absence of the Fg cross-linking cascade in pSynF. When aggregates were viewed without magnification, the pSynF aggregates were uniformly smaller than those formed in synovial fluid and in clinical cases where we have observed retrieval of more mucinous aggregates. It is of course probable that other proteins will have roles in subtly modifying the stability, antibiotic sensitivity, and morphology of the aggregates. We would suggest either the localization of additional proteoglycans found in synovial fluid or greater production of PIA, as well as perhaps additional proteins, that modifies the density of the aggregate. Possibilities would include collagen that binds to the same MSCRAMMs previously identified and Fn, a protein that decorates most extracellular matrices; all are present in synovial fluid. Interestingly, the Fg and Alb content of synovial fluid increases in the osteoarthritic joint . We simiWhile clinically relevant joint-lubricating fluids , as wellin vivo environment.Further emphasizing the similarity of these aggregates to biofilms, both synovial fluid and pSynF formed PIA-encased aggregates that were similar in morphology and apparent intensity of WGA staining. Furthermore, expression levels of PSM-α and -β reporter plasmids suggested that Fg, Alb, and HA are key mediators of bacterial behavior in synovial fluid, especially as similar magnitudes and kinetics of expression were noted in serum, synovial fluid, and pSynF. However, it is not clear if the lower viscosity, or perhaps the use of TSB as the reconstituting medium, led to a difference in P3 expression by MRSA USA300. Finally, we used P3 expression as a readout of Agr regulation. As Agr regulates quorum sensing, as well as the expression of a subset of virulence factors , it becoin vitro models of PJI and septic arthritis, as well as providing a more realistic medium for the development of antimicrobial strategies/materials for implants.In summary, we have identified Fg, Alb, and HA as critical components responsible for bacterial aggregation in synovial fluid and that together, as presented in pSynF, these proteins interact with MSSA and MRSA to recapitulate the majority of the properties of MSSA or MRSA in synovial fluid aggregates. Importantly, as we have previously shown, both serum and synovial fluid cause marked decreases in virulence factor expression, in contrast to many studies suggesting an apparent increase in Agr-controlled genes associated with infections . Our stuThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SK and DC contributed to the methodology, formal analysis, investigation, writing, review, and editing, visualization, and writing the final approved version. NZ contributed to the validation, formal analysis, investigation, writing, review, and editing, and writing the final approved version. PM contributed to the methodology, investigation, visualization, and writing the final approved version. SD contributed to the methodology, investigation, writing, review, and editing, and writing the final approved version. CP contributed to the validation, investigation, writing, review, and editing, and writing the final approved version. MH and AC contributed to the resources, writing, review, and editing, and writing the final approved version. TS contributed to the conceptualization, resources, writing, review, and editing, funding acquisition, and writing the final approved version. MO contributed to the methodology, resources, writing, review, and editing, visualization, funding acquisition, and writing the final approved version. NH contributed to the conceptualization, writing the original draft, writing, review, and editing, visualization, supervision, funding acquisition, and writing the final approved version. 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.


Cancer is a major reason for veterinary consultation, especially in companion animals. Cancer surveillance plays a key role in prevention but opportunities for such surveillance in companion animals are limited by the lack of suitable veterinary population health infrastructures. In this paper we describe a pathology-based animal tumour registry (PTR) developed within the Small Animal Veterinary Surveillance Network (SAVSNET) built from electronic pathology records (EPR) submitted to this network. From an original collection of 180232 free text (non-structured) EPRs reported between April 2018 and June 2019, we used specific text-mining methodologies to identify 109895 neoplasias. These data were normalized to describe both the tumour (type and location) and the animal . The resulting PTR, the largest of its kind for companion animals to date, is an important research resource being able to facilitate a wide array of research in areas including surveillance, clinical decision making and comparative cancer biology. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.15074079 In an adaptation of this method in Italy, national6 and regional8 TRs have offered free histopathologic diagnosis for practitioners operating in their respective areas. A similar process was used in the “Cancer in the Dog” project (1990–1998)9, in Norway, and further updated in the Danish Veterinary Cancer Registry (2005–2008)10, in which veterinarians were invited to submit their tumour diagnosis (TD) through a web-based application. Veterinary insurance databases have also been used12 to obtain data from insured animal populations and finally, more recently, researchers have sought to harness data available in individual electronic pathology records (EPRs). In 2015, records from three diagnostic laboratories in Switzerland were used to create the Swiss Canine13 and Feline14 Cancer Registries, with more than 85000 tumour cases; the largest PTR so far.One approach adopted in TRs in the US  and diagnosed more commonly by histology (63.4%) than cytology (36.6%). The most common tumours in dogs were lipomas (21.7%), mast cell tumours (13.1%), and histiocytomas (7.7%) and in cats, lymphomas (14%) and squamous cell carcinomas (11.1%).Figure To our knowledge, this is the largest and most comprehensive animal PTR at a national level providing a reliable tool for veterinary practitioners and researchers as well as a baseline from which further studies can be developed although being always aware of the aforementioned limitations of PRTs to perform surveillance strategies.Given the importance of companion animals as sentinels and models of human health, this registry and its future developments could play a significant role in comparative studies with human cancer registries under a ‘One Health’ approach.17), and from 120 of 121 UK postcode areas (only missing Hebrides), as well as Jersey, Guernsey and the Isle of Man. Each test result includes assay codes, test methodologies, sample descriptors, results (e.g. pathologist microscopic description) and pathologist interpretation as well as patient details including species, age, sex and a geographical locator based on the UK postcode of the submitting veterinary practice.This project used anonymized diagnostic test results submitted to the Small Animal Veterinary Surveillance Network (SAVSNET) at University of Liverpool between April 2018 and June 2019 by three UK diagnostic laboratories  and Batt Laboratories Ltd). During the study period and based on matching of postcodes, this included data from 2196 (48%) of the 4573 UK small animal veterinary practices in the Royal College of Veterinary Surgeons practice database , the sample taken  and a free text description of the pathology report including diagnosis, prognosis, clinical summary, histology and comments. From some laboratories, data for individual samples (same sample reference) were supplied in a series of consecutive rows that required prior concatenation based on the sample reference number. Table Data extraction from the free text pathology report . For instance, in a histology report, the diagnosis appeared after the word DIAGNOSIS, while on a cytology report it was written after the words CYTOLOGICAL INTERPRETATION.In order to facilitate explanation of the extraction process, an example of pre-extraction data is shown in Table Given that LL could be written in one of these three different sections of the EPR, we developed a search which looked for LL in each of the possible positions within it. A prioritization system was then established selecting LL positioned between DIAGNOSIS and CLINICAL DIAGNOSIS over a LL between CLINICAL HISTORY and HISTOLOGY which itself was prioritised over an LL written in the histology section between HISTOLOGY and COMMENTS.The positions of each of these key words  were identified using the Excel function SEARCH .The vast majority of such cases were identified as an individual diagnosis preceded by a number or a letter, as a delimiter .In particular, the curated reference TD list ),0)).Each nested array formula took the following general format: = INDEX: here we have included the terms low-grade, intermediate-grade and high-grade where recorded.COLUMN C- Grade 3 tier (Patnaik for MCT): here we have included the terms grade I, grade II and grade III where recorded.COLUMN D - Differentiation: this list includes terms related to the differentiation of the tumour such as “Benign”, “Malignant”, “Undifferentiated” or “Well- differentiated”.COLUMN E - Uncertain terms: this category contains terms that may be added to the TD when the pathologist has any doubt about the diagnosis such as “highly likely” lymphoma or “consistent with” lipoma.COLUMN F - Location: In this category we have included anatomical terms related to the tumour location although some caution must be considered since sometimes this is not technically the tumour location but rather the location where a first lesion was detected in the animal and motivated the first visit to the vet.19 using the VLOOKUP Excel function. The preferred terms were themselves either based on domain expertise, or for tumour types using the different tumour lists found in ‘Tumors of Domestic Animals’20. An alternative would have been to use WHO ICD-O terms, but these are not fully compatible with veterinary tumours at this time. Once a veterinary ICD-O has been finalised it would be relatively straightforward to code the PTR data to that format. Dog breeds were mapped to those recognised by the Fédération Cynologique Internationale (FCI) and the American Kennel Club (AKC) while cat breeds were mapped to those recognised by the Fédération Internationale Féline (FIFE) and the International Cat Association (TICA) augmented by recent additions based on popular hybrids (e.g. labradoodle).As a result of applying the aforementioned methodology, different ways of referring to the same kind of data were obtained, as exemplified by Table 19.Once both tumour and animal dataset were completely processed and normalised separately, they were merged using functions OPENXLSX and MERGE with RStudio software (RStudio Version 1.2.1335) by using a bespoke R scriptThe end result is a dataset with an easy to read structure as shown in Table 19.Figure 19 consists of 109895 rows tumours and 15 columns (columns A to O) which are described below.A.ReportRef: N = 93941 pathology reports (“R.” stands for report). It indicates the number of the pathology report (linked anonymised from the submitting laboratory report number). This value if repeated in different rows indicates those cases where reports contain multiple tumours.From the original 180232 pathology reports, 93941 reported at least one tumour while the other 86291 reports with no tumour were discarded.B.TumourRef: N = 109895 tumour references within the 93941 pathology reports. It indicates the reference of the tumour, so for example tumours R.10000-T.1 and R.10000-T.2 means that there are two different tumours in report R.10000.C.ResultDate: the date the tumour was reported by the lab.D.Species: 180 canine breeds or 39 feline breeds.E.Breed: breed of the dog  or cat .F.Gender: gender of the cat or dog including neuter status where known. From a total of 93941 pathology reports, 85435 were from dogs and 8506 from cats. Within dogs, 41570 female, 41574 male and 2291 unknown. Within cats, 4275 females, 3969 males and 262 unknown.G.17).Anonymous_PracticeID: indicates the practice where the sample was taken. During the study period and based on matching of postcodes , this included data from 2196 (48%) of the 4573 UK small animal veterinary practices in the Royal College of Veterinary Surgeons practice database  or histology (N = 69643).I.Tumours_in_the_report: the number of tumours a report contains. 1 tumour = 82479, 2 = tumours 16904, 3 = tumours 6066, 4 = tumours 2480, 5 = tumours 1210, 6 tumours = 756. Median 1 for both cats and dogs.J.Primary_tumour: indicates the specific name of the tumour . Top 3 cat  and dog .K.Grade_2_tier (Kiupel for MCT): indicates the 2 tiers grade for lymphomas and Kiupel for mast cell tumours.L.Grade_3_tier (Patnaik for MCT): indicates the 3 tiers grade for mammary carcinomas and soft tissue sarcomas and Patnaik for mast cell tumours.M.Differentiation: provides additional information about the diagnosis 12 terms used in total. Most common: “malignant”, “benign”, “well differentiated”.N.Location: indicates the tumour location on the patient. 88 locations in total. Top 3 cat  and dog O.Uncertainty_terms: this category contains terms such as “highly likely” or “consistent with” that may be added to the TD when the pathologist has any doubt about the diagnosis. Most common: “Consistent with”, “Possible”, “Probable”.The final SAVSNET PTR datasetThe final dataset describes a PTR that includes a list of 121 different types of tumours that appear at least 10 times in the database. However, within this 121 TD list there are six non-specific terms  which, in turn, either include other specific tumour types appearing less than 10 times such as for example some Leukaemias (included within the term Neoplasia_Tumours_others) or some Islet cell carcinomas (included within the term Carcinoma_others) as well as other tumours reported only using general terms such as “Mammary gland carcinoma” or “Rectal Adenoma” without additional information about the type or tumour it consisted. Additionally, some types of tumour such as multiple myelomas and plasmacytomas were aggregated under the term “Plasma cell tumour”. In all these cases, LL and differentiation may be particularly useful for indicating the tumour type. For example: from the 4838 “Epithelial tumour_others” found in the dataset, 42 are located in the liver. Further, one of them are said to be “Well differentiated” and one is said to be “Benign” further supporting the impression that they are both hepatocellular adenomas. Conversely, from these 1 of the 42 liver epithelial tumours, one is said to be “Malignant”; so this is more likely to be an hepatocellular carcinoma.In regard to LL; this information is derived either from the histology or more commonly from a transposition of the lesion description on the submission form into the pathology report, and has certain limitations. Firstly, therefore, the location may indicate a region of the body rather than a precise anatomical location. Three examples are that several lipomas are said to be located in the mammary gland according to the dataset due to the fact that they are reported as “lipomas close to the mammary gland” or “Lipomas: mammary gland region”. Given that these reports use the term “mammary gland” to set a LL instead of using other words such as “thorax” or “abdomen” some of these tumours are recorded in the PTR as LL ‘mammary gland’ when in fact they may have been overlying the gland or just in that general location. Secondly, when there are multiple tumours without a clear separation between them and their respective locations, an erroneous LL may rarely appear as, for example, “a seminoma in the head”. Finally, the user should be aware that anatomical structure and LL are sometimes not differentiated in the report for the same reason, hence, a tumour affecting a limb could in principle be affecting any of the structures of the limb, although in practice it is often evident from the tumour type which the most likely structure is.It must also be pointed out, concerning lipomas, that given the cells of these tumours are identical to those in normal adipose tissue, it is not possible to differentiate between lipoma and normal subcutaneous fat by cytology alone; this is a clinical decision. Consequently, readers are encouraged to check the Histo_Cyto column when it comes to considering such tumour type where the diagnostic procedure could impact the diagnostic accuracy.The ultimate goal of this system is to automate the collation of groups of tumour types for further review . With this in mind, we designed a technical validation to assess the accuracy of the text-mining procedure in identifying the correct TD from each EPR. To do this we compared the text mining results to a gold standard of expert opinion. Firstly, two experts, one a board-certified medical oncologist (DK) and the other a board-certified veterinary pathologist (LR), each reviewed a random sample of 200 unique EPRs with no overlap, recording their own TD; to avoid any possible bias, both reviewers were blinded to the results obtained by the text-mining procedure.Secondly, the assessment of the 400 expertly reviewed rows was compared to the output from the text mining procedure by a third expert (AE), a Professor of Veterinary Pathology, who was also blinded to the origin of both groups of results in such a way that he was unaware which of the two results were from the expert, and which were derived by text mining.Overall, for reports in which a single tumour was present (298 out of 400), 286 successful results were observed giving an accuracy of 96%. For the multiple tumour group (102 out of 400), the accuracy was 89% with 91 successful results observed.However, when considered separately, cytology and histology reports showed some differences in accuracy.In the single tumour group, which included 144 cytology and 154 histology reports, accuracy was 92% (133 successful results) and 99% (153 successful results) respectively.In the multiple tumour group, which included 72 cytology and 30 histology reports, accuracy was 88% (63 successful results) and 93% (28 successful results) respectively. Table Overall, there were 23 reports, shown in Table Reason 1- Lipomas. Reporting a lipoma was missed six times by text-mining because the original report did not include the word “lipoma” in the Cytological interpretation section but rather expressions such as “fat tissue aspiration” or “aspiration of lipid material”. In these cases, the experts determined that the most likely diagnosis was a lipoma based on information in other sections of the report including the clinical summary, the cytological description and the comments.Reason 2 - Missing tumours in reports with multiple tumours. In seven cases, reports containing multiple tumours were partially misclassified by text mining because delimiters between tumours were not used the usual way. For example, instead of using numbers as delimiters , the report may have quoted “Seminoma in both testicles” or “All four sites: Lipoma”. In these cases, the current text mining approach would only identify the first tumour type mentioned in the report.Reason 3 – Not detecting provisional diagnoses. In six cases, an NT or inconclusive diagnosis were misclassified by text mining because the report included expressions such as “…cannot exclude a melanocytic neoplasm”, “Lymphoma not excluded” or “Meibomian gland hyperplasia (DDx early Meibomian gland adenoma)”. In these particular examples, a diagnosis of a Melanocytic tumour, a Lymphoma and a Meibomian adenoma respectively were given wrongly.Reason 4 – Wrong location. Two reports were misdiagnosed because a wrong tumour location was pulled out. Firstly, a carcinoma in the perianal area (hepatoid carcinoma) was diagnosed by the data mining when the actual location were the anal sacs glands (apocrine glands), so the experts diagnosed an anal sac carcinoma instead of an hepatoid carcinoma. Secondly, in a multiple tumour report without a clear separation between the different lesions, a report containing an epithelial tumour in the thyroid gland and an inflammatory lesion in the abdomen was misdiagnosed as an epithelial tumour in the abdomen.Reason 5 – Incomplete diagnosis. Two reports were partially misdiagnosed because the complete diagnosis was not written in the report. One report was given the diagnosis of an Epithelial tumour  in the thyroid gland when the actual diagnosis was a Thyroid carcinoma. Equally, a diagnosis of a mesenchymal neoplasia was given when the correct diagnosis was a soft tissue sarcoma.21. Because of this, the data from this dataset cannot be extrapolated to the entire populations of dogs and cats in the UK due to the potential for systematic bias in the reporting and ascertainment.In spite of the large amount of information provided by the SAVSNET PTR and the wide geographic area (nationwide) from which these data are received, it should be pointed out that in this paper we are not providing any data or estimation about the reference population or population at risk which has been a key limitation to former TRs in the veterinary field over the last decades. As mentioned earlier, the SAVSNET PTR has received data from just three veterinary diagnostic labs so, consequently, we are not providing data on all the tumours diagnosed in the UK since not all veterinary diagnostic labs submit data to SAVSNET. Indeed, others have shown that tumour registries based on this kind of data suffer both from underreporting  and underascertainment 22.In other words, this is not a population-based tumour registry but a pathology-based tumour registry and, therefore, this data should not be used to calculate tumour incidence rates in the whole population nor should it be considered as a reliable resource to obtain conclusions or estimations about risks related to any breed or tumour type within the whole UK populations of dogs and cats. For example, within the total 93,941 reports presented in this dataset, 10,095 came from Labrador Retriever dogs. However, this breed is also considered the most common in the UK population of vet visiting dogsClearly, in the absence of clear denominator, it cannot be inferred that Labrador Retrievers are the most at risk of cancer in the UK.In this regard, the Small Animal Veterinary Surveillance Network is looking to produce population denominator surrogates using electronic health records of dogs and cats visiting first opinion veterinary practices and estimates of overall UK dog populations.23, it would be possible to perform simple statistical analysis to analyze the influence of the different variables  on the appearance of the different tumours within the dataset although with the caution of being always aware that any result obtained from this analysis would be referred and limited to the animals within the dataset and not to the whole population.Taking these limitations into account, the information presented in the dataset could however provide descriptions of the proportional distribution of tumour types within breeds and\or different neuter status or sex among animals included in our dataset. Additionally, as others have done before in similar research projectsThe final dataset can be fully manipulated in Excel, using simple functions like pivot tables, thereby allowing the association between factors such as sex or breed and tumour types to be readily explored within the cohort of animals included in the dataset.The SAVSNET tumour registry relies on information provided by diagnostic labs. All the data related to sex, neuter status, breed, etc., should be considered secondary data showing a lot of diversity given the large amount contained in the dataset. For that reason, a normalization process was performed in the Methods section.Readers should consequently consider that normalized secondary data may not be as accurate as primary data obtained directly from the researchers.Given that this is a tumour diagnosis-based database, and no unique ID for animals is provided, it may be possible that individual dogs or cats might have more than one sample of the same tumour in the database (for example because owners wanted a second opinion and decided to take another sample of the same tumour in a different veterinary practitioner). This would lead to multiple counting of the same tumour, breed, etc.In some cases, users may be interested in data related to the animals or regions presented in this dataset rather than in the tumours themselves and so, for this purpose, users can work at the level of 93941 pathology reports (n = 93941), rather than at the level of individual tumours (n = 109895).https://www.liverpool.ac.uk/savsnet/using-savsnet-data-for-research/ where assessment will be made based on objectives, publication strategy and track record. In some cases, an access fee may be chargeable. Those successful in their application will need to complete a data user agreement19 which details the necessary safeguards for these data.The histopathology reports on which the final published dataset is based cannot be made available in an open access format as they contain clinically and financially sensitive information relating to the diagnostic laboratory or veterinary practice, as well as rare references to animal names. However, access may be possible by reasonable request for use in line with SAVSNET´s overarching ethical approval from the University of Liverpool. Researchers wishing to access the raw data need to apply for access here Under SAVSNET’s ethical approval, owner consent is not required as SAVSNET does not collect any data that could identify them. Postcodes of the submitting practice for each test performed are collected; under our ethical approval, these postcodes cannot be published. Instead, we have described in the text the percent of veterinary practices as an indicator of coverage provided in the existing PTR and provided an anonymised practice code for each sample in the PTR itself to allow researchers to explore clustering of tumours by practice.


ALOX5 gene, and we reveal that ALOX5 mRNA expression was markedly reduced in lung adenocarcinoma tumours. We next utilised an oncogenic G12DKras lung adenocarcinoma mouse model to investigate the efficacy of AT-RvD1 in vivo. We show for the first time that AT-RvD1 reduces tumour growth in the lungs of G12DKras mice and alters the immune landscape in tumours by reducing the NLR.Aspirin-triggered resolvin D1 (AT-RvD1) is biosynthesised by leukocytes as a mechanism to resolve inflammation during infection and/or injury. Emerging studies reveal that AT-RvD1 also has anti-cancer properties associated with stimulating macrophage-mediated clearance of tumour debris. No study to date has investigated how AT-RvD1 influences the neutrophil to lymphocyte ratio (NLR) in lung cancer, an established marker of poor prognosis. The biosynthesis of AT-RvD1 is dependent on the n = 48) and control tissue (n = 20) by IHC. Tumour expression of SAA and ALOX5 were analysed by RTqPCR and an oncogenic G12DKras lung adenocarcinoma mouse model was used to investigate the in vivo efficacy of AT-RvD1 treatment. ALOX5 expression was markedly reduced in lung adenocarcinoma tumours. The SAA/ALOX5 ratio strongly correlated with TANs and was significantly increased in tumours harbouring an oncogenic KRAS mutation. AT-RvD1 treatment reduced tumour growth in G12DKras mice, which was accompanied by suppressed cellular proliferation within parenchymal lesions. In addition, AT-RvD1 significantly reduced the neutrophil to lymphocyte ratio (NLR), an established prognostic marker of poor survival in adenocarcinoma. This study identifies a novel molecular signature whereby elevated levels of SAA relative to ALOX5 favour accumulation of TANs. Furthermore, the ALOX5/5-LO enzymatic product, AT-RvD1, markedly reduced the NLR and suppressed tumour growth in G12DKras mice.Tumour-associated neutrophils (TANs) can support tumour growth by suppressing cytotoxic lymphocytes. AT-RvD1 is an eicosanoid that can antagonise neutrophil trafficking instigated by ALX/FPR2 ligands such as serum amyloid A (SAA). We aimed to establish whether SAA and ALOX5 expression associates with TANs and investigate the immunomodulatory actions of AT-RvD1 in vivo. MPO-positive neutrophils were quantified in tumour blocks from lung adenocarcinoma ( Long-term low dose aspirin may reduce the risk of developing lung adenocarcinoma , althougALOX5 gene. Both enzymes are expressed in leukocytes where aspirin-acetylated COX2 lipid metabolites are sequentially oxygenated by 5-LO, resulting in the production of AT-RvD1 [AT-RvD1 is biosynthesised by a multistep process involving aspirin-acetylated COX2 and the 5-lipoxygenase (5-LO) enzyme encoded by the  AT-RvD1 ,7. It is AT-RvD1 . Further AT-RvD1 . In cont AT-RvD1 ,11.highALOXlow molecular signal or endotype that is associated with increased neutrophil infiltration within the tumour microenvironment in lung adenocarcinoma patients. In addition, we investigated the therapeutic efficacy of AT-RvD1 in vivo, which significantly reduced lung tumour growth in a mouse model genetically engineered to conditionally express the oncogenic G12DKras allele. Our findings identify a novel avenue for therapeutically blocking pathogenic neutrophils in lung cancer.SAA can also impair cytotoxic T cells functionality by promoting the expansion of IL-10-expressing neutrophils  and acti2 sample of tumour tissue excess to diagnostic requirements was bisected with one piece snap frozen and one piece formalin fixed and paraffin embedded as previously described [n = 48). Control lung tissue biopsies (n = 20) were obtained from patients without malignant disease (n = 10) and from the adjacent tumour-free resection tissue (n = 10) from a subset of the adenocarcinoma patients.Surgically resected tumour tissue biopsies from patients with stage IA-IIIA NSCLC were obtained at the Royal Melbourne Hospital pathology department. A 1–2 cmescribed . Specimeescribed  were use® + Dual Link System-HRP kit, Agilent Technologies, Santa Clara, CA, USA). Slides were incubated for another 1 h at room temperature in blocking buffer specific for MPO  or SAA . Sections were then incubated with MPO (1:500 dilution) or SAA (1:40 dilution) antibody in PBS for 1 h at room temperature. Following 3 washes, slides were incubated at room temperature with labelled polymer-HRP, supplied in the EnVision+ kit and washed three times prior to incubation with SIGMAFAST 3,3′-Diaminobenzidine  for 3–5 min. Sections were then counterstained in Mayer’s Haematoxylin , dehydrated and mounted onto slides using entellan mounting medium . To obtain an unbiased analysis of tumour-infiltrating immune cells with objective data, all immuno-stained slides were subjected to whole slide microscope scanning under high-power magnification using the Olympus V120 virtual slide scanner. The entire tumour section was captured and the area positive for MPO was determined using the Olympus CellSens software and presented as the area positive relative to the entire tumour section on the slide.The following antibodies were used for the human tissue staining: rabbit polyclonal antibody against human MPO (Dako # A0398) and mouse monoclonal antibody against human Amyloid A . Serial control and tumour sections were submerged in preheated citrate buffer  and incubated at 95 °C for 20 min using a Decloaking Chamber . Endogenous peroxidase activity was quenched using the dual enzyme block for 5 min , rabbit monoclonal antibody against human CD3 (Abcam # ab16669), donkey polyclonal secondary antibody to goat IgG conjugated to Alexa Fluor 488 (Thermo Fisher Scientific # A32814) and donkey polyclonal secondary antibody to rabbit IgG conjugated to Alexa Fluor 568 (Thermo Fisher Scientific # A10042). Serial control and tumour sections of 5 μm thickness were deparaffinised in histolene and rehydrated in a series of graded ethanol steps. For heat-induced epitope retrieval, slides were submerged in preheated Tris-EDTA buffer  and incubated at 95 °C for 20 min using a water bath. Slides were then rinsed in PBS and tissue permeabilised (0.5% Triton X-100 in PBS) for 10 min. Slides were next incubated in a humidity chamber for 1 h at room temperature in blocking buffer . Sections were then incubated overnight at 4 °C with MPO (1:40) and CD3 (1:40) antibodies in blocking buffer (no glycine). Following 3 washes (PBS with 0.05% Tween 20), slides were incubated at room temperature in secondary antibodies (1:200) in 1:10 diluted blocking buffer (no glycine). After 3 washes, slides were coverslipped with DAPI-fluorescence mounting medium (Thermo Fisher Scientific # 00-4959-52) and imaged under high power magnification using an Olympus BX53 upright microscope. Ten random high-power fields of the left lung were manually quantified for MPO- and CD3-positive cells.http://www.cbioportal.org, accessed date 2 June 2021, Memorial Sloan Kettering Cancer Center, New York, NY, USA) was used to extract the data and present ALOX5 and SAA1/2 mRNA expression as z-scores relative to normal/control samples. In addition, gene expression of ALOX5 and SAA were stratified according to mutation status of KRAS, LKB1 and TP53. The OncoLnc database (www.oncolnc.org accessed date 2 June 2021) was utilised for Cox coefficient analysis between ALOX5 gene expression levels and survival in lung adenocarcinoma according to Kaplan-Meier estimates. The results are in whole or in part based on data generated by the TCGA Research Network (https://www.cancer.gov/tcga (accessed on 2 June 2021)).TCGA datasets (PanCancer Atlas project) were used to analyse RNA-Sequencing data on 507 lung adenocarcinoma tumour specimens. cBioPortal . The 50th percentile was used as a cut-off to differentiate high versus low expression of ALOX5 and SAA. Kras mutation status was assessed using the frozen biopsy genomic DNA and the ddPCR Kras screening kit . The kit was used in conjunction with the QX200 Droplet Digital PCR System  according to the manufacturer’s instructions to screen for seven Kras mutations including G12A, G12C, G12D, G12R, G12S, G12V and G13D. A cut-off of 0.2% mutant allele frequency was used in accordance with the reported mutation detection cut-off value for this assay [Total RNA and DNA was isolated from snap frozen biopsies using the AllPrep DNA/RNA kit  according to the manufacturer’s instructions. The concentration of nucleic acid was determined using a NanoDrop spectrophotometer. Total RNA was converted to cDNA as previously described ,20. Quanis assay .The human SAA sandwich enzyme-linked immunosorbent assay (ELISA) kit  was used to quantify SAA levels in serum in accordance with the manufacturer’s instructions. The absorbance was measured at 450 nm using the CLARIOstar . The concentrations of the samples were determined using a 4-parameter logistic regression analysis of the standard curve.G12D strain (maintained on a C57BL/6 × 129Sv background) carrying a Lox-Stop-Lox (LSL) sequence followed by the KrasG12D point mutation was used for this study. Lung adenocarcinoma was induced in 6-week-old male and female KrasG12D mice by intranasal inhalation of 5 × 106 plaque-forming units of Adenovirus Cre recombinase  as previously described [G12DKras mice were analysed as previously published [The Krasescribed ,24. AT-Rublished ,24. All t-tests were performed to compare two unpaired samples and the Wilcoxon matched-pairs signed rank test was used for paired analysis. A comparison of multiple groups was determined using the Kruskal–Wallis test followed by Dunn’s multiple comparison post-hoc tests. Data were presented as Tukey box plots. Spearman correlation was performed between two groups. Statistical significance for all analyses was determined by p-values less than 0.05.Data analysis and graphs were generated using GraphPad Prism 7.02 . Mann–Whitney two-tailed r = 0.51, p < 001).MPO-positive neutrophils were detected throughout the tumour section A and theLOWSAALOW, ALOX5HIGHSAALOW, ALOX5LOWSAAHIGH, ALOX5HIGH/AAHIGH) where there was a relatively even distribution of patient numbers  had a history of cigarette smoking, which is consistent with studies that have identified smoking as a risk factor for acquiring activating KRAS mutations [KRAS status, where levels were significantly increased in KRAS mutated but not wild-type tumour biopsies (KRAS status (KRAS mutated tumours but not in KRAS wild-type tumours (HIGHALOX5LOW patients was twofold higher in KRAS mutated tumours compared to KRAS wild-type tumours (5/17 = 29% vs. 5/31 = 16% respectively). Taken together, these observations suggest that acquisition of the KRAS driver mutation represents a molecular abnormality that will drive increased SAA expression in a tumour microenvironment where ALOX5 expression is reduced. SAAHIGHALOX5LOW tumours harbouring a wild-type KRAS genotype were also observed, albeit at a lower frequency, to suggest that this endotype is not restricted to KRAS mutated patients.Tumour biopsies were then genotyped for oncogenic is assay , 17/48 , adenoma and sporadic adenocarcinoma in situ (AIS) lesions throughout the lung parenchyma . There was a significant increase in the number of CD3-positive lymphocytes in the lungs of G12DKras mice treated with AT-RvD1 . BecausKRAS, TP53 or LKB1) can influence ALOX5 expression in tumour biospecimens. Using the large TCGA datasets, ALOX5 was reduced irrespective of tumour genotype with lowest expression observed in KRAS/LKB1 mutated tumours. LKB1 is encoded by the STK11 gene that is frequently altered in lung adenocarcinomas. STK11 mutations cause LKB1 protein truncation and loss of tumour suppressive function. LKB1 levels can also be reduced by promoter hypermethylation [ALOX5 gene is also altered by methylation as its promoter region contains eight proximal GC-boxes [LKB1 and ALOX5 levels in the lung cancer tumour microenvironment, although further work is needed to confirm this.Leukocytes normally express high levels of ALOX5/5-LO and lung tumours are enriched for leukocytes. Hence, the tumour microenvironment is likely reducing ALOX5 mRNA in leukocytes. We investigated whether common mutations . Our finding is highly consistent with the known biology of the IL-6/STAT3 pathway, which is essential for maximal expression of expression of SAA [KRAS does not appear to directly stimulate increased expression of SAA by transformed tumour cells. Lung tumours can be intrinsically pro-inflammatory with increased IL-6 levels driving signalling pathways that support carcinogenesis [SAA can also support tumour growth by activating neutrophils and myeloid-derived suppressor cells ,13. An in of SAA . Since Iogenesis ,39. We pG12DKras mouse model of lung adenocarcinoma to evaluate whether AT-RvD1 has anti-tumour properties in this pre-clinical model. We demonstrate that AT-RvD1 effectively reduced growth and proliferation of transformed cells as assessed by quantifying the area of parenchymal lesions and PCNA staining. It is also the first study to determine whether AT-RvD1 can alter the neutrophil to lymphocyte ratio, a prognostic marker of poor survival in NSCLC [Kras lung tumours and propose that the suppression of pathogenic neutrophils by AT-RvD1 promotes a more robust anti-tumour lymphocytic response.There are multiple recent studies demonstrating the tumour suppressive actions of SPMs such as AT-RvD1, which support its progression into the clinic. These studies show that AT-RvD1 acts via the ALX/FPR2 receptor expressed on leukocytes to reduce tumour growth. For example, RvD1 acts via ALX/FPR2 on macrophages to reduce tumour growth by promoting clearance of tumour cell debris and inhibiting macrophage-secreted proinflammatory cytokines ,9. SPMs in NSCLC ,15. We fin NSCLC . Moreovein NSCLC . We demoG12DKras mice. A limitation of our study is that AT-RvD1 was delivered immediately after the initiation of Kras transformation and that lesions were assessed at 6 weeks post-induction, where in situ adenocarcinomas are sporadic. Future studies should evaluate the efficacy of AT-RvD1 in the therapeutic setting where it is delivered to tumours that are already established and more advanced. This study does provide important preclinical proof-of-concept that AT-RvD1 can be further developed as a treatment approach for future clinical implementation. We also demonstrate for the first time that the NLR is elevated in parenchymal lesions of G12DKras mice and AT-RvD1 significantly reduced the NLR. Hence, we propose that AT-RvD1 can suppress the accumulation of TANs that are capable of inhibiting cytotoxic lymphocyte function within tumours. Future studies should focus further on comprehensively phenotyping neutrophils in the tumour microenvironment and establishing how AT-SPMs influence the phenotype of tumour-associated myeloid cells.We have identified that a subset of lung adenocarcinoma patients express higher levels of SAA relative to ALOX5 and that the SAA/ALOX5 ratio positively correlates with neutrophil infiltration. Our findings support the clinical application of AT-SPMs such as AT-RvD1 in lung cancer, whereby AT-RvD1 potently suppressed tumour growth in