Fluid resuscitation following burn injury must support organ perfusion with the least amount of fluid necessary and the least physiological cost. Under resuscitation may lead to organ failure and death. With adoption of weight and injury size-based formulas for resuscitation, multiple organ dysfunction and inadequate resuscitation have become uncommon. Instead, administration of fluid volumes well in excess of historic guidelines has been reported. A number of strategies including greater use of colloids and vasoactive drugs are now under investigation to optimize preservation of end organ function while avoiding complications which can include respiratory failure and compartment syndromes. Adjuncts to resuscitation, such as antioxidants, are also being investigated along with parameters beyond urine output and vital signs to identify endpoints of therapy. Here we briefly review the state-of-the-art and provide a sample of protocols now under investigation in North American burn centers. One of the most challenging aspects of caring for burned patients is the acute resuscitation. The profound inflammatory response generated by a burn far surpasses that seen in trauma or sepsis, and the resultant fluid needs can be extreme. There is a large and ever-increasing body of research devoted to refining strategies for acute burn resuscitation, and this article attempts to summarize some the most important recent findings in the field.After treating victims of the infamous Coconut Grove fire in 1942, Cope and Moore first postulated that burn resuscitation needs may have contributions from both the patient's body weight and the size of their burn [Ongoing research focuses on refining existing formulas to prevent complications of over-resuscitation. This includes devising novel means for titrating resuscitation, such as nurse-driven or computer-driven protocols. The composition of the fluids used in resuscitation has generated significant interest, with a particular focus on colloids and hypertonic saline. Pharmaceutical therapies that attempt to down regulate the inflammatory response such as vitamin C may have a role in acute resuscitation. Likewise, investigators are proposing the use of adjuncts such as plasmapheresis to remove inflammatory mediators from the bloodstream during resuscitation. The final topic inviting significant scrutiny is outcomes of resuscitation, both choosing the most appropriate outcomes to use and how to best measure these outcomes in clinical practice.The Parkland formula is simple to calculate and has a long record of widespread use in the burn community. This has not stopped researchers from frequently examining the efficacy of this formula, and also determining whether practitioners of this resuscitation formula are using it correctly.The most consistent criticism of the Parkland formula is that patients tend to receive more fluid than the formula would have predicted based on the patient's weight and %TBSA. Whether this is an inherent flaw of the formula or user error by practitioners is debated, but it is clear that fluid volumes used in resuscitation are higher than historical controls. This phenomenon was described by Pruitt as "fluid creep ." EngravOne factor playing into the trend of higher resuscitation volumes may be inadequate titration of fluids by physicians. Cancio et al. reviewed their experience with resuscitations using the modified Brooke formula, which predicts 2 ml/kg/%TBSA fluid volumes. An important finding was that clinicians directing the resuscitation were significantly less likely to reduce the rate of fluid infusion when urine output was high than they were to increase fluid rates when urine output was low . In an eTo further remove the element of human error, some centers have been incorporating computer-based resuscitation algorithms. Salinas et al. described a computer model for burn resuscitation that was utilized in the resuscitation of 32 burn patients that were compared to historical controls. Their protocol resulted in less 24-hour and 48-hour crystalloid volumes, and less total crystalloid volumes while in the intensive care unit. Volumes by body weight and by burn area were also significantly less for the computer resuscitation group. The computer protocol also helped patients to more effectively meet hourly goals for urine output . In a foHistorically, prevailing opinion had been that using colloid in the first 24 hours of resuscitation was contraindicated. It was thought that colloid would pass through the "leaky" capillaries in burn shock and exert an osmotic pull, drawing even more fluid into the interstitial space and worsening burn edema. However, investigators more recently are advocating the use of colloid in burn resuscitation, even in the first 24 hours.Lawrence et al. performed a retrospective review of 52 burn patients with greater than 20% TBSA burns. Twenty-six of these patients received albumin during their resuscitation, and 26 only crystalloid. As part of an institutional resuscitation algorithm, patients requiring more fluid volumes than predicted by the Parkland formula changed to an arm of the algorithm in which they got a third of their hourly fluid volumes as 5% albumin, with the other two-thirds given as lactated Ringer's (LR) solution. After colloid infusion began, patients quickly returned to predicted fluid rates and stayed at those lower volumes for the remainder of their resuscitation. Neither the colloid nor crystalloid group had any patients with abdominal compartment syndrome, though the colloid patients had more extremity escharotomies, likely related to their larger average burn size . In a prIn an attempt to prevent overresuscitation, some investigators have also started using hypertonic saline, alone or in combination with colloid. Belba et al. performed a prospective, randomized study of 110 burned patients, 55 of whom were resuscitated with LR according to the Parkland formula for adults and the Shriner's formula for children. The other 55 patients received a hypertonic saline lactate solution containing sodium (250 mEq/L) and lactate 120 mEq/L). The hypertonic group needed higher fluid rates initially, but both groups decreased to less than predicted by the Parkland formula over the first 24 hours. The hypertonic group used less fluid overall than the isotonic fluid group, but this difference was not statistically significant . Using a mEq/L. TThe extensive inflammation seen in burn injury causes free oxygen radical release, which worsens vascular permeability and subsequently causes significant peripheral edema. The loss of fluid into the interstitium results in higher fluid needs during resuscitation. It is though that the use of antioxidants during resuscitation may help scavenge these free radicals and attenuate vascular permeability.Tanaka et al. compared two groups of patients, 18 that were resuscitated with LR alone, and the other 19 patients receiving LR plus high dose ascorbic acid . They found average fluid needs of 3 ml/kg/%TBSA in the vitamin C group versus 5.5 ml/kg/%TBSA in the group resuscitated with LR alone. In addition, the vitamin C group had fewer ventilator days . Kahn etIn a murine model, Constantini et al. experimented with the use of pentoxifylline (PTX) after burns. After a 30% steam burn, PTX in saline alone was injected intraperitoneally into one group of mice and saline alone into another group. The group with PTX had decreased intestinal permeability and inflammation. In a secondary finding, they also noted a decreased incidence of acute lung injury in the PTX group. Although there are no existing human trials with PTX in burn resuscitation, it may have promise as an antioxidant immune modulator during acute resuscitation .In addition to using antioxidants, some centers have looked at using mechanical removal of inflammatory mediators from the bloodstream. Klein et al. reviewed the use of plasma exchange at their institution over a 5-year period, in which 37 burn patients underwent plasma exchange during their acute resuscitation, seven of whom received two treatments for a total of 44 plasma exchanges. These were severe burns with a mean %TBSA of 48.6%, and 73% of the patients had associated inhalation injury. There was no protocol for initiation of plasma exchange, but it was often prompted by reaching twice the predicted Parkland formula resuscitation volume. Average time to initiation of plasma exchange was 17 hours, and the average duration of therapy was 2.4 hours. Albumin (5%) was primarily used as replacement fluid, unless the patient had low fibrinogen or abnormal clotting factors, in which case fresh frozen plasma (FFP) was used. They found that plasma exchange decreased crystalloid administration by 28.3%. When adjusted for patient weight and %TBSA, the average post-exchange fluid resuscitation volumes dropped by 40%. After plasma exchange, the hourly fluid administration rates never returned to preexchange levels in any patient .Neff et al. performed a retrospective case-control study of 40 patients over a two-year period, all of whom had greater than 20% TBSA burns. Twenty-one of these patients underwent plasma exchange as part of their resuscitation, and they were matched with 19 contemporaneous controls. Plasma exchange was triggered by fluid volumes of 1.2 times that predicted by the Parkland formula, or by continued low urine output or hypotension in the face of escalating fluid rates. They found several physiologic benefits with plasma exchange, including a 24% increase in mean arterial pressure (MAP), a 400% increase in urine output, and a 25% reduction in intravenous fluid resuscitation rates need to maintain vital signs and urine output goals. Lactate levels also decreased, and they noted that an elevated admission lactate independently predicted the need for eventual plasma exchange [The second challenge in management of burn resuscitation is determination of optimal clinical endpoints. Traditionally, urine output has been used as the primary gauge of tissue perfusion during acute resuscitation. Greenhalgh recently published findings from a survey of American Burn Association (ABA) and International Society for Burn Injuries (ISBI) members regarding various topics in resuscitation. Respondents (94.9%) used urine output as a major index of successful resuscitation, with 22.7% using other monitors . DespiteParameters derived from transcardiopulmonary thermodilution using the PiCCO system have shown good correlation with values from a conventional pulmonary artery catheter in burned patients . The use2, and PaO2. They also simultaneously charted urine output, mean arterial pressure (MAP), and serum lactate. Third, these investigators measured burn wound perfusion with laser Doppler imaging and correlated the previous variables with observed changes in burn wound perfusion. Although changes in all variables were associated with changes in Doppler perfusion, they found that tissue pH and CO2, as well as gastric CO2, had the closest temporal relationship to changes in peripheral perfusion. Urine output, MAP, and lactate did change over time but tended to lag behind changes in peripheral tissue perfusion measured with laser Doppler imaging [Jeng et al. studied four patients with severe (average 58% TBSA) burns and shock, using a multisensory probe with three transducers. One transducer was placed in the subcutaneous tissue in a representative second-degree burn, another transducer in the stomach via a tonometric gastric tube, and a third into the bloodstream through a single-lumen femoral line. They then used these transducers to measure tissue pH, CO imaging .Though lactate may not give "real-time" information about resuscitation success, it does predict morbidity and mortality in burned patients. Cochran et al. reviewed 128 patients with an average of 41.7% TBSA and measured base deficit and lactate levels at 6-hour intervals. They found that the non-survivors had higher lactates at admission, 12, 18, and 24 hours than the group of survivors. Elevation of lactate in the first 48 hours was an independent predictor of mortality, but they were unable to demonstrate a specific threshold for clinical use. The authors caution that treatment should not be withheld based on any individual laboratory value .Burn resuscitation continues to be a complex and challenging phase of care for burn patients. The long-running trend of increases in crystalloid fluid volumes is now recognized by practitioners, and efforts are being made to reduce excess fluid administration when possible. Refinement in resuscitation protocols, as well as multiple adjunctive therapies, may help reduce excess crystalloid administration. Finding a more accurate measure of resuscitation success could allow better and faster responses to physiologic changes.American Burn Association in 2008. While there has been significant work examining alternatives to standard resuscitation practices, the ultimate consensus report retains emphasis on a crystalloid-based resuscitation utilizing 2-4 mL/kg/body weight/%TBSA during the first 24 hours. Fluid should be isotonic and titrated to maintain urine output of 0.5 to 1.0 mL/kg/hr in adults and 1.0-1.5 mL/kg/hr in children. Children may require additional fluid due to maintenance requirements. Incremental volume administration may be necessary in patients with significant full-thickness injury, delayed resuscitation or smoke inhalation  or young adults as defined. The starting point for fluid resuscitation is the standard Parkland formula utilizing lactated Ringer's at 2 to 4 mL/kg/% burn. Subsequent therapy is titrated based on urine output and vital signs. In large burns, our crystalloid limit of 100 mL/kg means that transition to colloids or vasoactive drugs occurs well before the conclusion of 24 hours of resuscitation. Note that this protocol is designed for utilization by the bedside nurse. However, if vasoactive drugs or colloids are considered, burn unit faculty are immediately engaged.Click here for fileFigure S2. Colloid Protocol and Pressor Protocol. Where patients are not successfully resuscitated using a simple crystalloid protocol, options include administration of fresh frozen plasma or albumin, which can be very valuable in children as dilution of serum albumin with crystalloids can rapidly occur. Vasoactive drugs are utilized in conjunction with a central venous catheter and measurement of central venous pressure. Bladder pressures are also monitored via the urinary catheter to identify intraabdominal hypertension and minimize the risk of intraabdominal compartment syndrome in patients receiving large resuscitation volume [n volume -34. FinaClick here for fileBox 2. Key Points. Resuscitation options in the burned patient.Click here for file


In this work a mathematical model describing the growth of a solid tumour in the presence of an immune system response is presented. Specifically, attention is focused on the interactions between cytotoxic T-lymphocytes (CTLs) and tumour cells in a small, avascular multicellular tumour. At this stage of the disease the CTLs and the tumour cells are considered to be in a state of dynamic equilibrium or cancer dormancy. The precise biochemical and cellular mechanisms by which CTLs can control a cancer and keep it in a dormant state are still not completely understood from a biological and immunological point of view. The mathematical model focuses on the spatio-temporal dynamics of tumour cells, immune cells, chemokines and “chemorepellents” in an immunogenic tumour. The CTLs and tumour cells are assumed to migrate and interact with each other in such a way that lymphocyte-tumour cell complexes are formed. These complexes result in either the death of the tumour cells  or the inactivation of the lymphocytes and consequently the survival of the tumour cells. In the latter case, we assume that each tumour cell that survives its “brief encounter” with the CTLs undergoes certain beneficial phenotypic changes.We explore the dynamics of the model under these assumptions and show that the process of immuno-evasion can arise as a consequence of these encounters. We show that the proposed mechanism not only shape the dynamics of the total number of tumor cells and of CTLs, but also the dynamics of their spatial distribution. We also briefly discuss the evolutionary features of our model, by framing them in the recent quasi-Lamarckian theories.Our findings might have some interesting implication of interest for clinical practice. Indeed, immuno-editing process can be seen as an “involuntary” antagonistic process acting against immunotherapies, which aim at maintaining a tumor in a dormant state, or at suppressing it.Biology Direct), M. Kimmel and A. Marciniak-Czochra.This article was reviewed by G. Bocharov (nominated by V. Kuznetsov, member of the Editorial Board of  Cancer research, both experimental and theoretical, in last 12 years has been deeply influenced by the paper “The Hallmarks of Cancer” by : i, with   the rate of formation of complexes  and close the system by applying appropriate boundary and initial conditions. As far as the boundary conditions are concerned, zero-flux boundary conditions are imposed on all state variables (apart from C): E, α, ρ and Ti, i=0,…,N. These boundary conditions are appropriate for the tumour-immune dynamics we are considering. For example, in BCL1lymphomas of the spleen tumour cells are spatially contained in the lymph tissue of the spleen, an elongated organ that, in mice, is characterized by a very strong basement membrane, which is only broken when the tumour cells switch to an “invasive phenotype”. Since here we are concerned with earlier stage dynamics of tumour cells in a dormant state evading the CTLs, it follows that the zero-flux boundary conditions are adequate for our particular model. As far as the initial conditions are concerned, we assume an initial front of naive tumour cells encountering a front of CTLs, resulting in the formation of C0complexes. We suppose that initially there are no non-naive tumour cells and hence no complexes involving them. No chemicals are initially present in the spatial domain. These assumptions yield: We initially consider the model in a fixed 1-dimensional domain [0,where Eais the baseline homogenous steady-state value of CTLs in the absence of tumour cells, and that Tais the the baseline homogenous steady-state value of the naive cells in absence of CTLs, whereas Ca is the maximum possible density of complexes resulting from initial values of E and T.Note that We simulated our system after nondimensionalizing it as shown in the Appendix, where one can also find the numerical values of the parameters. In addition to the baseline parameter set detailed in the Appendix, in the following sections all our simulations were performed assuming the following values for key parameters associated with the encounter of CTLs and tumour cells:  Moreover =1.3×10−7 and ki+ Since the average lifespan of a chimeric mouse is three years, and since we are interested in assessing the possibility  of the onset of immunoevasion of a tumour, all simulations (unless stated) represent an interval of length 1100 days ≈3 years.pN=0, and N=4 the onset of evasion is at t≈200 days, i.e. the tumour remains dormant for 200 days, which is a long period of time for a mouse. On the contrary, if N=10 then the immunoevasion is delayed even further, with onset at t≈500 days.In this first set of simulations, we set all the spatial components of the model to zero and consider only the reaction kinetics in order to ascertain whether the primary mechanism of evasion can be purely temporal. All simulations suggest that our model, with the parameter assumptions and values we used, is able to reproduce the onset of immunoevasion in a biologically realistic time-frame. Figure pN=0.5, and as in the previous figure, t≈425 days for N=4 and at t≈950 days for N=10.Figure pN=0.75, and as in the previous figure, in the lifespan of the mouse only for the case N=4. This suggests that in absence of changes in the parameter i) the late stages Tiare the most important to determine the onset of the evasion; ii) due to the finite lifespan of chimeric mice and to the slow rate of the transitions, the immunoevasion process requires that the maximum ability of genetic or epigenetic changes in a tumour cell upon forming a complex with a CTL , is reached in a small number of encounters.Figure pN=0.75, but in this case the parameters i) here the onset of immunoevasion is for N=4 at t≈250 days, i.e. it is considerably accelerated; ii) there is the onset of immunoevasion (at t≈550 days) also for N=10. Thus, this simulation suggests that the role of the decrease of the probability that a tumour cell is recognized by a CTL is important for the timing of the onset of immunoevasion. Moreover, the decrease of the parameters pi=constant=0.9997 and Figure pN=0, then the addition of the mechanism of decreasing However, as shown in Figure Finally, comparing the results of our simulations, in the cases where Max(T0) seems to be a decreasing function of pN. These results might be explained as follows: the decrease of the competition between all the tumour cells and the immune system embedded in the decrease of the parameters  Moreover, Before we present the computational simulation results of the new model in this paper, in Figures pN=0.75 and pN, we obtain a process that is identical to the baseline case for most of the time. Indeed, basically the left plot of this figure is identical to that of Figure9. However, after the onset of the evasion, the tumour cell density distribution changes significantly as can be seen in the left part of the domain. From these observed differences, we may say that in this modelling framework the effect of immunoevasion on the spatio-temporal dynamics is characterized by a return to a spatially homogeneous steady-state. This transition to the new spatial regimen is illustrated in more detail by the “time-slices” shown in Figure Figure Ti, is no more detectable at level of the total density of all tumour cells. However, this effect is again noticeable if we consider the density of T0 versus the density of T1 + ⋯ + TN of cells in each classes, i.e. In Figure A1(t) + ⋯ + AN(t). The effect of the chemorepulsion on each sub-population Aiis striking, although overall it is globally compensated (see the last plot). over time, as well as, in the last plot, the grand-total pN=0.5 and pN=0.5, probably due to the constancy of T0>T1 + ⋯ + TN. Note that at the end of the average lifespan of the mouse, the tissue is invaded to a large extent but to a lesser extent than in the case where pN=0.5 and t=0 to t=1100. Figure Figure pN=0.5 and pN and T0 and T1 + ⋯ + TN.Finally, Figure In this paper we have presented a novel mathematical model of the immune response to cancer, focusing on the specific spatio-temporal response of cytotoxic T-lymphocytes to tumour cells. We have developed and extended ideas originally formulated by  by propoNote that the model by  is basedIn this work we have dealt with the spatio-temporal interplay between tumours and a specific immune response from CTLs. We chose this approach because of the experimental evidence on the relevance of CTLs in determining tumour dormancy or the evasion of many important tumours such as melanomas, ovarian carcinomas and colorectal carcinomas, where the presence of infiltrating lymphocytes is a useful prognostic marker ,46. HoweConcerning spatial issues we also briefly mention that also in case of small dormant tumours the next generation of spatial models of tumour growth should better stress and investigate the interplay of tissue 3D geometry and the tumour vascularization with phenotypic changes in tumour cells. We have built our model based on the tumour dormancy mathematical model of ,16, wheri) a decrease in the probability pi of being lethally hit; ii) a decrease in the probability, embedded in piis needed to produce evasion, which does not occur in the case where pi remains constant at its baseline level inferred from the experimental data. However, the role of the parameters Our simulations suggest that the proposed mechanism is able to mimic various dynamics of immunoevasion during the lifespan of a mouse. We have also highlighted the differential spatiotemporal contributions to evasion due, respectively, to: Concerning the possible chemorepulsion of CTLs, our computational simulation results showed that, in our biological settings, although it does not affect the spatiotemporal dynamics of the total number of tumour cells, it has a remarkable influence on the spatio-temporal distribution of the different individual classes of tumour cells. Further analysis is needed to ascertain if, with different parameters, the effect of this factor can be different, and in order to understand the behaviour in the current setting.θi, pi, and p0 and As far as the key ‘immuno-evasion-related’ parameters such as 0+, from ,16) becaFrom a theoretical point of view, our model, although detailed and focused on a very specific aspect of immuno-oncology, and on some very specific mechanisms, is conceptually in line with the general theories by Bellomo ,37,41, wTo the best of our knowledge, the evolutionary nature of the immuno-editing process has been studied until now under the framework of the so-called “modern synthesis”, following which the environment (in our case the immune system) is not the “causative agency”  but a meOn the contrary, in our model the more immuno-resistant phenotypes may arise because of genetic or epigenetic causes - due to the interaction between the tumour cells and the immune system. This point of view, which is quasi-Larmackian , is in lθi∈), are triggered by the cell and its interplay with the “external world”, and that are beneficial for the cell [In stress-induced mutagenesis ,49, somethe cell ,49. As athe cell ,50,51. Fthe cell  that quathe cell ), which,As far as the epigenetic path is concerned, it is now known that there are inheritable phenotypic changes without underlying genetic variations ,49, and In this work we were interested essentially in the basic facts of the immune response to tumours. However, a number of immunotherapies have been proposed and also theoretically investigated  strategies deployed by those cells in order to reach their aim, are phenomenologically described by means of the model of the dependence of the various parameters on the classes of tumour cells, as well as in the macroscopic modelling of the chemorepulsion. This is a first step in a research effort for a more complete description of tumour cell immunoevasion, which will include the detailed modelling of the biological mechanisms underlying those and other specific evasion strategies. Thus, given the complex network of interplaying between inter-cellular and intra-cellular signalling, and given the various temporal scales  as well as the spatial ones , a more detailed model will have to be multiscale. This will involve a wide array of computational tools, from those typical of computational biology and bioinformatics, to more classical analytical and numerical methods of statistical mechanics and mathematical physics.i) space is scaled by adopting a reference value xa equal to the size of the domain in consideration and we assume xa=1cm; time is rescaled relative to the diffusion rate of CTLs by setting Before undertaking any computational simulations, we non-dimensionalise our model  by adopting the following scaling: ii) cellular densities are rescaled relative to the maxima of the respective initial conditions: Ea=Ca; iii) The concentrations of the chemoattractant and of the chemorepellent are rescaled relative to the baseline values αa and ρarespectively: where, we recall, By omitting the bars, for the sake of simplifying the notation, the proposed model becomes: where:  After the non-dimensionalization, the boundary conditions become (in 1-D): Ciequations, that which then imply, assuming some smoothness of the solution and the form of and the initial conditions take the form (again in 1-D):  where s/d1 and therefore this is the value we have taken for the initial density Eaof CTLs in the initial conditions. Similarly, in the absence of an immune response, the homogeneous steady-state density of the tumour cells is 1/β1 and this is what we take as the initial density of tumour cells Tain the initial conditions. Thus, when the fronts of the two cell populations meet, the maximum density of CTL-tumour cell complexes will be min and hence our choice for Ca. The function exp(−1000(x−l)2) was chosen to mimick the onset of sharp but continuous front. The chosen non-null initial conditions for the naive tumour and immune cells represent a front of tumour cells encountering a front of CTLs, resulting in the formation of CTL-tumour cell complexes. In the absence of a tumour, the homogeneous steady-state density of the CTLs is BCL1, which is an animal model for the study of tumour dormancy [To carry out the computational simulations of the proposed model, we used the baseline parameter set reported in , since tdormancy . In addidormancy . Thus, tHence, from the experimental data above, the non-dimensional values of parameters becomes: αi.e. Dρ=Dα, δρ=δα, μρ=μα, and ξ=γ.As far as the spatiotemporal dynamics of the chemorepellent is concerned, we assume that its diffusion coefficient and decay rate is the same as the chemokine Π0, wherease in the second table we directly provided the adimensional value μαΠ0=104, in line with [Note that in the first table above we did not provide the value of ine with ,16.The comments of the referees were reported in italics. All the three referees included minor comments on misprints or undefined parameters or other minor suggestions, which were all implemented. Thus, we only reported the comments of interest to the general readership.Note for the referees: following your suggestions the revised version now contains an Appendix.The paper presents a theoretical study of the tumour immuno-evasion from dormancy. To this end a mathematical model of reaction-diffusion-chemotaxis type formulated with PDE is proposed. The model considers the spatio-temporal population dynamics of interactions between tumour cells and cytotoxic T cells. The key assumption of the model reflecting recent biological insights into the pathogenesis of the solid tumour growth states that the tumour cell population is heterogeneous with respect to the parameters characterizing the outcome of the interaction with cytotoxic T lymphocytes. The interaction with CTL appears to acts as a selection force shaping the microevolution of the tumour. The heterogeneity assumption enters the model via parameterized dependence of the rate of transition of tumour cells from naive to more mature states, the efficacy of CTL mediated killing and the production of chemicals acting as attractants and repellors for CTLs. Numerical simulations with the model for various parameters combinations show that the immuno-evasion can result from the phenotypic heterogeneity of the tumour cells dynamically adapting to and shaped by the anti-tumour immune response.General remarks:1. The issue of spatio-temporal modelling of tumour growth is an area of active research in cell population dynamics . The related work on spatio-temporal modelling of tumour growth needs to be refereed to in the Introduction section.We fully agree, and we added the required references.Page 6: The authors make use of the Mass Action Law to describe the interaction between CTL and target cells via bilinear terms. Meantime, the reaction kinetics in crowded cell environments can differ from those assumed by the classical chemical kinetics. This issue need to be commented.This point is very important, and we commented it in the conclusions.Page 10: The parameter “1000” appearing in the initial functions has to be justified. Please, explain the choice ofCa.We added a full justification of the chosen initial conditions.Page 13: For the readership, the parameters of the model should be presented in a Table with columns specifying notation, biological meaning, units and uncertainty ranges.We added a table with average values of the parameters, as obtained in Ref. 13 by means of least squares algorithm, and their meaning.Page 13, line 20: Can the parameters equality assumption for the repellor and attractant be biologically justified?Unfortunately there is a lack of experimental results on this important data, so this is a pure assumption.Page 14, line -5: Please, elaborate more on the specific choice of the parameter values.We added a reference for this parameter. More in general all parameters in absence of immuno-editing were taken as in references ,16.Page 15-16: Figures8could be presented in a more compact way . The same applies to FiguresWe tried your suggestion, but the result was not less clear. Take also into the account that Biology Direct is purely online, thus there are no pages restrictions.Page 18: Section 2-Dimensional Domain is based upon simulations with the chemotaxis and chemorepulsion not included in the model. The value of the results of the 2D model based simulations is not straightforward. Please, either expand the section by considering the same set of processes as in 1D case or remove it.We removed it.Conclusions section: It would be interesting to have the authors opinion on whether the tissue 3D geometry and its vascularisation represent important structural constraints affecting the genomic or phenotypic changes in tumour cells that need to be considered in the next generation of the spatially resolved models of tumour growth?We fully agree, and we mentioned this important issue in the discussions.Finally, all suggestions in your minor remarks were implemented.The paper concerns an extension of Chaplain group’s model  of response of tumour cells to cytotoxic T-lymphocytes. The extension involves classes from naive through non-naive tumour cells, which have different ability to repel T-lymphocytes and other differing characteristics. The models consists of a complicated system of partial differential equations and is investigated purely by simulation.In this first investigation we employed numerical analysis for two main reasons: first, we feel the immuno-editing phenomena as mainly a transitory effect, and as such requiring mainly numerical analysis; second, we wanted to write a manuscript aimed to theoretical biologists with biological background, such as the general readership of ’Biology Direct’. However, we also outlined a more compact version of the proposed model where the phenotypes are described by means of a continuum. For this version we soon start a full mathematical analysis.In my opinion, stratification of tumour cells with respect to their immuno-naivete is a natural concept, which however, leads to many complications and ambiguities in model building. The paper only partially reviews the consequences of varying the hypotheses.In the above mentioned continuum version we should be able of better exploring these important issues.In particular, the 2-d model by the end of the paper seems superfluous, as it is not thoroughly investigated and seems to be plagued by boundary artifacts. I suggest removing it for the time being.Following you suggestions, and the suggestions of another referee, we removed it.I am not sure if the tumour immuno-editing can be considered a Lamarckian evolutionary process. Even if it is, I am not sure if this is important for the paper.We respectfully disagree with you on this point. The possibility that immuno-editing might be, at least in some cases, a Lamarckian process, where the interplay between the different kind of cells is the ’driver’, is of some interest, in our opinion.Page 2. I do not know if “extremely complex” implies “strongly non-linear” as the authors seem to claim.We removed this observation.Page 2.(bottom). It would be perhaps helpful to specify which cell types belong to the adaptive immune system and which to the innate one.Done.Page 5. “Since we are modeling a situation where immuno-evasion of the tumour cells is not considered …” Discussion of what this assumption really implies will be helpful.We removed the above quite ambiguous phrase. Indeed, we wanted to means that the baseline model, with the considered values of its parameters, referred to a case where the tumour is not able (without immuno-editing) to evade the immune control.Pages 9-13. Part of this material can probably go into an online Appendix.Done.The paper is devoted to modelling of spatio-temporal dynamics of interactions of cytotoxic T-lymphocytes with cells of a homogeneous avascular tumour. It takes into consideration a chemoattraction of the immune cells by the tumour and a chemorepulsion to chemicals produced by non-naive tumour cells. The model is based on the one presented in the paper “Mathematical Modelling of the spatio-temporal Response of Cytotoxic T-Lymphocyte to a Solid Tumour”  The novelty of the current work is in the assumption that the tumour cells, surviving encounter with the immune cells, undergo a functional change, which benefits their immune resistance. This resistance increases with each encounter until the maximal resistance is reached. The resulting system of coupled partial and ordinary di erential equations is solved numerically. The results are discussed in the biological context. I recommend the manuscript for publication in Biology Direct after some minor revisions related to the organisation of the paper. The issues, which should be taken into account before publication are :P.8, the second Equation: f and g are not introduced. Although model equations are nearly identical to those in reference , authors should motivate the term “proliferation and recruitment”.fC/(g + T).In the revision, we fully explained the meaning of the term like P.10: It would be helpful to have the initial conditions plotted.Since the shape of the initial conditions are elementry, and since another referee asked to add some written comemnts on this point, we did not include the requested plots.P.13: It should be mentioned how parameters are estimated.et al 1991 basically by means of mininmal least squares estimate algorithm.The parameters were estimanted in Kuznetsov Please motivate the choice of θ0; pN; how sensitive are the results to the choice of the parameters?θ0because we think that the probability of acquiring the fitter phenotype is small, in analogy with the small probability of surviving to an attack by a CTL. The value of et al 2004).We have choosen a small value for P. 28, Figure Why  and not ?800 days would have been a too long time with respect to 700 (100 days in mice correspond approximatively to 2300 days in men).Axis labels of the following figures are not readable: 9, 10, 11, 13, 15, 17, 20, 21.Concerning the readability of the axis labels, it probably depend on the fact that our eps figures were converted by the publisher in jpeg figures. In our original pdf figures they seems readable. We shall carefully manage this point in case the manuscript should be accepted.The rescaling of equations have mistakes. For example on p. 30, λshould beIndeed, it was a misprint. We apologize.whileCa=Ea, so what we wrote is correct.We had forgot to stress that I find the following phrase hardly readable “Finally, we note that the predicted effect on the tumour cells spatial distribution of the induction of chemorepulsion of CTLs is not detectable if we consider the total density of all tumour cells.” Please refomulate it.We reformulated it.The authors declare that they have no competing interests.AD and MC defined the mathematical model; MAT performed the simulations; AD, MC and MAT discussed the results; AD,MC and MAT wrote the manuscript. All authors read and approved the final manuscript.


The aim of adjuvant therapy in breast cancer is to reduce the risk of recurrence. Some patients develop metastases many years after apparently successful treatment of their primary cancer. Tumour dormancy may explain the long time between initial diagnosis and treatment of cancer, and occurrence of relapse. The regulation of the switch from clinical dormancy to cancer regrowth in locoregional and distant sites is poorly understood. In this review, we report some data supporting the existence of various factors that may explain cancer dormancy including genetic and epigenetic changes, angiogenic switch, microenvironment, and immunosurveillance. A better definition and understanding of these factors should allow the identification of patients at high risk of relapse and to develop new therapeutic strategies in order to improve prognosis. The term ‘dormancy’ was introduced in the first half of the 20th century by the Australian pathologist Rubert A. Willis in . Tumour Mechanisms of metastasis include the release of cancer cells from primary tumour; invasion of the surrounding stromal tissue, lymphatic, and blood vessels as CTCs; and then colonisation of target organs as DTCs. The fate of DTCs may have several scenarios: (1) spontaneous apoptosis or cell killing by recognition of immune effectors; (2) cell proliferation with clinical early relapse; and (3) state of tumour dormancy .Preclinical data have shown that tumour dormancy can occur both as a single dormant cell and as micrometastases. Single dormant cells are defined as cells undergoing cell cycle arrest and have the ability to develop mechanisms to evade immune surveillance , 5. OnlyFor reasons that are still not clear, after variable periods, even years after the diagnosis of primary tumour, dormancy ends and cancer cells start to proliferate causing late relapse. There are many factors that might explain the regrowth of cancer cells, hence regulating the entry in or exit from tumour dormancy: (1) genetic and epigenetic changes; (2) angiogenic switch; (3) immune system and immune evasion; and (4) microenvironment.et al . Th. Th26]. The cellular immune response plays a critical role for maintaining cancer dormancy. This is supported by the evidence that depletion of CD4+ and CD8+ T cells in mouse models causes escape of tumour cells from dormant state . In humaFinally, several immune cells are present in the tumour microenvironment, such as regulatory T-cells (TREG cells)  and tumoA better understanding of the influence of the immune system on dormancy could lead to the development of immunological therapies in order to prevent the progression of the tumour and then the switch from the state of dormancy to tumour growth.in vivo demonstrate that a loss of growth signals and cell-to-cell signalling attachments can lead to dormancy, suggesting that a microenvironment, with inappropriate cell contacts and signalling, can contribute to tumour cell dormancy . Indormancy  40, 41], 41In viThus, the proposed molecular mechanisms of the growth inhibition that occurs in dormancy involve either activation of the p38/MAPK pathways or inhibition of the ERK/MAPK pathways. Instead, the interaction of uPAR with fibronectin receptor and EGFR leads a shift from the state of dormancy to cell proliferation. The ratio of ERK to p38 is very important for dormancy; a high ERK to p38 ratio is linked to proliferation, whereas low ERK to p38 expression ratio is linked to growth arrest and dormancy .in vivo studies demonstrated that ATF6 alpha promotes survival of dormant tumour cells through the upregulation of Rheb and activation of mTOR signalling, independent of Akt [In addition, t of Akt . Finallyt of Akt  or anti-t of Akt .Several studies evaluated the prognostic value of CTCs or DTCs at the time of initial diagnosis and before the start and/or the end of adjuvant chemotherapy , 47. DTCP = 0.001) and overall survival (OS) (P = 0.001) worse than other groups of patients, regardless of ER status. These results suggest that resistant dormant cells have a proliferative and survival advantage; they may resist the conventional anticancer therapy and may start to proliferate even many years after the diagnosis of primary tumour. Why not all patients with detectable CTCs develop late recurrence, while some patients without detectable CTCs relapse, could be explained by the evidence that CTCs showed significant genetic heterogeneity.In a recent study, authors suggested a possible value of CTCs in predicting the risk of late relapse, defined as relapse that occurs at least two years after the end of adjuvant chemotherapy in 312 patients with operable BC at stage I–II–III either hormonal receptor positive or negative, who were disease free during the first two years of follow-up . The patP = 0.002 and P < 0.001, respectively) during the first five years following cancer diagnosis.It was also investigated the potential role of DTCs in predicting risk of relapse after the completion of adjuvant chemotherapy. Six hundred and seventy-six patients with operable BC stage I–III were analysed . Median The detection of these cells after therapy could be considered as an indirect evidence for the presence of dormant cells chemotherapy/ hormonal therapy resistant. However, not all patients with detectable DTCs or CTCs will experience late recurrence. Thus, additional prognostic markers are needed to define those patients are at high risk of late relapse.et al [In 2003, Clarke et al  first deet al . Accordiet al . These Cet al . CSCs exet al  and can et al .Moreover, some studies investigated the interactions between the microenvironment and CSCs. These cells could find (or create) a new specialised microenvironment or ‘tumour niche’ in secondary sites that generates extrinsic factors that control stem cell number, growth, and differentiation. It could be hypothesised that CSCs remain dormant in their niche either as solitary cells or as dormant micrometastases until they are activated by improper signalling from the microenvironment . FurtherSince early tumour detection is a critical determinant of survival in patients with cancer, the recognition of dormant tumours and/or cells and their possible eradication with targeted therapies is one of the major goals of care of BC survivors.Currently, there are no markers able to exactly predict the risk of late recurrence, and it is not possible to predict which dormant tumours and/or cells will eventually grow and which will stay dormant and will never switch to the rapidly proliferating phenotype . We do nIn order to overcome this medical need, it might be useful to develop gene signature as a means by which tumour behavior can be predicted. Several genes involved in many cellular pathways have been identified and have been associated with the conversion of dormant lesions into fast-growing tumours, and they could represent potential targets and markers for tumour dormancy. For example, different cellular and animal models showed that both MYC and Ras significantly affected the dormancy, through the regulation of expression of genes involved in angiogenesis . The ideet al [P < 0.0000001) [Kim et al  suggesteet al . The que0000001) . MoreoveThe identification of miRNAs associated with tumour dormancy might contribute to understand tumour dormancy and manipulation of their expression level could help us to delay or prevent the dormancy periods .et al [In addition to CTCs, DTCs, and CSCs, various efforts are being made to identify biomarkers in circulation that correlate with the presence of dormant tumours, such as platelet-associated PF-4 . Carlssoet al  identifiThe dormant phase of tumours is also a promising therapeutic target. Dormant tumour cells are refractory to current cancer therapies. In an experiment using mouse mammary carcinoma cell lines, treatment with doxorubicin reduced the size of large metastases but did not reduce the number of solitary dormant cells . AlthougAngiogenesis is another potential therapeutic target. Many anti-angiogenic drugs are currently approved in the treatment of metastatic cancer, such as Bevacizumab. To evaluate their capacity to influence dormancy, these agents should be tested in patients at high risk for relapse, without evidence of disease. Results from recent trials (AVANT and NSABP-08) failed to show a clear benefit from the antiangiogenic therapy bevacizumab when it was given as one year of adjuvant treatment in early-stage colon cancer , 67. In Immunological therapy might represent a possible strategy to prevent late recurrence. Most of the vaccines are tested in patients with metastatic disease. Therapeutic vaccines may be more effective in patients with microscopic disease or in adjuvant setting at high risk or relapse, because this therapy could prevent the loss of immunological surveillance and promote persistent dormancy. A limited number of adjuvant trials are in progress.kB ligand (RANKL), a potent inducer of osteoclast formation. RANKL binds its receptor, RANK, on the surface of osteoclast precursors and signals through the nuclear factor-kB (NFkB), and Jun N-terminal kinase (JNK) pathways induce the formation of osteoclasts and promote osteoclast survival. RANKL inhibitors (i.e. denosumab) block the activation, survival and differentiation of osteoclasts from their precursors resulting in complete absence of osteoclasts in the treated bone.Many dormant cancer cells metastasise to the bone marrow as blood flow is high in this area. The tumour cells also produce adhesive molecules that bind them to marrow stromal cells and bone matrix (osteo-niche). In 1889, Stephan Paget described for the first time this ‘seed-and-soil hypothesis’ of the mechanism of bone metastasis . The recTraditionally, bone-targeted agents are used as a therapy to prevent or reduce the incidence of skeletal-related events in patients with malignant bone disease. However, recent evidences suggest that they may act as antitumour agents , able toversus intermittent letrozole treatment after four to six years of adjuvant endocrine therapy, can reduce the risk of relapse in early-stage BC patients. These clinical trials in hormone-dependent BC suggest that dormant cells remain vulnerable to this therapy. The open question is to evaluate the risk and benefit of long-term therapy.Finally, patients with ER+ tumours might derive benefit from extended endocrine therapy. As discussed above, the initiation of metastatic growth is not equivalent to the dissemination of tumour cells . This suA better understanding of the regulatory mechanisms that govern the state of dormancy will help us to identify markers of early tumour progression and to detect early tumour cells prior to their rapid growth in order to treat tumours or recurrent cancer years before they become symptomatic. This could lead to a reduction in cancer mortality. Moreover, future studies on tumour dormancy could lead to the development of novel targeted strategies for eliminating dormant tumour cells or for maintaining dormant status of tumour cells, keeping the disease in a chronic state.


A Funding statement is incorrectly omitted from the article. The Funding statement should read: "This work was supported by JSPS KAKENHI Grant Number 23689071 [grant-in-aid for Young Scientists (A) (A.T.)], Grant Number 24791859 [grant-in-aid for Young Scientists (B) (T.Y.)], Grant Number 10294943 [grants B (K-H.S.)], and Grant Number 23592573 [grants C (Y.O.)] from the Ministry of Education, Science, Sports and Culture, Japan."


In the Funding section, one of the grant numbers from the National Cancer Institute was listed incorrectly. The grant number "CA 132706" should be "CA 134304."


Two novel selective 18F-phthalimido σ2 ligands, 18F-SIG343 and 18F-SIG353, were prepared and characterised for their potential tumour imaging properties.Sigma2 (σ18F-SIG343 and 18F-SIG353 was achieved via nucleophilic substitution of their respective nitro precursors. In vitro studies including radioreceptor binding assays in the rat brain membrane and cell uptake studies in the A375 cell line were performed. In vivo studies were carried out in mice bearing A375 tumours including positron emission tomography (PET) imaging, biodistribution, blocking and metabolite studies.Preparation of In vitro studies showed that SIG343 and SIG353 displayed excellent affinity and selectivity for σ2 receptors (Ki(σ2) = 8 and 3 nM, σ2:σ1 = 200- and 110-fold, respectively). The σ2 selectivity of 18F-SIG343 was further confirmed by blocking studies in A375 cells, however, not noted for 18F-SIG353. Biodistribution studies showed that both radiotracers had similar characteristics including moderately high tumour uptake (4%ID/g to 5%ID/g); low bone uptake (3%ID/g to 4%ID/g); and high tumour-to-muscle uptake ratios (four- to sevenfold) up to 120 min. Although radiotracer uptake in organs known to express σ receptors was significantly blocked by pre-injection of competing σ ligands, the blocking effect was not observed in the tumour. PET imaging studies indicated major radioactive localisation in the chest cavity for both ligands, with approximately 1%ID/g uptake in the tumour at 120 min. Metabolite studies showed that the original radiotracers remained unchanged 65% to 80% in the tumour up to 120 min.in vitro and in vivo characteristics. However, PET imaging indicated low tumour-to-background ratios. Furthermore, we were unable to demonstrate that uptake in the A375 tumour was σ2-specific. 18F-SIG343 and 18F-SIG343 do not display ideal properties for imaging the σ2 receptor in the A375 tumour model. However, since the radiotracers show promising in vitro and in vivo characteristics, longer scans using appropriate half-life isotopes and alternative tumour models will be carried out in future studies to fully validate the imaging characteristics of these radiotracers.The lead ligands showed promising  Comparisons of organ radioactivity concentrations (%ID/g) and TORs in the biodistribution studies or %ID/cc in PET imaging studies between the two radiotracers were performed using separate unpaired, two-tailed Student's t tests, matched organs and time points.Selected tumour-to-organ (tumour-to-blood and tumour-to-muscle) uptake ratios (TORs), were calculated by dividing the tumour radioactivity concentration (%ID/g) by the radioactivity concentration in the respective organ at time post hoc tests were performed for each radiotracer to determine any statistically significant changes in the uptake (%ID/g) amongst selected ROIs compared to the control.Meanwhile, for the blocking studies, separate within-group two-way ANOVAs [drug treatment  × regions] followed by Bonferroni's The synthesis of SIG343 and SIG353 and their respective nitro-radiolabelling precursor was via a condensation of the appropriate phthalic anhydride with either 4--yl)butan-1-amine for the SIG343 pair or 5--yl)pentan-1-amine for the SIG353 pair (Scheme H-yl)buta18F-SIG343 and 18F-SIG353 were synthesised by aromatic nucleophilic substitution of their respective nitro precursors, (1) and (2) (Scheme n = 13) and 18 ± 7% (n = 13) (non-decay corrected) for 18F-SIG343 and 18F-SIG353, respectively. Controlling the basicity of the reaction was an important parameter that affected the yield. Levels of potassium carbonate above 1 mg generally resulted in lower yields possibly by inducing hydroxide formation from trace amounts of water in the reaction, with subsequent ring opening of the phthalimido ring or acting as a competitor of the fluoride.Radiotracers  (1) and   to the σ1 and σ2 receptors and a number of other neuroreceptors for their selectivity. The radioreceptor binding studies showed high affinity and high selectivity for σ2 receptors, with 200- and 110-fold selectivity for σ2 receptors compared to σ1, for SIG343 and SIG353, respectively. The compounds did not show any cross reactivity for the other neuroreceptors examined . The Ki or IC50 values and logP values are shown in Table SIG343 and SIG353 were evaluated using competitive inhibition experiments to determine their binding affinity , peaked at 3% to 3.5% between 15 and 60 min for 18F-SIG353 and plateaued at 2% for 18F-SIG343 for the same period. Uptake slightly declined after 60 min to 1.6% to 2.3% for 18F-SIG343 and 18F-SIG353, respectively , haloperidol (non-selective σ1/σ2) and unlabelled SIG343 and SIG353 (σ2). One-way analysis of variance (ANOVA) analysis (treatment as a factor) indicated an overall significant variation in the cell uptake values amongst all treatment groups for 18F-SIG343 . Bonferroni’s post hoc tests revealed the cell uptake of 18F-SIG343 was significantly reduced by haloperidol  and unlabelled SIG343 , whereas (+)-pentazocine produced no significant blocking effect on 18F-SIG343 cell uptake compared to the controls  . In contrast, the uptake of 18F-SIG353 was significantly higher than 18F-SIG343 in the liver and pancreas for the same period. At 120 min, the uptake in the lungs, kidneys, tumour and heart was not different between the two radiotracers. The uptake in the ovaries was not different between the two radiotracers at any time point. In the brain, clearance was found to be more rapid for 18F-SIG353, whereas the lung clearance was faster for 18F-SIG343.Despite the similarities, the two radioligands exhibited some differences in the extent and the rate of uptake. For instance, a significantly higher uptake of 18F-SIG343 and 18F-SIG353 showed similar tumour-to-blood uptake ratios (eight- to ninefold at 120 min), whereas 18F-SIG353 had significantly higher tumour-to-muscle uptake ratios compared to that of 18F-SIG343 at any time point (0.0001 < Ps <0.05)   and unlabelled SIG343 or SIG353 (σ2) as competitors was observed in various tissues at 30-min post injection of the radiotracer.The degree of 18F-SIG343 organ distribution. Significant main effects of treatment , regions  and the significant interaction between the two factors  were also noted. Similar significant blocking was found in almost organs expressing σ receptor in both haloperidol- and unlabelled SIG343-treated groups including brain , liver , spleen , and lungs , respectively  examining the blocking effect of pre-administration of competing σ ligands on 18F-SIG353 regional distribution. Significant main effects of treatment , regions  and significant interaction between the two variables  were observed. Bonferroni post hoc revealed only significant blocking occurred in the liver  for both haloperidol and unlabelled SIG353 treatment groups, respectively. Uptake of 18F-SIG353, however, increased in the pancreas for the unlabelled SIG353-treated group   in the above-mentioned organs, at 120 min post injection.Following ultrasonic disruption and centrifugation of tissue, at least 81% of radioactivity from the tumour and brain cortex was recovered in the supernatant from samples collected at 15, 60 and 120 min. However, 18F-SIG343 and 18F-SIG353 was reduced from 60% at 15 min to 17% at 120 min. In urine, unchanged 18F-SIG343 was rapidly reduced to 13% at 15 min while only 5% of radioactivity was intact 18F-SIG353 at 15 min.In plasma, rapid decrease of unchanged radiotracers was observed; the percentage of unchanged 18F-SIG353 having higher uptake compared to 18F-SIG343, (P < 0.05) resulting low tumour-to-background contrast. Time-activity curves of 18F-SIG343 and 18F-SIG353 of selected organs are presented in Figure Results from the PET imaging studies showed high uptake in the chest cavity for both radiotracers, with continuous accumulation in the liver. Low uptakes were observed in the tumour, with 2 ligands, SIG343 and SIG353, from a series of phthalimide derivatives that have high affinity and are highly selective for σ2 receptors. From literature, it is interesting to note that the structure designed for the σ receptor also shows affinity for the related dopamine receptors [in vitro data indicated that the compounds have no affinity for dopamine receptors.In the current study, radioreceptor binding assays identified two lead σeceptors ,57. Howe2 receptor localisation in vivo, particularly in tumours, was explored. A number of cancer cell lines that express various concentrations of σ receptors have been described; however, the level of expression of the two σ subtypes was often not reported [2 receptors, respectively [2 receptors, approximately 100 times higher than that of the σ1 subtype [2 receptors and cellular proliferation was explored and demonstrated that proliferative cells have ten times more σ2 receptors than quiescent cells, although full down-regulation of the σ2 receptor was reached only several (>3) days after the cells had become quiescent [2 receptor appeared to be rather long (>12 h) [2 receptors in the cells, also to optimise signal-to-noise ratio in cell uptake studies. The results from the cell uptake inhibition studies are in agreement with the in vitro receptor binding data confirming that SIG343 has a higher selectivity for σ2 receptors, compared to SIG353.The potential of these tracers as imaging agents for identification of σreported . Amongstectively . The A37 subtype . The reluiescent . Thus, t (>12 h) . In our 18F-SIG343 and 18F-SIG353 were retained in the organs known to express high densities of σ receptors such as the brain, liver, kidneys, lungs, pancreas and ovaries. As the uptake of the radiotracers in the liver and pancreas was still increasing, it is possible that the terminal half-lives of these ligands are longer than 120 min which is beyond the last time point of the study. Lack of clearance of the tracers observed at 120 min may be attributable to either continuing accumulation of the radiotracer and/or its radioactive metabolite in the liver and pancreas.Biodistribution studies in tumoured mice indicated that statistically significant uptake of 18F-SIG343 compared to that of 18F-SIG353 was observed in the brain and the lung. However, the brain clearance was more rapid for 18F-SIG353, and the lung clearance was faster for 18F-SIG343. High to moderately high brain uptake of the radiotracers together with their appropriate lipophilicity indicated that the radiotracers are also suitable as imaging agent for receptors in the brain.Despite some similarities in distribution profiles and clearance in the organs, the two radiotracers exhibited some differences in the extent and the rate of uptake. For instance, a significantly higher uptake of 18F-SIG343 and 18F-SIG353, respectively, throughout the study and up to 120 min, showed promising tumour imaging potential for these radiotracers. Both radiotracers showed similar trends of increasing tumour-to-blood and tumour-to-muscle uptake ratios over time. Selective σ218F-radioligands for PET imaging from the literature were reported to have tumour-to-muscle ratios ranging from 2 to 8 at 120 min in various tumoured mouse models  or from the muscle (increasing tumour-to-muscle uptake ratios) over time. Bone uptake of both radiotracers was low and has steadily declined over time which indicates no significant in vivo de-fluorination and subsequent fluoride bone uptake.Moderately high and prolonged tumour uptake, 3% to 4% and 4% to 5% for 2 ligands as competitors as the σ2 specificity of these radiotracers had been previously established in naïve animals (manuscript in preparation). The competing drugs (1 mg/kg in 100 μL) including haloperidol (Ki(σ2) = 20 nM) and unlabelled SIG343 and SIG353 (Ki(σ2) = 8 and 2.7 nM, respectively) were administered 5 min prior to radiotracer injection. The concentration used deems to be in excess for all compounds used  which are at least 50 times greater than the Ki values of the radiotracers for σ2 receptors to ensure a complete saturation of receptor occupancy.In the blocking study, we used only selective σPs < 0.001) in both treatment groups  compared to controls for 18F-SIG343. However, this increased uptake phenomenon in the pancreas was only observed in the unlabelled SIG353-treated , but not in the haloperidol-treated group, for 18F-SIG353 (P > 0.05). It is possible that pancreas is a site of radioactive metabolite accumulation, and the metabolism is a dynamic process which would make it difficult to measure specific binding in this organ. This observation was also evident in, and supported by, the biodistribution data which showed that the uptake in the liver and pancreas was constantly increasing over time. Although there was no significant increase in radioactivity of the tracer in the blood or bone as a result of the blocking drug treatment (data not shown), based on the increased radiotracer uptake values over time in many organs expressing σ receptors, we hypothesise that the continuing increase in pancreas and liver uptake is likely due to an increase in radioactive metabolites, rather than specific σ uptake.It is interesting to note that the uptake in the pancreas were found to increase  and those of blocking studies (in vivo specificity) indicated that the pre-administration time of 5 min for these challenging ligands used in the current study may not be sufficient for the drugs exerting their pharmacological action. This is highly likely because in an in vitro system, the drugs have free access to cell membrane receptors and are only limited by their affinity, appropriate concentration used and the intrinsic efficacy. However, in an in vivo environment, in addition to those parameters mentioned above, the drugs are also limited by their pharmacokinetic properties before they can reach the target for receptor occupancy or saturation. In addition, the non-blocking of the competing drugs observed in the tumour in the present study could also be due to a number of variants such as the non-specific binding of the tracers in the tumour, internalisation or internal localisation of surface receptor-ligand complex in the tumour that was not readily blocked by the competing σ ligands when the radiotracers were administered. Other possibilities are pathophysiological status of the tumour in the animal model used including hypoxic fraction, proliferative or vasculature structure of the tumour, or even the pathophysiology of the diseased animals where the pharmacokinetics of both competing drug and radiotracer could also be compromised. Taken together, these factors could influence the absorption and distribution of competing drugs. Therefore, while competing σ ligands showed high binding affinity in the in vitro assay system, their pharmacokinetics may not be able to reach a sufficiently high concentration, or at a sufficient time, at the binding sites in solid tumours to exert their competing effects in our in vivo biodistribution study.Amongst the competing σ in rats  , resulting in low tumour-to-background contrast which hampered the potential of the radiotracers as tumour markers. PET images showed high radioactive uptake in the abdominal cavity; hence, high background activity which is consistent with the biodistribution data where high uptake in the liver, spleen, lung, heart, pancreas and kidney was observed. The discrepancy in the results between in vivo PET and ex vivo biodistribution studies may be attributed to several reasons, including methodological differences in the techniques employed or method quantification/analysis of the images.Unlike the biodistribution, PET imaging of in vivo studies gives an absolute index in binding which means that it reflects specific and non-specific binding in targets , radioligand present in the systemic blood circulation and possibly radioactive metabolites in the tumour and blood circulation [18F-SIG343 and 18F-SIG353 have not been fully assessed. However, results of the metabolite studies indicated that the intact radiotracers were 65% to 80% in the tumour; therefore, the fate of the remaining fraction of the radiotracers cannot be fully concluded until the metabolites are identified and quantified. The second factor is the potential effect of inhalation anaesthesia used in in vivo studies, but not in ex vivo studies, which may also interfere with the kinetics of the radiotracer, especially in terms of tissue equilibrium and the extent of tissue perfusion [ex vivo studies compared with approximately 5 to 16 MBq in PET studies. High radioactivity retention in the tail (approximately 20%ID/cc) observed for both radiotracers indicated a misinjection possibility which could result in less radiotracers available for the systemic circulation, hence low radiotracer uptake in the target. Taken together, these factors could contribute to the low uptake of the radiotracers in the tumour and to the discrepancy in results between ex vivo and in vivo studies.Firstly, methodological issues of data analysis should be considered. The percentage of injected dose (%ID/g) which is conventionally used in culation , as the erfusion . Finally18F and considering that the amount of radiotracers in the tumour remained constant, the issue of low tumour-to-background contrast could be resolved.Future studies using longer scans (>120 min) and longer half-life radioisotopes compared to 2 receptor is overexpressed in a variety of human tumour cell lines and is a biomarker for tumour cell proliferation, making it an attractive target for the development of new radiotracers for tumour detection and assessment of proliferative status using PET and SPECT imaging. Two lead compounds from a series of novel phthalimido compounds, SIG343 and SIG353, showing high affinity and selectivity for the σ2 receptor in vitro were selected for further development of the 18F-labelled PET tracer. 18F-SIG343 and 18F-SIG353 were evaluated in tumour-bearing mice, and their potential to image the σ2 receptor in vivo was investigated. Biodistribution studies showed high uptake and suitable kinetics of both radiotracers in organs known to express σ receptors. Relatively high tumour-to-blood and tumour-to-muscle uptake ratios were observed for both 18F-SIG343 and 18F-SIG353. Furthermore, we were able to demonstrate that 18F-SIG343 and 18F-SIG353 remained moderately stable in vivo. However, blocking studies could not fully confirm that the uptake of 18F-SIG343 and 18F-SIG353 in the tumour was σ2-receptor-specific. PET images showed low tumour-to-background contrast and high radioactivity localisation in the abdominal cavity for both radiotracers making them less ideal candidates for PET tumour imaging in the current animal model. Continuing the work in this direction, full potential of these radiotracers will be revisited in more appropriate models using different designed PET/SPECT protocols and suitable radioisotopes of longer half-lives to address the current low tumour-to-background contrast issue.The σThe authors declare that they have no competing interests.in vitro and in vivo studies, participated in analysis and interpretation of data, and revised the manuscript. All authors read and approved the final manuscript.VN carried out radioreceptor binding studies, analysis and interpretation of data, performed statistical analysis, and drafted the manuscript. TP synthesised and radiosynthesised the compounds, carried out metabolite studies and revised the manuscript. CF synthesised the compounds. PB, ED, GR and NH participated in the biodistribution and blocking studies. IG developed the HPLC metabolite study's method. AA analysed the PET data. FM wrote the animal ethics protocols and participated in the biodistribution and blocking studies. MG supervised the research group. AK contributed to conception of the study. RS carried out the VN is a BPharm  and PhD  graduate. He is holding the current position as radiotracer evaluation pharmacologist-in-charge. His main interests include radiotracer evaluation, drug pharmacodynamics/pharmacokinetics and G-protein signalling pathway investigations.


Excess fluids may be harmful in critically ill patients. We aimed to evaluate the cumulative fluid balance during 7 days in patients with septic shock after recovery from shock.n = 20) and Group 2 >4.4 l (n = 20).A prospective and observational study in septic shock patients. Patients with MAP >65 mmHg and lactate <2.0 mEq/l were included <12 hours after weaning from vasopressors. Daily fluid balance was registered during 7 days after the enrollment. Patients were divided into two groups according to the full cohort's median cumulative fluid balance administered during the period of shock (use of vasopressors) calculated on study day 1: Group 1 ≤4.4 l (P <0.001 for both). Time for recovery from shock was predictive of receiving larger volume of fluids . After zeroing fluid balance on study day 2, 7 days cumulative fluid balance continues to increase in both groups (Figure Cumulative fluid balance was 1.6 ± 1.8 l in Group 1 and 10.2 ± 4.1 l in Group 2 and 8.5 ± 5.3 l in Group 1 and 18.5 ± 7.9 l in Group 2 on study day 8 (After recovery from septic shock we notice a huge accumulated fluid balance. A more positive fluid balance was associated with a more prolonged length of stay in the ICU and in the hospital.


In most previous studies, neutrophil gelatinase-associated lipocalin (NGAL), measured immediately following cardiac surgery, has been demonstrated to predict postoperative increases in serum creatinine and decline in urine output. In patients undergoing cardiac surgery, postoperative fluid overload is a typical complication. In this study, we investigated the early postoperative value of NGAL to predict subsequent fluid overload, a potentially modifiable risk factor in these patients.We studied 100 adult cardiac surgery patients assigned to the control arm of a randomized controlled trial. Urine and serum were sampled immediately after admission to the ICU. Urine NGAL was measured on the ARCHITECT laboratory platform (Abbott Diagnostics) and serum creatinine using an enzymatic assay. Postoperative fluid overload was defined as positive fluid balance with >10% excess of preoperative body weight within 48 hours. An area under the curve of the receiver-operating characteristics (AUC-ROC) of 0.5 indicates the predictive ability equaling the toss of a coin and >0.7 of a useful biomarker.P = 0.010. A positive fluid balance predicted in-hospital mortality with AUC 0.94 (95% CI 0.83 to 0.99), sensitivity 100% and specificity 80% at a cut-off >6 l. Urine NGAL predicted fluid overload (AUC-ROC 0.80 (95% CI 0.64 to 0.93)) and mortality (AUC-ROC 0.88 (95% CI 0.78 to 0.97)). Serum creatinine did not predict fluid overload (AUC-ROC 0.51 (95% CI 0.24 to 0.78)) or mortality (AUC-ROC 0.61 (95% CI 0.16 to 0.99)).Postoperative fluid overload was present in 15% of patients with a mean positive fluid balance of 12 ± 9 kg. Patients who survived the hospital stay had a lower positive fluid balance of 2.8 l (25th to 75th percentiles: 1.5 to 5.5) compared to patients who died (23.0 l (25th to 75th percentiles: 14.5 to 24.0)); Fluid overload frequently occurs during the first 48 hours after cardiac surgery and is strongly correlated with postoperative mortality. Early postoperatively measured urine NGAL is a good predictor of fluid overload and mortality whereas measurement of serum creatinine at the same time equals the toss of a coin. Early NGAL-guided adjustments of fluid management might reduce organ edema and potentially improve patient outcomes after cardiac surgery. Our findings should be validated in larger patient cohorts.


The number of a grant in the Funding statement is incorrect. In addition, funding organizations are incorrectly omitted from the Funding statement. The Funding statement should read: "This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (Grant No. 2013M3A9C4078138)."


In this narrative review, an overview is given of the pros and cons of various crystalloid fluids used for infusion during initial resuscitation or maintenance phases in adult hospitalized patients. Special emphasis is given on dose, composition of fluids, presence of buffers  and electrolytes, according to recent literature. We also review the use of hypertonic solutions.We extracted relevant clinical literature in English specifically examining patient-oriented outcomes related to fluid volume and type.A restrictive fluid therapy prevents complications seen with liberal, large-volume therapy, even though restrictive fluid loading with crystalloids may not demonstrate large hemodynamic effects in surgical or septic patients. Hypertonic solutions may serve the purpose of small volume resuscitation but carry the disadvantage of hypernatremia. Hypotonic solutions are contraindicated in (impending) cerebral edema, whereas hypertonic solutions are probably more helpful in ameliorating than in preventing this condition and improving outcome. Balanced solutions offer a better approach for plasma composition than unbalanced ones, and the evidence for benefits in patient morbidity and mortality is increasing, particularly by helping to prevent acute kidney injury.Isotonic and hypertonic crystalloid fluids are the fluids of choice for resuscitation from hypovolemia and shock. The evidence that balanced solutions are superior to unbalanced ones is increasing. Hypertonic saline is effective in mannitol-refractory intracranial hypertension, whereas hypotonic solutions are contraindicated in this condition. The debate on fluid treatment of the surgical and critically ill patient is ongoing, partly because of insufficient data, historical and geographical differences, and poor training -3. This This review aims to describe the place of various crystalloid fluids in the treatment of adult hospitalized patients, in a narrative manner for educational purposes, leading through various clinical conditions and considerations by evaluation of various studies. We first discuss normal fluid and electrolyte composition of the body, balances and doses of crystalloid fluids, before embarking upon a discussion of relative pros and cons, under various circumstances, of the variously composed crystalloid solutions currently available. We will not discuss any experimental animal-derived nor pediatric data and studies. We end with a discussion of specific electrolyte repletion.Table In hospitalized or critically ill patients, however, fluid and electrolyte requirements may be increased following fever, vomiting, diarrhea, blood/plasma loss (trauma and surgery) and polyuria. For fever, formulas are available to estimate insensible losses, which approach 10 ml/kg per 1°C rise in temperature above 37°C [Assessing hypovolemia is hard in hospitalized patients and many methods, from simple physical to complex physiological, have been described. Hypovolemia is often, rightfully or erroneously, considered in case of hypotension, low filling pressures, low central or mixed venous hemoglobin oxygen saturation, or need of isotropic or vasopressor drugs, which are clinical triggers for fluid infusion, even though this may benefit the patient only in the presence of preload and fluidresponsiveness, irrespective of circulating plasma volume. Fluid responsiveness is defined by an increase in stroke volume or cardiac output by a fluid challenge or, preferably, passive leg raising to increase venous return. Incorporation of this strategy into decision making on fluid loading, even in a patient in shock, may help to prevent potentially harmful overhydration.Although not beyond doubt, severe hypovolemia may include the interstitial and eventually even the intracellular space, and repletion of these volumes can be accomplished by intravenous fluid administration ,9. HypovRepletion of lost plasma should ultimately include all components, to prevent electrolyte and acid–base derangements, even independently of hypovolemia- and shock-induced alterations in production and elimination of acids and shifts in electrolyte distributions between intra- and extravascular spaces. One should take into account the electrolyte status of a patient before choosing an optimal fluid type for resuscitation and maintaining euvolemia.In this section, we will discuss fluid doses and types from a ‘crystalloid’ perspective. Crystalloid fluid loading may decrease mortality in hypovolemic patients, for instance after hemorrhage , but theIn contrast to crystalloids, colloids remain in the circulation and increase intravascular volume by raising colloid osmotic pressure. They therefore have a three- to fourfold greater (sustained) plasma volume expanding and hemodynamic effects compared to crystalloids, for a given infusion volume ,11-16. AIndeed, it is likely that the simpler the hemodynamic targets are, like static hydrostatic pressures, the less they reflect plasma volume, and the less strictly they are used to guide resuscitation, the lower is the anticipated volume difference between resuscitation fluid types . This laAlthough fluid therapy is essential in the treatment of shock and tissue hypoperfusion, overhydration and grossly positive fluid balances are, generally, considered harmful by contributing to organ dysfunction after sepsis, trauma or surgery, raising the question which fluid type and dose can optimize hemodynamics and volume status, with the lowest risk of fluid overloading -34. CrysThe propensity of crystalloid fluids to increase pulmonary edema formation, even in face of increased pulmonary vascular permeability and edema (in sepsis) at baseline, has not been confirmed, provided that fluid loading was done in the steep (rather than the plateau) portion of cardiac and pulmonary vascular filling . The tenCompared to isotonic crystalloids, relatively small volumes of hypertonigc solutions may suffice in restoring euvolemia and hemodynamics without grossly expanding the interstitial space, in hypovolemia and shock ,13,30. RHypertonic lactate solutions are also available and, like other hypertonic solutions, may have positive inotropic effects on the heart ,46. HypeDosing and guiding emergency or perioperative crystalloid fluid therapy is still surrounded by controversy. In spite of the less plasma volume expanding and hemodynamic effects of crystalloids versus colloids, for a given fluid infusion volume, a restricted  fluid policy has been suggested to result in less complications than a standard or more liberal fluid strategy, regardless of the principal type of solution used, in randomized studies in surgical patients ,33,49-55However, too restrictive fluid therapy may result in under-resuscitation and potentially associated adverse sequelae, again including gastrointestinal hypoperfusion, anastomotic leaks, nausea, vomiting, and others, which can be prevented in part by more liberal  fluid loading ,54,59-63Hence, the debate on fluid volumes and hemodynamic targets is confounded by the type of fluid, with increasing doubts on the safety of synthetic colloids, at least in sepsis and perhaps not in trauma and surgery, thereby favoring crystalloids ,67. AlsoConcluding this section, one should give crystalloid fluid challenges as a first choice on clinical grounds, for resuscitation from hypovolemia or shock, by utilizing rapid infusions of 500 to 1000 ml, provided that the patient is likely to be fluid responsive at baseline or indeed responds to fluids during loading. In the maintenance phase, measurable losses augmented by 1 l per day for insensible losses mostly result in infusion of about 2 to 2.5 l per day. For resuscitation and maintenance, special considerations obviously apply to brain injury, trauma and surgery, when restriction of fluids and electrolytes may be preferred to limit a positive fluid balance, and to cardiac failure and edema-forming states, when a negative fluid balance rather than maintaining the balance is aimed at.We will now discuss varying crystalloid fluid types and their properties. Normal or physiological saline was first described and applied in 1831, and since then 0.9% sodium chloride (NaCl) is the most widely used intravenous fluid in the hospital, in spite of doubts on its ‘normal’ or ‘physiological’ properties . Adding Commonly applied crystalloid solutions of osmolality, cationic and anionic composition are described in Table in vivo effects on acid–base balance are profoundly different [The chloride content of normal saline (0.9% NaCl), a so-called unbalanced solution, that is 154 mmol/l, is much higher than found in plasma (101 to 110 mmol/l) and higher than the so-called balanced solutions as (modified) Ringer’s or Hartmann’s, where some of the anions are buffering lactate, acetate, or gluconate instead of chloride . The strifferent . Indeed,ifferent ,78-86.Although the use of saline may result in organ dysfunction  and may contribute to gastrointestinal symptoms after surgery, the true detriments of hyperchloremic acidosis in clinical studies remain uncertain . Furtheret al., in a large retrospective study, using propensity-matched samples, recently found that use of normal saline resulted in a higher mortality than using balanced solutions during and following surgery (5.6% versus 2.9%), preventing hyperchloremic metabolic acidosis as well [However, Shaw  as well . In addi as well ,89.Finally, we caution against the use of potassium-containing balanced solutions in anuric patients with renal insufficiency since the infused potassium cannot be excreted ultimately.Hepatic uptake results in gluconeogenesis, an energy costing process. Oxidation is the fate of 30 to 60% of infused lactate, even in septic and cardiogenic shock, thereby sparing other substrates . LactateRinger’s acetate solution closely resembles Ringer’s lactate. Acetate, a component of Ringer’s acetate and other solutions, is, similarly to lactate, not metabolically inert. It enters the tricarboxylic acid cycle and is combusted, thereby consuming oxygen by partly replacing other substrates. Indeed, when infused, about 90% is oxidized. Acetate as a buffer, so that, upon infusion, the bicarbonate concentration and pH rises, similarly as when equimolar bicarbonate is infused, in acidosis following diarrhea or renal insufficiency . Also, aper se may be associated with increased need for renal replacement therapy [On the one hand, crystalloid solutions better preserve diuresis and renal function, and prevent need for renal replacement therapy than colloids in the critically ill , but not therapy ,76.et al. compared in a randomized controlled trial balanced fluids with normal saline as intraoperative fluid replacement during renal transplant surgery, and showed that only saline decreased pH, bicarbonate and base excess [Recent evidence suggests that this may also depend on the type of crystalloid administered and the evidence for a renal protective effect of balanced  crystalloids is cumulating. Hypotonic Ringer’s lactate infusion in healthy volunteers is more rapidly excreted via urine than normal saline (requiring hours), but this can be attributed in part to hypotonicity of the fluid rather than prevention of hyperchloremia, even though normochloremia may protect against a fall in measured renal (and gastric) blood flow associated with hyperchloremia ,81,83,97e excess . This ane excess ,92.et al., mentioned above [P<0.001), even after adjustment for confounders. The incidence of tubular injury and acute kidney injury in sodiumbicarbonate-treated cardiac surgery patients may be lower than when equimolar saline is used [In contrast, a recent large sequential cohort study suggests that the use of a chloride-restricted fluid regimen of (modified) Ringer’s lactate solution  did not decrease mortality, but decreased acute kidney injury and need for renal replacement therapy . In the ed above , adminis is used ,100. Howex vivo, a hypercoagulable state at 20 to 40% dilution of blood and decreased coagulability at 60% dilution [Another controversy relates to alleged immune effects of balanced versus unbalanced solutions . It has dilution ,104.In hospitalized and surgical or critically ill patients, electrolyte disturbances are common ,105. SolDysnatremia is often observed in the ICU setting and is associated with higher in-hospital mortality. Hypertonic saline is used in the urgent treatment, when indicated, of hyponatremia ,109. GluHypocalcemia is also relatively frequent in postoperative and critically ill patients and may have adverse consequences . It is lContinuous renal replacement therapy is a well known risk factor of hypophosphatemia in the critically ill. The latter may also be a feature of refeeding and hypophosphatemia is thus relatively frequently encountered in the critically ill patient  . It may Crystalloid fluids are the fluids of choice for expanding and maintaining plasma volume in hospitalized patients with hypovolemia or shock, although relatively large volumes have to be administered to increase circulating plasma volume and tissue oxygen delivery. Overzealous crystalloid administration increases the risk for fluid overload with hypernatremia, peripheral and pulmonary edema, and compartment syndromes. Hence, both under-resuscitation and overhydration should be avoided by careful monitoring. The evidence that balanced solutions are superior, particularly in helping to prevent acute kidney injury and when isotonic, to unbalanced ones is increasing. Hypotonic solutions are generally contraindicated in conditions with or at risk for cerebral edema. In the maintenance phase of non-oral fluid infusion, solutions which more resemble plasma water composition than normal saline are also to be recommended. We finally recommend serum sodium-guided treatment by hypertonic saline for mannitol-refractory cerebral edema and intracranial hypertension.Ca2+: Calcium; HCO3: Bicarbonate; HP04: Phosphate; HSO4: Hydrogen sulfate; ICU: Intensive care unit; K+: Potassium; Mg2+: Magnesium; Na: Sodium; SAFEstudy: Saline versus albuminfluid evaluation study.ABJG and CI received an unrestricted research grant from B Braun Medical  for this study. CI has received research grant and speaker fees from Fresenius , Baxter HealthCare  and B Braun Medical.AS and ABJG brought about initial set-up, literature research and writing of the manuscript. CI provided comments and recommendations, and improved the quality and clarity of the manuscript. ABJG was the supervising author. All authors read and approved the final manuscript.