During the last decades studies addressing the effects of exposure to Extremely Low Frequency Electromagnetic Fields (ELF-EMF) have pointed out a possible link between those fields emitted by power lines and childhood leukaemia. They have also stressed the importance of also including in the assessment the contribution of frequency components, namely harmonics, other than the fundamental one. Based on the spectrum of supply voltage networks allowed by the European standard for electricity quality assessment, in this study the exposure of high-resolution three-dimensional models of foetuses to the whole harmonic content of a uniform magnetic field with a fundamental frequency of 50 Hz, was assessed. The results show that the main contribution in terms of induced electric fields to the foetal exposure is given by the fundamental frequency component. The harmonic components add some contributions to the overall level of electric fields, however, due to the extremely low permitted amplitude of the harmonic components with respect to the fundamental, their amplitudes are low. The level of the induced electric field is also much lower than the limits suggested by the guidelines for general public exposure, when the amplitude of the incident magnetic field is set at the maximum permitted level. It is well known and widely accepted that exposure to Extremely Low Frequency (ELF) electromagnetic fields (EMF) causes well-defined acute biological effects on the nervous system, which include in particular nerve stimulation as well as the induction of retinal phospenes ,2,3,4. Fi.e., causes of introduction of harmonics) are spreading due to the development of new technologies, which use non-linear loads, in common electrical appliances and industrial equipment. This spread, besides impairing the quality of the supplied electrical power and producing damage to the devices, can increase consistently the amount of delivered power and thus increase the levels of exposure in an uncontrolled way [However, many authors have stressed the possible influence on ELF health effects of frequency components other than the fundamental 50 Hz one . To that purpose, the networks are clustered into three classes, as a function of the root mean square (rms) value of the supply voltage of the fundamental component: Low Voltage (LV), Medium Voltage (MV) and High-Voltage (HV). Quantitatively, EN 50160 [So far, only a few studies have dealt with the estimation of the harmonic components of a uniform magnetic field. The scarce available data  doesEN 50160  limits tIn this study, as worst-case exposure scenario, the amplitude over time of the public transmission and distribution networks voltage is set as the time summation of the fundamental component to all the harmonic components up to the twenty-fifth, each one set at the maximum percentage of the fundamental permitted by the standard. Considering the need to keep the time cost of the computational procedure reasonable (see below) and that extremely low amplitude components are expected to minimally contribute to the exposure, harmonics mandated to be of maximum amplitude lower than the 3% of the fundamental (2.5% for HV) have been neglected in our analysis. A, , can be calculated from the scalar potential φ by:Simulations of the exposure to a magnetic field as described in the section above, have been conducted with the Magneto Quasi-Static low frequency solver of the simulation platform SEMCAD X v. 14.8.4  , based oThe exposure of three anatomical models of pregnant women and corresponding foetuses at 3, 7 and 9 months of Gestational Age (GA), has been simulated here. Those models were obtained from insertion of the corresponding models of foetus in the “non-pregnant” woman model “Ella” of the Virtual Family , partialB and the foetal position  in the bone tissue  and in the whole body of foetus, for each GA. In details: -front, for the 3 months GA (mGA) foetus;B-lat, for the 7 mGA foetus;B-front, for 9 mGA foetus.B where Bfront, Blat, and Btop means front-to-back, lateral (left-to-right) and top-to-bottom exposure, respectively. In the case of the 3 mGA foetus, the posture that induces the highest electric field E in the bone tissue and in the whole body of foetus corresponds to the foetus with the head up [The orientation of the uniform magnetic field position  have bee head up .et al. [-i.e., dermis [The skin conductivity was set to 0.1 S/m for all frequencies to take into account the higher conductivity of deeper granular tissue, , dermis .-Placenta and umbilical cord were modelled as blood because of their high perfusion .-The conductivity of the amniotic fluid was set to 1.28 S/m, 1.27 S/m and 1.10 S/m at 3, 7 and 9 months GA, respectively, and taken constant across all frequencies ,46.-An average value between grey matter and white matter conductivities was assigned to medulla oblongata, pons and midbrain tissues (as in ).-The lowest conductivity among the ones of the woman’s brain tissues was assigned to the foetal brain from a conservative point of view, to obtain higher electrical conductivity contrast with the embedding cerebrospinal fluid (CSF) (as in )-i.e., 650 Hz).The foetal bone conductivity was set to 0.35 S/m as in  taken as-et al. [Similarly, fat conductivity was set at 0.12 S/m, estimated as a blend between fat and muscle conductivities, according to the suggestion of Peyman et al. , relatedThe dielectric properties were set according to the frequency of the harmonic component under simulation. For the fundamental component at 50 Hz, the dielectric properties have been set as in . Since tet al.  databaseet al. ,42,43,44rms) of the electric field averaged over cubes of 2 × 2 × 2 mm3 was calculated, as suggested by the ICNIRP exposure guidelines [3 of contiguous tissue cannot be found , the averaging has been performed over a smaller volume that includes only the specific tissue under analysis. The analysis was then accomplished by extracting the peak, the 99th and the 50th percentile  of the E distribution of each tissue. The whole body foetus Epeak, E99th and E50th are here considered as the maxima Epeak, E99th and E50th found over all the tissues.The exposure assessment was based on the estimation of the electric field induced both in the whole-body foetus and in each foetal tissue taken separately. In particular, the root mean square  is the magnetic flux density at the frequency j, and R,jB (μT) is the reference level of the magnetic flux density at the same frequency j. Considering that the relationship between the amplitude of B50Hz and the one of the other harmonics is based on the relative percentage as shown in 50Hz can be easily computed by Equation (2). The expanded equation becomes: jB is calculated taking into account the relative percentage of the harmonics content with respect to the fundamental  as allowed by the EN 50160  according to jE (mV/m) is the internal electric field strength induced at frequency j, and EL,j is the induced electric field strength restriction at frequency J.  and bladder (E50th), skin (Epeak and E99th) and liver (E50th at 50 Hz) and brain (E50th at harmonic frequencies) and skin (Epeak and E99th) and spinal cord  for 3, 7, 9 months GA foetuses.The values reported were found in the fat  and amo99th over the CNS tissues, for all frequencies and across all the GAs were found in the brain at all the GA . The maximum induced E99th over all other tissues were found over fat , skin  and skin  for 3 mGA, 7 mGA and 9 mGA, respectively. As expected, the electric field levels at the fundamental components due to the HV electricity transmission networks are higher than those ones due to the LV&MV electricity distribution networks, whereas an opposite behaviour has been found for the harmonic components. The comparison with the exposure standards was also analysed in terms of maximum fraction of the Basic Restrictions proposed by the ICNIRP guidelines [The peak of the electric field is always at the fundamental frequency of 50 Hz and increases progressively with the stage of pregnancy. In particular, it ranges from 0.94 and 3.92 mV/m over CNS tissues of the head and from 3.35 to 9.1 mV/m over all other tissues, passing from 3 mGA to 9 mGA, respectively. The maximum EAs one can see from this figure, all foetuses are exposed to levels far below the Basic Restrictions  for bothThe main outcome of this study is to provide a full picture of the EMF exposure of three realistic models of foetus at different gestational ages to electricity transmission and distribution networks. In particular, the source of EMF is here modelled taking into account the maxima spectral harmonic characteristics of the networks as provided by the quality standard EN 50160. It is important to note that the exposure scenario modelled in this study is worse than the one due to a realistic power supply network. Indeed, the few data available in literature ,48,49) s,49 s48,4The first evidence coming from the analysis of the exposure to the full harmonic spectrum of the electricity transmission and distribution networks derives from the extremely low relative amplitude between the main component at 50 Hz and its harmonics (here ranging from 2.5% to 6.0%). Indeed, the induced electric fields due to the harmonic components are far lower than those ones induced by the main frequency at 50 Hz. As an example, in the foetal bone (one of the tissues considered of interest in leukaemia studies) and considering a source at the limit of the quality standard EN 50160  Figure , the relAgain, this strong reduction of induced fields with frequencies can be also seen when the characteristics of the sources are set also considering the limit of the exposure levels (ICNIRP reference levels ) as showpeak, E99th and E50th, has been found of 2.3, 2.5 and 1.5, respectively passing from 3 to 7 months GA, and about 3.2, 3.4 and 2.2, respectively, passing from 3 to 9 months GA , as expected, are also the most superficial ones for all GA and the frequencies considered  strongly depend on the combination of foetal position and size and magnetic field polarization. At 3 months GA they are bladder, spinal cord, kidney and spleen, at 7 months GA brain, liver, skin and SAT, at 9 moths GA again brain, skin, SAT, lung and spinal cord. At 7 mGA, the maximum E50th change position depending on frequency, passing from the liver (at 50 Hz) to the brain (at harmonic frequency); this is due the impact of the different choice of dielectric properties values  of these two tissues, which then affect mildly the whole electric field distribution. As for the whole-body electric fields, the levels of Epeak, E99th and E50th show an increasing trend with the stage of pregnancy. Looking specifically at the bone tissue, the increase with foetal growth is even more conspicuous, suggesting that the foetuses close to the birth are exposed to larger induced electric fields. In particular, on average, along frequencies, it is equal to about 3.4, 3.2 and 4.0 for Epeak, E99th and E50th, respectively passing from 3 to 7 months GA, and about 4.6, 3.4 and 5.7 for Epeak, E99th and E50th, respectively, moving from 3 to 9 months GA.As to the tissue dosimetry, the most exposed tissues  discussed above, the results of Liorni and colleagues apply also in this study.Finally yet importantly, the results of this study, as for any result obtained from computational assessment studies, are affected by a variable degree of uncertainty. Liorni and colleagues  discusseBesides that, the major source of uncertainty linked to the results of this study is the representativeness of the pregnant woman models and the tissue parameters of the whole population included in the process of risk assessment.To this purpose, the accuracy of the fetal anatomy was already discussed by  where soAs to the dielectric properties, the main source of uncertainty is given by the intrinsic variability due to the tissue inhomogeneity and to the large difference in tissues structure and composition. The measurement of the dielectric properties of foetal tissues here used, have been performed by Gabriel and colleagues , who obsOther considerations and uncertainty assessment relative to the pregnant woman models are discussed in ,39,53,55In conclusion, this study showed that in the exposure to the magnetic fields generated by electricity transmission and distribution networks, the harmonic components add some contributions to the overall level of the electric field induced in foetal tissues. However, although these contributions increase with frequency and cannot be neglected, their amplitude is low, due to the extremely low permitted amplitude of the harmonic components with respect to the fundamental one, even in the worst-case scenario here considered. The levels of the electric field in the foetus tissues are also extremely lower than the limits suggested by the ICNIRP 2010 guidelines  for gene


The aim of this study was to investigate the protective effect of erythropoietin (EPO) against acute myocardial injury and its underlying mechanisms. Mice (n=146) were randomly divided in a double-blind manner into four groups, sham, Rocephin, EPO and sepsis, and mortality was observed on the seventh day after cecal ligation and puncture. In addition, a total of 252 rats were randomly divided into three groups, sham, EPO and sepsis, and indicators of cardiac function, inflammatory mediators and serum creatine kinase levels were assessed. Mitochondrial membrane potential, cell apoptosis and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) p65 expression levels were detected using flow cytometry. Following intervention with EPO, the mortality rate in mice with sepsis was significantly reduced and the cardiac function of septic rats was significantly improved. In addition, the levels of inflammatory mediators, serum creatine kinase and apoptosis and the myocardial mitochondrial membrane potential and expression of NF-κB p65 in cardiac tissue were all improved following EPO treatment, and the differences between the results for the sepsis and EPO groups were statistically significant (P<0.05). These findings suggest that EPO reduces the myocardial inflammatory response in septic rats, attenuates the reduction in mitochondrial membrane potential and inhibits myocardial cell apoptosis by reducing NF-κB p65 expression, and therefore exerts a protective effect in the myocardium. Sepsis is a systemic inflammatory response syndrome  caused bIn myocardial injury during sepsis there is a decline in cardiac function. This decline in cardiac function is an important mechanism for refractory hypotension, which seriously affects the prognosis of sepsis. For the treatment of sepsis, the timely administration of antibiotics can effectively kill pathogens to reduce the rate of mortality . In the Cytokines can act in either a pro- or anti-inflammatory manner. The group of proinflammatory cytokines has been shown to include tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-8, while the anti-inflammatory cytokines, which inhibit the inflammatory response, include IL-10. The interactions and dynamic balance between pro- and anti-inflammatory cytokines play an important role in maintaining the stable internal environment of the body; in certain cases an imbalance can result in an increase in the inflammatory response and organ dysfunction, leading to the occurrence of MODS .et al , and the protective effect of EPO was investigated. The effects of EPO were studied from a physiological, pathological and molecular perspective, and indicators of cardiac function, the inflammatory response, serum creatine kinase (CK), myocardial histopathology and cardiomyocyte apoptosis were detected to determine whether EPO exerted a protective effect on the heart and other major organs  in sepsis. The molecular mechanism of EPO was also investigated to provide a theoretical basis for clinical therapeutic applications.A total of 146 male, clean-grade Kunming mice  and a total of 252 healthy male, clean-grade Sprague Dawley (SD) rats (weighing between 210 and 250 g) were used in the study. All the animals were provided by the Experimental Animal Center of Hebei Medical University , were raised according to the requirements of clean-grade mice/rats and had free access to water. This study was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Third Hospital of Hebei Medical University.The grouping of animal models was divided into two parts: i) Grouping of healthy Kunming mice, which were used for the assessment of mortality rates following CLP surgery and ii) grouping of healthy SD rats, which were used for the observation of pathophysiological indicators following CLP surgery.For the first part of the study, 146 Kunming mice were randomly divided in a double-blind manner into four groups: Sham , Rocephin , EPO intervention  and sepsis . A total of six mice succumbed to mortality and were, therefore, not included. For the observation of pathophysiological indicators, 252 healthy SD rats were randomly divided in a double-blind manner into three groups: Sepsis , EPO intervention  and sham . Of the 252 rats, 12 succumbed to mortality due to factors, including anaesthesia and temperature.The animals were allowed to acclimate for one week subsequent to purchase and were fasted overnight before the start of the experiment, but with free access to water . The sepsis animal model was created using line CLP in accordance with a previously reported method .Following the establishment of the sepsis model, EPO at a dose of 1,000 IU/kg was immediately and intraperitoneally injected into the animals in the EPO group and Rocephin at a dose of 1 mg/20 g was immediately intraperitoneally injected into the mice in the Roc group. In the Sham group, the abdominal cavity of the animals was opened and the abdominal incision was sutured once the cecum had been located; however, the cecum was not punctured.max) and the maximum rate of left ventricular pressure decline (−dP/dtmax) were calculated using the physiological recorder associated with Chart 4.0 software .Following the CLP procedure, SD rat models from each group were taken at 0, 3, 6, 12 and 24 h. A catheter connected to a pressure transducer was inserted into the left ventricle of the heart via the common carotid artery. The pressure curve of the left ventricle was recorded, and the left ventricular systolic pressure (LVSP), the maximum rate of left ventricular pressure rise  was obtained from the inferior vena cava for the detection of cardiac troponin I (cTnI), C-reactive protein (CRP), TNF-α, IL-6, IL-10, CK, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) using ELISA, in accordance with the manufacturer’s instructions.6cells/ml.SD rat models from each group were taken 24 h after the CLP procedure, and the fresh tissues  were used to prepare single cell suspensions. The preparation of single cell suspensions was performed, as follows. Fresh tissues were placed onto a 120 mesh stainless steel net  above a petri dish. The tissues were cut using ophthalmic scissors and gently rubbed with the scissors whilst rinsing with normal saline. The suspensions in the petri dish were filtered with a 300 mesh copper mesh filter  to remove cell clumps. The cell suspension was collected and precipitated by centrifugation at 829 × g for 2 min. The precipitate (single cell suspension) was collected, the supernatant was removed and the concentration was adjusted to 1×10Apoptosis was detected as previously described . A total6 cells/ml, and the cells were incubated for 30 min at room temperature in the dark. The cells were then washed with10 ml phosphate-buffered saline (PBS). The supernatant was subsequently removed and analyzed using flow cytometry (Epics of America XL II Flow Cytometry instrument).A total of 100 μl rhodamine 123 was added to 0.1 ml single cell suspension with a concentration of 1×106 cells/ml, and the cells were incubated for 30 min at room temperature in the dark. The cells were then washed with 10 ml PBS and the supernatant was removed. A total of 100 μl secondary antibody working solution of goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase was added, and the cells were incubated for 30 min at room temperature in the dark. A total of 10 ml PBS was then added to the cells, prior to centrifugation. The supernatant was subsequently discarded to remove any unbound fluorescent secondary antibody, and 0.1 ml PBS filtered with 500-mesh copper mesh was added prior to detection using a flow cytometer. Background and negative controls for the primary and secondary antibodies were established when determining protein immunofluorescence markers. The samples were analyzed using flow cytometry (Epics of America XL II Flow Cytometry instrument).A total of 100 μl rabbit anti-rat NF-κB p65 primary antibody was added to 0.1 ml single cell suspension with a concentration of 1×10Tissue sections were prepared using the conventional wax block method and stained with hematoxylin and eosin, prior to being observed and photographed under a light microscope.The data were analyzed using the SPSS 13.0 statistical software package  and all data are presented as the mean ± standard deviation. The inter-group differences were tested using the bilateral t-test for independent samples. P<0.05 was considered to indicate a statistically significant difference.Three hours after surgery, the sham-operated mice returned to a normal state. The rates of mortality in the mice in the Roc and EPO groups at 24 and 48 h were significantly reduced compared with those in the CLP group (P<0.01) .max and −dP/dtmax values in the SD rats with sepsis were significantly decreased compared with those in the sham-operated rats (P<0.01). Rats in the CLP group exhibited decreases in the LVSP, +dP/dtmax and −dP/dtmax after 3 h, with the lowest values observed after 6 h. The values obtained in the CLP group were significantly lower than those obtained in the Sham group (P<0.01). The LVSP +dP/dtmax and −dP/dtmax values of the rats in the EPO group demonstrated partial restoration and were significantly higher compared with the values of the rats in the CLP group at 3, 6, 12 and 24 h (P<0.01)  .As shown in Three hours after the CLP, the levels of TNF-α and IL-6 in the rats in the CLP and EPO groups peaked and were significantly increased compared with those in the rats in the Sham group (P<0.01). The levels of TNF-α and IL-6 in the rats in the CLP and EPO groups then progressively decreased and reached the lowest value 24 h after surgery; the difference between the two groups was significant (P<0.01).Six hours after the CLP, the levels of IL-10 in the rats in the CLP and EPO groups peaked and were significantly increased compared with those in the rats in the Sham group (P<0.01). The levels of IL-10 in the CLP and EPO groups then showed a downward trend .As shown in Levels of CK increased 3 h after the CLP and then decreased at 6 h, prior to increasing again at 12 h and showing a continued increasing trend. Twelve hours after the CLP, the CK levels in the SD rats in the CLP and EPO groups were significantly higher than those observed in the Sham group (P<0.05).Following the CLP, the levels of AST and LDH in the SD rats increased progressively. Twelve hours after the CLP, statistically significant differences were observed in the levels of AST and LDH between the rats in the EPO group and those in the Sham group (P<0.01), and the serum AST and LDH levels were significantly lower in the rats in the EPO group than those in the rats in the CLP group (P<0.01) .The results showed that 24 h subsequent to the CLP, the early and late apoptosis rates of the major organs  of the CLP and EPO groups were significantly increased compared with those in the Sham group (P<0.01). The early apoptosis rate of the EPO group, however, was significantly lower than that of the CLP group (P<0.01) .Twenty-four hours after the CLP, significant differences were observed in the mitochondrial membrane potential of the major organs  between the CLP/EPO groups and the Sham group (P<0.01). When the CLP group was compared with the EPO group, the mitochondrial membrane potential of the major organs was shown to be significantly reduced (P<0.01). In the results from the flow cytometric analysis, the fluorescence peaks of the rat organ cells from the CLP and EPO groups were significantly shifted to the left when compared with those from the Sham group, i.e. the number of cells with low fluorescence intensity increased. This indicated that the mitochondrial membrane potential was significantly reduced (P<0.01).Following treatment with EPO the fluorescence peaks of the rat cells from the EPO group were significantly shifted to the right compared with those from the CLP group, i.e. the number of cells with strong fluorescence intensity increased. This demonstrated that the reduction in mitochondrial membrane potential was significantly attenuated in the EPO group (P<0.01) .Twenty-four hours after the CLP, a significant difference was observed in the NF-κB p65 expression in the major organs  in the CLP and EPO groups compared with that in the Sham group (P<0.01). Comparing the EPO group with the CLP group, the expression of NF-κB p65 was significantly reduced (P<0.01) .Pathological sections of the rat hearts 24 h after the CLP are shown in Pathological sections of the rat livers 24 h after the CLP are shown in Pathological sections of the rat kidneys 24 h after the CLP are shown in Pathological sections of the rat lungs 24 h after the CLP are shown in In the first part of the present study, mice treated with Rocephin (Roc group) were used as a positive control group. The results showed that treatment with EPO significantly reduced the mortality of septic Kunming mice. In the second part of the study, involving SD rats, pathological alterations in the major organs  were observed in sepsis; however, following treatment with EPO the pathological alterations in the organs were significantly reduced, indicating that the use of EPO may improve the prognosis of sepsis and MODS, and therefore reduce mortality.max are sensitive indicators of myocardial systolic function and −dP/dtmax is a sensitive indicator of myocardial diastolic function. In the present study it was demonstrated that the LVSP, +dP/dtmax and −dP/dtmax were significantly reduced in septic SD rats compared with those in sham-operated rats (P<0.01). In the present study, the observation of cardiac hemodynamics suggested that +dP/dtmax, −dP/dtmax and LVSP of the left ventricle in the septic rats was significantly reduced compared with that of the normal control group. The significant decrease of LVSP, +dp/dt max and −dp/dt max index of the CLP group appeared at 3 h and reached the lowest point at 6 h, which was significantly lower compared with that of the control group (P<0.01). The +dp/dtmax, −dp/dtmax and LVSP of the EPO rats was gradually restored. These results demonstrated that EPO assisted in the maintenance and recovery of cardiac systolic and diastolic function in rats with sepsis.Hemodynamic indicators of the heart can comprehensively reflect the degree of heart damage following sepsis and the protective effect of drug intervention. Among these indicators, LVSP and +dP/dtcTnI is an indicator that has been found to reflect the degree of myocardial injury after sepsis and the protective effect of drug intervention with high sensitivity and specificity ,23. In tIn the present study, pathological sections of the major organs  were observed. Under the light microscope, a large number of ruptured cardiac myofilaments, myocardial cell edema and severely damaged and disordered muscle fiber structures were observed in the heart 24 h after the CLP, as well as the infiltration of inflammatory cells into the myocardial tissue. Following treatment with EPO, the pathological damage to the heart was significantly reduced, and the degree of damage to the other major organs  was also significantly reduced, suggesting that in the early treatment of sepsis, attention should be paid to the protection and support of the functions of the heart and other organs in order to prevent the occurrence of multiple organ failure and to reduce the sepsis-associated mortality rate.CRP is an acute phase reactive protein expressed when acute inflammation occurs in the body; in the clinic, it is often used to monitor infectious diseases. The prognosis of sepsis and the inflammatory state of the body may be reflected by the level of serum CRP. In the present study, CRP was significantly increased 6 h after CLP, which further suggested that an inflammatory response occurred in the animal models. The levels of CRP decreased following treatment with EPO.The results of this study demonstrated that, 3 h after the CLP, the levels of serum TNF-α and IL-6 in the CLP and EPO groups were significantly increased compared with those in the Sham group; however, the peak of the EPO group was lower compared with that of the CLP group. This suggests that excessive cytokine release occurred during the progression of sepsis and that a significant inflammatory response was present in the body during the early stages of sepsis. As an anti-inflammatory substance, EPO inhibits the release of TNF-α and IL-6 and therefore inhibits the excessive inflammatory response. As a result, EPO reduces the pathological damage caused by the inflammatory response of tissues and organs and acts to protect the myocardium. This is conducive to the prevention of the occurrence and development of MODS during sepsis, and therefore improves the prognosis of sepsis. In the present study, the serum levels of IL-10 were significantly increased at 3 h and reached a peak at 6 h, whilst the serum levels of TNF-α and IL-6 at 6 h were significantly reduced compared with those at 3 h after the CLP (P<0.05). After 12 h, the serum levels of IL-10 gradually decreased, and the serum levels of TNF-α and IL-6 also showed a declining trend, indicating that the inflammatory factors of septic rats were decreased by varying degrees. IL-10, as anti-inflammatory cytokine, inhibits the synthesis of proinflammatory cytokines, thus helping to reduce the systemic inflammatory response and restore the balance of the inflammatory response.The results of this study demonstrated that, 24 h subsequent to sepsis injury, the early and late apoptosis rates of the heart, liver, kidney and lung cells in the EPO and CLP groups were significantly increased compared with those in the Sham group. The early apoptosis rate of the cells in the EPO group following injury was significantly lower than that of the cells in the CLP group. These results suggest that EPO may inhibit cardiomyocyte apoptosis following sepsis, and the administration of exogenous EPO may reduce cardiac apoptosis caused by septic damage.A potential mechanism underlying the inhibition of myocyte apoptosis by EPO is that EPO acts through the mitochondrial pathway. In the present study rhodamine 123 was used to detect alterations in mitochondrial membrane potential in the three groups using flow cytometric analysis. The results of the study confirmed that the mitochondrial function in the major organs was affected in sepsis, and that mitochondrial membrane potential was decreased. In the EPO group, the fluorescence peak was significantly shifted to the right in the rats cells of the major organs  and the fluorescence intensity of the cells was increased compared with the CLP group. This indicates that, in the EPO group, there was a significant attenuation in the reduction of mitochondrial membrane potential; thus, apoptosis was inhibited. These results suggest that EPO inhibits apoptosis through the mitochondrial pathway.In the present study, the activation of NF-κB was examined by determining the expression levels of NF-κB p65 protein. The results demonstrated that the expression of NF-κB p65 protein in septic rats in the EPO and CLP groups was significantly increased compared with that in the rats in the Sham group. Following treatment with EPO, the expression of NF-κB p65 protein was significantly lower than that in the CLP group. The inflammatory pathology of the organs and tissues  in the septic rats was also significantly reduced. Combining the findings for the levels of TNF-α, IL-6 and IL-10, as well as the expression of CRP and NF-κB p65 protein, the results suggest that EPO decreased the expression of its downstream inflammatory mediators by inhibiting the activation of NF-κB, and, as a result, reduced the inflammatory damage to the heart tissue of septic rats. In addition, levels of the serum anti-inflammatory factor IL-10 were increased following intervention with EPO, which promoted the restoration of the anti-inflammatory balance; this was one of the mechanisms of inflammatory damage mitigation in the tissues of septic rats.These anti-inflammatory and protective effects of EPO cells are consistent with previous studies in animal models of sepsis and inflammation. The pre-and post- use of EPO have been observed to inhibit injury. In an animal model of necrotizing pancreatitis, EPO reduces sepsis-induced acute lung injury and exhibits improved maintainence of microvascular cell integrity . In the In evaluating a new treatment program, the comparable side effects require consideration. The data of patients with renal failure indicated that, the risk of thrombotic events increased when hemoglobin exceeded 12g/L ,26. The In conclusion, sepsis is caused by the induction of the inflammatory cascade by endotoxin, resulting in the excessive release of inflammatory mediators from the body and leading to an imbalance of pro- and anti-inflammatory factors. This inflammatory response activates NF-κB. Therefore, treatment targeting only one or two types of inflammatory mediators is ineffective; in order to effectively inhibit the disease development and improve prognosis, intervention should be targeted against the transduction signals from the source. Therefore, NF-κB, the central factor of multiple inflammatory pathways, is an important target for treatment, and blocking the activation of NF-κB may prevent further deterioration caused by the disease. The results of this study showed that NF-κB is involved in the processes leading to the inflammation-induced tissue damage in septic rats. EPO inhibits NF-κB and regulates and promotes the anti-inflammatory balance, thereby alleviating tissue injury in septic rats and exhibiting a protective role. This provides a molecular mechanism of action for the application of EPO in the clinical treatment of sepsis. However, the potential side effects and inadequacies of EPO highlighted in the present study should be considered in the evaluation of a new treatment program. Data from patients with renal failure and acute myocardial infarction showed that the risk of thrombotic events was increased when the concentration of hemoglobin was >12 g/l ,26. Furt


ATP13A2, PARK2, PARK7, PINK1, LRRK2, SNCA, ALDH1A1, PDHB, PPARGC1A, and ZNF746 genes in the peripheral blood of patients was carried out using reverse transcription followed by real-time PCR. A statistically significant and specific increase by more than 1.5-fold in the expression of the ATP13A2, PARK7, and ZNF746 genes was observed in patients with PD. Based on these results, it can be suggested that the upregulation of the mRNA levels of ATP13A2, PARK7, and ZNF746 in untreated patients in the earliest clinical stages can also be observed in the preclinical stages of PD, and that these genes can be considered as potential biomarkers of the preclinical stage of PD.Parkinson's disease (PD) is a widespread neurodegenerative disorder. Despite the intensive studies of this pathology, in general, the picture of the etiopathogenesis has still not been clarified fully. To understand better the mechanisms underlying the pathogenesis of PD, we analyzed the expression of 10 genes in the peripheral blood of treated and untreated patients with PD. 35 untreated patients with PD and 12 treated patients with Parkinson's disease (Hoehn and Yahr scores 1-2) were studied. An analysis of the mRNA levels of  Parkinson's disease (PD) is a widespread neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons of the midbrain . RestingTo date, several studies of transcriptome alterations have been carried out in the postmortem brains of patients with PD. The patients analyzed in those studies were in the last and most severe stages of the disease and received active medication –9; thereVDR, HIP2, CLTB, FPRL2, CA12, CEACAM4, ACRV1, UTX, and SRRM2) exhibited changed expression in the blood of patients with PD [Because it is very difficult to study endogenous processes that occur directly in the brain of patients with PD during the early symptomatic stages, one of the main approaches that is used to bypass such difficulties is the investigation of changes in gene expression in the fraction of peripheral blood lymphocytes. These cells have several features that are specific to DA neurons, for example, as they produce dopamine receptors and enzymes involved in the synthesis of dopamine –13. Rece with PD , 22. AltATP13A2, PARK2, PARK7, PINK1, LRRK2, and SNCA) and genes that exhibited a change in expression that may be associated with the pathogenesis of PD . Previously, it was shown that the expression level of ALDH1A1 is high in mesencephalic neurons but that the mRNA level of this gene is decreased in patients with PD [ PDHB encodes a subunit of pyruvate dehydrogenase and is associated with mitochondrial functioning. It was shown that an increase in the concentration of pyruvate in the plasma is specific to patients with PD [ PPARGC1A, the PGC-1α protein, is a transcriptional activator of mitochondrial biogenesis that, at the same time, protects neurons from oxidative stress by suppressing reactive oxygen species [ ZNF746 is highly expressed in the substantia nigra of the brain of mice, and its protein product, PARIS, is involved in mitochondrial biogenesis via the regulation of PGC-1α [To understand better the mechanisms underlying the pathogenesis of PD, we analyzed the expression of 10 genes in the peripheral blood of two groups of patients with PD  who were in stages 1-2 of the disease . The genes that were chosen for this investigation were selected based on an analysis of data published previously. The list included genes involved in the development of monogenic forms of PD  were diagnosed with PD at the Research Center of Neurology, Russian Academy of Medical Sciences (RAMS), and did not have family history of PD. All patients with PD were selected and studied according to the international Unified Parkinson's Disease Rating Scale (UPDRS) and Hoehn and Yahr scores , 31. TheAll patients were examined for the presence of frequent mutations in all genes responsible for monogenic forms of PD by using SALSA MLPA P051 Parkinson mix 1 probemix and SALSA MLPA P052 Parkinson mix 2 probemix .In this work, the following groups of patients with PD were studied: (1) untreated patients with the sporadic form of PD  in the moment when blood was taken ; (2) treated patients with the sporadic form of PD  . The mean age ± SD at disease onset was 58.6 ± 7.4 years (range: 46–71), and the mean age at enrollment was 59.2 ± 7.1 years (range: 47–73). Patients with PD had no severe concomitant diseases, such as severe cardiovascular disease, cancer, or diabetes mellitus. Treated patients with PD received different medications , either as monotherapy or in various combinations. Forty-four neurologically normal age-matched individuals and 21 patients with different neurological disorders , Charcot-Marie-Tooth disease (18.4 ± 3.6), Wilson's disease (22.8 ± 5.2), and cerebellar ataxia (34.4 ± 3.7)) from the same population were studied as controls.All blood samples were collected with the informed consent of the investigated persons. The study was approved by the Ethics Committee of the Research Center of Neurology.Blood samples were collected from subjects at 8 a.m. before eating and stored at +4°C for no more than 2 h before RNA isolation. Total RNA was isolated from peripheral blood using a ZR Whole-Blood Total RNA Kit . RNA quantities were determined by spectrophotometry using a Quant-iT RNA BR Assay Kit and a Qubit fluorimeter . RIN values were measured by using The Agilent 2100 Bioanalyzer system  and were above 8.The gene expression analysis was carried out using the reverse transcription reaction and real-time PCR with TaqMan probes. Single-stranded cDNAs were produced in three replicates using 50 ng of total RNA, a specific reverse primer , and theThe sequences of gene-specific primers and probes are presented in Real-time reactions were performed using an ANK-32 machine  and PCR reagents . Thermal cycling was performed as follows: (1) 600 s at 95°C and (2) 45 cycles of 50 s at 95°C and 20 s at 60°C. POLR2F and PSMA5 transcripts were used as reference genes to normalize gene expression data [For each cDNA, the reaction was replicated thrice.ion data . ThreshoThe sequences of the primers and probes were designed using Beacon designer 7.0 software .R = 2−ΔΔCp [U test with “Statistica for Windows 8.0” ) and MS Excel 2010 (Microsoft). ROC curves and PCR efficiency and specificity were obtained by using and Real Statistics Resource Pack of MS Excel 2013 (Microsoft).Relative levels of the transcripts in the test groups were calculated as = 2−ΔΔCp . The lev ALDH1A1, ATP13A2, LRRK2, PARK2, PARK7, PINK1, PDHB, PPARGC1A, SNCA, and ZNF746 was performed in the peripheral blood of treated and untreated patients at the early stages of PD . These transcripts were also analyzed in the control neurological disease group, which included patients with different neurodegenerative diseases , to assess the specificity of the observed changes of transcript levels in the context of the pathogenesis of PD. During the work, it was shown that the representation of the transcripts of PARK2 and PPARGC1A in human lymphocytes was below the level of detection of the method used in this investigation. The results of the expression analysis of the remaining genes are shown in An analysis of changes in the mRNA levels of the genes ATP13A2, PARK7, and ZNF746 genes was observed in patients with PD. These changes were specific to PD, because the expression levels of these genes in the neurological control group did not differ from the respective levels of expression in healthy controls. Changes in the levels of transcripts were observed in the groups of untreated and treated patients with PD. It should be noted that the changes in the transcript levels of ZNF746 were not statistically significant in treated patients; however, the dynamics of changes were similar to those observed in untreated patients with PD.As it can be seen from  SNCA in patients with PD, by over fourfold; however, these results are not specific to PD, because a significant downregulation of this gene was also found in the neurological disease control group. The results of the expression analysis of the PINK1 gene were also interesting. There was no significant change in the expression of this gene in the group of patients with PD; however, a dramatic decrease in its mRNA levels (by more than fourfold) was observed in the neurological disease control group.Furthermore, the analysis showed a statistically significant decrease in the expression levels ofTo date, a large number of studies of the transcriptome have been conducted in patients with PD at advanced stages of the disease, as well as in the postmortem brain material of patients with PD. In such studies, patients were receiving active medication treatment; therefore, the differences discovered may be associated with the medication and the presence of concomitant diseases in patients –9, 15–17It should be noted that only those patients who lacked the most frequent mutations associated with PD were selected for the analysis of the levels of the transcripts of the genes investigated and that the cause of the disease was unknown. Thus, two groups of patients with PD  were formed for the study, as well as the neurological disease control group, which included patients with ALS, Wilson's disease, and so forth. The selection of patients for the neurological disease control group was carried out so that the pathogenesis of diseases of the patients included in this group was very different from that of the patients included in the PD group and that it was directly related to the central nervous system and the processes of neurodegeneration. SNCA was found both in the group of patients with PD and in the neurological disease control group. It is known that SNCA is a presynaptic protein that is actively involved in the function of different types of neurons [ SNCA levels observed here may be associated with the general patterns of development of pathogenic processes in various neurodegenerative disorders. Furthermore, a statistically significant decrease in the levels of the transcript of PINK1 was shown for the neurological disease control group. PINK1 is one of the key regulators of mitochondrial biogenesis [ PINK1 observed in our work either was associated with changes in mitochondrial functions, or may have led to their disruption, which in turn may cause the development of neurodegeneration.Nonspecific changes in the levels of the transcripts of two genes were identified in this analysis: a significant decrease in the level of the main transcript of neurons . Thus, togenesis . For exaogenesis , was sho ATP13A2, PARK7, and ZNF746 genes were the most interesting. A statistically significant increase in the mRNA levels of these genes was observed in the group of untreated patients with PD but not in the neurological disease control group. These changes were specific, and these genes may play an important role in the pathogenesis of PD, including the early clinical stages of the disease. The elevated levels of the transcripts of these three genes were not related to the therapy of patients because the observed changes in treated patients were similar to those observed in untreated patients with PD.The data obtained in the analysis of changes in the levels of the transcripts of thePARK7 and ATP13A2 are genes that are responsible for monogenic forms of PD and are associated with the development of the hereditary form of PD; however, there are no published data on changes in their expression levels in patients with PD. The increase in the mRNA level of PARK7 may indicate a compensatory phenomenon that develops in response to oxidative stress in cells, as it has been shown that the product of this gene, the DJ-1 protein, is able to protect the cells via autoxidation [ ATP13A2 encodes a lysosomal ATPase, and changes in its mRNA levels may indicate disturbances in processes that are associated with the removal of harmful components from cells [xidation . ATP13A2om cells . ZNF746 gene in the group of untreated patients with PD. Although the role of this gene in the pathogenesis of PD is not clear, there is evidence of a high level of its protein product, PARIS, in the striatum and substantia nigra [α and a transcriptional coactivator of the peroxisomal receptor, which is involved in the activation of mitochondrial biogenesis and also undergoes ubiquitination by Parkin. PARIS degradation via Parkin triggers the expression of PGC-1α and the subsequent activation of mitochondrial biogenesis [ ZNF746 can affect the inhibition of mitochondrial biogenesis via the repression of PGC-1α, leading to the development of neurodegenerative processes.However, the greatest changes in expression levels were observed for theia nigra . PARIS iogenesis . Thus, i ATP13A2, PARK7, and ZNF746 in untreated patients in the earliest clinical stages can also be observed in the preclinical stages of PD and that these genes can be considered as potential biomarkers of the preclinical stage of PD. However, further studies are needed to examine this hypothesis, especially in patients in the preclinical stage of PD.Based on these results, it can be suggested that the upregulation of the mRNA levels of


The target of the Fourth Millennium Development Goal (MDG-4) is to reduce the rate of under-five mortality by two-thirds between 1990 and 2015. Despite substantial progress towards achieving the target of the MDG-4 in Iran at the national level, differences at the sub-national levels should be taken into consideration.The under-five mortality data available from the Deputy of Public Health, Kermanshah University of Medical Sciences, was used in order to perform a time series analysis of the monthly under-five mortality rate (U5MR) from 2005 to 2012 in Kermanshah province in the west of Iran. After primary analysis, a seasonal auto-regressive integrated moving average model was chosen as the best fitting model based on model selection criteria.The model was assessed and proved to be adequate in describing variations in the data. However, the unexpected presence of a stochastic increasing trend and a seasonal component with a periodicity of six months in the fitted model are very likely to be consequences of poor quality of data collection and reporting systems.The present work is the first attempt at time series modeling of the U5MR in Iran, and reveals that improvement of under-five mortality data collection in health facilities and their corresponding systems is a major challenge to fully achieving the MGD-4 in Iran. Studies similar to the present work can enhance the understanding of the invisible patterns in U5MR, monitor progress towards the MGD-4, and predict the impact of future variations on the U5MR. The under-five mortality rate U5MR) is a key pointer of child well-being including health status, and is also a broad indicator of social and economic progress ,2. It isMR is a kIran is a country that has experienced considerable reductions in the U5MR over the past decades . In IranThe main purpose of this study was to detect any statistically significant features in the monthly U5MR in Kermanshah province from 2005 to 2012 and find an appropriate time series model that could adequately explain variations in the monthly U5MR. The study period of 2005 to 2012 was chosen because the child mortality data were available to the authors only for these years at the time of conducting the study.2 and 69.7% urbanization rate) with mainly Kurdish ethnic background [Kermanshah province is an undeveloped province located in the west of Iran and comprising 14 counties. Based on the population and housing census data of 2011, the population of Kermanshah province was 1,945,227 residents  in the provincial health jurisdiction. In this mortality digital file, under-five death records were collected from hospitals, health centers and health houses located in the province. There are few studies on sub-national child mortality in Iran, but a published report by the Iranian Ministry of Health showed that in 2010, more than 91% of mortality in the neonatal period and more than 63% of mortality 1 to 59 months after birth occurred in Iranian hospitals . The freBox-Cox transformation for assessment of stability in the data variance (p=0.20 for the null hypothesis of θ=1) shows that variance of the U5MR series is constant over time and no variance-stabilizing transformation is required. The Holt–Winters smoothing was applied to the U5MR series to examine any trends in the data. The time series plot of the original and the smoothed U5MR data are shown in p, d, q) 6 models with different combinations of SARIMA  and  orders were fitted to the U5MR series. Based on the corresponding AIC and BIC values of the fitted models, the best-fitting model was SARIMA  6 with the smallest value of AIC=526.2 and BIC=533.9.The portmanteau Q-test  and the Bartlett’s periodogram-based test  for white noise reject the null hypothesis of no serial correlation among observations of the differenced U5MR. To check the structure of such correlations, the sample autocorrelation function (ACF) and partial autocorrelation function (PACF) plots of the differenced U5MR series are shown in (A) and (B) of 2=3.64, p=0.16) accept the normality assumption of the model residuals. Finally, the ARCH-LM test for heteroscedasticity in variance of the model residuals accepts  the null hypothesis of no auto-regressive conditional heteroskedasticity effect. Therefore the overall goodness-of-fit of the model was evaluated.According to All analyses were performed using STATA/SE version 12.0   and the t, tx, is determined byAccording to the best-fitting model, the U5MR at month tz is a white noise process with standard deviation where The six months ahead (short-term) predictions of the U5MR and their corresponding 95% prediction intervals based on the fitted SARIMA model are reported in 6 was selected as the best fitting model and model assessment and goodness-of-fit tests showed that the model can adequately explain the fluctuations in U5MR. Finally, predictions six months ahead of December 2012 and their corresponding prediction intervals were obtained based on the fitted model.In this paper, time series analysis of monthly U5MR data in Kermanshah province in the west of Iran was conducted. According to our findings, the U5MR in Kermanshah province during the study period had a stochastic increasing trend. After primary analysis and applying necessary data adjustments, a SARIMA  Mortality data sources in developing countries, such as Iran ,18,19, oBesides the possibility that the U5MR has been under-reported, another limitation of this study is that the available data cover a rather narrow time period of eight years due to restrictions in data availability. The third limitation of the study is that, although the model adequacy was evaluated by various statistical methods, the capability of the model for forecasting in short time periods and the accuracy of the predicted values by model are not assessed. The uncertainty of the predictions is assessed only by prediction intervals. However, it should be noticed that generating accurate estimates and predictions of child mortality is a considerable challenge for developing countries ,2,4,10.Despite its limitations, the greatest strength of the present study is that it is the first study in Iran that uses time series analysis in order to mathematically model and predict an out-of-sample U5MR. However, additional data from other parts of the country are needed to generalize the results to the national level. In 2015, the MDG-4 target for the Iranian U5MR is 19 deaths per 1,000 live births ,11, as wTo achieve the MDG-4 on time, reducing under-five mortality inequities among Iranian provinces is an important priority. It seems that one of the major challenges ahead in fully achieving the MDG-4 in Iran is qualitative and quantitative improvement of under-five mortality data collection in health facilities and their corresponding systems. In general, for generating reliable mortality statistics at the sub-national levels it is necessary to improve the reporting and registering of child deaths by health facilities and to make sure that all child deaths that occur in health facilities are of critical importance. This is also crucial in understanding and measuring the impact and effectiveness of plans and interventions in this field. Since the definition of the U5MR indicator has not changed during the recent years, analyzing the invisible patterns and statistical modeling of this indicator within recent years is valuable and may enable health policy-makers to monitor the progress of Iranian child health status in order to evaluate the efficacy of health care systems. Such findings may be useful as they enhance the understanding of the current underlying patterns in the U5MR and monitoring of progress towards the MDG-4. Moreover, using studies similar to the present work, it is possible to predict the impact of future changes on this important child health indicator. Surely, with the expansion of study time periods and improvement of data collection methods, more accurate results will be obtained in the future.


Relapsing Remitting Multiple Sclerosis (RRMS) patients treated with interferon beta (IFN beta) can develop neutralizing antibodies (NAbs) that reduce treatment efficacy. Several clinical studies explored the association of NAb+ status with increased disease activity.The aim of this study was to estimate the cost of RRMS patients who develop NAbs while treated with IFN beta by the Italian National Healthcare Service (NHS) and the Italian Society perspectives.The clinical data derived from a published observational study on 567 RRMS Italian patients treated with IFN beta. The management cost data derived from the published literature. Cost data were inflated to Euro 2014.The annual direct cost to treat a patient was estimated in €15,428 in the NAb+ cohort and €14,317 in the NAb- cohort. The annual societal cost was estimated in €33,890 and €30,790 in NAb+ and NAb- patients, respectively. The cost increase related to the NAb+ status was €3,100 in the Italian societal perspective and €1,111 in the Italian NHS perspective.The results of this economic evaluation suggest the presence of an association between NAb+ status and increased costs for the management of RRMS in Italy. Further pharmacoeconomic research will be needed to confirm this first result. The worldwide prevalence of Multiple Sclerosis (MS) is estimated at about 30 cases per 100,000 persons, with maximum values  in Northern Europe and North America. Data from the Italian Multiple Sclerosis Association (AISM) reported about 72,000 people with MS in Italy . DM. DM18]. In the cohort of 546 patients, 75 were NAb+ (13.7%) and 471 were NAb- (86.3%) . The NN-The comparison of the EDSS scores in time and of the number of relapses between the NAb+ and NAb- cohorts was performed on the NN matched population with a time horizon ranging from 6 months to 30 months after the baseline. After this date more than 15% of the NAb+ cohort was lost to follow-up (at month 36 only 56 patients over 67 were still on study) . To the In the NN-matched cohort, the annual direct cost to manage a cohort of NAb- RRMS patients resulted of €14,317. The same direct cost for the NAb+ patients was €15,428, with an increase of €1,111 per year specifically related to the development of NAbs. This increase was due to the increased number of relapses (68.4%) and to the increased need for healthcare resources (31.6%). In the perspective of the broad Italian society the total social cost increase related to the development of NAbs was €3,100 per patient-year Annual total costs were lower for the NAb- vs. NAb+ patients on all parameters. As a sensitivity analysis, the same cost analysis was repeated in the overall unmatched population. The differences in costs linked to the development of NAbs were less enhanced than in the NN-matched cohort, due likely to casual differences in baseline characteristics between the NAb- and NAb+ cohorts. . HoweverThe objective of this study was to estimate the costs of the RRMS patients treated with IFN beta who develop NAbs. To this purpose the study was based on real world clinical data derived from an observational cohort of RRMS patients from a previously published observational study  and on lWe have extrapolated these finding to the entire population of RRMS patients treated with IFN beta in Italy. In Italy a total of 72,000 MS patients is estimated  and abouThis economic evaluation is funded on the clinical data of the University of Bari study  that conFurthermore, a relapse increases the level of EDSS (momentary or definitive), worsening the disability, which is linked to a reduction in the patient's QoL and increased costs.The cost of illness study from Ponzio et al. showed that the severity of MS and poor QoL are strongly correlated with higher costs. The study reported that every additional EDSS point increases the total cost by 48% and that every improvement in EQ-5D scale determines a 15% decrease in total cost. The same study showed that the total annual mean costs per patient increased from €22,750 at an EDSS score of 0–3 to €63,047 at an EDSS≥7 [To our knowledge, this economic evaluation is the first study attempting this assessment. However in 2014 a study was published that, with a different method, explored a similar concept . The WalSame limitations can be ascribed to our economic evaluation, of whom the most relevant is undoubtedly the fact that the cost increase linked to the onset of NAbs was not directly observed in the same cohort of patients. The real world data of University of Bari study , in factAs for most of the pharmaco-economic evaluations, further research is needed to overcome the limitations of the present study and confirm our conclusions.The results of our economic evaluation confirm that the complication of the clinical picture, which occurs with the onset of NAbs during IFN beta therapy may have important economic implications, which can be crucial for policy decisions aimed at increasing the quality of life for patients as well as at promoting a more efficient use of economic resources.


This paper proposes a new methodology to simultaneously select the most relevant SNPs markers for the characterization of any measurable phenotype described by a continuous variable using Support Vector Regression with Pearson Universal kernel as fitness function of a binary genetic algorithm. The proposed methodology is multi-attribute towards considering several markers simultaneously to explain the phenotype and is based jointly on statistical tools, machine learning and computational intelligence.The suggested method has shown potential in the simulated database 1, with additive effects only, and real database. In this simulated database, with a total of 1,000 markers, and 7 with major effect on the phenotype and the other 993 SNPs representing the noise, the method identified 21 markers. Of this total, 5 are relevant SNPs between the 7 but 16 are false positives. In real database, initially with 50,752 SNPs, we have reduced to 3,073 markers, increasing the accuracy of the model. In the simulated database 2, with additive effects and interactions (epistasis), the proposed method matched to the methodology most commonly used in GWAS.The method suggested in this paper demonstrates the effectiveness in explaining the real phenotype (PTA for milk), because with the application of the wrapper based on genetic algorithm and Support Vector Regression with Pearson Universal, many redundant markers were eliminated, increasing the prediction and accuracy of the model on the real database without quality control filters. The PUK demonstrated that it can replicate the performance of linear and RBF kernels. Single nucleotide polymorphisms (SNPs) are an abundant form of genomic variation, which differ from rare variants  and the This study aims to propose a method that can simultaneously evaluate several SNPs in relation to the phenotype described by a continuous variable, unlike case-control dichotomous phenotypes addressed to the majority of GWAS studies. With this, there are three immediate benefits relative to standard methodology: one relating to the various levels of the phenotypes, the other by complex simultaneous interactions that may occur between the various markers and, finally, the use of evolutionary computation to properly select the main SNPs. Recently, some studies are being conducted with continuous phenotypes and Support Vector Regression (SVR) as in , 9] and and9] anWith the evolution of new chips for cattle, with densities from 500,000 to 800,000 markers and if the genetic structure is defined by an underlying large number of small QTL, the formation of large data sets for training and precise phenotypic measures are needed to perform the improvement of the accuracy in increasing the density of SNPs . ConsequIn this section, the two main techniques, SVR and GA (Genetic Algorithm), used in the construction of the suggested method will be discussed to demonstrate their potential advantages.The first version SVM for regression was proposed in 1996 by Vladimir N. Vapnik, Harris Drucker, Christopher J. C. Burges, Linda Kaufman and Alexander J. Smola . This tef, described by Equation 1, which maps variables of the input space in the variable output space, minimizing the Expression 2.Let the set where w and b, respectively, the slope and intercept of the hyperplane to be estimated from Expression 2.Let 2 Lnorm of the vector where where,C is called the regularization constant and reflects the balance between complexity f and the amount of greater deviations that will be tolerated   6] on thFor the calculation of PTA milk, only the genetic effect is considered, eliminating all other environmental effects. Like this, the explanation of PTA from molecular marker information is consistent. According to Table The generated model is described by Equation (12).error is a normal random variable with mean 0 and standard deviation 5, L1 = (SNP1 == 2), L2 = (SNP10 == 1), L3 = (SNP20 == 3), L4 = (SNP30 == 3), L5 = (SNP40 == 3), L6 = (SNP50 == 2), L7 = (SNP60 == 2) and Y is simulated phenotype. The beta coefficients were set as Y of Equation (12) is continuous. The simulated databases 1 and 2 formed by the genotype-phenotype can be reconstructed from the sup file Where The generated model is described by the Equation (13).error is a normal random variable with mean 0 and standard deviation 1, L1 = (SNP4 != 2)3 & (SNP3 != 1), L2 = (SNP5 == 3), L3 = (SNP12 != 1) & (SNP9 == 3) and Y is simulated phenotype. The beta coefficients were set asWhere 3The SNP1 != 2 symbol means that the SNP1 does not have the largest allele frequency represented by 2.In the histogram of Figure The simulated phenotype 1 was constructed from multiple simulations of the betas of Equation (1) to introduce an asymmetry similar to milk PTA as shown in Figure The simulated 2 phenotype has empirical distribution which apparently does not fit into any theoretical probabilistic model as seen in Figure The real and simulated phenotypes 1 have outliers above the average in Figures For comparison of the filters used to select the most important markers were created two database: one without and one with quality control (QC). There has been no standard preprocessing in the data without QC such as call-rate, MAF and Hardy-Weinberg equilibrium (HWE). The purpose of this was not to eliminate SNPs with small effects alone, which when combined with other SNPs, are important in the description of PTA milk.In the case of simulated databases, no QC filters were applied because the MAF has been defined in such a way to be greater than 10% and less than 40%. Since, the threshold is 5%, all SNPs generated will satisfy it.The parameters used by the Illumina software referring to genotype calls are standard. As for the database with QC, the applied filters simultaneously were call-rate>=0.95, MAF > = 0.05 to HWE > = 0.05/56,947, being this value the Bonferroni significance and 56,947 is the quantity of SNPs in the original database. After applying the filters described above, 22,799 markers for applying the selection method established in this study remained. If the filters were applied separately, 9,158 markers would be eliminated by call rate, 25,764 by MAF and 1,091 by HWE.In the sample without quality control, 6,192 markers showed no information due to errors in reading the chip Ilummina Bovine 56k, leaving 50,755 markers. From them, 3 markers had no allelic variation, so they were disregarded, totaling 50,752 for subsequent analysis. In the final database consisting of 50,752 SNPs, there are 265,788 missing values, thus the same number of imputations was carried out.For the construction of most significant groups of markers, we used the Spearman correlation coefficient, because it has some advantages over the Pearson correlation coefficient, namely: does not assume that the relationship between the two variables is linear, does not significantly change the outcome in the presence of outliers and does not require that the variables have probability distributions determined a priori ,20. Brien is the sample size, -9, index 2, the p-value 10-8, and so on, until the index 8 which is for the largest p-value 10-2, as shown in Table -1 the performance was the same of the group with p-value 10-2 and the number of markers has greatly increased. Thus, the behavior of the accuracy of the PUK increased to the p-value 10-2 and remained constant for higher p-values.Where For the simulated databases, we assessed 17 groups of markers, because the behavior of the performance of PUK increased to a certain point and then began to decrease, possibly due to noise generated by highly correlated markers.The SVR regression was used to assess the explanatory power of the groups of markers in relation to the two simulated phenotypes and PTA for milk. This choice was due to its great flexibility, because the SVR does not assume linearity of the model (as long as they adopt non-linear kernel), nor normality of residuals and easily adapts to the high data dimensionality.C and ε of SVR models were tested with linear kernel. For the other two kernels, besides the 2 previous parameters, it was the tested specific parameter γ for the RBF kernel and the specific parameters ω and σ for the PUK. For all SVRs models, several simulations were carried out for each subset of SNPs.Various parameters For comparison of SVR models, we used cross-validation with 10-folds on each of the 8 sets of data in Table Based on group 8 of the real database that generated the highest average and lowest standard deviation of the correlation, a wrapper, based on a binary genetic algorithm (GA) with fitness given by the cross-validation MSE is applied to a second selection of markers. The entire methodology used in this second variable selection is based on  and 16]16].As the number of combinations between 6,512 markers is extremely high, we used a GA to find the "best" markers in a skilled computational time, while not guaranteeing the uniqueness of the solution "optimal" found. Thus, the objective of the GA is to check the possibility of eliminating some markers of the best set generated from the first selection, as it is believed that the GA can better assess the interactions between markers than elimination made through filters quality control standards such as call-rate, HWE, MAF and LD.The parameters adopted for the GA used for selecting SNPs were: probabilities of crossover and mutation equal to 0.6 and 0.033 respectively, population size and number of generations equal to 20 for real database. For the simulated database without epistasis (simulation 1), the population size was increased to 100 and the number of generations to 500, because the number of markers simulated was only 1,000, consuming less processing time. The selection technique known as elitism was used to keep the best individual of the previous generation in the next generation.arff, to be evaluated in the GUI called Experimenter. The SNPRelate was used to generate the graphs of the matrices of LD between markers.Both the wrapper SVR with GA as all models of SVR were performed in Weka software version 3.7.9 . All theFollowing the results of the methodology applied to both simulated and real basis bases will be presented in addition to the comparison of our results with the standard method. In addition, all files generated from the first selection for the two simulated databases (simulations 1 and 2) are in sup file -10 to 10-1, however, when applied Bonferroni correction, SNPs 20, 30 and 40 are lost because they have adjusted p-value equal to 1. But these markers showed non-significant raw p-values, so they were not excluded by the Bonferroni correction. An important observation is that since the proposed method constructs intervals of p-values (raw or adjusted), SNPs with p-values close to 1, but smaller, they can be selected. However, when the Bonferroni correction adjusts the raw p-value of the marker matching it to 1, the method will never select it. If the threshold of the p-value is equal to 0.05, only the SNP5 would be eliminated. Nevertheless, if the same threshold is considered, we would have at least 119 SNPs as can be seen in line 9 of Table From Table According to Table Although the average correlation PUK in the group 12 of Table The PUK showed the best result and the group 10 is the set with the least number of markers and higher average correlation as shown in Table The filter GA reduced approximately 10 times the number of SNPs, increasing the average correlation from 0.73 to 0.75, and maintaining the standard deviation for kernel PUK as shown in Table Figure According to Table By Tables When we evaluated the model consisting only of 5 relevant SNPs, the average correlation of the PUK was 0.99 with standard deviation 0.01, that is, the best result among the 3 kernels reviews. According to Table Therefore, if the group 16 is chosen to be analyzed, one can use the GA to reduce the number of markers and improve the performance of the PUK, aiming to converge to the average correlation of 0.99 correct model, but we did not perform this analysis because the proposed method evaluates the subset with the smallest number of SNPs with the highest average correlation.-2.The first analysis was related to the accuracy of SVR models with linear kernels, radial basis function and Pearson VII Universal. From Table According to Table The Table In Figure C and γ, there was an extensive search grid. However, neither of the two studies extracts from groups, constructed from the p-value, the "best" subset explanation for the phenotype and this was accomplished by applying the technique of variable selection wrapper based on the prediction error of the SVR as fitness GA.In relation to the work of  and 6],,6], therThe PUK kernel proved to be robust to capture the behavior of linear and RBF kernels, as long as the appropriate parameters are used. Therefore, on the methodology proposed in this paper, it is necessary to assess the performance of the SVR with PUK kernel. However, regardless the kernel adopted, the mathematical formulation of the SVR brings a disadvantage regarding the biological interpretation.The method developed in this study indicated that the 68 most significant markers of group 1 without QC has low predictive power and low accuracy compared to PTA milk and the subgroup with 3,073 markers showed high prediction and greater accuracy. This may indicate that the PTA milk is a phenotype that is influenced by several markers with small effects on it, besides the possibility of epistasis and dominance, however, such genetic effects cannot be proven by the method suggested in this work.The SVR model with kernel PUK of groups 8 with and without QC showed high predictive power even in the presence of non-normality in the dependent variable PTA for milk, and have similar performance, and accuracy. However, when applied the filter of the GA, the best subset generated from group 8 without QC was higher both in prediction and accuracy as compared to group 8 with QC. This fact seems to show that the group 8 without QC has sufficient and necessary for the explanation of the phenotype and the group 8 with QC has markers necessary, but not sufficient.The method developed in this work demonstrated robustness, because the initial set of markers without QC was composed of approximately 50,752 markers and reached up to 3,073 at the end of the selection process, ensuring good accuracy and high accuracy for the SVR model with PUK kernel. At the base without QC, the method found 1,073 markers in total 22,799 SNPs, maintaining the same average correlation. This fact may indicate a trait with polygenic effect as in . In addiIn the simulated database 1, the suggested method found 21 SNPs, 5 belonging to the group of 7 most important, while the standard method indicated only 1. Thus, the polygenic effect was mapped very accurately, however, to find only 7 markers, it is necessary to perform a sensitivity analysis on the parameters of the GA  in an attempt to eliminate the noise of the 16 false-positive SNPs.In the simulated database 2, the standard and proposal methodologies indicated only SNP3 as relevant marker, where 5 SNPs is the correct number. Thus, we have only chance to find the top 5 SNPs, analyzing the subgroup 16 with 7,934 SNPs, however, this group showed one of the lowest correlations. This indicates that the proposed method needs to be improved in scenarios with epistasis. But, the first selection by the p-value was not satisfactory, suggesting future work for the adoption of GA directly in the database without any filter, but with the introduction of LD, physical distance between markers, p-value, HWE, MAF, call-rate in the fitness of the GA in order to improve the convergence of GA to the "optimal" subset of markers.Another point to be analyzed in depth is SNPs that were not eliminated in the database without QC and can bring a high level of noise through the imputation used, inflating the explanatory power of SVR in 8 group.The standard filters  used in the database with QC seem to delete markers essential to explaining the phenotype PTA milk. From this study it is necessary to understand which filters are responsible for eliminating the most relevant SNPs. Therefore, the results obtained are promising for the application in GWAS, because most of the works in this area apply standard filters for preprocessing the database. Furthermore, the Bonferroni correction excluded essential markers in the simulated database, indicating that this "protection" for the inflation of type I error can impair association studies.No change in the values of real and simulated phenotypes in order to make your normal distribution was necessary because the PUK showed 0.95  or not based on this metric. But that will be developed later.The authors declare that they have no competing interests.p-value ranges for the construction of SVR; RVS  provided the real database; MVGBS  contributed to the genomic approach and the application of the quality control filters; WA  is the project leader, proposed the general approach of this study and the use of SVR and GA for the second selection markers and other computational intelligence techniques described in the future works. All authors read and approved the final manuscript.FCO  proposed to use the Spearman correlation instead of simple linear regression and SVR for evaluating groups of markers making the model multi-attribute; CCHB  is the team leader in this work, gave valuable contributions to the analysis of the method and on the possibilities for future works; FNA  revised the text and contributed to the methodology of this study; FFS  proposed to carry out the initial selection based on Simulated databases 1 and 2. R scripts for generating the genotype and phenotype from a linear model with only additive effects (simulation 1) and additive effects with epistasis (simulation 2). Furthermore, it was made the first selection of markers in data frames for later conversion into weka ARFF format used in the second selection by GA with SVR in Weka. Format: RAR Size: 3,45 KBClick here for fileDatabases after the first selection of markers first selection in the simulations 1 and 2. Format: RAR Size: 6,92 MBClick here for fileAdditional explications of the files contained in the sup file 2. Format: DOC Size: 51,5 KBClick here for file


The effect of the ITS-90 on previously calibrated sources for realizing CIE illuminants is negligibly small.2The differences between ITS-90 and IPTS-68 above 1235 K are described. It is shown that none of the following CIE definitions or recommendations require revision because of the introduction of the ITS-90: International Lighting Vocabulary definitions; CIE Standard Illuminants A, D This introduces a redundancy of the new scale, whereas all previous scales were unambiguously defined in terms of the gold point alone. However, the text of the ITS-90 states that “the From the above, the following relationships between the ITS-90 and the IPTS-68 are noted:The activities of the CIE which relate to temperature and could conceivably be affected by the adoption of the ITS-90 fall in the domain of thermal radiation and its applications in radiometry, photometry, and colorimetry. The published CIE output in these areas can be classified into two broad areas: definitions and recommendations. In what follows, it will be shown that none of these definitions and recommendations requires revision because of the introduction of the ITS-90.The output of the CIE contains a large number of definitions of the fundamental concepts, physical quantities, and technical terms used in the areas of light and lighting. These definitions have traditionally been collected in the International Lighting Vocabulary, which is now available in its fourth edition  as a joiThe definitions published by the CIE comprise, as a special class, “standards” in the form of uniquely defined data which form the basis of internationally accepted standard systems. These include “standard illuminants” for applications of colorimetry requiring the use of spectral power distributions which are representative of typical lighting conditions ,5. These“It is recommended that the following … luminous sources be adopted for the general colorimetry of materials:“Source A. A gas-filled lamp of color temperature 2848 K …c2, of Planck is taken equal to 14,350 micron degrees.“For calculations of the spectral energy distribution the constant, “Source A. The spectral energy distribution of this source shall be considered for all colorimetric applications as that of a blackbody at the temperature 2848 K.”CIE Standard Illuminant A is representative of domestic tungsten lighting and was originally defined as follows .“It is c2 but preserving the relative spectral distribution of illuminant A. This was achieved by shifting the color temperature assigned to it, rather than computing new spectral distributions for the original color temperature of 2848 K. Accordingly, the spectral values of CIE standard illuminant A are to be considered independent of the International Temperature Scale and require no adjustments in view of the adoption of the ITS-90. The color temperature assigned to standard illuminant A is a descriptive parameter which depends on the value of c2 assigned in the International Temperature Scale. Its value is approximately 2856 K on the IPTS-68 and remains the same on the ITS-90 because the value of c2 has not been changed.This definition was independent of the International Temperature Scale of 1927, which was in effect at the time. After the introduction of the International Temperature Scale of 1948, and once again after the IPTS-68 was introduced, the CIE followed a policy of adopting the assigned values of 65 are consistent with the IPTS-68 value of c2, and are also not affected by the introduction of the ITS-90 because c2 was not changed. Likewise, the correlated color temperature of illuminant D65, which is given as 6504 K on the IPTS-68, remains unchanged.This standard is representative of average daylight and is given in the form of numerical values that have been derived by Judd, MacAdam, and Wyszecki  from exp50 D55. and D75, have been defined in addition to standard illuminant D65 to represent daylight with approximate correlated color temperatures of 5000, 5500, and 7500 K. Additionally, illuminants F1 through F12 have been defined to represent typical fluorescent lamps. The published relative spectral distributions of these additional illuminants [The CIE has also defined illuminants which do not have the status of primary CIE standards but are useful for special purposes . Illumin2 is usually specified in these recommendations. However, the change from IPTS-68 to ITS-90 temperatures does not affect any of these recommendations because the value of c2 remains the same in the new scale.The CIE recommendations  concerniThe CIE has recommended specific sources ,5 which For example, the CIE recommends that Standard Illuminant A be realized by means of a gas-filled tungsten-filament lamp known as “Source A” which is operated at a correlated color temperature (2856 K on the IPTS-68) equal to the color temperature associated with Standard Illuminant A. This definition requires no change with the introduction of the ITS-90 because the color temperature associated with Standard Illuminant A has not changed.65. However, the CIE has recommended a method [At present, no source is recommended for realizing CIE Standard Illuminant Da method  for asseIlluminants B and C can be realized by combining Source A with specially formulated liquid filters . The speThere are no CIE recommendations for realizing the D and F illuminants mentioned in section 2.1.3.T68 will have the ITS-90 kelvin temperatureAlthough none of the CIE recommendations for laboratory realizations of CIE Standard Illuminants are affected by the adoption of the ITS-90, it should be noted that sources that have previously been calibrated in accordance with these recommendations do not exactly produce the standard spectral distributions. Because the ITS-90 gold-point assignment is closer to the thermodynamic temperature of this fixed point, a source calibrated on the IPTS-68 and operated at a given radiance temperature


The Manual Ability Classification System (MACS) has been widely used to describethe manual ability of children with cerebral palsy (CP); however its reliabilityhas not been verified in Brazil. To establish the inter- and intra-rater reliability of the Portuguese-Brazilversion of the MACS by comparing the classifications given by therapists andparents of children with CP. Data were obtained from 90 children with CP between the ages of 4 and 18 years,who were treated at the neurology and rehabilitation clinics of a Brazilianhospital. Therapists  classified manualability (MACS) through direct observation and information provided by parents.Therapists and parents used the Portuguese-Brazil version of the MACS. Intra- andinter-rater reliability was obtained using unweighted Kappa coefficient (k) andintra-class correlation coefficient (ICC). The Chi-square test was used toidentify the predominance of disagreements in the classification of parents andtherapists. An almost perfect agreement resulted among therapists , as well as with intra-rater (therapists), with Kapparanging between 0.83 and 0.95 and ICC between 0.96 and 0.99 for the evaluator withmore and less experience in rehabilitation, respectively. The agreement betweentherapists and parents was fair . The Portuguese version of the MACS is a reliable instrument to be used jointly byparents and therapists. Parents signed an informed consent form(HCRP no. 12469/2008).To assess the reliability of the MACS, ninety children with CP, aged between four and18 years, were included. The children were treated at the Occupational Therapy orPhysical Therapy Service of the Rehabilitation Center or at the Neurology OutpatientClinic of HCFMRP-USP between September 2010 and October 2011.This was a convenience sample and the inclusion criteria were: to be diagnosed withCP, regardless of the type or severity of the motor disability, to be aged between 4and 18 years, and to understand simple commands. The exclusion criteria were lowvision or blindness and/or epilepsy.Two evaluators collected data simultaneously at separate locations. Evaluator 1 (E1)was an undergraduate occupational therapy student and Evaluator 2 (E2) had nine yearsof experience in the rehabilitation of children with CP .None of them were familiar with the MACS and both received training before the startof data collection through an instructional video produced by the authors of theclassification. They also classified 10 children who were not included in theresearch sample. Any questions concerning the selection of the MACS level wereclarified with Dr. Eliasson by email. A research assistant (E3), also an occupationaltherapy student, was responsible for filming each child while handling objects, aswell as for filming the parents' report about how their children commonly used theirhands to manipulate objects in their daily activities as described below. E3 was alsoresponsible for the selection of videos for the intra-rater reliability test.E1 observed each child performing the following tasks: 1) Opening a puzzle box oranimal memory game box; 2) Emptying the contents of the box onto the table; 3)Handling the pieces of a puzzle or memory game; 4) Placing the pieces back in thebox; 5) Closing and opening a zipper; 6) Buttoning and unbuttoning a jacket; 7) Usinga spoon to scoop up food (e.g. yogurt or soft cake); 8) Drinking from a cup; 9)Building a tower with 5 blocks; 10) Pounding pegs using a toy hammer; 11) Performinga graphic activity; 12) Putting marbles and/or beads into a jar. After observing thechild, E1 also obtained additional information from the parents concerning thetypical performance of the children at home and school, in relation to the use oftheir hands, such as playing, feeding, and using materials at school. E1 classifiedeach child, considering the observation of his/her performance, in combination withthe information collected from the parents about the child's use of their hands intheir performance of daily activities; this was the first assessment of Evaluator 1(A1E1). Each child's task performance and the parents' report were filmed by E3 to beused in inter- and intra-rater reliability tests.While E1 observed the child performing the tasks, E2 read the Portuguese-Brazilversion of the MACS to the parents, who had to select a single level of the MACS toclassify their child. E2 informed the parents that, in order to select the level ofthe MACS, it was necessary to consider the manual ability of their children whenplaying, drawing, writing, and handling objects during feeding and dressing. Whenparents provided examples of the manual ability of their children, it was reinforcedthat they should select the level that best described the manual ability. If theywere unsure about which level they should select, the difference between the twolevels was read to them. E2 simply wrote down the MACS level that the parentsselected without any clarification.Two weeks later, in an attempt to minimize memory bias (i.e. remembering theclassification level chosen by parents), E2 classified the children according to theMACS levels by analyzing the videos of the children's observation and the parents'information about the children's performance in daily activities, which was the firstassessment of Evaluator 2 (A1E2).The second assessment (intra-rater reliability) took place one month after the firstone. Both evaluators (E1 and E2) watched the videos separately in order to classifythe children's manual ability level. This stage included 30 videos selected by E3(corresponding to 33% of the sample) from various age groups and MACS levels based onthe initial classification of the E2.Parent and therapist classifications were compared using the first assessmentperformed by E2 (A1E2).The MACS is an ordinal scale with five levels. Descriptive statistics were used tocharacterize the participants. Ordinal data were analyzed using percentage agreementand Cohen's unweighted Kappa (k) to examine parent-therapist agreement and intra- andinter-rater agreements. By using the Intra-class Correlation Coefficient (ICC) it waspossible to make comparisons with other studies.The following criteria were used for the Kappa coefficient interpretation: valuesbelow zero (poor), between 0.00 and 0.20 (slight), between 0.21 and 0.40 (fair),between 0.41 and 0.60 (moderate), between 0.61 and 0.80 , and between0.81 and 1.0 The average age of the children was 7.58 years, ranging from 4 to 17.91 years of age.Most were diagnosed as children with spastic bilateral CP (n=61), followed by unilateralspastic (n=17), dyskinetic (n=9), and ataxic (n=3) CP. Regarding gender, the sample wasequally distributed.The children with unilateral CP were classified by the occupational therapist (E2) asMACS levels I or II (35% in level I and 65% in level II). Most of those with bilateralCP were classified as level I (31.1%), although the other children from this group weredistributed over the other MACS levels . In rela2=1.57; p=0.67), two of them between levels I and II,one between II and III, three between levels III and IV, and one between levels IVand V.It can be noted that there were seven disagreements that were not predominant atspecific levels  classified the manual ability of 30 children with cerebralpalsy in the initial assessment and after one month. The results show almost perfectagreement between both evaluators. E1 had two disagreements between levels I and IIand two others between levels III and IV . E2 (the researcher with the most experience in the neurology field)had only one disagreement between levels I and II of the MACS .Fair agreement was observed between the classification performed by the occupationaltherapist (E2) and the classification performed by the parents . 2=36.18;p=0.001).It is possible to verify that there were 46 classification agreements between parentsand the therapist (E2) and 44 disagreements. Of these, 21 occurred between levels Iand II, 9 between levels II and III, 7 between levels III and IV, and 3 betweenlevels IV and V. There were also four disagreements in more than one level betweentherapist and parents, two of them between levels I and III and the others betweenlevels I and IV. It can be noted through the chi-square test for adherence that thedisagreements between levels I and II are the most predominant  by means of inter- and intra-raterreliability tests concerning the MACS, with an almost perfect reliability ICC=0.97;K=0.90). Similar results were found in the validation study of the MACS in Sweden;K=0.90.,,,,In this study, video analysis was used for the children's performance while handlingobjects and the parents' reports was used for the evaluators' classification. Moststudies concerning the MACS reliability verified by healthcare professionals have usedthe direct observation of the children in the clinical context and have shown positiveresults for the age group proposed by the MACS (4 and 18 years of age),The intra-rater reliability was considered almost perfect for both evaluators, with onlyone disagreement for E2 and four disagreements for E1, who had less experience. Onlythree studies were found in the literature involving intra-rater reliability  of the MACS as part of the process of cultural adaptation and reliabilityand they were conducted in Korea,In our research, we used a similar method (direct observation of the child and parentinterview), but the second assessment to evaluate intra-rater reliability was made bywatching the video recordings of the initial assessment. This procedure has been used inother studies to evaluate intra-rater reliabilityRegarding the reliability between therapists and parents, there was substantialagreement, with disagreements predominantly between levels I and II. The resultsobtained in this research are similar to Morris et al.In our research, the evaluator simply read the MACS booklet to the parents. If theyprovided examples of the children's handling of the objects at home and asked whetherthose examples corresponded to a certain MACS level, the evaluator provided no responseso as not to influence the parents, but instead emphasized the need for them to chooseone of the levels that was closer to the manual ability of the children, and repeatedthe levels and/or topics, differentiating the levels if necessary.,Among the studies that had higher rates of agreement, parents and physical therapistsread the MACS booklet and the parents' questions concerning the classification wereanswered by the researcherIt should be acknowledged that, in its descriptions, the MACS uses terms like "quality","speed", and "independence", which are more commonly used by healthcare professionals intheir clinical practice and which can be interpreted differently by parents andtherapists. In addition, these terms have to be considered in relation to the child'sage,,Other aspects that can be related to disagreements between therapists and parents'classification is that healthcare professionals, when using classifications like theMACS, tend to evaluate the children in clinical environments, which are designed toallow their optimal functioning and minimize the negative effects of theenvironmentHowever, taking into consideration the limitations of the MACS as mentioned, such as theuse of terminology directed to healthcare professionals and the complexity of the manualability concept, it is suggested that therapists provide information to the parentsabout the concepts present in the MACS, examples concerning the terminology used, andthat they answer their questions to assist with the classification. In addition, parentsand therapists should be able to jointly classify the manual ability of children withCP, as the thinking process conducted by the parents in relation to their children'smanual ability and the communication of information to healthcare professionals canprovide better personal understanding and improve self-confidenceWithin the limitations of the study is the fact that the classification of the MACS wasdone by parents based only on the reading of the MACS booklet and no explanation aboutspecific terms or clarification of questions was provided by the researcher. Furtherstudies about content validation and stability of the MACS by therapists and parents areneeded, as well as the comparison of the classification by therapists and parents whohad or had not been trained through the institutional MACS video.Therefore, we conclude that the MACS has been shown to be a reliable classification thatcan be used by health professionals with different levels of experience and by parents.Even if therapists and parents perceive the manual ability of children with CPdifferently, the information from both sources should be complementary. The use of theMACS in clinical practice can be an important starting point to set intervention targetsfor the children in partnership with the family. In addition, the health professionalsshould give examples/information about manual ability and clarify the parents' questionsabout the description of the MACS levels in order to enhance their understanding of theclassification.