Thus, the morphology and gene expression of endothelial cells following partial hepatectomy or shear stress in vitro, to physiological laminar shear stress forces was associated with translocation of vascular endothelial cell growth factor receptor-2 (VEGFR-2) and neuropilin-1 from perinuclear and faint cytoplasmic distribution to plasma membrane and cytoskeletal localization. Under these conditions, VEGFR-2 co-stains with VE-cadherin. Unlike VEGFR-2, the nuclear localization of VEGFR-1 was not affected by shear stress. Quantification of the above receptors showed a significant increase in VEGFR-1, VEGFR-2 and neuropilin-1 mRNA following shear stress.Following partial hepatectomy blood flow-to-liver mass ratio reached maximal values 24 hrs post resection. Concomitantly, large fenestrae (gaps) were noted. Exposure of liver sinusoidal endothelial cells, Our data suggest a possible relation between elevated blood flow associated with partial hepatectomy and the early events occurring thereby. Furthermore, following shear stress both receptors alternate from perinuclear and faint cytoplamic orientation to adhere to cytoskeletal components and cell membrane. These changes coincide with the behavior of the adherence junction proteins VE-cadherin and β-catenin.Following partial hepatectomy (PHx) the remaining liver is transfused by normal blood volume, thereby exposing liver sinusoidal endothelial cells (LECs) to excess hemodynamic forces. These forces have been noted as an early event leading to liver restoration in rats -3; howevSeventy percent of PHx is associated with cell proliferation and a gradual increase in liver mass (data not shown). Nine days post-hepatectomy close to 80% of the original liver weight was restored. PCNA labeling index peaked at 48 hrs thereby returning to preoperative values. Concomitant with liver resection an immediate increase in blood flow to the remnant liver was evident, reaching a maximum of 2.5 fold at 24 hrs Fig. . ElevateThe effects of partial hepatectomy and the associated shear stress developing as a result of excessive blood flow to the remnant liver were evaluated with the aid of scanning electron microscopy. Special emphasis was given to the influence of these forces on the surface of liver sinusoids and intactness of the endothelial lining.Under normal conditions, liver lobule sinusoids show an intact endothelial lining, consisting of LECs with flattened processes perforated by small fenestrae. These fenestrae measure 0.15–0.2 μm in diameter and are arranged in groups, sieve plates Fig. . As earlTransmission electron microscopy was used to study in great detail the above alterations. Fig. . Control2/15 minutes), a strong cytoplasmic presence was evident, with a clear tendency to adhere to cytoskeletal components. VEGFR-1 displayed nuclear localization, which was unchanged when shear stress was applied.Purified LECs grown in culture retained their characteristic sieve plates (data not shown). Following 4 hrs of incubation at 37°C and extensive washing, the cells demonstrated nuclear localization of NFκb suggesting an active state. To avoid activation, purified LECs were grown in feeding medium containing 0.25% FCS for 4 hrs, extensively washed and left for 12 hrs before further used. Under these conditions, more than 94% of the cells exhibited cytoplasmic NFκb which was re-localized to the nucleus following shear stress (data not shown). LECs displayed perinuclear and cytoplasmic localization of VEGFR-2 and neuropilin 1 . Co-staining of VE-cadherin and VEGFR-2 Fig.  exhibitsReal time RT-PCR was used to quantify the amount of mRNA of all receptors before and after shear stress Fig. . The resLiver regeneration is associated with an increased expression of a diverse number of genes including immediate early genes, delayed genes, cell cycle and DNA replication and mitosis genes ,5. Some Endothelial cells lining liver sinusoids are likely to be the first to sense changes in shear stress. Those cells are unique as they have no typical basal lamina. Moreover, the cells are fenestrated allowing free passage of chylomicrons, lipoproteins, hormones, growth factors and proteases . The sizin vivo is, at the moment, beyond our reach. Therefore, the present study examines the effects of increased blood flow following PHx on the morphology of LECs. We also follow the gene expression and protein distribution in LECs exposed to controlled shear stress in vitro. These forces mimic to the best of our ability physiological conditions.Exploring the effects of shear stress on LECs Following 70% PHx an immediate ultrastructural change was noted in the form of fused fenestrae and gaps. Their number increased significantly in both periportal and pericentral areas Fig. ; yet, exIn our experiments Fig. , maximalGiven the increase in blood flow to the liver immediately after PHx, it is likely that the "damage" caused to LECs is the result of excessive shear stress to which the cells are exposed. Interestingly, injections of large volume at a short time, hydrodynamic injections  inflict Shear stress conditions can artificially be applied using the cone and plate apparatus . We haveLECs exhibited nuclear staining of VEGFR-1. This localization was not affected by shear stress conditions. VEGFR-2 and neuropilin-1 present a similar pattern of perinuclear and faint cytoplasmic presence. Following shear stress conditions the two receptors seemed to adhere to membrane and cytoskeletal components.Neuropilin-1 is an isoform specific receptor for VEGF-165 , VEGF-E Hemodynamic forces play a major role in restructuring blood vessels by modulating endothelial structure and functions such as increased permeability to macromolecules or damage to endothelial cells . Therefoin vitro induces in endothelial cells membrane translocation of VEGFR-2 and neuropilin-1. It is conceivable that under shear stress conditions a complex consisting of VEGFR-2/neuropilin-1 and adhesion molecules forms. Such a complex may well be formed following the elevated blood flow associated with partial hepatectomy, playing a role in the early signals leading to liver regeneration.In summary, the present study documents an increase in blood flow to remnant liver following PHx. This change is associated with an elevated number of endothelial cell gaps in both periportal and pericentral areas. Shear stress Male Sprague-Dawley rats weighing 300–325 g were used. PHx was performed on 5 animals under light anesthesia by removing the right lateral and median lobes. At diffLiver regeneration was monitored using liver mass and PCNA. Liver mass was calculated by weighing the removed lobes following surgery and the regenerating liver at the indicated time point. For PCNA immunostaining, specimens were fixed in paraformaldehyde, embedded in paraffin and sliced. Sections were incubated with anti-PCNA followed by biotin conjugated secondary antibody. The binding of anti-PCNA was monitored using avidin-peroxidase and amino ethyl carbazol as a substrate .Five rats were anesthetized and placed on a temperature-controlled table. Following tracheotomy and saline infusion an ultrasound sensor was fixed to the portal vein. Portal blood flow was monitored by ultrasound flowmetry and automatically recorded .3 blocks in 1.5% glutaraldehyde, in 0.12 M sodium cacodylate buffer. Following fixation, blocks were submerged in 1% osmium tetroxide, dehydrated in ethanol and embedded in Epon. Semithin (1 μm) sections were cut and stained with 1% toluidine blue solution. For detailed EM-study, 50–80 nm ultrathin sections were stained first with uranyl acetate and then with lead citrate. For SEM, dehydrated blocks were dried with hexamethyldisilazane and subsequently broken in liquid nitrogen, mounted on stubs and sputter coated with a thin layer of 20 nm gold [Tissue samples were prepared according to standard protocols . Briefly nm gold . Morphom nm gold . The UTH nm gold ,24,27. FLECs were isolated using a modification of the procedure described by Braet et al.  and Smed2 were enforced for 5 or 15 minutes at which time the cells were washed and either used for immunofluorescence or RNA extraction.LECs grown on plastic collagen-coated slides were subjected to shear stress forces produced between a stationary base plate and a rotating cone . High-leCells were fixed in 2% paraformaldehyde followed by 1% triton paraformaldehyde solution. The slides were then immersed in blocking solution and stained with either anti VEGFR-1, VEGFR-2, neuropilin-1, VE-cadherin, β-catenin or NFκb. Cy2 or rhodamine (TRITC) conjugated secondary antibodies were used.RNA was extracted from LECs by the RNAeasy kit  according to manufacturer's protocol and treated with DNase.RNA samples were reversed transcribed and amplified using the QuantiTect SYBR Green RT-PCR kit (Qiagen) and appropriate primers at concentrations of 90 nM to 125 nM. The one-step RT-PCR was carried out at a Rotor-Gene 2000 real time cycler . The thermal cycling conditions included 95°C for 15' followed by 45 cycles of amplification at 94°C 20", 60°C 15–30", 72°C 15". Samples were monitored after elongation by SYBR Green dye binding to the amplified double stranded DNA at 72°C–78°C. All samples were amplified in duplicates and each experiment was repeated twice. Quantitation was carried out using a standard curve. The Rotor-Gene analysis software was used for the calculation of the amount of each RNA sample.Significance was determined with the Mann Whitney two-sided U-test. Differences were considered significant when when p ≤ 0.05.FB and GS conceived the design and coordination of the study and drafted the manuscript and assessed LEC ultrastructure, MS carried out cell isolation, shear stress experiments and immunofluorescence. MP carried out Real time PCR and participated in animal procedures and drafting the paper. SB carried out portal blood flow evaluation. NK participated in animal procedures, NR participated in design and coordination. All authors read and approved the final manuscript.Israel Science Foundation 537/01, Chief Scientist's Office of the Israel Ministry of Health 5002, Mars-Pittsburgh Foundation for Medical Research 182-012, Rappaport Family Institute Fund. This research was partially supported by the "Fund for Scientific Research-Flanders" (grant N° 1.5.001.04N (F.B.)). F.B. is a postdoctoral researcher of the "Fund for Scientific Research-Flanders".


Laser Interstitial ThermoTherapy (LITT) is a well established surgical method. The use of LITT is so far limited to homogeneous tissues, e.g. the liver. One of the reasons is the limited capability of existing treatment planning models to calculate accurately the damage zone. The treatment planning in inhomogeneous tissues, especially of regions near main vessels, poses still a challenge. In order to extend the application of LITT to a wider range of anatomical regions new simulation methods are needed. The model described with this article enables efficient simulation for predicting damaged tissue as a basis for a future laser-surgical planning system. Previously we described the dependency of the model on geometry. With the presented paper including two video files we focus on the methodological, physical and mathematical background of the model.In contrast to previous simulation attempts, our model is based on finite element method (FEM). We propose the use of LITT, in sensitive areas such as the neck region to treat tumours in lymph node with dimensions of 0.5 cm – 2 cm in diameter near the carotid artery. Our model is based on calculations describing the light distribution using the diffusion approximation of the transport theory; the temperature rise using the bioheat equation, including the effect of microperfusion in tissue to determine the extent of thermal damage; and the dependency of thermal and optical properties on the temperature and the injury. Injury is estimated using a damage integral. To check our model we performed a first in vitro experiment on porcine muscle tissue.We performed the derivation of the geometry from 3D ultrasound data and show for this proposed geometry the energy distribution, the heat elevation, and the damage zone. Further on, we perform a comparison with the in-vitro experiment. The calculation shows an error of 5% in the x-axis parallel to the blood vessel.The FEM technique proposed can overcome limitations of other methods and enables an efficient simulation for predicting the damage zone induced using LITT. Our calculations show clearly that major vessels would not be damaged. The area/volume of the damaged zone calculated from both simulation and in-vitro experiment fits well and the deviation is small. One of the main reasons for the deviation is the lack of accurate values of the tissue optical properties. In further experiments this needs to be validated. Laser radiation is now used routinely in surgery to incise, coagulate, or vaporize tissues. The laser light power is converted into heat in the target volume with ensuing coagulative necrosis, secondary degeneration and atrophy, and tumour shrinkage with minimal damage to surrounding structures . The useClinical studies have yet to demonstrate that LITT is practical for the palliation of hepatic and nasopharyngeal tumours e.g. -9). The . The 9])Modelling the laser-tissue interaction is beneficial for the analysis and optimisation of the parameters governing planned laser surgical procedures. Nevertheless, we still lack an adequate model that grants accuracy. Most of the models suggested depend greatly on simplifications of the real problem, either in the geometry they offer or in the system of equations they use. Some models, which use the bioheat equation, neglect the role of the changes in the tissue properties during temperature elevation process , which dThis paper describes in detail the bases for a modelling method to simulate the effect of LITT for the treatment in various indications near large vessels, such as the carotid artery in the neck region. We thereby propose the use of LITT, frequently applied in the treatment of liver tumours , in moreThe actual response of tissue to laser irradiation is a time-dependent phenomenon. Initially, there are thermal and possibly photochemical changes of the tissue at the molecular level. Next are changes in tissue perfusion caused by thermally induced vascular relaxation and/or vessel damage. Heat deposited at the application site is transferred to adjacent structures. This may be desirable for coagulation purposes – or it may cause unexpected thermal damage to otherwise viable tissues adjacent to the irradiation site. The rate of heat transfer depends on the composition and organization of tissues involved. Blood perfusion during and after irradiation has significant effects on the size of the damage zone.We discuss in this paper our mathematical approach, its considerations and restrictions. In the main part we present the mathematical and physical backgrounds used to achieve the model. Then we present and discuss the results of our simulation in comparison with the results of our in-vitro experiment.Our model of LITT considers both optical and thermal effects. It is based on calculations describing the light distribution using the diffusion approximation of the transport theory; the temperature rise using the bioheat equation, including the effect of microperfusion in tissue to determine the extent of thermal damage, and the dependence of thermal and optical properties on the temperature and the injury. Injury is estimated using a damage integral, which depends on the temperature elevation history. The order and flow of the modelling steps are described in the following sections in detail.The head and neck area consists of complex anatomical structures in close proximity. In sonographic 3D volume datasets of the neck area the sternocleidomastoideus muscle and the neck vessels  serve as leading structures . Because3 shown in Fig. One can easily segment the carotid artery from 3D sonographical, MRI or CT volume datasets. For the segmentation of our 3D ultrasound dataset Fig  we used Commercially available laser applicator fibres for thermotherapy frequently have a water jacket to cool the surface. The applicator is assumed to be a cylinder, and the cooling effect is implemented as a boundary condition at the diffuser surface.The tissue surrounding the vessel is treated as a homogeneous muscle tissue. According to the geometry described using a mesh is generated to perform a finite element method calculation Fig. . The modIn most tissues, both absorption and scattering are present simultaneously. A mathematical description of the absorption and scattering characteristics of light can be performed analytically or by using the transport theory. Transport theory has been extensively used when dealing with laser-tissue interactions. Furthermore, experimental results have confirmed its validity in most cases .2 10600 nm laser is less than 350 μm. This leads us to the possibility of applying light diffusion approximation to the transport theory , D the diffusion coefficient [cm], and Q the source term [W cm-3]. μa is the absorption coefficient and μ's the reduced scattering coefficient in tissue. The roman number (I) indicates the position in Fig. μs is described by μ's = μs (1-g), with g being the anisotropy factor incorporating the effects of directionally dependent scattering.erm W cm-. μa is tμa for visible and for near infrared radiation ranges between 0.001 mm-1 < μa < 10 mm-1 for biological tissues. While for the scattering coefficient μs is in the order of 1 mm-1 <μs < 100 mm-1  is the tissue average volumetric blood perfusion rate , and cb the specific heat of blood [J kg-1 C-1]. The coefficients ρ, k and wb are functions of temperature T.where As a basis for the optical and thermal parameters for the simulations, we used values published by Mueller et al. . EspeciaIn order to make the model adaptable to individual shapes of segmented vessels, we considered the geometry of a large vessel as a volume in which an incompressible fluid (blood) flows. The direction of the blood flow and the initial speed profile are implemented as boundary conditions. The incompressible Navier-Stokes equation for the blood (Newtonian fluid) reads:η is the dynamic viscosity [kg s m-1], ρ the density [kg m-3], u the velocity field, p the pressure [N/m2], and F a volume force field such as gravity.Here, u = u(r)) and eq. 5 is reduced to the following:Implementing the Navier-Stokes equation in the model allows us to present a time-periodic change in the blood flow rate, i.e., to simulate the beat cycle effect in the vessel. The main effect here on the result of the simulation lies in the accuracy of the estimated heat elevation in the tissue: A continuous blood flow has a different profile than the cycled flow , which yields a different final cooling effect. For vessels away from the heart, the pumping cycle does not clearly appear; it tends to be a normal laminar flow. In this case  in a large vessel the bioheat equation becomes:The heat convection between tissue and a large vessel occurs as a direct energy transfer rather than perfusion. The vessel is a heat sink in the treated volume. Therefore, the perfusion term in the bioheat equation has to be modified to consider heat conduction and blood flow. A new term, the so-called ood flow . ConsideThe thermal damage in cells and tissue is described mathematically by a first-order thermal-chemical rate equation, in which temperature history determines damage. Damage is considered to be a unimolecular process, whereby native molecules transform into a denatured/coagulated state through an activated state leading to cell death ,19. DamaA [s-1] is the frequency factor, Ea [J/mole] the activation energy, R [J mole-1 K-1] the universal gas constant, and T [K] the temperature. C and C are the concentrations of the undamaged molecules at the beginning and at time τ, respectively. Damage Ω  is a function of the observer's definition of damage. In [The activation energy mage. In  a limit Heat capacity is assumed to be constant over a wide temperature range. The temperature dependence of thermal conductivity and density is taken into consideration by the following linear approximations   and leads to reduction in penetration depth. The actual property set is calculated from the actual damage value as well as the optical properties in the native and coagulated tissue states  . In the first loop step Ω is zero, and it starts to increase according to the rise in temperature, i.e., the different optical properties have as their starting point native tissue and as end point coagulated tissue. The actual value lies between both limits as determined according to Ω.• In the second step, the temperature distribution in the tissue caused by laser energy deposition is estimated by solving the two bioheat equations for tissue and large vessel. The source term in both equations is defined by the absorbed energy at each mesh point Fig.  from the) from the Navier-Stokes equation (either eq.5 or eq. 6). In our solved model, because we suggested treatment as taking place in the neck near the carotid vessel, we considered to use eq. 6 for obtaining the speed field, which is valid for laminar flow.• A by-step here is the estimation of the blood speed field .• After estimating the heat distribution and the damage value, we perform a backward step to calculate the new values of the properties according to eq. 9 through eq. 12, which are updated in the equations set for the next loop iteration.tn and a corresponding sequence of values for the dependent variable  so that each φn, Tn,... approximates the solution at tn [hn = tn+1 - tn so that the estimated error in the numerical solution is controlled by a specified tolerance [t in this case) [For our calculations we used FEMLAB's standard mesh generator with its default settings for modelling . The meson at tn . Modern olerance . The "flis case) . There iLight is considered to be emitted from an interstitial fibre with a fibre-diameter of 1 mm; it was modelled as an isotropically radiating cylindrical source Fig. .In the real treatment a cooling process using a special cooling catheter is performed to keep the temperature at the surface of the applicator low, preventing damage at its surface. A special boundary condition at the applicator surface should be applied in order to simulate this cooling effect. In the model this is realized by setting the outer surface temperature of the applicator to a constant value .At the modelled volume surfaces the insulation boundary conditions, optical and thermal, are used,n is the outward unit vector normal to the surface. This means the gradients of light fluence rate and of temperature vanish at the surface. Even though this condition is more suitable for light fluence rate, as small amount of radiations reach the surface, but in general the temperature and light fluence rate will not be constant. The need to set this condition in this way is because that the numerical solver demands defined fixed boundary conditions, which sometimes do not agree with the real situation.where wb over the entie tissue is set to 1.4·10-6kg of blood s-1 cm-3 for T < 60°C and to 0 when T ≥ 60°C, which is to be considered as a normal result of stopping the blood perfusion according to temperature elevation in the tissue [η, to 3.5·103kg·s·m-1. To evaluate the damage, Ω, the activation energy Ea is set to 670000 J/mol and the frequency factor A to 9.4·10104s-1 [According to the NCRP-data  the perfe tissue . In orde10104s-1 ,35. Dama10104s-1 .UltraSPARC IIIi processor.The simulation takes around 2 hours on Sun Blade 2000 with Solaris 9 OS, 6 GB RAM and 3. The tube inner diameter is 5 mm and the outer diameter is 7 mm. The blood and the laser cooling liquid had the room temperature of 21.4°C while the sample itself 17.6°C. A laser power of 30 W and blood average flow speed of 40 ml·s-1 were used. We measured the exact distance between the laser applicator and the tube edge, 3 mm, at the end of the experiment after performing the cut in the sample. We fixed our application time to 300 s.We performed a single in-vitro experiment to check our model. The setup is shown in Fig. In the simulation model we omit the perfusion term, as there is no perfusion to be considered in-vitro. We simulated the tube (blood vessel) with diameter of 6 mm. All other experimental conditions are implemented in the model as they are in the experiment.Because of the lack of data, which describe the properties of the porcine tissues, in all literature available to us, we used the same data presented in Table The highest temperature and widest damage are reached in front of the centre of the applicator. Taking into account the perpendicular surface to the applicator at centre, which is the most critical in the volume as all effects participate together: applicator, vessel, and blood flow, we complete the comparisons of the results between the experiment and the simulation using this plane. Hence, we made a cut in the probe at the level equivalent to this plane.Coagulation of tissue is immediately apparent and always indicates lethal thermal effect . Anyhow,A comparison in the z-direction needs an up-down cut (y-z plane) through the applicator position perpendicular to the tube/vessel, which was not possible after the cut in x-y direction.In  we preseFig. Fig. The dimensions of the damage zone, which may be considered the target goal of the simulation, can be calculated directly by producing grided axes in all the 3D and 2D results as well as with routines written especially for this aim.Fig. Fig. 2, while our model shows a damaged zone of 2.1*1.45 cm2. We obtain calculation errors of 5% in the x-axis parallel to the blood vessel, and of 20% in the y-direction perpendicular to the vessel. This deviation happens mainly due to inaccurate optical properties values.Fig. To date most simulation models of LITT have used the Monte-Carlo method (MC) to calculate the light distribution, then combine its results with Finite Difference method (FDM) to calculate the heat distribution. Because of its formulation, this combination fits very well for a radially symmetric problem. A weakness arises, however, when dealing with asymmetric volumes in real human anatomy. Arbitrarily curved surfaces separate the different tissues. Consequently, calculations from the FDM becomes so complex that errors start to appear in the results presented stemming from the dependency of FDM on dividing the volume into voxels. One way to overcome this is to increase the voxels' number. Indeed, this leads to less error at the tissue-separating surfaces, although it increases the resources and calculation steps, making the procedure inconvenient. In principle, combining MC and FEM (instead of FDM) is possible theoretically, and seems to be promising as it overcomes the latter problems, but to our knowledge has not implemented yet. From another perspective, MC solution converges to the exact solution of the transport equation only when the number of traced photons increases infinitely ,21, whicBecause we are dealing with an asymmetric geometry, we chose the FEM. It allows us to define and refine the mesh in the volume of interest in order to obtain more precise results. Furthermore, using a FEM mesh we are able to adapt individually the mesh for each patient's dataset. Never the less, it was not necessary to combine methods, FEM is used for all equations.The model we propose depends on the following considerations:• The coupling of a set of time-dependent equations, which simulate the whole process of the LITT treatment Fig. . The set• We consider the functional dependence of the various tissue properties at the various spatial and temporal points, according either to the tissue type, or temperature, or the damage value – or even a combination thereof.• We take into account the irreversible changes in the tissue stemming from the treatment as they directly affect the solution of the set of equations.Our model remains a mathematical model, meaning errors could appear from the considerations and simplifications made to realize it. Generally, such errors appear because of the following reasons:• The inaccuracy of the optical, thermal, and damage properties are main point in the model's set of equations. In fact, these properties play a key role in the accuracy of the model's results. Many methods have been presented to calculate these properties ,19,21, b• An error appears because of machine performance limitations: The available memory limits the number of mesh nodes and the degrees of freedom (DOF) used to build the model. This causes a deviation from an otherwise accurate result . On the • Absolute tolerance: All numerical methods have an allowed error (absolute tolerance) that reflects the criterion of the convergence. Normally, different solvers use different tolerances. In our model we used the FEMLAB's default tolerance value of 0.01 which leads to a final error of 1%, considered as a reasonable value for modelling.One way to follow these errors and deviations from a real treatment result is to estimate them and to eliminate their effects from the final results of the model. This can be realized and implemented in the model by adding an error-correcting factor from the first degree (or even higher) in the set of equations correcting the result of each equation at each time step. These corrective factors should be measured practically by comparing the results of the model and the results from real experiments on test tissues or probes.μa for both native and coagulated different biological tissues are close. Baring that in mind, and knowing that the value of the scattering coefficient μs becomes normally, for biological tissue, 10 times greater than its starting value, i.e. native state, we can judge that as soon as the damage zone appears and the moving from native to coagulated state according to eq. 11, eq. 12, and eq. 13 the deviation in the calculations will increase as well.Our experiment shows a deviation of 5% in x-direction and 20% in y-direction. As the main reason for this deviation we propose inaccurate values for the optical tissue properties. Fig. Thus, accurate values of the different tissue properties, and especially the optical properties are key points in obtaining realistic results from the simulation. One promising technique for determination of optical properties was presented by Dam et al. . There mFinally, beside the error obtained from the optical properties, which affect all directions, both cutting the tissue with scalpel and the opening induced a tissue movement. This movement is a reason for deviation, especially in y-direction, as we perform the cutting in this direction.For several years now LITT has been a well-known and approved therapy system for tumour ablation in the liver and some other anatomical regions. Minimally invasive LITT procedures use a Nd:YAG 1064 nm laser. Therapy planning, however, remains unsolved and is still a challenging issue. Today's simulations are based on symmetric geometries. Without exact therapy planning systems, the usage of LITT is limited to homogeneous tissues or the respective surgeon's experience.The finite element technique proposed in this paper can overcome both limitations. We propose a model to validate in the future the LITT method in other anatomic regions.The model enables the efficient simulation for predicting the damaged zone induced with the diffuser of the LITT. The simulation is performed for tissue ablation near vessels, though obviously FEM is not limited to this. Exemplarily, we implemented the model for tissue ablation near the carotid artery in the neck region using an approximation for the artery shape. We describe the bases necessary to calculate the effects of the temperature rise caused by the absorption of light energy in the tissue, using the bioheat equation and including the cooling effects of vessel blood flow and micro-perfusion in tissue in order to determine the extent of thermal damage. The shape of the carotid artery is derived from a real segmented geometry based on, but not limited to, 3D ultrasound.Experimentally, we performed a laser irradiation in a porcine muscle tissue sample. The results of our model diverge between 5% to 20% from the lesion obtained in the experimental work. From the authors' point of view two major reasons can be identified. The lack of accurate data describing the thermal and optical properties leads definitely to deviations. Furthermore the cut of the probe with scalpel induces a certain tissue shift, especially in the y-direction.Anyhow, more experiments with different conditions are necessary to be able to carry out a statistical study and find the exact origin of the deviation, and, if necessary, define an error correction factors and add them to equation set. But that does not set aside the desire of accurate values for the properties of the tissue.From another hand, still our model practical, it presents a step in using segmented data as basis for much more detailed surgical therapy planning. Combining LITT and adequate planning system could increase both the anatomical application range and the quality of therapy procedures.Animated gif file, the Geometry. The animated gif shows the 3D ultrasound volume together with the carotid artery segmented using 3D Slicer software [software . The movClick here for fileAnimated gif file, The heat distribution and the damage zone in the volume. The video stream demonstrates the temperature rise inside the tissue. The video stream shows where, how, and when this damage appears. The damage zone is shown in grey colour. The gif file can be played using the internet browser.Click here for file


Biologists studying the evolution of mate choice have their work cut out for them. Not only is there little agreement on how best to determine the interplay between mate choice and fitness, there's not even consensus on how to estimate fitness.  Why females fall for captivating males that give them nothing but trouble is especially puzzling. Such males tend to contribute their genes and little else, leaving the female to spend precious resources bearing and raising her young, efforts that often cut her life short. Of course, females are not all innocence and light in the mating game: some black widow species, for example, famously make dad a post-coitus snack. Still, females typically incur more costs than males in rearing offspring, especially when they choose flashy mates. So why do they do it?One model holds that females put up with deadbeat dads because the benefits, though indirect, outweigh the costs: that is, attractive males are more likely to grace their offspring with good genes that increase survival (“I'm so fit I can afford to waste energy on this excessively exuberant tail”) or with sexy traits that confer mating success . Another model argues that selection for such indirect benefits is much weaker than direct selection on genes that affect mate preference and thus is likely to exert little influence on mating preference.In a new study, Megan Head and her colleagues navigate this intellectual minefield by studying the mating behavior of crickets. The authors paired females with either “attractive” or “unattractive” males (see below) and measured a variety of fitness components to estimate the overall fitness consequences of the various unions. Female crickets, they found, pay a high price for mating with attractive males. But when the fitness consequences for their sons and daughters are taken into account, mate costs are balanced by, and may even be outweighed by, the indirect benefits of spawning offspring with elevated fitness. This benefit stems in large part, the authors argue, from siring sexy sons.How does one distinguish lothario from loser in the cricket world? By running a cricket tournament, of course. For crickets to mate successfully, the female must mount the male so their genitalia align. Noting that females produce more eggs for males they mount quickly, the authors use time to mount as a measure of male attractiveness.In the first round of the tournament, Head and colleagues paired males with a randomly assigned female; after mounting, but before copulation, the couples were separated. This continued until half of all females had mounted a male.  In round two, a new female was randomly assigned to each male. Males that had been mounted in the first round and remounted in the second were deemed “attractive.” Males rebuffed in the first round that remained unmounted longest in round two were “unattractive.” Females were randomly assigned to males that were either attractive or unattractive. Equivalent males were swapped out every seven days to control for any individual quirks that might bias the results.To estimate the total fitness of the participants, the authors measured both direct and indirect fitness components, such as female hatching success and reproductive effort (egg number and size), as well as sons' attractiveness and the number of eggs laid by daughters. Females that mated with attractive males produced daughters that laid more eggs within a given time and sons that were more attractive, though they had lower survival. Thus, by evaluating both the direct effects of female lifetime fecundity and the indirect effects of offspring fitness, the authors determined the net consequences of a mating strategy. And once again, it's mom's sacrifices that keep things on track.With this approach, Head and colleagues bridge the gap between empirical studies of mating choice evolution, which rely largely on rate-insensitive measures (such as counting grandchildren), and theoretical studies, which typically use rate-sensitive measures. Their results suggest that there may be selection for choosing costly mates and that generating a reliable analysis of the fitness consequences requires a long view: look at the reproductive success of mom's sons and daughters before judging her bad taste in mates.


Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA.P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified.We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase.We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of  Pichia pastoris is well established as expression host for heterologous proteins )Pichia pet al. [et al. [et al. [Astatotilapia burtoni) for the heterologous hybridisation with cDNAs from eight different fish species, some of them closely related (other cichlids) and others more distantly related . They conclude that significant results are obtained even with distantly related species, however, the number of positive spots declines with the phylogenetic distance, as strikingly does the degree of measured regulation.To circumvent this problem, heterologous hybridisation to commercially available DNA microarrays might be conceivable. In fact, the successful non-homologous probing to microarrays has been reported recently. These studies cover a wide variety of organisms, including bacteria , a yeastet al.  report,  [et al.  compared [et al.  compareda priori. When cDNA of the non-conventional yeast Zygosaccharomyces rouxii was probed by cross-hybridisation to Saccharomyces cerevisiae GeneFilters, only 155 ORFs out of the Z. rouxii genome could be reproducibly detected [Z. rouxii cells and 38 genes behaved differently than the S. cerevisiae homologues, indicating that the information gained is limited but nevertheless useful. As judged from 26S ribosomal DNA sequences, Z. rouxii is assumed to be more closely related to S. cerevisiae than P. pastoris, but still, all three belong to the hemiascomycetes [While yeasts share many morphological and physiological similarities, they represent a very heterogeneous group of fungal organisms, and a high degree of gene sequence similarity cannot be assumed detected . Anyhow,omycetes .P. pastoris, there is no simple way to assess the degree of gene sequence similarity between P. pastoris and S. cerevisiae. However, in many of the few genes sequenced, stretches of high similarity (score >75% over a length of at least 250 bp and more) can be identified. Most of the known genes belong to the carbon and energy metabolism or contribute to amino acid or protein synthesis. Another group of P. pastoris genes with known sequence belongs to pathways specific for methylotrophic yeasts. For these genes there are no homologues present in S. cerevisiae. Evidently, this respective fraction of the P. pastoris genome would remain unevaluated by heterologous hybridisation.As there are not many genes characterised for P. pastoris, and to identify genes that are specifically regulated upon a shift from glycerol to methanol as carbon and energy source. This shift is of particular interest since one of the specific features of methylotrophic yeasts is the tightly regulated methanol metabolism, which has been utilised for the construction of strong and tightly controlled expression vectors. The methanol induced promoter of the alcohol oxidase 1 (AOX1) gene, which is repressed by many carbon sources such as glucose, glycerol or ethanol, is widely used for heterologous gene expression in P. pastoris. Accordingly, methanol is often used as the carbon source that induces the production of heterologous proteins. In addition to heterologous protein induction, the shift of the carbon source to methanol causes major structural and physiological changes within the cell. The enzymes for methanol metabolism are synthesised de novo and some of them are translocated into peroxisomes. Strikingly, peroxisomes can fill most of the cellular volume and AOX1 alone can account for up to 35% of the total soluble protein [P. pastoris [P. pastoris, we used a strain expressing human trypsinogen under control of the AOX1 promoter, under conditions that strongly influence the physiology of the host cells, as previously described [The main aims of this work are to verify whether a heterologous DNA array approach allows to obtain useful information for  protein . Additiopastoris . Hence, escribed ,16. A seS. cerevisiae DNA microarrays with P. pastoris cDNA results in significant data. Obviously, the intensity of a signal will depend both on the amount of the specific mRNA in the sample, and the sequence similarity with the respective gene of S. cerevisiae. Therefore, we compared the signals obtained from four microarrays hybridised with P. pastoris cDNA with four microarrays hybridised with S. cerevisiae cDNA (as a control), obtained from shake flask cultures.Before analysing differential gene expression data, it was our intention to verify whether the heterologous hybridisation of P. pastoris. In contrast, by hybridisation with S. cerevisiae cDNA about 46 % of all genes remained undetected.To estimate the overall potential to obtain data, and the degree of loss of information, the total number of genes giving significant values, and those determined to be under a given threshold were compared table . In averP. pastoris for which sequences were deposited in the GeneBank database for sequence similarities to the S. cerevisiae genome, and determined the number of significant spots on 6 microarrays. 66 % of the signals derived from genes with high similarity  were significant, while of the moderately to weakly similar genes only 28 % of the signals were significant. This indicates that a high sequence similarity of a sub-sequence within a gene is sufficient for efficient hybridisation. Considering that the signal intensity will depend on sequence similarity and length, but also on mRNA abundance, it becomes obvious, however, that a distinct minimum threshold of similarity cannot be defined.We analysed those genes of P. pastoris cDNA.It was expected that the number of positive spots would be lower for heterologous hybridisation as compared to homologous hybridisation, but the relatively high number of significant values obtained in our experiment is very promising to achieve useful and new information from this technique. Nevertheless, we sought statistical evidence for the biological significance of hybridisation signals obtained with S. cerevisiae and P. pastoris respectively  were evaluated, showing very high correlation coefficients of 0.97 and 0.98, for ly table . The corP. pastoris experiment, showing that the standard deviations do not vary over a wide range of signal intensities, which is in concordance with the results of Moody et al. [Since usually the data of two samples on one microarray are to be compared, it was important to evaluate the reproducibility, expressed as the standard deviations of both values of each spot on microarrays hybridised with identical samples. Fig. y et al. . 96 % ofP. pastoris cDNA with that from S. cerevisiae cDNA (cells grown under the same conditions). A highly significant correlation (r = 0.72) was observed . A similar observation was made for more distantly related fish species [Finally, the global regulation pattern of ons Fig. . The cor species .As an example for differential gene expression in this study, the level of transcripts during the methanol induction phase of a lab scale fermentation was compared to that during the glycerol feeding phase, both at pH 5.0, and at pH 3.0. Figure S. cerevisiae.Of all genes that gave significant signals, we report those that showed significant differences upon the shift from glycerol to methanol under at least one condition (pH), and that have a defined function in Unsurprisingly, genes involved in the core carbon metabolism show a significantly different expression during methanol and glycerol metabolism table . Almost Table MET3, MET16 and MET17 were upregulated on methanol, the homologues to MET6, SAM1, SAM2 and SAH1 were downregulated. As shown in figure MET6 (5-methyltetrahydrofolate-homocysteine S-methyltransferase), which catalyses the transport of activated methyl groups from 5-methyl-THF to homocysteine. These methyl groups are then passed on via S-adenosyl-methionine by a variety of S-adenosyl-L-methionine-dependent methyl transferases, many of them being involved in ribosomal subunit biogenesis, rRNA and tRNA-processing, mRNA capping and nuclear export – once again stressing the lower demand for protein synthesis rate upon growth on methanol.Only a few amino acid biosynthesis genes appeared to be regulated table , indicatSNZ1 and SNZ2, were differently regulated at pH 5 and pH 3, being repressed on methanol at pH 5 and induced at pH 3, while SNZ3 was repressed at pH 5, but did not yield a significant value at pH 3. However, the reported high sequence similarity of the S. cerevisiae SNZ genes will not enable a reliable differentiation of their regulation on microarrays. For S. cerevisiae it is reported that the highly homologous products of the SNZ gene family are involved in vitamin B6  synthesis [et al. [SNZ genes have been reported to be induced both by thiamine and pyridoxal depletion [SNZ and THI genes, since among the genes utilising thiamine-pyrophosphat (TPP) as cofactor, transketolase (TKL1) and pyruvate decarboxylase  are upregulated while PDB1 (pyruvate dehydrogenase beta-subunit) and presumably also α-ketoglutarate dehydrogenase (belonging to the TCA cyle) and DHAS (belonging to the methanol utilisation pathway) are downregulated.Other significantly regulated groups of genes table  belong tynthesis . Zeidler [et al.  postulatepletion . However2O2 and peroxide radicals, tended to be upregulated. This was expected due to the higher amount of oxidative stress during methanol utilisation.Thioredoxin-related genes appeared to be regulated upon shift from glycerol to methanol, too table . Those bP. pastoris homologous sequences to produce the probes  of the cells. The cells of the culture at pH 5.0 showed a pHi of 7.1 at the end of the glycerol phase, and of 7.2 the end of the methanol phase, whereas the pHi of both samples of the cultures at pH 3.0 was 7.3. Thus, no changes of the pHi were observed between cultures grown at the chosen pH values, which is in contrast to the behaviour of S. cerevisiae, where the pHi appears to be more dependent on the external pH [Surprisingly, only minor differences in transcriptional regulation were observed between cultures grown at pH 5.0 and pH 3.0. Of course it has to be considered that not the direct effects of a shift in external pH was observed. Still, one could expect a different set of genes to be significantly regulated upon a shift to methanol metabolism at the different pH values. At pH 3.0 a decreased yield in biomass was detected as compared to cultures at pH 5.0, which is consistent with the observation of Hohenblum et al.  stating ernal pH ,20.P. pastoris by probing S. cerevisiae DNA microarrays.We could successfully demonstrate that it is possible to obtain new and valuable information about transcriptomic regulation in Specific regulation upon a shift from glycerol mineral medium to methanol mineral medium under conditions similar to a production process were analysed. As major effects we recognised a downregulation of TCA cycle genes, and a transcriptional decrease of genes related to ribonucleotide and ribosome synthesis. Furthermore, the supply of activated methyl groups via adenosyl methionine was reduced, indicating a decreased ribosome and tRNA synthesis, which is not surprising since the specific growth rate is significantly decreased during the methanol feed phase in comparison to the glycerol feed phase. Correspondingly, a downregulation of the energy metabolism upon methanol induction appears reasonable.SNZ genes and the histone genes, but a plausible hypothesis for the differential pH dependent regulation was not found. Interestingly, also the intracellular pH did not change between the different external conditions, indicating a major difference in pH regulation between P. pastoris and S. cerevisiae.Only a few genes were differentially regulated when comparing expression differences between growth on glycerol and growth on methanol of a culture grown at pH 5.0 to one at pH 3.0. Among the few genes found are the Unless stated otherwise, all chemicals were purchased from Merck Eurolab, and all enzymes for DNA manipulation were purchased from MBI Fermentas.P. pastoris strain X33 (Invitrogen), a wild type strain which can grow on minimal media without supplements. The identity of the strain in use was verified by partial 26S ribosomal DNA sequencing (data not shown). The selection mechanism was based on the Zeocin™ resistance of the transformation vector. Transformation of the strain was carried out with a plasmid derived from pPICZαB (Invitrogen), containing the gene for human trypsinogen 1 [AOX1 promoter of P. pastoris and the α-factor leader sequence of S. cerevisiae for product secretion. The selected strain was of the methanol utilisation positive (mut+) phenotype, which means that it is fully capable to metabolise methanol as the sole carbon source.The expression strain used in this study was inogen 1 . pPICZαBS. cerevisiae CEN.PK 113-5D  [As a control strain we used a, ura3) .Shake flask cultures were performed at 28°C in YPD medium .660 of 0.3 from a pre-culture grown over night.The cells were inoculated to an OD660 for S. cerevisiae: 0.89, P. pastoris: 1.2).For the yellow experiment the samples were taken in exponential phase after 5 h of growth . All chemicals for PTM1 trace salts stock solution were from Riedel-de Haën, except for biotin (Sigma), and H2SO4 (Merck Eurolab).6.0 g CuSO3PO4 (85 %), 0.6 g CaSO4• 2H2O, 9.5 g K2SO4, 7.8 g MgSO4• 7H2O, 2.6 g KOH, 40 g glycerol, 4.4 ml PTM1 trace salts stock solution.23.7 ml H1 trace salts stock solution632 g glycerol (100 %) and 12 ml PTM1 trace salts stock solution988 ml methanol (100 %) and 12 ml PTM-1 air, which was supplemented with oxygen (up to 25 %) after the begin of the fed batch. The temperature was 25°C, and the pH was controlled with NH3 (25 %).The dissolved oxygen was controlled at DO = 30 % with the stirrer speed (600 – 1200 rpm). Aeration rate was 100 l h3 (25 %). The batch phase of approximately 32 h was followed by a 4 h fed batch with glycerol medium (feed rate 15.6 ml h-1), leading to a dry biomass concentration of approximately 40 g l-1. Then, the feed with methanol medium was started with a feed rate of 6.4 ml h-1. The fermentation was terminated 14 h after the methanol feed start. The pH was 5.0 during batch, and either kept at 5.0 throughout the fermentation, or decreased to 3.0 at the beginning of the glycerol fed batch. The final dry biomass concentration was 51.4 g l-1 at pH 5.0, and 46.7 g l-1 at pH 3.0.Before starting the fermentation, the pH of 1.2 l batch medium was set to 5.0 with NHSamples were taken at the end of the glycerol fed batch phase and at the end of the methanol fed batch phase, respectively, as depicted in figure The cell pellets were re-suspended in 10 × the volume of TRI-reagent (Sigma) and frozen.The samples were thawed on ice and after addition of acid washed glass beads the cells were homogenised in a Ribolyser (Hybaid Ltd.) for 2 × 20 sec, in between cooling on ice. After addition of chloroform, the samples were centrifuged and the total RNA was precipitated from the aqueous phase adding isopropanol. The pellet was washed 2 × with 70% ethanol, dried and re-suspended in RNAse free water. mRNA was isolated using the MicroPoly(A)Purist mRNA purification Kit (Ambion) according to the manufacturers protocol.5 μg of mRNA and 0.5 μg of oligo dT primer were mixed in 7 μl of water, incubated for 5 min at 70°C and subsequently at 42°C for about 3 min. The following components were added to 5 μl of said reaction mixture: 4 μl reaction buffer (5 x) for SuperScript II reverse transcriptase (Invitrogen), 2 μl dTTP (2 mM), 2 μl dATP, dGTP, dCTP (5 mM), 2 μl DTT (100 mM), 2.5 μl RNasin  and 2 μl FluoriLink Cy3-dUTP (1 mM) or 2 μl FluoriLink Cy5-dUTP  respectively, and 1 μl SuperScript II reverse transcriptase  to result in a total of 19.5 μl. The mixture was incubated for 1 h at 42°C. After addition of further 200 U SuperScript II reverse transcriptase the mixture was incubated for another 1 h at 42°C. 7 μl of 0.5 M NaOH/50 mM EDTA were added and the mixture was incubated at 70°C for 15 min. The reaction mixture was neutralised by addition of 10 μl Tris-HCl pH 7.5 (1 M). The labelled cDNA of the two corresponding samples were pooled and purified with Qiaquick purification columns (Qiagen) according to the manufacturer's protocol.The cDNA microarrays used for this study were Hyper Gene Yeast Chips from Hitachi Software Engineering Europe AG. According to the manufacturer, about 0.1 to 0.3 ng of PCR amplified cDNA (approximately 200 bp to 8000 bp) were spotted onto a poly-L-lysine coated glass slide and fixed by baking, succinic anhydride blocking and heat denaturation.Labelled cDNA was resuspended in about 70 μl of 5 × SSC/0.05% SDS, heat denatured at 95°C for 3 min and cooled on ice. SDS crystals appearing were dissolved by short and slight warming and the mixture was gently applied to a Yeast Chip according to the scheme presented in table The cover glasses were removed in 2 × SSC/0.1% SDS and the chips were washed consecutively for 5–10 min each in 2 × SSC/0.1% SDS, 0.5 × SSC/0.1% SDS, and 0.2 × SSC/0.1% SDS at RT. The chips were centrifuged at 600 rpm for 3 min in order to dry them. The washing conditions were chosen according to the manufacturer's manual. We have tested less stringent washing conditions which led to higher background without increasing the number of positive signals.Images were scanned at a resolution of 50 μm with a G2565AA Microarray scanner (Agilent) and were imported into the GenePix Pro 4.1 (Axon Instruments) microarray analysis software. GenePix Pro 4.1 was used for the quantification of the spot intensities. Each appearing gene spot was averaged. The data set was then imported into GeneSpring 6.1 (Silicon Genetics) for further normalisation and data analysis.S. cerevisiae, and 1.5 fold for P. pastoris, see results) on both parallel independent microarrays were judged as significantly regulated.All of the values of each channel on each chip were divided by their respective median for normalisation. Subsequently, the median intensity of all 84 TE spots  deduced from each value, and all spot values less then the standard deviation of said 84 threshold values were considered to be not significant and were set to the value of the standard deviation. To determine induction or repression of gene activity, the normalised signals on each spot were compared, and all genes showing a signal difference exceeding the threshold  on correlation, Spearman's correlation coefficients were calculated as well. As these differed only slightly from Pearson's coefficients, they are not shown.P. pastoris against S. cerevisiae in order to compare the average fold change observed for both yeasts.Linear regression analysis was performed with the regulation intensities of i was determined as described by Valli et al. [4 cells were measured per analysis, using PBS as the sheath fluid. Carboxy SNARF-4F fluorescence emission was measured through a 585/21 BP filter (FL2) and a 670 LP filter (FL3). Threshold settings were adjusted so that cell debris were excluded from data acquisition. The ratio of the two fluorescence intensities is a measure for the internal pH. Calibration was performed with amphotericin B (Sigma) perforated cells as described in Valli et al. [The pHi et al. . Essentii et al.  at pH 3.i et al. .et al. [Northern blot analysis was essentially performed as described by Sambrook et al. . In shorP. pastoris DNA using the following primers: actin: gttccagccttctacgtttctattca and acggagtactttctttctggtggag; SAH: agctgaacttgattttggacgac and acttgaggcttgatgttgctgac; Met17: tgcatcaatggtcacggtaaca and tggtgagtagagtagtaaggagcaatga. The probe for human trypsinogen was prepared as described in [The probes were PCR amplified from genomic ribed in . The proPre-hybridisation and hybridisation were performed in high SDS hybridisation buffer at 42°C.The blots were washed twice at RT with 2 × SSC/0.1 % SDS and two times at 68°C with 0.5 × SSC/0.1 % SDS. Staining of the blots was performed using anti-Digoxigenin-alkaline phosphatase Fab Fragments (Roche) and the CDP Star chemiluminescent reagent (Tropix) according to the manufacturer's protocol. The images were taken with a Lumi imager F1 (Boehringer Mannheim).i determinations. MM ran and analysed the fed batch fermentations. DP participated in the design of this study, and in data interpretation. DM participated in the design of this study, and in data interpretation, performed the statistical analyses, and drafted part of the manuscript. All authors read and approved the final manuscript.MS and PB designed and performed the microarray hybridisation experiments. MS and BG analysed and interpreted the microarray data, and drafted the manuscript. MV performed the pH


There has been an increased interest in the role of a human rights framework to mobilize resources for health.specific health problems one might want to do something about. Specifically, it is not possible to argue that a particular allocation of scarce health care resources should be changed to a different allocation, benefiting other groups. Lack of access to health care services by some people only shows that something has to be done, but not what should be done.This paper argues that the human rights framework does provide us with an appropriate understanding of what values should guide a nation's health policy, and a potentially powerful means of moving the health agenda forward. It also, however, argues that appeals to human rights may not necessarily be effective at mobilizing resources for The somewhat weak claim identified above together with the obligation to realize progressively a right to health can be used to mobilize resources for health. During the past few years there has been an increasing interest in attempts to utilize a human rights framework to argue that we have obligations, in one way or another, to do something about the human suffering among the large number of poor in the world. The argument is that the suffering of the poor is a violation of their human rights, and the international human rights instruments place an obligation on us to do something about it. The exact details of the argument vary among different commentators, but they all have in common this basic argument structure. Rarely, however, are we provided with any details about exactly how one should understand particular violations of human rights or exactly how one arrives at recommending a particular action to rectify the alleged violation of a human right. Nevertheless, many are confident that a linkage to human rights will prove useful when we want to mobilize resources for the world's poor. In the words of Paul FarmerOf course, it is easy to demand more resources; what is hard is to produce them. But if social and economic rights are acknowledged as such, then foundations, governments, businesses, and international financial institutions-many of them awash in resources-may be called on to prioritize human rights endeavors that reflect the paradigm shift advocated here , p. 244.specific health problems one might want to do something about.In this paper I want to examine this claim as it relates to health: How can one use a human rights framework to mobilize resources for health? If we want to use this framework presumably we can make demands on the basis of two types of reasons. Those who lack access to resources can argue that the total amount available to promote health should be increased, or they can argue that the allocation of available resources should be made in a different way, giving them access to health care services, but denying other, less deserving groups access. One may, of course, also wish to make both demands. In this paper I shall argue that the human rights framework does provide us with an appropriate understanding of what values should guide a nation's health policy, and a potentially powerful means of moving the health agenda forward. I shall, however, also argue that appeals to human rights may not necessarily be effective at mobilizing resources for One may understand the claim that "health is a human right" in at least three different ways. First, we may simply want to say that health is important, that we should all do what we can to promote health, and we may even expect that referring to health as a human right might produce an emotional response in our audience, motivating them to action. If this is what we want to do, and if claiming that "health is a human right' does in fact produce this type of response, then utilizing a rights framework can indeed be expected to mobilize resources. While it is undoubtedly true that this strategy will sometimes be effective, its effectiveness will more often than not depend on the immediate reaction to the deprivation of a particular group, and that group's ability to elicit sympathy for their cause, rather than a legitimate policy response where all competing claims have been taken into account. Second, we may want to make a moral claim of a particular type. There is an extensive philosophical discussion of how one should understand the concept of "a right" in general, and a "right to health" in particular. While not denying the importance of this discussion, it does not have much relevance to the problem addressed in this paper: does one have a reason to believe that the claim that "health is a human right" will mobilize resources for health? Even if we take the strongest claims to be true, for example that there are moral obligations on each one of us to do something quite specific to improve other people's health, we still have to provide an account of how to translate these moral obligations into effective action. That is why I want to limit my discussion to an understanding of "health as a human right" as those specific legal obligations on states that arise out of international law. International law in this context refers primarily to The Covenant on Social, Economic and Cultural Rights, but also the General Comment by the UN Committee on Economic, Social and Cultural Right on article 12  of the International Covenant on Economic, Social and Cultural Rights, and the first report of the recently appointed Special Rapporteur with a mandate to focus on the right to health . In addiThe issue to be addressed is therefore: Can one on the basis of international law argue that a person's right to health is violated if that person is denied access to health care services or the underlying determinants of health on economic grounds; that is, either because that person does not have sufficient resources herself to pay for the services or the state claims that it does not have sufficient resources to pay for the necessary services. If one could establish that such cases are violations of international law, then one would have an effective way of mobilizing resources for health based on a human rights framework.If one adopts this approach, one challenge is, in the words of the Special Rapporteur, that "although there is a growing national and international jurisprudence on the right to health, the legal content of the right is not yet well established (#39)". Some indication of how one should understand the right to health in international law is nevertheless provided in the key, authoritative documents and in the court cases that have been decided on the basis of right to health challenges. Some of the statements made in the General Comment might indeed lead one to believe that a state has strong legal obligations to provide sufficient resources to ensure adequate health for all. It is said, for example, that "health facilities, goods and services must be affordable for all. Payment for health care services, as well as services related to the underlying determinants of health, have to be based on the principle of equity, ensuring that these services, whether privately or publicly provided, are affordable for all, including socially disadvantaged groups (12 1 iii). One could take this to mean that it prohibits denial of health care services on economic grounds. Other statements seem to support this claim " [F]unctioning public health and health care facilities, goods and services, as well as programmes, have to available in sufficient quantity within the State party (12(1)). Central to the General Comment is the principle of non-discrimination: "...the Covenant  proscribes any discrimination in access to health care and underlying determinants of health ... on the grounds of race, colour, .... health status ... and civil, political, social or other status" (18). This is reinforced by the special rapporteur: "Accordingly, international human rights law proscribes any discrimination in access to health care, and the underlying determinants of health, on the internationally prohibited grounds, including health status, which has the intention or effect of impairing the equal enjoyment of the right to health" (26).Taken together, these statements seem to give a strong endorsement to the claim that it is a violation of a person's health rights to deny him treatment on the grounds that treating that person is too expensive for the state. If one denied treatment to persons who happen to have diseases that are expensive to treat, one does indeed discriminate on the basis of "health status", one of the prohibited grounds, and one does not ensure equal access to health care services to all.There is, however, one important qualification to this claim. Although the state has an obligation to provide health care in "sufficient quantity", "the precise nature of the facilities, goods and services will vary according to numerous factors, including the State party's developmental level" (12 1). Furthermore, "the Covenant provides for progressive realization and acknowledges the constraints due to the limits of available resources" (30). It is, therefore, in spite of the strong statements that everyone should be assured access to health care, in principle legitimate for a state to claim that it can deny access to health care to patient groups who happen to have diseases that are expensive to treat.A state's claim that it does not have sufficient resources to provide access to health care or its determinants to a particular group because of its costs can, of course, be challenged. We would then need some principled way of adjudicating between the competing claims: on the one hand those of the group denied health care access claiming that its health rights are violated and on the other hand those of state claiming that its resources can be utilized better elsewhere. The official documents on how we should understand a right to health do not provide us with much guidance on how one should adjudicate between such competing claims. As we shall see in the next section, neither apparently do the cases which have been brought forward as violations of a right to health.The two recent South African cases provide a particular striking example of the challenge of using a reference of a right to health or the courts to mobilize resources for health. The South African constitution, article 27 (1) gives everyone a right to have access to health care services, including reproductive health care and in article 27 (2) it says that the state must take reasonable legislative and other measures, within its available resources, to achieve the progressive realization of these rights. These national provisions, of course, reflect the ones in the UN Covenant. In November 1997, the Constitutional Court of South Africa decided on a case involving the scope of such a "right to health" . The cas"It [article 27(3)] provides reassurance to all members of society that accident and emergency departments will be available to deal with unforeseeable catastrophes which could befall any person, anywhere and at any time" (section 51)andThe applicant suffers from chronic renal failure. To be kept alive by dialysis he would require such treatment two to three times a week. This is not an emergency which calls for immediate remedial treatment. It is an ongoing state of affairs resulting from a deterioration of the applicant's renal function which is incurable" (section 21)Although the appellant did not appeal the decision on the basis of the South African Constitution's article 27 (1) giving everyone a right to have access to health services, the Court did discuss this matter. The Court pointed out that it is not disputed that the Department of Health"does not have sufficient funds to cover the cost of the services which are being provided to the public ... This is a nation-wide problem and resources are stretched in all renal clinics throughout the land. Guidelines have therefore been established to assist the persons working in these clinics to make the agonising choices which have to be made in deciding who should receive treatment and who not" (section 24).The Court further maintained that the current guidelines are justified by the fact that more patients would benefit from the limited resources available than by any alternative use of resources, and that "it has not been suggested that these guidelines are unreasonable or that they were not applied fairly and rationally when the decision was taken by the Addington Hospital that the appellant did not qualify for dialysis" (section 25).The second case involves use of nevirapine to prevent perinatal HIV transmission,6. As isThe South African government had claimed that providing nevirapine to pregnant women would be too costly in terms of infrastructure, in particular provision of testing and counseling, and that its safety and efficacy had not been sufficiently demonstrated in a South African context. The Supreme Court disagreed with both of these claims. The issue was "whether it was reasonable to exclude the use of nevirapine for the treatment of mother-to-child transmission at those public hospitals and clinics where testing and counseling are available". Regarding the safety and efficacy issue, the court cited scientific opinions which made these claims completely unreasonable.The Court did not address the issue of an appropriate allocation of resources. Accepting the Court's decision would not require the South African government to allocate additional resources to health care delivery nor would it require it to re-allocate existing resources for health:A potentially lifesaving drug was on offer and where testing and counseling facilities were available it could have been administered within the available resources of the state without any known harm to mother and child.In fact, the Court, as in the dialysis case, rejected any role for the courts in resource allocation decisions, instead adopting a criterion of evaluating government policies on the basis of a criterion of "reasonableness", from a previous right to housing case. In this previous case, the court would not decide "whether other more desirable or favourable measures could have been adopted, or whether public money could have been better spent" (41). A If one therefore follows the views of the South African Court regarding a right to health, it would seem to be difficult to use the courts to challenge a particular allocation of health care resources. The Court felt that the provincial administrations should make decisions as to how funds for health care should be spent and that courts should be "slow to interfere with rational decisions taken in good faith by the political organs and medical authorities whose responsibility it is to deal with these matters" (section 29).And"courts are not the proper place to resolve the agonising personal and medical problems that underlie these choices. Important though our review functions are, there are areas where institutional incapacity and appropriate constitutional modesty require us to be especially cautious. Our country's legal system simply cannot replace the more intimate struggle that must be borne by the patient, those caring for the patient, and those who care about the patient. The provisions of the bill of rights should furthermore not be interpreted in a way which results in courts feeling themselves unduly pressurized by the fear of gambling with the lives of claimants into ordering hospitals to furnish the most expensive and improbable procedures, thereby diverting scarce medical resources and prejudicing the claims of others" (section 58).One might, of course, want to criticize this view of the role of the courts, as has been done by Darrel Moellendorf. MoellendThe central issue with regard to differential access to health care services is, on what basis one can claim that a state does not spend a sufficient amount of resources on health care, relative to its general wealth, and on what basis can one claim that a state does not use the resources it devotes to health care appropriately. If a state does not allocate sufficient resources to health care, or uses its available resources inappropriately, a citizen could claim that the state violates her right to health when a particular health care intervention is denied her, and when that intervention would be available to her if the state increased its allocation to health care to an acceptable level, or re-allocated resources within the health care sector to an acceptable mix of interventions.in principle entitled to make judgments about when a particular treatment is too expensive to provide within its healthcare systems, given available resources, we have to find a way to examine claims about appropriateness, and provide criteria for how these judgments are to be made. At least in the two South African cases the Court has been reluctant to get involved in this type of questions.Focusing attention on this set of questions is important because they are at the heart of debates about health care access, in both resource rich and resource poor settings. We want to know whether denying a person access to a life-saving intervention because it is too expensive for a given health care system is a denial of his right to health. This can be the typical high cost therapies in rich countries, such as bone marrow transplantation, or it can be antiretroviral treatment for HIV in resource poorer countries. If we assume that a state is It should come as no surprise that this is the case. As the ever growing literature on the ethics of health care resource allocation demonstrates, there is no clear consensus about how one should balance the various legitimate concerns and values involved in making these kinds of decisions. Judgments will have to be made, and, unless there are grave violations of due process, or a gross misapplication of the principles of resource allocation, it seems unlikely that one could make a principled criticism of a particular decision that could be used as a basis for a claim against the state. This is probably why the courts have been reluctant to criticize allocation judgments made in good faith by government bodies.negative conclusion, I shall now nevertheless a strategy that shows that appeals to health as a human right might nevertheless be used to argue that denial of care on economic grounds is a violation of that right.In spite of this largely everyone, there should be equal access to health care, and health care should be affordable for all. It is fairly easy, and uncontroversial, to establish that a policy such as the one accepted during apartheid in South Africa, of denying access to health care services on the basis of race, is a violation of a right to health. It is more difficult to agree on how we should understand the principle of non-discrimination on the basis of social and health status. One might be tempted to conclude that the principle of non-discrimination on the basis of social status implies a right for everyone to access the same bundle of health care services. An unqualified acceptance of that claim, however, would obviously be unacceptable: there are going to be health care services which provide very little benefit, but which are hugely expensive, which may be accessible to the very rich, but which should not by any account be included in the bundle of health care services accessible to all, even by the most egalitarian standards of justice. We therefore need some account that will specify which health care services should be accessible to all, even within a rights approach to health care. In order to give a satisfactory account of that problem, we again need an appropriate value framework for prioritization. It seems to me that there is nevertheless something we can say, even before we have a fully worked out framework for prioritization, if we take a right to health approach. If a range of health care services, known to be effective in significantly preventing premature death or significantly increasing quality of life, is available to a substantial portion of a country's population, but is not available to a particular group of people, such as the very poor, this is a violation of the right to health of the members of that group. The qualifiers indicate that a lot of work needs to be done to give precise content to this claim, but for the purposes of this discussion, this somewhat vague claim will be sufficient. It is easy to identify examples: If antiretroviral treatment for HIV is available to most people in country, but is not available to people in the lowest 1% income bracket, we can conclude that there is a prima facie violation of their health rights.The central guiding principle in the international health and human rights documents is that of non-discrimination. It is prohibited to deny a person access to health care or its determinants on the basis of characteristics such as race or religion, but also, as we saw above, on the basis of social and health status: health care services should be accessible to Although one might agree that such a principle identified here does indeed follow from the international human rights documents, there are some obvious objections if one's aim is to show that an international human rights framework can be effective at mobilizing resources for health. First, if one accepts this principle of non-discrimination based on social status, why should one not accept a principle of non-discrimination based on health status, and argue analogously that if a small group of persons is denied access to health care services because of the illness that they happen to have, at the same time as the majority of the population has access to health care for their illnesses, this is an illegitimate form of discrimination? Second, can not a state use the same principled objection to mobilization of resources in cases of discrimination based on social status, that its limited resources are better used elsewhere? Third, are any real policy alternatives ruled out by this principle, or is it not so general that all types of health policies is compatible with it.The first criticism says that if we find it objectionable that the poor are treated differently from the rich with regard to health care access, why should we not find it objectionable that those who happen to have late stage breast cancer are treated differently from those who happen to have disease that can be treated more cheaply?Why could not a person with late stage breast cancer argue successfully that a decision not to fund the known effective treatment she suffers from is of the same type as a decision not to fund treatments the poor suffer from?One response to this objection would be that decisions regarding what constitutes a non-discriminatory policy are different in these two cases. When deciding whether who to give access to health care services in general, we do not discriminate on the basis of what illnesses people have: we look at other characteristics, such as gender, age, ethnic background, social status, and income level. When deciding what bundle of health care services to provide within a nation's health care system, other considerations are relevant. We would then look at the effect of the various interventions, their costs, how each individual benefits from the intervention, and what the cumulative benefit for the population is. A discriminatory practice in the first type of decision would be to single out a particular income group for preferential treatment, while a discriminatory practice of the second type of decision would be to single out a particular illness for preferential treatment. It would not be discriminatory to distribute resources on the basis of, for example, health effects of the various interventions. The reason why we have more problems in identifying inappropriate discrimination on the basis of health status is that there is less agreement on how we should identify interventions that have the same effect on health status across illness groups.The second objection is more difficult to answer. Let us assume that there is a country with a government that tries to allocate resources justly. Although the government tries its best, there continues to be lack of access to health care services to the poor. The government has also done its best in raising the resources allocated to health care, but they have still not been able to fund life-saving interventions for a particular group of patients. Because of its generally sound economic policies, the country has experienced more than the expected economic growth over the past few years, and there is a budget surplus. The government now has to decide what to do with the additional money: Should they remove continued barriers of access to the poor, or should they ensure funded for the neglected disease? We assume that the government cannot do both. It seems to me that the principle of non-discrimination within a human rights approach to health care does not provide us with guidance about how we should go about solving this type of problem.progressive realization of a right to health. This places an obligation on the country to do something to increase access to health care services during periods of economic growth. On that basis we could justifiably criticize government inaction, but we would not be justified in proscribe a particular action.In spite of this problem, there is one answer we can give, albeit a limited one, but still with a potential for identifying some inadmissible policies this imaginary country might want to consider. Within the human rights framework there is a recognition that any given country cannot fulfill its obligations immediately, but there is an obligation of The third objection is that the principle of non-discrimination is so general that it would not rule out any particular health policy. This is not the case. One currently fashionable proposal is that governments should identify an essential package of health services based on a criterion of cost-effectiveness. This package should be available to everyone, regardless of ability to pay. Those who are wealthy would be able to, or indeed encouraged to, pay for additional health care services using their own funds. WHO has called this proposal "the new universalism". Classic universalism would obligate governments to provide everything medically useful to everybody. How should we evaluate this proposal in terms of a human rights approach to health care delivery?Let me first examine the arguments in favor of this proposal. First, it is quite clear that the policy is pro-poor, in the sense that it would increase access to health care services for the poor compared with what is currently available to them. Data from many country shows that government subsidies today go disproportionally to the wealthy to pay for interventions of low cost-effectiveness, while the poor suffer from conditions which require interventions of high cost-effectiveness. Requiring that governments fund a package of services of high cost-effectiveness available to everyone would therefore shift government resources from the rich to the poor. Second, according to the principle of non-discrimination within the human rights tradition, governments should not discriminate against a particular group of people in terms of access to health care services. This policy seems to satisfy that requirement. The basic package is accessible to everyone, and the government does not subsidize health interventions for non-poor if they are not also subsidized for the poor, unlike what is the case in many countries today. For these arguments, see ,10. The Although many countries cite equity as the reason for strong government controls, public sector-controlled policies do not have a good track record on equity. In Indonesia, for example, the rich receive almost three times as much public health care as the poor. In China in the early 1980s, rural households-almost 80 percent of the population-received just 29 percent of public health spending. In Tanzania the richest fifth of the population use more than twice as many government hospital beds and more than four times as many outpatient services as the poorest fifth. In Côte d'Ivoire less than one-quarter of the rural poor who were sick received any form of medical care, as compared with half of the urban rich. In Peru only 20 percent of the poor received care, versus 57 percent of the rich. In general, when government expenditures are concentrated on urban areas and on hospitals rather than on basic services, the results are highly inequitable, governments are essentially subsidizing the rich. This conclusion only follows, however, if the principle of government non-discrimination only applies to the proportion of public resources which goes to different population groups. If the non-discrimination principle is limited in this way, and if the public system is available to everyone regardless of ability to pay, a two-tiered system where the wealthy have access to a much broader range of health care services because they are able to pay for the additional services from their own income is not in conflict with a right to health. If, on the other hand, one takes the position that a right to health should be understood in terms of equality of access to health services in general, then an explicit acceptance of at least some types of two-tiered systems is not in accordance with a right to health.There are a number of considerations which speak in favor of this expanded notion of government responsibility. A government does not only have an obligation to provide or to finance health care services. In fact, in recent discussions about health sector reform, these types of government obligations have been de-emphasized and instead there has been a focus on a government responsibility to regulate, facilitate and ensure that people have access to health care services, whether they are provided or financed by government, non-profit organizations or for profit companies. If one takes this role seriously, focusing only on how government resources are spent is at best only one component of government obligations to fulfill the right to health of their population. Persistent inequalities of access to health care, whether they are caused by imbalances in public resources, or caused by differences in income between population groups, should therefore be regarded as an essential concern of equity and rights oriented policies.particular corrective to that violation of health as a human right. All one can say is that the state has an obligation to do something about the injustice.I have in this paper argued that appeals to health as a human right are not going to be helpful if we want advice on how we should allocated resources among different patient groups. Many of the most difficult problems of resource allocation are therefore not addressed by a human rights approach. However, I have also argued that some inequalities of access can be criticized: One can say that it is a violation of human rights if one group systematically has less access to health care services compared with other groups. There is, however, not much more on can say; in particular, one cannot proscribe a something has been done when the next periodic report is done to and they would have to take steps to improve access in some respects. While this may seem to be a modest requirement, it can, together with appropriate political pressure, lead to significant, positive changes in health care systems. But it is undeniable that it is much less than what many who advocate a human rights approach to health care access hope can be achieved by utilizing this framework. It is also true that it is generally recognized that the reporting requirements, at least with regard to health, are not very effective. Strengthening the reporting and monitoring mechanisms of the UN system with regard to health should therefore be the highest priority for those who want to emphasize a human rights approach to health sector reform.This result may not be very satisfactory for those who are concerned about doing something about lack of access to health care. However, if one takes the requirement of progressive realization seriously, together with the periodic reporting requirement, one might conceivably have a powerful basis for criticism of government policies. Governments are obligated to report to the UN, and, as I have shown, there is a basis on which one can criticize them for not fulfilling their health rights, also with regard to their economic policies. This creates an obligation on countries to show that The opinions expressed are the author's own. They do not reflect any position or policy of the National Institutes of Health, Public Health Service, or Department of Health and Human Services.The pre-publication history for this paper can be accessed here: