Background: Fluid overload is common in critically ill children and is associated with adverse outcome. Therefore, restricting fluid intake may be beneficial. This study aims to study the feasibility of a randomized controlled trial (RCT) comparing a conservative to a standard, more liberal, strategy of fluid management in mechanically ventilated pediatric patients with acute respiratory tract infection (ARTI).Methods: This is a feasibility study in a single, tertiary referral pediatric intensive care unit (PICU). Twenty-three children receiving mechanical ventilation for ARTI, without ongoing hemodynamic support, admitted to the PICU of the Emma Children's Hospital/Amsterdam UMC between 2016 and 2018 were included. Patients were randomized to a conservative  or standard  fluid strategy, which was kept throughout the period of mechanical ventilation.Results: Primary endpoints were adherence to fluid strategy and safety parameters such as calorie and protein intake. Secondary outcomes were cumulative fluid intake (CFI) and cumulative fluid balance (CFB) on day 3. In the conservative group, in 75% of the mechanical ventilation days patients achieved their target fluid intake. Median [25th−75th percentiles] calorie intake over all mechanical ventilation days was 67.9 [51.5–74.0] kcal/kg/day in the conservative vs. 67.2 [58.0–75.2] kcal/kg/day in the standard group (p = 0.878). Protein intake was 1.6 [1.3–1.8] gr protein/kg in the conservative and 1.5 [1.2–1.7] gr protein/kg in the standard group (p = 0.598). No adverse effects on hemodynamics or electrolyte imbalances were noted. Mean (±SD) CFI on day 3 was 262.3 (±58.9) ml/kg in the conservative group vs. 360.5 (±52.6) ml/kg in the standard fluid group (p < 0.001), which did not result in a lower CFB.Conclusions: A conservative fluid strategy in mechanically ventilated children with ARTI seems feasible, without limiting metabolic needs. However, in our study a conservative fluid strategy surprisingly did not reduce the degree of fluid overload. This study aids the design and sample size calculation of a future larger multicenter RCT, in which we need to redefine the target fluid strategy, possibly by even further fluid restriction and early initiation of active diuresis.Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT02989051. Fluid overload is a major problem in critically ill patients and is gaining increasing attention in both research and clinical practice. Importantly, body fluid mostly accumulates early in the disease process , 2, whicAs evidence in the pediatric population up to this point is exclusively mounted through observational studies, it becomes clear that there is a great need for prospective testing in children , 11. HowWe performed a study to determine the feasibility and safety of a larger multicenter RCT comparing current fluid maintenance protocols with an early conservative fluid management strategy. Primary endpoints were adherence to fluid strategy and safety parameters. We specifically chose a highly prevalent patient cohort of mechanically ventilated critically ill children with acute respiratory tract infection (ARTI), not necessarily characterized by major hemodynamic instability and capillary leak, in order to steer toward a general PICU fluid management protocol.This is a single center feasibility study in patients admitted to the PICU of the Emma Children's Hospital/Amsterdam UMC, The Netherlands. Our PICU is a 12-bed, tertiary unit, serving the greater Amsterdam area in The Netherlands. Patients were enrolled between September 2016 and April 2018 mostly by the primary researcher with the help of the attending physicians in the PICU.Patients were randomized in a 1:1 allocation ratio to a conservative fluid regimen or a standard, more liberal, fluid regimen in randomly permuted blocks of lengths 4 and 6 with the use of an encrypted and automated website . A Safety Monitoring board was set up to monitor monthly progress and possible relation with adverse events in both study arms.t-test with a 0.05 two-sided significance level. In literature, sample size in pilot studies is recommended to be a minimum of 12 participants per treatment arm , as this one of the primary outcomes for the future larger set-up and a parameter most often used in literature to describe fluid overload. Earlier work by our group showed a mean (±SD) CFB3 of mechanically ventilated patients with acute respiratory failure due to severe bronchiolitis of +97.9 (±49.2) ml/kg. Aiming for a 50% decrease in CFB3 in patients who receive a conservative fluid treatment vs. standard treatment, a sample size of 17 in each group will have 80% power to detect a difference in means of 50% (the difference between a group 1 mean of 100 ml/kg and a group 2 mean of 50 ml/kg) assuming that the common standard deviation is 49.2 using a two group ment arm  confirmiPatients below 10 years of age were eligible when they were intubated and mechanically ventilated for a (suspected) ARTI, with an anticipated duration of mechanical ventilation of at least 72 h at enrolment. Enrolment into the study protocol needed to be fulfilled within 12 h after start of mechanical ventilation.Exclusion criteria were use of previous or maintenance diuretic treatment at enrolment, ongoing (fluid) resuscitation at enrolment, acute kidney injury with the need for renal replacement therapy and patients in need of a particular fluid regimen .Age-dependent fluid strategies are defined by international guidelines for healthy children , 20. YetData were prospectively collected from the patient clinical data management system. Allocation to one of the fluid arms was not blinded for the attending physicians, dietitians or researchers, as we wanted fluid and feeding calculations to be as accurate as possible in both fluid arms and it is difficult to blind for a difference in fluid volume.Adherence was observed for every study day for each patient and median adherence over all study days was calculated in a percentage of the total number of study days. Reasons for non-adherence were examined for the conservative fluid strategy as this is the most challenging intervention with respect to limiting fluid volume for medication and nutrition. Safety parameters were calorie and protein intake, the need for interventions such as fluid boluses or administration of diuretics. Calorie and protein intake were assessed daily with the help of an experienced dietician. To assess hemodynamic stability in either fluid strategy, blood pressure, and heart rate were monitored continuously. In order to evaluate electrolyte imbalances, available sodium, potassium, and chloride assays were checked for too low or high values. An electrolyte imbalance was scored if the levels of the respective electrolytes were too high or low for more than 1 day consecutively during admission . To assess the occurrence of acute kidney injury (AKI) the KDIGO AKI criteria were used  by using2]/SpO2) × 100, only when SpO2 was below 97% (Secondary outcomes were (cumulative) fluid intake, fluid output and fluid balance, which were collected daily for the whole period of mechanical ventilation. Fluid intake included intravenously administered fluids and medication, parenteral and enteral feeding, oral medication, and blood products. Fluid output included urine output, stools, gastric aspirate, blood loss, fluid loss from drains, and losses from other body cavities. Fluid balance was calculated over every 24 h as intake minus output per kilogram (kg) body weight on admission. The CFB3 was calculated, as most fluid overload occurs in the first 72 h of start of mechanical ventilation and is therefore one of the most used parameters in literature to describe fluid overload . In the elow 97% .The primary and secondary outcomes were slightly altered from earlier trial protocol, because the most important outcomes are adherence and safety parameters for this feasibility study. However, for a future, larger RCT, fluid intake and balance will be of more significance. As this is a feasibility study with small numbers, these fluid intake and balance cannot be studied as (primary) outcome measures.p-value of 0.05 was considered as statistically significant. Statistical analyses were performed using SPSS Statistics (V.25) and R statistical programming software . Confidence interval of the difference for non-parametric analyses were calculated using CIA  software. Fisher's exact test was used for categorical variables and proportions, as expected counts were lower than 5. Data are expressed as means (±SD), medians [25–75th percentiles] or proportions as appropriate.All analyses were performed according to the intention-to-treat principle. As this was a feasibility study, all analyses listed are exploratory and used to inform future trial designs in this area. A two-sided software  and the istics V. and R stDuring the study period 37 patients were screened for enrollment. In th percentiles 0.8–10.1] and was distributed evenly among the treatment groups. There was no meaningful difference in the sex distribution between fluid strategies. Patients had a median [25–75th percentiles] admission weight of 5.5 kg [3.9–8.2], which was also similar in both fluid strategy groups. There were no other important differences of disease severity parameters, such as the PIM2 score or OSI, between the fluid strategy groups . The main reasons for non-inclusion were because parents declined participation. Three patients were missed, and two patients were not asked for permission because the attending physician considered the emotional burden of their child's disease already too demanding to ask for consent. During 75% of the study days, mechanically ventilated children in the conservative group received fluid according to their target intake. There was no significant difference in adherence between both fluid management groups .p = 0.878 for calorie intake, p = 0.598 for protein intake).Calorie and protein intake were not significantly different between fluid strategy groups and reached a median of 67.9  kcal/kg and 1.6 [1.3–1.8] gr protein/kg in the conservative and 67.2  kcal/kg and 1.5 [1.2–1.7] gr protein/kg in the standard group . Importantly, a conservative fluid strategy did not lead to worsened hemodynamic response over time, as measured by mean blood pressure, during the mechanical ventilation period . Change in blood pressure and heart rate was also assessed by the delta (change from day 1 to day 3) which similarly showed no significant difference . Cumulative parenteral fluid intake on day 3 consisted of mean (±SD) 88.8 (±29.6) ml/kg vs. mean 115.6 (±39.7) ml/kg in the conservative and standard fluid strategy, respectively , translating into a percentage of 34 and 32% of total CFI on day 3 in either group. Urine output was significantly higher in the standard group  of 262.2 (±58.9) ml/kg in the conservative group, vs. 360.5 (±52.6) ml/kg in the standard fluid group . There was no significant difference between fluid strategies in oxygenation failure over time, as measured by OSI .The duration of mechanical ventilation was 5.1 [25–75th percentiles 4.7–7.0] days in the conservative group and 4.3 [25–75th percentiles 2.7–5.4] days in the standard fluid group  and/or major capillary leak. Apparently, these patients are still able to maintain adequate diuresis and fluid homeostasis. Yet, in an earlier study we found that this specific patient group also suffers from fluid overload which is associated with prolonged duration of mechanical ventilation . NeverthIn order to set up a multicenter RCT in the future, we need to take a close look at the needed sample size to obtain a sufficiently precise answer whether a conservative fluid strategy could provide clinical benefit in this pediatric population. We consider 1 day a minimum clinically important difference (MCID) in duration of ventilation. For this study we obtained a common SD of the parameter of ventilation duration of 1.8. With a significance level of 0.05 and power of 80%, this would require 52 patients per group. In an earlier retrospective study of mechanically ventilated patients with ARTI, we found a SD of 3.0. When taking this SD into account, with similar MCID, a sample size of 143 patients per group would be needed. Of course we have to take into account that if a future RCT were to include a more heterogeneous group of patients, an even wider spread in mechanical ventilation duration is expected and the sample size needed may increase even further. Another side note which is essential to consider, is that in this study we did not yet succeed in influencing the CFB3, which will be the primary goal for any future RCT since the CFB3 is the marker associated with adverse outcome in earlier literature. Although we might be able to accomplish this with further fluid restriction, we were not able to take this into account while calculating abovementioned sample size.We believe that the main strength of our study is that it is pragmatically designed study, using a standardized protocol that was based on current clinical practice in critically ill children, that are prone to various risks of fluid overload. Due to this design, implementation into the clinical practice of potential study results is both relevant and feasible. Second, we were able to select a homogeneous study population with a circumscript disease entity that is frequently seen in PICU settings. Finally, we were able to prove feasibility of a larger RCT on this topic. A limitation of our study is the small sample size, Yet, according to the published literature on minimal sample size for pilot studies we did obtain sufficient patients per group , 18. NevElaborating on the data from this study, the next question is how to set up the different fluid strategies for a future multicenter RCT. This study suggests that a conservative fluid management strategy is feasible and safe both hemodynamically and with regard to nutritional intake. Moreover, the standard, liberal fluid strategy led to higher diuresis but similar fluid balances, making it a potential harmful form of overtreating our patients. This study implies that we really need to work on further fluid restriction  to decrease the CFB, as that seems to be our primary goal given the association with adverse outcome in literature. We propose a conservative fluid regimen which consists of compensation for insensible losses, which are significantly lower in mechanically ventilated patients , and a sIn conclusion, a conservative fluid strategy in mechanically ventilated children with ARTI seems feasible, without limiting metabolic needs. Future recommendations would be to conduct a large multicenter RCT in this specific patient population, be it with an adapted protocol of further fluid restriction and early start of active diuresis treatment.The datasets generated for this study are available on request to the corresponding author.This study was approved by the Medical Ethical Review board of the Amsterdam UMC [NL55053.018.16]. Written informed consent for participation was obtained from parents or guardians of eligible patients.SI designed the study, included and randomized patients, and optimized their fluid intake accordingly, analyzed and interpreted the data and wrote the manuscript. VG helped design the study specifically focusing on the calorie and protein intake requirements. Moreover, he contributed in writing the manuscript. MD focused specifically on designing the calorie and protein intake requirements and provided advice during the study on how to optimize energy intake during study participation. JL interpreted the data and provided major contribution in writing the manuscript. RB designed the study, interpreted the data and was a major contributor in writing the manuscript. JW designed the study, interpreted the data and also provided major contribution in writing the manuscript. All authors read and approved the final manuscript.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.


Varicella zoster virus (VZV) is the causative agent of chickenpox (varicella) and shingles (herpes zoster). VZV and other members of the herpesvirus family are distinguished by their ability to establish a latent infection, with the potential to reactivate and spread virus to other susceptible individuals. This lifelong relationship continually subjects VZV to the host immune system and as such VZV has evolved a plethora of strategies to evade and manipulate the immune response. This review will focus on our current understanding of the innate anti-viral control mechanisms faced by VZV. We will also discuss the diverse array of strategies employed by VZV to regulate these innate immune responses and highlight new knowledge on the interactions between VZV and human innate immune cells. Varicella zoster virus (VZV) is a medically important human herpesvirus and infections are extremely common, with seroprevalence rates >90% in most populations around the world. Primary VZV infection causes chickenpox (varicella). The virus then establishes life-long latency in sensory neurons from where it can reactivate years later to cause shingles (herpes zoster), which is typified by a skin rash with a dermatomal distribution. Following herpes zoster rash resolution, many individuals continue to experience severe neuropathic pain, termed post-herpetic neuralgia (PHN), that can persist for months to years .VZV is a member of the alphaherpesvirus family and is closely related to herpes simplex virus type 1 (HSV-1). VZV is genetically stable, a property which is demonstrated by little nucleotide variation between isolates . The VZVin vitro utilizing human cells and infection of human tissue xenografts implanted in severe combined immunodeficient (SCID-hu) mice, as well as observations from the simian varicella virus (SVV) infection of non-human primates, which has been used to model VZV infection in vivo  and lungs. Dissemination of infection may result in a number of serious complications, including VZV encephalitis, cerebellar ataxia, demyelinating neuropathy, myelitis, and pneumonia , 21.During primary infection, despite a robust immune response, VZV is not completely eliminated from the host but rather the virus gains access to neurons in the sensory ganglia and establishes a life-long latent infection –24. The Reactivation from latency causes herpes zoster (shingles), a neurocutaneous disease which occurs in 10–20% of seropositive individuals and involves anterograde axonal transport of virus from the reactivating ganglia to the innervating dermatome –33. The Herpes zoster rash development is often preceded by a prodrome of dermatomal pain and is clinically characterized by a unilateral cluster of lesions typically across a single dermatome, accompanied by localized pain of varying intensity, and neuritis. The cutaneous lesions contain infectious virus and provides another reservoir for virus transmission to other susceptible individuals . OccasioHerpes zoster has the potential to severely impact an individual's quality of life. The most common complication of herpes zoster is PHN which is a pain persisting for months to years after herpes zoster rash resolution . PHN occProgrammed cell death is a critical component of the intrinsic and innate immune response, as it allows for the rapid elimination of damaged or infected cells . Viral iIn the context of VZV, apoptosis has been the most comprehensively investigated programmed cell death pathway. Apoptosis contains distinct biochemical pathways, which are highly complex and involve an energy dependent cascade of molecular events , 48. ThrInterestingly, VZV has been shown to modulate apoptosis in a cell type specific manner. Specifically, VZV induces apoptosis in multiple skin cell types such as MeWo cells  and humaVZV has also been shown to induce apoptosis in immune cells such as T cells, B cells, and monocytes –56, howeIn contrast to some skin cell types and immune cells, VZV does not induce apoptosis in neurons . This waThe ability of VZV to protect neurons from apoptosis induction was attributed to ORF 63, using a recombinant virus which was able to express only one copy of the diploid ORF63 gene . HoweverCell type specific modulation of apoptosis is a crucial component of VZV research due to its link to VZV pathogenesis. As VZV establishes life-long latency in neurons of the DRG, the inhibition of apoptosis in neurons is critical for viral maintenance of latency and the establishment of reactivation . In contOther VZV gene products have also been shown to play a role in apoptosis inhibition. For example, VZV ORF66 inhibits apoptosis in T cells, as evidenced by T cells undergoing apoptosis more readily when infected with a virus in which ORF66 protein expression is impaired . InvestiIt is clear VZV encodes multiple ORFs with anti-apoptotic mechanisms, demonstrating the importance of modulating apoptosis for viral replication and spread. Interestingly, when expressed alone or deleted from VZV, these genes have an anti-apoptotic effect in cell types where VZV is known to induce apoptosis. It will be important to determine whether these gene products delay the onset of apoptosis in vulnerable cell types during VZV infection as this could be a critical component of VZV pathogenesis in the skin and during reactivation. Furthermore, it would be beneficial to determine whether VZV can protect against other forms of cell death, as when apoptosis is inhibited other cell death forms such as necroptosis can occur to limit viral spread . HSV-1 hThe innate immune response to VZV involves the recognition of viral pathogen associated molecular patterns (PAMPs) via PRRs, which triggers inflammatory cytokine secretion and/or cell death. Of the Toll-like receptors (TLRs), Wang and co-workers demonstrated that exposure of monocytes to VZV induced TLR2 and NFκB dependent secretion of interleukin (IL)-6. Furthermore, this report suggested sensing of VZV involved cell-surface TLR2 binding to virion envelope glycoproteins . RecentlTLR3 is known to be expressed in human neurons and peripheral nerve Schwann cells , 83, thuRetinoic acid-inducible gene I (RIG-1) is a cytoplasmic PRR which senses both RNA and DNA viruses and can result in the production of the type I IFN response . KnockdoMonocytes and other myeloid cells are also able to sense virus through NLRs, which trigger a pro-inflammatory response through inflammasome formation . InteresAnother intrinsic defense mechanism limiting VZV infection in human skin is the formation of promyelocytic leukemia (PML) cages in infected epidermal cells, which trap VZV nucleocapsids resulting in inhibition of virion assembly . Wang anInterferons (IFNs) are key anti-viral cytokines that mediate their activity through the induction or upregulation of a suite of interferon stimulated genes (ISGs), which have a range of anti-viral activities . Recogniin vivo observations. More than 30 years ago a clinical trial to evaluate the efficacy of IFNα in inhibiting VZV infection in children suffering from cancer indicated that IFN treatment could limit the dissemination of severe varicella lesions  to modulate type I IFN signaling and viral replication . Multiplin vivo and in vitro, with VZV infected pDCs significantly inhibited in their capacity to produce IFNα after stimulation with a TLR9 agonist  have the capacity to secrete significant amounts of IFNα following appropriate stimulation . Work fr agonist . More re agonist    T cells . These f T cells . The imp T cells .in vitro showed no significant decrease in cell viability or evidence of apoptosis (in vitro MDDC infection model to demonstrate that the VZV vaccine strain (V-Oka) and virulent VZV clinical isolates equally infect these immune cells  , 127, exin vivo . We extenfection . FurtherVZV infection of human DCs has been shown to result in the modulation of cell-surface receptor phenotype and immune functions. Mature MDDCs, like their immature counterparts, are also susceptible to productive VZV infection  which reMoreover, VZV has been reported to reduce cell-surface expression of apoptosis receptor Fas on infected immature and mature MDDCs, whereas surface levels of MHC II remain unchanged . HoweverVZV infection of pDCs and epidermal cells has been observed to occur in the absence of an increase in the type I cytokine IFNα production , 113. ThInterestingly, Gutzeit and colleagues reported the secretion of signature Th1 cytokines (IFNγ and IL-12) was enhanced following infection of MDDCs with (v-OKA) but blocked by a VZV clinical isolate. This impairment of IL-12 secretion was shown to be due to a viral disruption of signaling downstream of TLR2, and proposed to be most likely caused by a VZV glycoprotein within the virion envelope . Thus, VMonocytes and macrophages play a key role in pathogen sensing, immune defense against infection and are important players in resolving inflammation . These c+ cells from varicella patients harbor detectable copies of VZV ORF62 and VZV gB transcripts . Expression of CD16 (FcγRIII) on NK cells allows engagement of IgG antibodies bound to a target cell, which typically occurs during anti-pathogen immune responses. VZV infected and bystander NK cells, however, potently downregulate cell-surface expression of CD16 , which whallenge . A thirdhallenge , which whallenge , potentiin vitro characterization of inhibited NK cell function by VZV.Not only does VZV regulate detection of infected cells, we have recently shown that it directly impairs NK cell function . Both inin vitro that VZV diminishes NK cell secretion of both IFNγ and TNF (Lastly, an important function of NK cells is the secretion of immune modulating cytokines. In relation to the control of VZV, IFNγ, and TNF are readily secreted by NK cells and have strong inhibitory effects on VZV replication , 172. Th and TNF  (Figure  and TNF . This seVZV has co-evolved with the human host for millions of years . In thatThere are still many areas of VZV encoded innate immunity manipulation that warrant further investigation. For example, exploring whether VZV protect against other forms of cell death, as when apoptosis is inhibited other cell death forms can occur to limit viral spread. Additional study will be required to fully define the role of type III IFNs during VZV infection. Specifically it will be of interest to understand the distinct activities of type I, II, and III IFNs in regulating infection as this will potentially dissect the roles played by distinct IFNs in regulating infection during different phases of the viral lifecycle. Despite VZV being shown to modulate immune functions of different DC subsets, the molecular mechanisms and VZV proteins directly responsible for these immune evasion strategies has yet to be elucidated. Finally, recent data showing NK cells and other immune cells within PBMC compartment can be infected with VZV provides an avenue to gain a deeper understanding of the impact VZV infection has on immune cell functions and the importance of these cells in viral pathogenesis.Modulation of the innate immune response ultimately effects the formation and effectiveness of the adaptive immune response. Therefore, it is clear VZV can modulate components of the intrinsic, innate and adaptative immune response to ensure viral dissemination and the establishment of life-long latency. It is critical to dissect the mechanisms of this immunomodulation to provide important insights into VZV pathogenesis which will likely be of benefit when designing new generation vaccines and anti-virals. Furthermore, the study of herpesvirus modulation of immune responses also enhances our general understanding of the complexity of the human immune system.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


The shale dispersion test (rolling test) is a common procedure that is used to measure the interactions between drilling fluids and shales. The shale rolling test depends on the moisture content of the shale, the shale composition, the viscosity of the test fluid, the rotation speed of the rollers, and the test temperature. The rheological behavior of the test fluid has the strongest influence on test results. The data was generated experimentally, shale samples from Agbada formation Niger-Delta was used. These shale samples were cored at a depth of 2000 ft and 3400 ft. Water based mud that will minimize shale dispersion and swelling of shale was formulated. The dispersion test was conducted, and it involves exposing a weighted quantity of sized shale to the formulated mud in roller-oven. This test is used to design fluids and screen the effectiveness of inhibitor additives to maintain the integrity of the cuttings and minimize the interaction of fluids with the shale sections during the drilling and completion operations. The swelling test was conducted and the linear expansion adopted because it is the most representative of the increase seen by the wellbore but was measured in the direction perpendicular to the bedding plane as this is the direction of swelling into the wellbore. Specifications TableValue of the data•These data describe the rheological behavior of the test fluid as the strongest influence on the test results.•The data can be used to design fluids and screen the effectiveness of inhibitor additives to maintain the integrity of the cuttings.•These data can be applied when analyzing the interaction of fluids with the shale sections during drilling operations.•The data analyzed the behavior of drilling cuttings in drilling fluids as they travel up the annulus.12.Dispersion tests were carried out using various drilling fluids for Shale samples in Well A and B at 120 °F. Percentage recovery, a measure of shale recovered after dispersion tests were calculated. 2. The results are in agreement with the literature, that K+ and Ca2+ ions are added to the water-based muds to inhibit the clay from dispersing, to stop it from breaking up when attacked by an aqueous solution +) associated with the clay in the shale, creating a more stable rock that is able to resist hydration. The results show that the dispersed solids in the fluids were large. The dispersion is a function of changes in the structure of the shale matrix and in the bound and crystalline water caused by the hydration of the cores The Well A cored at a depth of 2000 ft, is made up of 20% Quartz and 52% Clay. It contains other non-clay minerals such as feldspar and carbonates in minimal amounts. The bulk of the clay content consists of illite and mixed clays with a small amount of smectite. The existence of smectite indicates the probability of some swelling and dispersion in aqueous solution. Well B cored at a depth of 3400 ft, is composed of 22% Quartz and 51% Clay. It also contains negligible amount of non-clay minerals such as feldspar and carbonates. Zero smectite levels indicate low swelling tendencies.+ and Ca2+ ions are added to the water-base muds to inhibit the clay from dispersing, to stop it from breaking up when attacked by an aqueous solution. These ions commonly replace the sodium ion (Na+) associated with the clay in the shale, creating a more stable rock that is able to resist hydration. The results show that the dispersed solids in the fluids were large. The dispersion is a function of changes in the structure of the shale matrix and in the bound and crystalline water caused by the hydration of the cores.The results are in agreement with the literature, that KThe plastic viscosity (PV) and yield point (YP) were calculated before and after dispersion. A comparison of PV values before and after hot-rolling the shale samples showed distinct outcome with the salt/bentonite fluids. The salt/bentonite fluids showed an increase in PV when hot-rolled at 120 °F; which indicates shale dispersion. Yield point for the salt/bentonite fluids showed a decrease in YP after hot-rolling at 120 °F. YP is a function of electrostatic forces between fluid particles in motion Further analysis of the dispersion tests involved the measurement of the fluid rheology before and after the tests. Values of shear stress (τ) and shear rate (γ) were calculated and presented in 2The dispersion test involves exposing a weighed quantity of sized shale pieces (2–4 Sieve Opening Millimeters) to a formulated fluid in a conventional roller-oven cell. The test provides long-term exposure of the shale to the fluid under mild agitation. Under such conditions, dispersion of the shale into the fluid will occur depending on the tendency of the shale to disperse and the inhibitive properties of the fluid. Shale dispersion is a process by which shale cuttings disintegrate into smaller sizes. It is a function of mechanical factors such as shear and chemical factors such as hydration  and shear rate (γ) were calculated using the following equations:τ=shear stress (lbf/ft2)iθ=dial reading at ith rpmN=spring factor=1For shear rate,γ=shear rate (1/s)RPM=viscometer rotational speedFor shear stress,2)}, when used in the drilling fluid have proved to be beneficial in stabilizing shale formations. These additives lower the water activity of the drilling fluid, which leads to higher osmotic pressure gradients. Also, solute mobility is low in shale, which increases the membrane efficiency of the shale/drilling fluid system. As a result, the effective osmotic pressures generated could be strong enough to offset the hydraulic mud over-balance, which indeed could lead to shale stabilization through dehydration. Also, the main function of viscosifier is to increase the viscosity of the mud filtrate, and thus, reduce the hydraulic flow and pressure penetration into shale formations. They also lower the water activity of the drilling fluid and thus generate osmotic potentials Salt additives {Potassium Chloride (KCl), Sodium Chloride (NaCl), Calcium Chloride (CaClLinear Swelling tests were carried out on Shale samples from Well A and B using Water Base-mud and deionized water. Both shale samples swell in deionized water after immersion for 24 h. They also showed reactivity when placed in all the test fluids. Maximum linear swell for this shale was obtained in the deionized water.


Tumours rapidly ferment glucose to lactic acid even in the presence of oxygen, and coupling high glycolysis with poor perfusion leads to extracellular acidification. We hypothesise that acidity, independent from lactate, can augment the pro-tumour phenotype of macrophages.We analysed publicly available data of human prostate cancer for linear correlation between macrophage markers and glycolysis genes. We used zwitterionic buffers to adjust the pH in series of in vitro experiments. We then utilised subcutaneous and transgenic tumour models developed in C57BL/6 mice as well as computer simulations to correlate tumour progression with macrophage infiltration and to delineate role of acidity.Activating macrophages at pH 6.8 in vitro enhanced an IL-4-driven phenotype as measured by gene expression, cytokine profiling, and functional assays. These results were recapitulated in vivo wherein neutralising intratumoural acidity reduced the pro-tumour phenotype of macrophages, while also decreasing tumour incidence and invasion in the TRAMP model of prostate cancer. These results were recapitulated using an in silico mathematical model that simulate macrophage responses to environmental signals. By turning off acid-induced cellular responses, our in silico mathematical modelling shows that acid-resistant macrophages can limit tumour progression.This study suggests that tumour acidity contributes to prostate carcinogenesis by altering the state of macrophage activation. Levels of tissue oxygen, metabolic by-products, nutrients and hormones modulate these cellular interactions that, in turn, can regulate tumour progression.8 In particular, infiltration by macrophages can promote tumour progression and poor outcome in solid malignancies when their presence is associated with a tumour-promoting phenotype reminiscent of interleukin (IL)-4-driven activation.9 The pro-tumour phenotype of these tumour-associated macrophages (TAMs) can be affected by several aspects of the tumour microenvironment (TME).10 These include cytokines and antibodies produced by lymphocytes and tumour-derived cytokines/chemokines that promote macrophage infiltration and polarisation.13 Abnormal metabolic factors can also aggravate the phenotype of these cells. For example, hypoxia augments the immunosuppressive ability of TAMs,14 while lactic acid induces tissue remodelling though expression of vascular endothelial growth factor (VEGF) and arginase I.15 Whether acidic pH, as an independent entity from lactate,16 alters macrophage polarisation within tumours is not clear, hence, we sought to investigate the impact of tumour acidosis on the phenotypic characteristics of macrophages in vitro using zwitterionic organic buffering agents. We then used a series of mouse models to correlate tumour progression with macrophage infiltration and to delineate the role of acidity in prostate cancer. We then reiterate our findings using an agent-based mathematical model that simulate how pH affects the ability of macrophages to control tumour growth.Tumours are infiltrated by populations of myeloid cells that regulate tumourigenesis through their ability to mediate immunosuppression, matrix remodelling, angiogenesis, local invasion and metastasis.5 TRAMP-C2 cells. This concentration of sodium bicarbonate in drinking water is well tolerated and provides the required buffering effect as described earlier.17 No anaesthesia, analgesia or surgical procedure were needed to administer the sodium bicarbonate solubilised in water. To ensure the health status of animals, mice weight and water consumption were monitored twice a week (data not included). Tumour growth was evaluated weekly by measurement of two perpendicular diameters of tumours with a digital calliper. Individual tumour volumes were calculated as volume = [π/6 × (width)2 × length]. To collect tumours, mice were euthanised using carbon dioxide inhalation in their home cages, followed by cervical dislocation to ensure death on the 35th–42nd day tumour cell postinjection. Solid tumours were then excised and processed for flow cytometric analysis and immunohistochemistry (IHC) as will be described later. Male transgenic adenocarcinoma of the mouse prostate (TRAMP) mice was obtained from The Jackson Laboratory. Male TRAMP spontaneously develops autochthonous prostate tumours following the onset of puberty due to the expression of the oncoprotein SV40 T antigen (TAg) under transcriptional control of the rat probasin promoter.18All mice were maintained in accordance with Institutional Animal Care and Use Committee (IACUC) standards followed by the Moffitt Cancer Research Center . Mice have free access to water and food, housed in pathogen-free cages containing wood shavings and bedding in a 12-h light/dark cycle, with controlled room temperature (RT). All animals and cell lines were male or male derived, respectively, since this study is mainly investigating prostate cancer. For bone marrow isolation, C57BL/6N (C57BL/6NHsd), aged 8–12 weeks, male mice were purchased from Envigo. For the subcutaneous prostate cancer model, mice were randomly assigned to experimental groups and then provided with 200 mM sodium bicarbonate in their drinking water  starting on the fourth day prior to subcutaneous injections with 5 × 10Male-derived murine TRAMP-C2 and TRAMP-C3 prostate cancer cell lines were purchased from ATCC, maintained and cultured according to their suggested protocols.20 In brief, bone marrow was flushed from femurs and tibias of male C57BL/6N mice and cultured for 6–7 days in complete macrophage medium  and 20 ng/ml macrophages colony-stimulating factor (M-CSF) at 37 °C. Pro-inflammatory macrophages were induced by exposing BMDMs to 50 ng/ml interferon (IFN)-γ and 10 ng/ml lipopolysaccharide (LPS) in complete macrophage medium. Anti-inflammatory macrophages were stimulated by exposure to 10 ng/ml IL-4 in complete macrophage medium.21 Control macrophages (M0) were cultured for the same period in medium alone. Prostate cancer-associated macrophages were induced by incubating BMDMs with 30% 72 h-conditioned medium from either TRAMP-C2 or TRAMP-C3 cell lines. To detect the effect of tumour microenvironmental acidity, macrophages were induced according the previous protocol but with further supplementation of media with the zwitterionic organic buffers PIPES and HEPES (25 mM each) and adjustment of the pH to either 7.4 or 6.8.22Bone marrow-derived macrophages (BMDMs) were generated as described previously.Recombinant mouse IFN-γ, M-CSF and IL-4 were obtained from R&D Systems. Sources of conjugated antibodies were as follows: inducible nitric oxide synthase (iNOS)-Alexa Fluor 488 (eBioscience), CD206-Alexa Fluor 647 (AbD Serotec), CD45-APC and MHCII-BV21 (BD Biosciences), and F4/80-PE, Ly6C-APC/Cy7 and CD11b-PE/Cy7 (BioLegend). Sources of unconjugated antibodies were as follows: anti-MRC1 (CD206) and anti-iNOS (Abcam). Source of chemical was as follows: Rhodamine Phalloidin (Life Technologies). Griess reagent (Promega) was used to measure nitrite level. Click-iT 5-ethynyl-2′-deoxyuridine (EdU) pacific blue flow cytometry assay kit (Life Technologies) was used to measure cell proliferation. Proteome profiler mouse cytokine array panel A or XL Cytokine Array ARY028 (R&D Systems) were used to detect change in level of cytokines in culture media. All reagents, kits and chemicals, unless otherwise stated, were used according to the manufacturers’ instructions. Other chemicals unless specified were purchased from Sigma-Aldrich.25 Primers sequences are provided in .24 For gene expression analysis by NanoString nCounter, cell lysates were hybridised to the 770-gene murine PanCancer Immune Profiling Panel according to the manufacturer’s protocol (NanoString Technologies). Briefly, 10 µl of Ambion Cells-to-Ct buffer (Thermo Fisher Scientific) was added to a cell pellet and a 5.0-µl volume of lysate was hybridised to the NanoString reporter and capture probes in a thermal cycler for 16 h at 65 °C. Washing and cartridge immobilisation were performed on the NanoString nCounter PrepStation, and the cartridge was scanned at 555 fields of view on the nCounter Digital Analyser. The resulting RCC files containing raw counts were reviewed for quality and normalised in the NanoString nSolver analysis software v3.0, followed by exportation and analysis.RNA was extracted using the RNeasy Isolation Kit (Qiagen). RT-qPCR was then carried out using the iTaq Universal SYBER Green One-Step Kit (Bio-Rad) using primers specific for macrophage activation markers selected according to a previously published lists.Cells were collected, washed and incubated at 4 °C in staining buffer , 2% bovine serum albumin (BSA)) containing the indicated surface antibodies. For intracellular staining, cells were fixed, permeabilised and stained using the BD Cytofix/Cytoperm Fixation/Permeabilisation Kit (BD Biosciences) according to the manufacturer’s instructions. Cells were then washed with staining buffer and subsequently analysed. Data were recorded on a LSR II Flow Cytometer (BD Biosciences) and analysis completed using the FlowJo software. Additional details are included in Cell lysates with equal amounts of proteins (20–35 μg) were electrophoresed through 4–15% TGX Gel, then electrophoretically transferred to nitrocellulose membrane (Bio-Rad Laboratories). Membranes were then incubated with the specified antibodies diluted according to the manufacturer’s instructions. Membranes also were incubated with anti-α-tubulin or anti-glyceraldehyde 3-phosphate dehydrogenase as loading controls. Immunoreactive proteins were visualised with an appropriate peroxidase-conjugated secondary antibody.Macrophages cultured on chamber slides were washed twice with PBS, fixed in 3.8% formaldehyde for 20 min and permeabilised with 0.1% Triton X-100 for 5 min. Cells were washed twice with PBS, blocked with 2% BSA in PBS for 1 h and subsequently incubated with CD206 antibody (1:800) overnight at 4 °C. Cells were washed 3 times with PBS and incubated with appropriate fluorescent-labelled secondary antibodies at RT for 1 h. Images were visualised using Leica TCS SP8 laser scanning microscope (Leica Microsystems).The histological specimens were embedded in paraffin, sectioned (4-μm slices) and stained with haematoxylin & eosin. For IHC, slides were stained using a Ventana Discovery XT automated system . Briefly, slides were deparaffinised on the automated system with EZ Prep solution (Ventana). Enzymatic retrieval method was used in Protease 1 (Ventana). The rabbit primary antibodies that react to F4/80, α-smooth muscle actin (α-SMA) and CD206  were used at 1:400, 1:250 and 1:1200 dilutions, respectively, in Dako antibody diluent (Agilent) and incubated for 60 min. The Ventana OmniMap Anti-Rabbit Secondary Antibody was used for 8 min. The detection system used was the Ventana ChromoMap Kit, and slides were then counterstained with haematoxylin, followed by dehydrated and cover-slipping.2 of tissue. The normalised distances were then subjected to histogram analysis to determine the percentage of cells that fall into 10 µm/mm2 bins representing areas of high macrophage abundance and higher acidity.Histology slides were scanned using the Aperio™ ScanScope XT with a ×200 (0.8 NA) objective lens at a rate of 5 min per slide via Basler tri-linear-array. For TRAMP-derived prostate tissue analysis, images and their meta-data were then imported into the Definiens Tissue Studio v4.0 suite. Each slide was then segmented into several tissue regions with stroma and gland being the main point of interest using the composer function in the software. The individual marker areas were then scored in terms of the intensity of F4/80, α-SMA and collagen. A pathologist (J.D.) was consulted to quality control that each tissue was correctly segmented into the regions of interest as shown in Supplemental Fig. https://gdac.broadinstitute.org/). The expression estimates were derived using RSEM (Accurate transcript quantification from RNASeq) method.26 In Fig. x + 1). The 333 primary prostate tumours and associated clinical information, including reviewed Gleason score, were retrieved from the TCGA PRAD333 publication.27 Box and scatter plots were generated in MATLAB R2017a (MathWorks Inc.).The correlation of macrophage-related genes and glycolysis-related genes in a prostate cancer cohort was computed using level 3 gene expression estimates from the RNA-Sequencing in the TCGA PRAD database, extracted and hosted by Firehose DB . For the mathematical model, Mantel–Haenszel test in the R package “survival” was used.34 To identify changes in macrophage phenotype using NanoString, differentially expressed genes with p < 0.05 were ranked by fold change with a cut-off of 1.5 or 2.35 Statistical parameters, including the value of n, mean ± SEM and statistical significance, and the tests used are reported in the figures and/or figure legends.Unless otherwise indicated, we used unpaired 36 The consequent lactic acid production was shown to aggravate highly immunosuppressive microenvironment through shaping macrophage phenotype in lung cancer and melanoma.15 Based on that, we questioned whether highly glycolytic phenotype correlates with macrophage infiltration or phenotype in late-stage prostate cancer. Interestingly, analysing publicly available data of human prostate cancer revealed that CSF1R is expressed at higher levels in intermediate- and late-stage prostate cancers  in late-stage prostate cancer , as shown in Fig. 37 It is unknown whether the change in extracellular pH independent from changes in extracellular lactate concentration can modulate macrophage polarisation in prostate cancer.Advanced stages of prostate cancer adopt a high glycolytic phenotype that correlated with poor prognosis.ers Fig. . In addi38 To test whether an acidic tumour milieu can influence macrophage phenotype, we used zwitterionic buffer-based medium to stimulate BMDMs using IFN-γ/LPS and IL-4 for 24 h at pH 7.4 or 6.8. Under these conditions, acidic pH did not affect viability of stimulated macrophages at 24 h post-activation  as well as angiogenesis-associated genes  in IFN-γ/LPS activated macrophages, in addition to a global decrease in the inflammation score (Fig. Macrophages are highly plastic immune cells that display a range of phenotypic and functional properties.lot Fig. . In linelot Fig. . Multi-alot Fig. . To expaore Fig. . These rArg1. However, this effect was dramatically enhanced when the cells were activated at pH 6.8 (Fig. Cd206 mRNA expression and protein levels in macrophages as measured by RT-PCR and flow cytometry, respectively (Fig. To examine whether extracellular acidity could alter activation status of TAMs, we first activated BMDMs with tumour cell-conditioned medium at either pH 7.4 or 6.8. At pH 7.4, TRAMP-C2-conditioned medium significantly increased the expression of 6.8 Fig. . Similarely Fig. . BMDMs aely Fig. . In addiely Fig. .Fig. 3Ex+CD11b+ macrophages. As shown in Fig. We next evaluated whether the phenotypic shift in macrophages would alter their function in vitro. TRAMP-C2 cells were incubated in acidic or neutral media in the presence or absence of non-polarised macrophages for 24 h, and tumour cell proliferation was measured via EdU uptake after gating out F4/803 as an accepted experimental approach to neutralise tumour acidity. As shown in Supplemental Fig. Arg1 and Fcgr2b (Fig. Cd206 and Arg1 by single reaction RT-PCR (Fig. + fibroblasts corresponded with tumour progression, with the highest infiltration coincident with loss of fibromuscular tunica, disease progression from prostatic intraepithelial neoplasia lesions to high-grade adenocarcinomas and invasion (Fig. 3 for 28 weeks, starting at 4 weeks of age. Prostate tissue isolated from buffered TRAMP mice showed lower infiltration of F4/80+ macrophages into the stromal compartment compared to controls (Fig. To determine whether tumour acidity was a contributing factor to the phenotype of TAMs in vivo, we treated TRAMP-C2 subcutaneously injected mice with 200 mM ad lib NaHCOr2b Fig. . In a sePCR Fig. . In agrePCR Fig. . We secoion Fig. . Algoritols Fig. . Furtherols Fig. . TogetheDespite the effect of neutralising tumour acidity on prostate carcinogenesis and its impact on the phenotype of TAMs, it was unclear whether these were functionally related, as acidic pH is thought to impact a range of other biological processes within tumours. To test whether acid-responsive macrophages can enhance tumour progression, we developed an in silico agent-based model Fig.  that allTo our knowledge, this experiment can only be conducted in silico, as it is not possible to experimentally “turn off” a macrophage’s response to extracellular pH. The model complements the in vitro experiments, which were conducted in extreme and constant conditions. However, in this model, the changes in pH, cytokine concentrations and spatial co-localisation of macrophages with tumour cells creates a dynamic environment in which macrophages change phenotypes over time. This ever-changing environment thus determines macrophages’ ability, or lack thereof, to control or eradicate the tumour. Given that the model was parameterised using the experimental data, these simulations simulate the interactions in a way that is not feasible with in vitro techniques at present.p in Eq. p in Eq. Arg1 and Ccl2 expression. The time to tumour takeover can be visualised using Kaplan–Meier curves (Fig. p < 0.001, as calculated using Mantel–Haenszel test. The results from these simulations suggest that acid released by tumour cells can create a protective niche capable of directing the functional role of macrophages, thereby increasing tumour growth and decreasing time to progression.In this model, tumour acidity emerges from increased glycolytic metabolism in combination with poor perfusion, and it affects macrophage phenotype as modulated between two extremes states Fig. . Two sceves Fig. . The tum39 One of these metabolic abnormalities is upregulation of glycolysis, even under aerobic conditions. High rate of glycolysis provides malignant cells with proliferative privilege by facilitating uptake and incorporation of nutrients into the growing biomass.40 Metabolic by-products of glycolysis, such as lactic acid, also cause a heterogeneous acidification of the extracellular space, which can results in immunosuppressive nature of the TME.41 Unlike studies that combine lactate and H+ ions as single functional entity named “lactic acid”, we identified an independent role of tumour-generated acidity in driving TAMs phenotype, which in turn can contribute to tumour progression.Tumours undergo metabolic transformation that rewires cellular metabolism to promote tumourigenicity, immune evasion and disease recurrence.Arg1 and Cd206 that are usually associated with a tumour-promoting role for this population. Finally, we noted an association between tumour progression, acidosis and the presence of macrophages in prostate cancer progression in mice and human disease and utilise an in silico agent-based model to delineate a role for acidosis in regulating macrophage phenotype and tumour progression. Cumulatively, these results suggest that tumour acidosis is an important factor that dictates the pro-tumour functionality of macrophages in prostate cancer.In the current investigation, we propose a scenario in which acids generated by glycolytic cells alter the phenotype of TAMs, creating a permissive niche for cancer progression in prostate cancer. Using zwitterionic organic chemical buffering system, our data show that acidic pH alter the activation state of macrophages incubated under polarising conditions, directing the cells towards a functional state similar to the pro-tumour phenotype often ascribed to TAMs. Furthermore, we demonstrate that buffering tumour acidosis alters the activation state of TAMs, with a significant reduction in genes such as Arg1 expression by macrophages essential for lung cancer and melanoma growth.15 In addition, Carmona-Fontaine et al. have demonstrated that lactate cooperates with hypoxia to induce the expression of ARG1 in macrophages. Through the employment of an agent-based model, they also showed that hypoxia-responsive macrophages induce faster tumour growth.42 However, there is limited information regarding how acidity, independent from those metabolic factors, can influence properties of macrophages in TME. Only recently, Toszka et al. identified a role of tumour acidity independent from lactate in driving growth of melanoma cell line B16 in cAMP-dependent manner.43 In the current study that investigates prostate cancer, we also provide evidence that acidic pH, independent from lactate, can promote the pro-tumour polarisation of macrophages, including enhanced tumour cell proliferation, loss of cytotoxicity and release of angiogenic factors. In silico modelling also demonstrated that modulation of the macrophage phenotype by acidity was a significant driver of tumour progression and immune suppression.Lactic acid produced by tumour cells was reported earlier to polarise macrophages into an M2-like phenotype, with 46 An immuno-conjugate that integrates urease enzyme with the ability of pH alkalisation in TME is currently undergoing clinical trials in lung cancer but has not been tested in prostate cancer.48 Despite the efficacy of those modalities evident in preclinical studies, no studies had been conducted to investigate their impact on TME. Among the many future therapeutic applications of the current study is to test whether pH-sensitive macrophage-specific immuno-conjugates or nano-systems that specifically target acidic areas rich in macrophages can reduce immunosuppression and increase T cell infiltration. Those approaches hold the promise of improving efficacy of T cell immunotherapeutic strategies in prostate cancer.Acidic TME is a promising target for tumour-specific imaging and therapy. For example, pH-responsive peptides and pH-sensitive nanotechnology-based systems were shown to improve the efficacy and specificity of cancer therapeutics and diagnostics.Supplemental Information


Cushing’s syndrome (CS) is a set of clinical symptoms which occur as a result of hypercortisolemia. Endogenous ACTH-dependent CS related to an ectopic ACTH-secreting tumour constitutes 12%–17% of CS cases and is one of the most common causes of paraneoplastic syndromes. This study presents a case of a 31 year-old man with diabetes, hypertension, rosacea, purple stretch marks and hypokalemia. Findings of diagnostic procedures include high concentrations of cortisol and ACTH, pituitary microadenoma and a tumour in the anterior mediastinum. Dynamic hormone tests determined the source of excess hormone secretion and ectopic ACTH-dependent CS was diagnosed. Due to increasing symptoms of superior vena cava syndrome, an emergency resection of almost the whole tumour was performed, with only a small part of the upper pole left because of the proximity of large vessels and a risk of damaging them. On the basis of histopathological tests, an atypical carcinoid tumour of the thymus was identified. Immediately after the surgical procedure, there was a significant reduction of clinical and laboratory traits of hypercortisolemia, yet, during the 46 weeks of postoperative observation, despite chemotherapy, the progression of residual masses of the tumour occurred with metastases and increased hormone indices. The presented case shows and discusses the differentiation of ACTH-dependent hypercortisolemia and its causes, difficulties in surgical therapy and chemotherapy, as well as prognosis for atypical carcinoid of the thymus, which is a rare disease. Cushing’s syndrome (CS) is a set of clinical symptoms which occur as a result of hypercortisolemia. An excessive concentration of cortisol in the body can have either exogenous or endogenous aetiology. In most cases, its origin is iatrogenic. The incidence of this rare disease is 0.7–2.4 cases in a million of the population in a year ,2. ThereA smoking 31-year-old man with 1.5-year history of poorly controlled hypertension treated with angiotensin inhibitor and calcium channel blockers and a 6-month history of diabetes with metabolic imbalance treated with intensive functional insulin therapy and with metformin was admitted to the Clinic of Endocrinology, Diabetology and Internal Medicine of the Regional Specialist Hospital in Olsztyn. The patient was admitted as he manifested clinical and laboratory features of hypercortisolemia and body weight loss of about 18 kg in 6 months, swelling of lower limbs, decreased muscular strength/power, mood changes and lower back pain.2, WHR 0.86, conjunctival hyperaemia, oedema, erythema and lividity of the skin on the neck and face, dilated neck veins, spread papulopustular rosacea, purple stretch marks on the hips and thighs, amyotrophy of proximal limb muscles  . After fThe first descriptions of CS concerning ACTH-secreting tumours not located in the pituitary gland come from 1928 . The terThe patient was diagnosed with ACTH-dependent CS on the basis of hormone tests: high concentrations of ACTH and cortisol with a rigid circadian rhythm and no suppression of cortisol secretion in 2 mg dexamethasone suppression test. Imaging tests showed both a tumour of the anterior lobe of the pituitary gland as well as a massive tumour in the mediastinum. Both lesions could have led to hypercortisolemia, thus, differentiation of ectopic and pituitary overproduction of ACTH was made. Both very high concentrations of ACTH as well as no response in the CRH test unambiguously confirmed ectopic ACTH secretion. Moreover, a significantly increased concentration of chromogranin A suggested neuroendocrine nature of the tumour in the mediastinum, which was confirmed in the histopathological tests of postoperative material, in which traits of an atypical carcinoid of the thymus were found. The ACTH ectopy was ultimately confirmed by normalization of its concentration on the second day after the operation. A neurogenic tumour of the thymus is a rare malignant primary carcinoma of the thymus  which taA CT scan of the chest with contrast is a test of choice in the case of neuroendocrine tumours of the thymus. It reveals a large irregular or patchy mass in the anterior mediastinum, with a heterogenous contrast enhancement and foci of necrosis in the central section of the tumour, at times with microcalcifications. Histological differentiation of neuroendocrine tumours of the thymus includes three categories: low grade of malignancy (well differentiated G1 neoplasms), intermediate grade of malignancy (moderately differentiated G2 neoplasms), and high grade of malignancy (poorly differentiated G3 neoplasms). The majority of thymic tumours belong to the group of well differentiated neoplasms . ImmunohUntil 2016, the total number of cases of neuroendocrine tumours of the thymus was about 400 , while aThe treatment of choice in cases like the one presented here is early total operative resection  which, iACTH-dependent hypercortisolemia requires deep diagnostics to know the real aetiology of the disease. When diagnosing ectopic ACTH secretion syndrome, it should be remembered that its cause may be very rarely occurring atypical carcinoid tumour of the thymus—a neoplasma with extremely poor prognosis. The procedure of choice is complete surgery, which is only possible in some cases. Adjuvant treatment is chemotherapy, radiotherapy, and in the case of expression of somatostatin receptors, SST receptor analogues. The grade of histological malignancy, the size of the tumour, non-complete resection, no expression of SST receptors, distant metastases, progression of residual mass of the tumour and concentrations of particular hormones can be additional very unfavourable prognostic factors, as in the presented case.


Despite their promising thermo-physical properties for direct solar absorption, carbon-based nanocolloids present some drawbacks, among which the unpleasant property of being potentially cytotoxic and harmful to the environment. In this work, a sustainable, stable and inexpensive colloid based on coffee is synthesized and its photo-thermal properties investigated. The proposed colloid consists of distilled water, Arabica coffee, glycerol and copper sulphate, which provide enhanced properties along with biocompatibility. The photo-thermal performance of the proposed fluid for direct solar absorption is analysed for different dilutions and compared with that of a traditional flat-plate collector. Tailor-made collectors, opportunely designed and realized via 3D-printing technique, were used for the experimental tests. The results obtained in field conditions, in good agreement with two different proposed models, show similar performance of the volumetric absorption using the proposed coffee-based colloids as compared to the classical systems based on a highly-absorbing surface. These results may encourage further investigations on simple, biocompatible and inexpensive colloids for direct solar absorption. Current devices for solar energy conversion into thermal energy mostly rely on indirect absorption of the sunlight. This technology exploits a (selective) surface absorber with both high solar absorptance and low thermal emittance (i.e. low radiative heat loss), which efficiently captures the incident sunlight and then transfers the resulting thermal energy to a carrier fluid by conduction2. The efficiency of such indirect solar energy absorption is generally limited by the high surface temperature of the absorber with respect to that of the carrier fluid, which results in major convective heat losses with the surrounding environment. To overcome this issue, a promising alternative is represented by the direct absorption of sunlight, where a fluid serves both as solar energy absorber and heat carrier3. The advantage of this latter technique lies in the reduction of both convective and radiative heat losses, since the temperature peak is shifted from the absorber surface (indirect absorption) down to the bulk region of the carrier fluid (direct absorption)4. In this view, the solar absorbing fluid must have proper thermal and optical properties.Along the renewable backbone of the global energy mix, solar energy stands as one of the most promising resources to help reducing fossil fuel consumption and mitigate greenhouse gas emissionsblack fluid consisting of 3.0 g/l of India ink in water was experimentally investigated for direct solar thermal energy absorption5. The observed performance was encouraging, and the potential of direct solar absorption in terms of technological simplification paved the way, in the later decades, to an intense research activity on such fluids. In recent years, the so-called nanocolloids , namely stable suspensions of nanoparticles in a base fluid, have attracted great attention for their use in direct solar absorption. These fluids are characterised by a suspended phase with the ability to confer specifically-enhanced photo-thermal properties to the base fluid10; thus, if opportunely designed, nanocolloids are expected to have a promising potential in solar-to-thermal energy conversion13.In a pioneering work, a 14 and their properties extensively investigated19. Disagreement between experimental results in the literature has been reported and then investigated by several authors22, suggesting that more research is needed to clarify the underlying mechanisms at the basis of their modified properties. A considerable number of the studied nanocolloids rely on carbon nanoparticles dispersed in water or other liquids. Different nanoparticle types have been investigated, such as single- and multi-walled nanotubes25, graphite26, nano-horns29, or carbon powder in water30. However, the increasing use of carbon nanoparticles may lead to major environmental concerns31 and biological risks, because of their (cyto)toxicity33. In this sense, biocompatible (nano)colloids may represent a more sustainable and safe-by-design alternative to carbon-based nanosuspensions.A large number of different nanocolloids for solar energy conversion have been proposedIn this work, we propose an inexpensive, easy-to-prepare and environmentally-friendly colloid based on distilled water and Arabica coffee. This biocompatible colloid includes a small concentration of copper sulphate, to prevent biofouling, and glycerol, to lower the freezing point and allow its utilisation in cold climates. Optical characterization has been carried out for different dilutions, showing a good energy storage capability of the proposed fluid. Tailored collectors have been designed and realized, to test the photo-thermal performance of the coffee-based colloids as compared to classical indirect absorption based on a selective surface. Field tests, in good agreement with two different proposed models, show that the optimal dilution in terms of optical efficiency can be found as the one that guarantees the best energy storage capability during operation. Similar performance between the direct absorption using the proposed colloids and indirect absorption using a selective surface has been found, and this represents an encouraging result to a more in-depth engineering of the solar collectors for further enhancement of the conversion efficiency.This section illustrates, at first, the proposed coffee-based colloids and their optical characterization. Successively, we report on their photo-thermal performance  obtained via experimental field tests and numerical models.34. Among the different types of coffee makers, here we use moka, an aluminium coffee maker for stovetops available in different sizes  images in Fig. 36. Glycerol is chosen in place of ethylene glycol because of its non-toxicity. Finally, 2 ppm (parts per million) of copper sulphate (CuSO4) are added to reduce the risk of algae or moulds formation in the fluid. Five variants of the proposed colloid are considered: (i) the previously obtained solution, consisting of coffee, 30% wt. glycerol and 2 ppm CuSO4, which will be abbreviated as G30 from here onwards; (ii) a 1%, 10%, 20% and 50% volume fraction of G30 in distilled water, named respectively G30w1, G30w10, G30w20 and G30w50  is defined asαλ) and scattering (κλ) coefficients at a given wavelength λ37. Fig. λ = 300–2600 nm). The results show an extremely intense optical extinction coefficient for the G30 fluid, reaching nearly 850 cm−1 at λ = 300 nm, and a slightly lower secondary peak at 324 nm. These features can be attributed to coffee, as pure water shows a high transparency in this spectral region. The height of these peaks decrease for increasing dilutions of G30 in water, as expectable. All fluids show a monotonically decreasing long tail with larger wavelengths, until all the curves overlap at around λ = 1200 nm, and present further peaks of extinction coefficient at λ = 1450, 1900 and beyond 2500 nm. The curve for a 0.05 g/l suspension of nanohorns in water27 is also reported for comparison.The optical properties of the proposed colloids are experimentally characterised in terms of extinction coefficient and stored energy fraction. The extinction coefficient is a characteristic optical property of the material, which is inversely related to the mean penetration distance of light into the medium. The extinction coefficient (27Iλ the incident solar irradiance and x the penetration distance into the fluid (path length). Figure λmin = 300 nm and λmax = 2600 nm, respectively; whereas, the solar irradiance Iλ has been computed using both Planck’s black body distribution38  and, to provide the comparison, the AM1.5 standard spectrum with an overall power of 1000 W/m2. Note that the overall power in the considered range of wavelengths is recovered by an attenuation constant equal to either 0.77 (Planck’s black body) or 1.12 (AM1.5 standard spectrum). At x = 3 mm, for example, EF reaches 90% for G30 colloid, while it is 48% and 22% for G30w10 and G30w1 samples, respectively (in case of Planck’s black body distribution). The 0.05 g/l suspension of nanohorns in water27 presents an intermediate energy storage capability, being between the G30 and G30w10 colloids. For clarity, the same results for the G30w20 and G30w50 colloids are reported in a separated figure  fraction can be computed asT), the global solar irradiance (I0) and the ambient temperature (Ta) are constantly monitored by an electronic acquisition system. The optical efficiency of the collectors is then computed ascp the specific heat of the considered fluids , Anet the net exposed surface. Note that this latter area is approximately 10% lower in case of direct absorption, as it corresponds to the net exposed surface of the flowing fluids with respect to that of the exposed selective surface , while the optical efficiency through energy conservation for the two cases . The mean and maximum relative errors obtained by the model on the outlet temperature are respectively 2% and 4% with respect to the experimental values. Figure The colloids are tested at three different flow rates, namely 0.138, 0.276 and 0.414 ml/s, using the experimental set-up shown in Supplementary Fig. Numerical models have been developed and validated against the experimental data. In particular, we developed: (i) a one-dimensional model based on electrical analogy; and (ii) a two-dimensional Computational Fluid Dynamics (CFD) model. Outlet fluid temperatures are very well recovered in all cases, with an average 2% and maximum 6% relative errors with respect to the experimental data. Globally, the major source of error (7% maximum) is found on the temperature of the borosilicate glass, which affects global losses towards the ambient and thus the optical efficiency of the collectors.Tin = Ta = 25 °C), irradiance (I0 = 1000 W/m2) and wind speed (vw = 1 m/s), to assess the performance of the collectors independently from variable external conditions. The results obtained, coherently with the experimental evidences, show increasing power gain in all cases with the liquid flow rate, as well as with the G30 volume fraction for the volumetric absorption  = 9.6089 T +3501.8, with coefficient of determination R2 = 0.997. For water, previously reported values have been adopted for the specific heat as a function of temperature48. For the different dilutions of the G30 fluid in water, weighted means are considered.The specific heat capacity of the G30 fluid has been measured at the Department of Applied Science and Technology (DISAT) of Politecnico of Torino, using Differential Scanning Calorimetry (DSC). This technique allows to obtain the specific heat capacity as a function of temperature for the tested sample49, with a slight sedimentation generally observed50. In order to avoid any effect of unstable (micro-nano)particles on the optical properties, fluids were left sedimenting for two days before the characterization. The spectral transmittance is firstly measured using a double-beam UV-VIS-NIR spectrophotometer PerkinElmer Lambda900 with variable cell length. The spectral extinction coefficient is then calculated from the experimental transmittance via Beer-Lambert law using a method that allows to correctly remove unwanted reflectance artefacts52.The extinction coefficient of the proposed colloids has been computed at the National Institute of Optics (CNR-INO) in Florence. Coffee presents a very complex chemical composition, which includes e.g. polysaccharides, proteins, lipids and lignin2; whereas, the channels for the fluid flow are 6 mm deep and 22.5 mm wide, and the effective exposed surface is 90.6 cm2. The same collector geometry is employed for both direct solar absorbtion and indirect surface absorption. In the former case, the channels are filled with the coffee-based colloid and are covered by a 6.5 mm thick borosilicate glass , while in the latter case they are filled with water (heat carrier fluid) and covered by a selective surface absorber  and the borosilicate glass on top. TiNOX®Energy is an optimised material able to reduce radiative heat losses thanks to a low thermal emission coefficient (4%) in the infrared range, while providing 94% absorption in the range of solar wavelengths. The collectors are manufactured by means of 3D-printing technique, using ABS filaments . In order to reduce the porosity of the ABS plastic and avoid fluid leakages, Nano-Seal 180 W is applied to the prototypes. A tailored silicon gasket  is molded and, after a one-day stand at ambient temperature, assembled between the fluid channels and the borosilicate glass of the volumetric absorber. A flexible framework produced in TPU 95 A via 3D-printing (Ultimaker 3) is positioned on top of the borosilicate glass, to avoid the risk of cracking during the closure of the assembly, which is realized using 16 M5 bolts  of Politecnico of Torino. The collectors are designed using a CAD software, see Supplementary Fig. ϕ = VG30/(VG30 + Vw), from which, the required mass of the G30 fluid is easily obtained asThe different dilutions of the colloids are prepared in terms of the G30 volume fraction in water ρG30 = 994 kg/m3, assumed constant with respect to temperature. The density of water (ρw) has been computed using the equation reported by Kell48 at room temperature (Ta = 20 °C). The density of the dilutions is computed as a weighted mean of the components. Temperatures are sampled using K-type thermocouples, connected to a data acquisition system . In the experimental set-up, the ambient temperature and that of the glass cover are constantly monitored, besides the inlet and outlet temperatures of the flowing fluids. The wind speed is provided by the meteorological station at Politecnico di Torino. The fluids are pumped through the collectors by a peristaltic pump , which provides a constant volumetric flow rate within a 0.5% error. A thermostatic bath  is used to control the inlet temperature of the fluids at the collectors. Thermo-physical properties of the fluids are evaluated at the average temperature between inlet and outlet sections of the collectors.The experimental density of the G30 fluid is 53Dh the hydraulic diameter of the glass cover and ka the thermal conductivity of air. The radiative heat-transfer coefficient between the glass cover and the sky is evaluated asεc the infra-red emission coefficient of the cover (black body approximation), Tc,u its average upper temperature, Tsky = −25 °C and σ the Stefan-Boltzmann constant. The conductive resistance in the glass cover is sc/kc . Assuming the lateral walls of the channel as adiabatic and a constant heat flux condition at the upper and lower boundaries, the Nusselt number for the laminar flow of the fluid through the rectangular channel is Nu = 854. The convective heat-transfer coefficient between the solid walls and the fluid can be then computed asTf is the average fluid temperature, kf(Tf) the thermal conductivity of the fluid and Dh the hydraulic diameter of the channel. All thermodynamic properties are averaged along the length of the channel.The solar collectors can be modeled as an equivalent one-dimensional thermal network. Based on electrical analogy, each loss term is modeled as a thermal resistance. Starting from the top layer of the prototype, the convective and radiative losses from the borosilicate glass cover to the ambient are modeled as parallel resistances. The convective heat transfer coefficient between ambient and glass cover is evaluated asFor the volumetric absorber, the equivalent thermal transmittance at the top side of the receiver is then computed asSirr is obtained from a simplified version of the Radiative Transfer Equation (RTE), which neglects the contribution of scattering and thermal emission38, asλmin = 300 nm and λmax = 2600 nm are the minimum and maximum wavelengths analysed, τc is the transmission coefficient of the glass cover, Iλ the irradiance corresponding to the Plank distribution for a black body55 and H the height of the channel. The first term on the right-hand side accounts for the absorption during the first pass, while the second for the absorbed radiation which results from the reflection at the bottom of the channel (1 − αabs with αabs = 0.4). Finally, the model equation isW the width of the channel and Tw,l the average temperature of the ABS material at the bottom. Since Tw,l is initially unknown, Eq. sabs/kabs  and the convection from the ABS to the ambient The radiative source term mentclasspt{minima53 and the radiative effect is obtained considering two facing surfaces asTc,l and Ts are respectively the lower temperature of the cover and of the surface, εs is the infra-red emission coefficient of the surface. Again, the conductive resistance of the ABS material and the convection from the ABS to the ambient are considered. Thus, the equivalent transmittances towards the ambient areFor the surface absorber, the surface-to-cover convective and radiative coefficients are treated as parallel resistances. The convective effect Sirr = I0τcαs, being αs = 0.95 the absorption coefficient of the selective surface in the solar spectrum. In order to model the surface-to-fluid heat transfer, we define an efficiency factor as53D = A/L  is the inter-axial distance between two adjacent channels and F is the standard fin efficiency (see reference53 for details). The model equation for this case yieldsIn this case, the source term is ® over the channel length. The mean and maximum relative errors obtained by the model on the outlet temperature are respectively 2% and 4% with respect to the experimental values.The two models are implemented and solved iteratively in MATLAB® Multiphysics 5.3. The length of the fluid domain (L) is equal to the full fluid path through the collector (404 mm), and the height (H) equal to that of the channel (6 mm). The width (W), used as a reference for pre- and post-processing, is equal to that of the channel (22.5 mm). The steady, incompressible Navier-Stokes-Fourier system is solved, to obtain the temperature field.A two-dimensional model of the collectors is set-up using COMSOLρf the density of the fluid, cp,f and kf respectively its specific heat and thermal conductivity, and Sirr a volumetric source term which accounts for the incoming irradiance. This latter term corresponds to the divergence of the radiative flux and, in general, is obtained by solving the Radiative Transfer Equation (RTE)38 in the domain; however, in this study the incident solar radiation is considered collimated (and scattering is neglected), and the source term is obtained asIλ the reference AM1.5 solar spectrum, αabs the absorption coefficient of the fluid (considered as a gray medium4) and μλ the extinction coefficient. In this case, we adopt a mean value for the extinction coefficient obtained asλmin = 300 nm and λmax = 2600 nm. The effect of the borosilicate glass cover on top of the domain and of the ABS material at the bottom of the channel are modeled using appropriate boundary conditions:εf = 1) and that radiative losses are neglected from the ABS material to the ambient. The terms sc/kc and sabs/kabs account for the conductive resistances of the borosilicate glass cover and of the ABS material respectively.For the volumetric absorption (radiation in participating media), the energy equation reads asεs = 0.04 in the radiative loss term.For the surface absorption, the solar radiation is partly reflected and partly absorbed by the selective surface. Heat is transferred to the fluid by conduction, so Eq.  does notThe domain is discretized using a proper number of quadratic cells  and a direct solver, namely the Parallel Direct Sparse Solver (PARDISO) is adopted to numerically solve the governing equations. Independence of the numerical solution from the computational grid has been verified.The mean and maximum relative errors obtained by the model on the outlet temperature are respectively 2% and 6% with respect to the experimental values.56: statistical uncertainty of the experimental measurements (A-type), and uncertainty due to the offset/calibration of the experimental instruments (B-type). The population mean and standard deviation are computed from the statistical samples using the Student’s t-distribution and Chi-squared distribution57; thus, the A-type uncertainty on the optical efficiency of Eq. . For the B-type uncertainty, we consider the offset/calibration error on the input measurements for the following independent quantities ψ = {Tin, Tout, Ta, Io, vw}. Then the B-type uncertainty is computed as57ψi. According to the supplier datasheets, the standard errors are: ±0.4 °C on temperatures  due to the offset of the thermocouples; ±5% on the irradiance (I0) due to the offset of the pyranometer. In order to be conservative, we also assume quite a large uncertainty on the wind speed provided by the meteorological station at Politecnico di Torino, that is ±0.5 m/s. Finally, the global uncertainty , of the selective surface (αs ± 5%) and on the reflection coefficient of the borosilicate glass (τc ± 5%). The former uncertainty on the ABS material has been assumed in a conservative way based on typical values reported for white plastics58, while the latter two uncertainties have been adopted based on the supplier datasheets. The uncertainties are provided after feeding 1,500 normally-distributed random inputs to the 1D model, and the standard deviation of the model output distribution is shown by the error bars of the model in Fig. In the same Fig. Supplementary information.Stability of coffee-based colloids.