Continuous hemofiltration model using porcine blood for comparing filter life.

The purpose of the present study was to establish a continuous hemofiltration model using porcine blood to compare filter life. Continuous hemofiltration (CHF) experiments were performed using an in vitro hemofilter evaluation system utilizing porcine blood containing trisodium citrate in addition to nafamostat mesilate as anticoagulants. The lifetime of the hemofilter was evaluated using the transmembrane pressure and the pressure drop across the hemofilter at varying trisodium citrate concentrations. The porcine blood used in this experiment was considered to be in a slightly hypercoagulable state because of the continuous contact with non-biological materials and calcium inflow from substitution fluid. Blood containing 7 or 8 mM of trisodium citrate and nafamostat mesilate could be effectively used to compare the lifetimes of hemofilters utilized under the same conditions. In this CHF model using porcine blood, the plugging of the hollow fibers occurred shortly after the plugging of the membrane pores. In conclusion, a CHF model using porcine blood can be established by adjusting the concentration of trisodium citrate added to the blood.

Evaluation of the Biocompatibility of Dialysis Membranes.

BACKGROUND: Improvements in the biocompatibility of dialysis membranes have reduced biological responses elicited by blood-membrane interactions. In this article, recent technological developments in dialysis membranes with regard to biocompatibility and recent progress in the evaluation of the biocompatibility of dialysis membranes are reviewed. SUMMARY: The focus of investigation into dialysis membranes in recent years has focused on not only membrane materials, but also their surface textures, which have been changed, for example, by coating with vitamin E or by changing the amount and type of hydrophilizing agents used. Research and development is directed at altering the chemical and physical properties of membrane surfaces to suppress biological responses that are particularly elicited as a result of platelet activation. To develop membranes with excellent biocompatibility, biocompatibility should be evaluated on a like-for-like basis under conditions that are similar to those in clinical settings. Evaluation using actual dialyzers can be performed using porcine blood, platelet-rich plasma isolated from porcine blood (and platelet-rich plasma with leukocytes), or suspension of neutrophils isolated from porcine blood or cultured human monocytes. KEY MESSAGES: Highly biocompatible dialysis membranes can be developed when the overall correlations among biological reactions are examined by integrating all data on biological responses elicited by blood-membrane interactions or mutual interactions among blood cells.

Effects of increased surface coverage of polyvinylpyrrolidone over a polysulfone hemofilter membrane on permeability and cell adhesion during continuous hemofiltration.

The purpose of the present study was to evaluate the adhesiveness of blood cells and the solute removal performance change of modified polysulfone membranes which have increased polyvinylpyrrolidone (PVP) coverage over their surface. Continuous hemofiltration (CHF) experiments for 24 h were carried out using an ex vivo hemofilter evaluation system to compare a modified polysulfone hemofilter (SHG) with the conventional polysulfone hemofilter (SH). The 25 and 50 % cutoff values of the sieving coefficient of dextran after CHF and the protein concentration in the filtrate was higher in SHG, indicating that less fouling occurred in the SHG membrane. Adhesion of blood cells after 24 h of CHF was significantly higher in the case of SH than in the case of SHG. Blood cell adhesion and membrane fouling were reduced with the use of a polysulfone membrane modified with increased PVP coverage over the surface.

Secular changes in relative leg length confound height-based spirometric reference values.

BACKGROUND: Most but not all data from different ethnic groups fit the Global Lung Function Initiative (GLI) spirometric reference model. This study investigates to what extent discrepancies are caused by secular changes in body proportions. METHODS: FEV1 and FVC from 20,336 healthy Japanese subjects (13,492 women) aged 17 to 95 years were compared with GLI-2012 reference values for Europeans. Data on the sitting height/standing height ratio (Cormic index) in 17-year-old students, collected from 1949 to 2012 in successive birth cohorts, were used to assess secular changes in body frame. The cohort-specific Cormic index was used to assess how variation in body frame affected pulmonary function. RESULTS: FEV1 and FVC were lower than GLI-2012 reference values, with values progressively falling until age 35 to 40 years and then rising to European levels in the elderly. The Cormic index rose until 1942, then fell, with a nadir in the 1970s, before rising again until 1995. Nearly one-half of the spirometric variability from predicted values could be explained by differences in the Cormic index between birth cohorts. CONCLUSIONS: In low-income countries, improving health conditions are likely to drive increases in height and changes in relative leg length similar to those observed in Japan and, thus, to a change in body frame. This implies that height-based prediction equations for such populations will need to be periodically updated.

A mathematical model to predict protein wash out kinetics during whole-lung lavage in autoimmune pulmonary alveolar proteinosis.

Whole-lung lavage (WLL) remains the standard therapy for pulmonary alveolar proteinosis (PAP), a process in which accumulated surfactants are washed out of the lung with 0.5-2.0 l of saline aliquots for 10-30 wash cycles. The method has been established empirically. In contrast, the kinetics of protein transfer into the lavage fluid has not been fully evaluated either theoretically or practically. Seventeen lungs from patients with autoimmune PAP underwent WLL. We made accurate timetables for each stage of WLL, namely, instilling, retaining, draining, and preparing. Subsequently, we measured the volumes of both instilled saline and drained lavage fluid, as well as the concentrations of proteins in the drained lavage fluid. We also proposed a mathematical model of protein transfer into the lavage fluid in which time is a single variable as the protein moves in response to the simple diffusion. The measured concentrations of IgG, transferrin, albumin, and ß2-microglobulin closely matched the corresponding theoretical values calculated through differential equations. Coefficients for transfer of ß2-microglobulin from the blood to the lavage fluid were two orders of magnitude higher than those of IgG, transferrin, and albumin. Simulations using the mathematical model showed that the cumulative amount of eliminated protein was not affected by the duration of each cycle but dependent mostly on the total time of lavage and partially on the volume instilled. Although physicians have paid little attention to the transfer of substances from the lung to lavage fluid, WLL seems to be a procedure that follows a diffusion-based mathematical model.

Identification of EGFR expression status association with metastatic lymph node density (ND) by expression microarray analysis of advanced gastric cancer.

Metastatic lymph node density (ND) has been reproducibly proven to be a prognostic factor in gastric cancer. The molecular mechanisms that underlie this aggressiveness are underexplored. Here, we aimed to identify molecules associated with this unique phenotype. Tumor specimens from patients with stage III gastric cancer with high or low ND (n = 4 for both) were compared at the mRNA level using Affymetrix microarray (harboring 54,675 genes). The expression data were prioritized, and genes that correlated with ND were selected. Ultimately, the EGFR was validated as such a candidate molecule in patients with primary advanced gastric cancer who underwent standard treatment (n = 167). Expression data of the microarray were prioritized based on gene expression ratio and frequency of gene expression. The first priority genes to be selected were genes that are known to be amplified in cancer, which included NKX2.1, CHST9, CTNND2, SLC25A27, FGFR2, EGFR, and PTGER1. Of these genes, the EGFR gene was of particular interest. EGFR expression in primary gastric cancer was examined using immunohistochemistry (IHC). The Student's t-test elucidated a significant difference in EGFR expression between IHC 2+/3+ and IHC 1+ according to ND (P = 0.0035). The Chi-square test also indicated a significant difference between high and low levels of EGFR immunohistochemical staining (IHC2+/3+ and IHC1+, respectively) and ND status (P = 0.0023). According to the least squares method, as ND increased, the risk that EGFR staining levels changed from IHC 1+ to IHC 2+ also increased. In this study, we determined that high EGFR expression may underlie the aggressive mechanism of advanced gastric cancer with high ND.

Hydrogen preconditioning during ex vivo lung perfusion improves the quality of lung grafts in rats.

BACKGROUND: Although the benefits of ex vivo lung perfusion (EVLP) have been globally advocated, the potentially deleterious effects of applying EVLP, in particular activation of proinflammatory cascades and alteration of metabolic profiles, are rarely discussed. This study examined proinflammatory events and metabolic profiles in lung grafts on EVLP and tested whether preconditioning lung grafts with inhaled hydrogen, a potent, cytoprotective gaseous signaling molecule, would alter the lungs' response to EVLP. METHODS: Rat heart-lung blocks were mounted on an acellular normothermic EVLP system for 4 hr and ventilated with air or air supplemented with 2% hydrogen. Arterial and airway pressures were monitored continuously; perfusate was sampled hourly to examine oxygenation. After EVLP, the lung grafts were transplanted orthotopically into syngeneic rats, and lung function was examined. RESULTS: Placing lung grafts on EVLP resulted in significant upregulation of the messenger RNAs for several proinflammatory cytokines, higher glucose consumption, and increased lactate production. Hydrogen administration attenuated proinflammatory changes during EVLP through upregulation of the heme oxygenase-1. Hydrogen administration also promoted mitochondrial biogenesis and significantly decreased lactate production. Additionally, in the hydrogen-treated lungs, the expression of hypoxia-inducible factor-1 was significantly attenuated during EVLP. These effects were maintained throughout EVLP and led to better posttransplant lung graft function in the recipients of hydrogen-treated lungs. CONCLUSIONS: Lung grafts on EVLP exhibited prominent proinflammatory changes and compromised metabolic profiles. Preconditioning lung grafts using inhaled hydrogen attenuated these proinflammatory changes, promoted mitochondrial biogenesis in the lungs throughout the procedure, and resulted in better posttransplant graft function.

Reference values for spirometry, including vital capacity, in Japanese adults calculated with the LMS method and compared with previous values.

BACKGROUND: Reference values for lung function tests should be periodically updated because of birth cohort effects and improved technology. This study updates the spirometric reference values, including vital capacity (VC), for Japanese adults and compares the new reference values with previous Japanese reference values. METHODS: Spirometric data from healthy non-smokers (20,341 individuals aged 17-95 years, 67% females) were collected from 12 centers across Japan, and reference equations were derived using the LMS method. This method incorporates modeling skewness (lambda: L), mean (mu: M), and coefficient of variation (sigma: S), which are functions of sex, age, and height. In addition, the age-specific lower limits of normal (LLN) were calculated. RESULTS: Spirometric reference values for the 17-95-year age range and the age-dependent LLN for Japanese adults were derived. The new reference values for FEV(1) in males are smaller, while those for VC and FVC in middle age and elderly males and those for FEV(1), VC, and FVC in females are larger than the previous values. The LLN of the FEV(1)/FVC for females is larger than previous values. The FVC is significantly smaller than the VC in the elderly. CONCLUSIONS: The new reference values faithfully reflect spirometric indices and provide an age-specific LLN for the 17-95-year age range, enabling improved diagnostic accuracy. Compared with previous prediction equations, they more accurately reflect the transition in pulmonary function during young adulthood. In elderly subjects, the FVC reference values are not interchangeable with the VC values.

Transcriptional regulation by infliximab therapy in Kawasaki disease patients with immunoglobulin resistance.

BACKGROUND: Infliximab (IFX), a known monoclonal antibody against tumor necrosis factor-α (TNF-α), is used to treat Kawasaki disease (KD) patients with intravenous immunoglobulin (IVIG) resistance. The transcriptional modulation of inflammation following IFX therapy has not been reported in KD patients. METHODS: We investigated the transcript abundance profiles in whole blood obtained from eight IVIG-resistant KD subjects treated with IFX therapy using microarray platforms and compared them with those in initially IVIG-responsive subjects. A pathway analysis was performed using WikiPathways to search for the biological pathways of the transcript profiles. Four transcripts changed by IFX therapy were subsequently validated using quantitative real-time polymerase chain reaction. RESULTS: The pathway analysis showed the reduced abundance of transcripts in the nucleotide-binding oligomerization domain, matrix metalloproteinase (MMP), and inflammatory cytokine pathways and the increased abundance of transcripts in the T-cell receptor, apoptosis, TGF-ß, and interleukin-2 pathways. Additionally, the levels of four transcripts (peptidase inhibitor-3, MMP-8, chemokine receptor-2, and pentraxin-3) related to KD vasculitis and IVIG resistance decreased after IFX therapy. CONCLUSION: The administration of IFX was associated with both the signaling pathways of KD inflammation and several transcripts related to IVIG resistance factors. These findings provide strong theoretical support for the use of IFX in KD patients with IVIG resistance.

Hypoxia enhances the induction of human amniotic mesenchymal side population cells into vascular endothelial lineage.

Human amniotic mesenchymal side population (hAM-SP) cells have pluripotency and weak immunogenicity, and have promising roles in the field GAPDH of regenerative medicine. The aim of the present study was to determine whether hypoxic conditions induce the differentiation of hAM-SP cells into the vascular endothelial lineage. Mesenchymal cells were isolated from enzyme-treated amniotic membranes and stained with Hoechst 33342. The hAM-SP cells were negatively sorted by FACS and cultured in induction medium containing vascular endothelial growth factor (VEGF) under normoxic (20% O2) or hypoxic (1% O2) conditions for 1 or 2 weeks. The expression of endothelial markers such as kinase domain region (KDR), fms-like tyrosine kinase (Flt)-1, von Willebrand factor (vWF), vascular endothelial (VE)-cadherin and human vascular cell adhesion molecule (VCAM) at the gene and protein level was evaluated by real-time PCR and fluorescent immunostaining, respectively. The gene expression of KDR, Flt-1, VE-cadherin and vWF peaked after 2 weeks of culture. The protein expression of KDR and VE-cadherin was also enhanced after 2 weeks of culture under hypoxic conditions. To confirm the involvement of hypoxia-inducible factor (HIF) in the induction under hypoxic conditions, the expression of genes which are known to be upregulated by HIF was analyzed by DNA microarray. The expression of these genes increased under hypoxic conditions. hAM-SP cells cultured under hypoxic conditions differentiated into the vascular endothelial lineage, probably due to upregulation of the gene expression associated with angiogenesis through activation of the HIF system.

Breathing nitric oxide plus hydrogen gas reduces ischemia-reperfusion injury and nitrotyrosine production in murine heart.

Inhaled nitric oxide (NO) has been reported to decrease the infarct size in cardiac ischemia-reperfusion (I/R) injury. However, reactive nitrogen species (RNS) produced by NO cause myocardial dysfunction and injury. Because H2 is reported to eliminate peroxynitrite, it was expected to reduce the adverse effects of NO. In mice, left anterior descending coronary artery ligation for 60 min followed by reperfusion was performed with inhaled NO [80 parts per million (ppm)], H2 (2%), or NO + H2, starting 5 min before reperfusion for 35 min. After 24 h, left ventricular function, infarct size, and area at risk (AAR) were assessed. Oxidative stress associated with reactive oxygen species (ROS) was evaluated by staining for 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal, that associated with RNS by staining for nitrotyrosine, and neutrophil infiltration by staining for granulocyte receptor-1. The infarct size/AAR decreased with breathing NO or H2 alone. NO inhalation plus H2 reduced the infarct size/AAR, with significant interaction between the two, reducing ROS and neutrophil infiltration, and improved the cardiac function to normal levels. Although nitrotyrosine staining was prominent after NO inhalation alone, it was eliminated after breathing a mixture of H2 with NO. Preconditioning with NO significantly reduced the infarct size/AAR, but not preconditioning with H2. In conclusion, breathing NO + H2 during I/R reduced the infarct size and maintained cardiac function, and reduced the generation of myocardial nitrotyrosine associated with NO inhalation. Administration of NO + H2 gases for inhalation may be useful for planned coronary interventions or for the treatment of I/R injury.

Activation of platelets upon contact with a vitamin E-coated/non-coated surface.

The purpose of this study was to determine the effects of a vitamin E-coated surface on platelet activation, focusing on the interactions among the vitamin E-coated surface, platelets and leukocytes. Platelet-rich plasma (PRP) or PRP containing leukocytes (LPRP) was used. No difference was observed in platelet activation between PRP and LPRP for a vitamin E-coated membrane, meaning that platelet activation triggered by leukocytes was suppressed in plasma coming in contact with a vitamin E-coated membrane, while the membrane itself directly induced platelet activation. The antioxidant capacity of the vitamin E-coated membrane in contact with PRP or LPRP was partially reduced, but sufficient residual capacity remained. The in vitro experiments using an oxidized vitamin E-coated surface revealed that P-selectin expression and superoxide anion production in the platelets and platelet adhesion were induced by contact with the oxidized vitamin E-coated surface. We conclude that contact with a vitamin E-coated surface reduces platelet activation mediated by superoxide anions, probably by reducing superoxide anions, but during the process of the reduction, the vitamin E-coated surface itself becomes oxidized, which again causes platelet activation. The beneficial effects of a vitamin E-coated dialyzer in respect of platelet activation were counteracted by the formation of oxidized vitamin E.

A novel method of preserving cardiac grafts using a hydrogen-rich water bath.

BACKGROUND: Exogenously administered hydrogen exerts cytoprotective effects through anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms in various disease settings, including organ transplantation. Our objective in this study was to evaluate the efficacy of a novel cold storage device equipped with a hydrogen-rich water bath. METHODS: The study used an established rat heterotopic transplantation model. Syngeneic heart grafts from elderly donors (60- to 70-week-old Lewis rats) or allografts from adult donors (12-week-old Brown Norway rats) were exposed to prolonged cold preservation. The cardiac grafts were stored in plastic bags containing Celsior, which were immersed in the cold water bath equipped with an electrolyzer to saturate the water with hydrogen. The cardiac grafts then were heterotopically engrafted into Lewis rat recipients. RESULTS: In both experimental settings, serum troponin I and creatine phosphokinase were markedly elevated 3 hours after reperfusion in the control grafts without hydrogen treatment. The grafts exhibited prominent inflammatory responses, including neutrophil infiltration and the upregulation of messenger RNAs for pro-inflammatory cytokines and chemokines. Myocardial injury and inflammatory events were significantly attenuated by organ storage in the hydrogen-rich water bath. The grafts stored using the hydrogen-rich water bath also exhibited less mitochondrial damage and a higher adenosine triphosphate content. CONCLUSIONS: Hydrogen delivery to cardiac grafts during cold preservation using a novel hydrogen-supplemented water bath efficiently ameliorated myocardial injury due to cold ischemia and reperfusion. This new device to saturate organs with hydrogen during cold storage merits further investigation for possible therapeutic and preventative use during transplantation.

Rapid deconjugation of SN-38 glucuronide and adsorption of released free SN-38 by intestinal microorganisms in rat.

One of the dose-limiting toxicities of irinotecan hydrochloride (CPT-11) is delayed-onset diarrhea. CPT-11 is converted to its active metabolite, SN-38, which is conjugated to SN-38 glucuronide (SN-38G). SN-38G excreted in the intestinal lumen is extensively deconjugated by bacterial ß-glucuronidase, resulting in the regeneration of SN-38, which causes diarrhea. However, the deconjugation of SN-38G by the intestinal microflora remains to be clarified. This study aimed to investigate the microbial transformation of SN-38G by an anaerobic mixed culture of rat cecal microorganisms. Concentrations of SN-38G and SN-38 were then determined using high-performance liquid chromatography. Complete deconjugation of SN-38G to SN-38 in the mixed cultures was observed within 1 h of incubation, with 62.7% of the added SN-38G being found in the supernatant. Approximately 80.4% of the SN-38 in the supernatant was bound to protein, and the remaining 19.6% was detected as active free SN-38. In total, only 12.3% (19.6 × 62.7%) of the SN-38G added to the test tube was found in the supernatant in the ultrafiltrable free form, indicating that approximately 90% of the SN-38G added to the growth medium either remained adsorbed onto the pelleted fraction or occurred in a protein-bound form in the supernatant. The remaining 10% of the SN-38G added to the growth medium existed in the unbound form, the form capable of causing damage to the intestinal membrane. In conclusion, these results indicated that the greater part of the SN-38 produced from SN-38G by the action of bacterial ß-glucuronidase is rapidly adsorbed onto intestinal bacterial cell walls or dietary fibers in pelleted fraction, and only 10% remains in the ultrafiltrable unbound form in the intestinal luminal fluid.

Ectopic expression of a T-box transcription factor, eomesodermin, renders CD4(+) Th cells cytotoxic by activating both perforin- and FasL-pathways.

During viral infection, CD8(+) cytotoxic T lymphocytes (CTL) play a central role to eliminate viruses by destructing virus-infected cells utilizing two cytolytic pathways, i.e., perforin/granzyme pathway and FasL-Fas pathway. It has been shown that effector functions of CTL are critically controlled by two T-box transcription factors, T-bet and eomesodermin (Eomes), although their precise activities in constructing CTL functions are not fully understood. To investigate the functional potency and activities of Eomes, the effects of ectopic expression of Eomes in two terminally differentiated murine CD4(+) Th lines, on their effector functions were analyzed. The results showed that in Eomes-transfected Th hybridoma, cell surface FasL expression upon Con A stimulation was markedly enhanced, although perforin expression was not induced. In normal, non-transformed Th2 cells, introduction of Eomes elicited perforin expression, and also augmented FasL up-regulation. Interestingly, cyotlytic activity of Eomes-transfectant was more efficient than that of perforin-transfected Th2 cells which expressed high levels of perforin and granzyme B mRNA, indicating that Eomes may play additional roles other than preparation of these cytolytic effector molecules. In contrast, stimulation-induced CD154 up-regulation, one of the typical helper T cell characteristics, was repressed in Eomes-transfectant. Collectively, these results suggest that Eomes may not only be involved in perforin/granzyme expression but also play various functions, including FasL up-regulation, to develop the characteristics of CD8(+) CTL. These studies have also suggested that introduction of Eomes alone was sufficient to convert the functions of fully differentiated Th cells toward those of CTL.

Prevalence of topoisomerase I genetic mutations and UGT1A1 polymorphisms associated with irinotecan in individuals of Asian descent.

Topoisomerase I (TOP-I) mutations have been shown to be correlated to irinotecan resistance in vitro. However, the prevalence of TOP-I germline mutations has yet to be systematically elucidated. On the other hand, polymorphisms of UGT1A1 have been shown to be associated with CPT-11 toxicity in clinical situations. The primary aim of this study was to investigate the prevalence of mutations in the TOP-I exons associated with CPT-11 resistance, including untreated cancer tissue. A secondary aim was to confirm the less frequent UGT1A1*28 and more frequent UGT1A1*6 in individuals of Asian descent compared to Caucasians and individuals of African descent. The prevalence of 5 reported TOP-I mutations in exons was investigated in volunteers (n=236) using DNA sequencing of the PCR products. The prevalence of TOP-I mutations in untreated lung cancer tissues (n=16) was also investigated. Additionally, 3 UGT1A1 polymorphisms, UGT1A1*6, *27 and *28, were investigated in volunteers (n=126). There were no mutations of TOP-I in any of the 236 subjects or in the untreated lung tissues. Among 128 subjects, the distribution of homozygous polymorphisms of UGT1A1 was: UGT1A1*28 in 3 (2.4%) and UGT1A1*6 in 4 (3.2%) subjects, and co-occurrence of heterozygous polymorphisms for both UGT1A1*6 and UGT1A1*28 in 4 (3.2%) subjects, and for UGT1A1*27 and UGT1A1*28 in 1 subject (0.8%). The Hardy-Weinberg deviation test showed there was no significant deviation from the equilibrium, and the association analysis indicated no significant linkage between UGT1A1*6 and UGT1A1*28. In conclusion, TOP-I genetic mutations correlated to CPT-11 resistance were not detected in any of the subjects and untreated lung cancer tissues. Less frequent UGT1A1*28 and more frequent UGT1A1*6 were confirmed in East Asian individuals compared to Caucasians and individuals of African descent. Linkage disequilibrium was not detected between UGT1A1*6 and UGT1A1*28.

X-ray radiation causes electromagnetic interference in implantable cardiac pacemakers.

BACKGROUND: X-rays are not thought to cause electromagnetic interference (EMI) in implantable cardiac pacemakers. However, x-ray radiation during computed tomography (CT) scanning has been reported to cause EMI in some implantable cardiac pacemakers. The objectives of this study were to identify the location within the pacemakers where x-ray radiation causes EMI and to investigate the association of EMI with the x-ray radiation conditions. METHODS: We verified the location where x-ray radiation caused EMI using a CT scanner and conventional radiographic x-ray equipment. An inhibition test and an asynchronous test were performed using five types of implantable cardiac pacemakers. RESULTS: X-ray radiation inhibited the pacing pulses of four types of implantable cardiac pacemakers when the body of each implantable cardiac pacemaker, containing a complementary metal-oxide semiconductor (CMOS), was scanned using a CT scanner. We confirmed that x-ray-induced EMI depends on the x-ray radiation conditions, that is, the tube voltage, tube current, x-ray dose, and direction of x-ray radiation, as well as the sensing thresholds of the implantable cardiac pacemakers. CONCLUSIONS: X-ray radiation caused EMI in some implantable cardiac pacemakers, probably because the CMOS component was irradiated. The occurrence of EMI depended on the pacemaker model, sensing threshold of the pacemaker, and x-ray radiation conditions.