Development of a 3-D energy-momentum analyzer for meV-scale energy electrons.

In this article, we report on the development of a time-of-flight based electron energy analyzer capable of measuring the 3-D momentum and energy distributions of very low energy (millielectronvolt-scale) photoemitted electrons. This analyzer is capable for measuring energy and 3-D momentum distributions of electrons with energies down to 1 meV with a sub-millielectronvolt energy resolution. This analyzer is an ideal tool for studying photoemission processes very close to the photoemission threshold and also for studying the physics of photoemission based electron sources.

Healthcare resource utilization and costs associated with long-term corticosteroid exposure in patients with systemic lupus erythematosus.

Objective To evaluate the association between exposure to oral corticosteroids and future healthcare resource utilization and costs for patients with systemic lupus erythematosus. Methods Adults diagnosed with systemic lupus erythematosus (index date) between 1 January 2008 and 30 June 2013 and naive to oral corticosteroids with continuous health plan enrollment for ≥6 months pre- and ≥5 years post-index were identified from a large health plan claims database. Per-patient monthly average daily dose of oral corticosteroids (prednisone or its equivalent) was calculated for the first 2 years post-index to categorize patients into four steroid exposure cohorts: low (≤5 mg/day), medium (6-20 mg/day), high (>20 mg/day) and no steroids. Differences in healthcare resource utilization and total healthcare costs during the third year post-index across corticosteroid exposure cohorts were modeled with adjustment for baseline characteristics. Results The study included 18,618 systemic lupus erythematosus patients (163 high dose, 1127 medium dose, 6717 low dose and 10,611 no steroids). Compared to low-dose corticosteroid users, high-dose corticosteroid users were more likely to have emergency room visits (39.3% vs. 29.7%; p = 0.0085) and to be hospitalized (21.5% vs. 12.3%; p = 0.0005). After adjustment for baseline characteristics, they also had significantly greater average annual total healthcare costs (US$60,366 vs. US$18,777; p < 0.0001). A 1 mg increase in corticosteroid average daily dose was associated with 1.07 times the average annual costs after adjusting for baseline characteristics ( p < 0.0001). Conclusion Long-term high-dose oral corticosteroid use was associated with significantly greater future healthcare resource utilization and costs. Judicious reduction in daily steroid dose may decrease the imminent economic burden associated with high-dose steroid use in systemic lupus erythematosus.

Impact of haemophilia with inhibitors on caregiver burden in the United States.

Inhibitor development complicates haemophilia treatment and may impact caregiver burden. Compare overall burden of caregivers of children with/without inhibitors in the United States using a novel disease-specific questionnaire and the previously validated CarerQol. An on-line questionnaire with six burden domains (i.e. emotional stress, personal sacrifice, financial burden, medical management, child's pain, and transportation) and three visual analogue scales (VAS) was developed based upon a targeted literature review and previous survey findings. The study sample consisted of caregivers of children with haemophilia. The total burden score was calculated by summing the six individual burden domain scores. Higher scores represented greater burden. Descriptive statistics was performed to examine the sample characteristics. The Wilcoxon rank-sum test was performed to compare burden by inhibitor status. All variables were considered significant at P < 0.001. A total of 310 caregivers completed the survey; 30 of them reported caring for a child with an inhibitor. A majority of caregivers of children with inhibitors were mothers (80.0%) and between 35 and 44 years of age (56.7%). Caregivers of children with inhibitors reported significantly higher median total burden scores (99.0 vs. 76.5, P < 0.0001) and median burden-VAS scores (5.5 vs. 3.0, P < 0.0001), as compared to those caring for children without inhibitors. A similar trend was seen across all the six burden domains, with greatest difference in the median burden scores observed in the 'personal sacrifice' (3.2 vs. 2.0) and 'transportation' (3.3 vs. 2.3) domains. Burden of caregivers should be considered when assessing the psychosocial aspects of managing patients with inhibitors.

Understanding the experience of caring for children with haemophilia: cross-sectional study of caregivers in the United States.

Congenital haemophilia is an inherited bleeding disorder typically diagnosed at birth or shortly thereafter. Haemophilia imposes a significant burden on patients and their caregivers. The aim of the study was to quantify the overall burden of haemophilia on caregivers in the USA using a novel disease-specific questionnaire and the previously validated CarerQol. Targeted literature review and a previous survey conducted by the authors was used to develop an online questionnaire with six burden domains of interest to caregivers (emotional stress, financial, sacrifice, medical management, child's pain and transportation) and several visual analogue scales (VAS). Content validity of the questionnaire was confirmed by three haemophilia caregivers. The study sample consisted of caregivers of children with haemophilia identified via a previously developed opt-in research database. Descriptive statistics were employed for demographic and clinical characteristics; a generalized linear model (GLM) was used to identify factors influencing caregiver burden. A total of 310 caregivers completed the survey (45.5% response rate). Most of the participating caregivers were mothers of a child with haemophilia (88%), between 35 and 44 years of age (48%), and with a college education or a postgraduate degree (63%). 'Child's pain' was identified as the most burdensome domain to caregivers (median score = 3.50 out of 5), followed by 'emotional stress' (2.67), 'financial' (2.40), 'transportation' (2.33), 'sacrifice' (2.17) and 'medical management' (2.00) domains. Although higher income exhibited a protective effect, episodes of bleeds, current presence of an inhibitor and lower caregiver productivity in the past month negatively affected caregiver burden per GLM results. Training and educational programs should potentially be developed to address caregiver burden.

Cholelithiasis induced in the Syrian hamster: evidence for an intramucinous nucleating process and down regulation of cholesterol 7 alpha-hydroxylase (CYP7) gene by medroxyprogesterone.

This report reviews previously published studies from our laboratory and shows some recent morphological data obtained with scanning and transmission electron microscopy regarding gallstone formation and alteration of the gallbladder epithelium in the Syrian hamster model. Both male and female hamsters were treated with female sex steroids (estradiol alone, estradiol and medroxyprogesterone, medroxyprogesterone alone) during one month. The results show that the Syrian hamster is a good model to study bile changes, gallbladder structure changes, including gallstone formation, and the regulation of cholesterol metabolism at the molecular level. Arguments in favor of this animal model are presented and, during gallstone formation, epithelial cell changes, anionic mucus secretion, and formation of gallbladder luminal deposits can be demonstrated. Recent molecular biology observations related to the effect of female sex steroids on liver cholesterol 7 alpha-hydroxylase (CYP7) gene suggest that progestin alone or primed by estrogen down regulates CYP7 transcription and activity. In addition, progesterone in cell culture systems has been shown to enhance intracellular accumulation of free cholesterol by increasing its uptake and synthesis and by decreasing its esterification by inhibiting the activity of acylcoenzyme A: cholesterol acyltransferase. Non-esterified cholesterol is free to migrate to the extracellular spaces and may contribute to nucleation within the bile. It is suggested that these effects of progesterone on cholesterol metabolism combined with the CYP7 gene down regulation, physical changes in the mucus and the hypomotility of the gallbladder and biliary ducts result in hypersaturation of cholesterol in the bile which favors gallstone formation.

Ultrastructural aspects of human gallbladder epithelial cells in cholelithiasis: production of anionic mucus.

The surface epithelium of 28 gallbladders removed during elective cholecystectomies and pathology collection was studied ultrastructurally. Focusing on 10 of the 28 cases that were diagnosed as cholecystitis, we found that the epithelium displayed numerous apical mucous granules and bulging apical apices. Mucous granule changes included 1) hyperproduction of secretory granules of neutral type containing an electron-dense proteinaceous spherule, similar to that described in other mucus-producing glands of the digestive system, and 2) production of anionic, osmiophilic secretory mucus. Other alterations of the surface epithelial cells included the production of bizarre surface appendages resembling primitive cilia without axoneme and epithelial excrescences.

Purification and characterization of human tissue prokallikrein and kallikrein isoforms expressed in Chinese hamster ovary cells.

We report here the expression of recombinant human prokallikrein and kallikrein in engineered Chinese hamster ovary cells transfected with a human genomic gene encoding preprokallikrein. At high expression levels, recombinant prokallikrein, an inactive proenzyme form, is predominantly secreted into the culture medium. Upon chromatographic separations, the inactive prokallikrein as well as the mature kallikrein after thermolysin activation of the proenzyme can be prepared to apparent purity. Both prokallikrein and kallikrein can be further separated into two distinct high- and low-molecular-weight isoforms. Kallikrein preparations are fully active in standard kallikrein activity assays such as esterase activity and kinin release from kininogen. Both kallikrein and prokallikrein display multiple molecular forms with differences in both molecular sizes and charges. The structural differences in high- and low-molecular-weight kallikreins or prokallikreins were found to be due to glycosylation, with the high-molecular-weight species glycosylated at three Asn-linked sites and the low-molecular-weight species at two of the three Asn-linked sites. The multiply charged kallikrein isoforms are derived from different numbers of sialic acids attached at the detected Asn-linked carbohydrates. In comparison with kallikrein, prokallikrein appears to show a significant decrease in the magnitude of near uv-circular dichroism bands, suggesting a change in local conformation. This conformational change correlates with the loss of activity in proenzyme due to the presence of propeptide.

Effects of sex steroids on the Syrian hamster liver.

The primary objective of this research project was to study the role of sex steroids in the pathogenesis of cholelithiasis using the Syrian hamster as a model. In addition to the morphological examination of the gallbladder epithelium, we thought it imperative to observe the changes induced in the biliary tract in response to the sex steroid treatment. This report focuses on the morphological changes induced in the liver. The hamsters were randomly divided into 4 groups, control (C), estrogen-treated (E), estrogen and medroxyprogesterone-treated (E+MP), and medroxyprogesterone-treated (MP) groups. The E group hepatocytes demonstrated proliferation of the smooth endoplasmic reticulum, lipofuscin-like granules, aggregates of glycogen rosettes, and dense bodies. Lipid droplets in the hepatocyte cytoplasm as well as the nuclei were detected in this group. E+MP combined treatment induced an exacerbation of all the changes observed in the E group, furthermore, there appeared to be a disruption of the hepatic parenchymal architecture. The MP-treated group also exhibited the architectural changes observed in the E+MP group, but also showed sinusoidal dilation. In response to MP alone, the fatty changes in the liver appeared to be accentuated. A striking feature induced in response to MP treatment, was a focal area suggestive of adenomatous changes.

Ultrastructural changes of female Syrian hamster cystic duct epithelium as a result of sex steroid treatment.

In view of the lack of sufficient data regarding the morphology of the cystic duct and the extensive focus on the gallbladder, a preliminary examination of the cystic duct response to female sex steroid treatment was conducted to follow up a detailed ultrastructural study of the gallbladder epithelial response to a similar treatment. As observed in the gallbladder epithelium, the cystic duct epithelial cells of nulliparous Syrian hamsters demonstrate morphologic changes in response to female sex steroid treatment. Control (C) cystic duct epithelial cells are covered by short microvilli and each cell appears to exhibit a single vestigial cilium. Estrogen (E)- and estrogen + medroxyprogesterone (E + MP)-treatments induce differential duct cell morphologic changes. These changes are the result of steroid treatments in the significant decreasing sequence E > E + MP > C for nuclear volume, indentations and perinuclear lysosomal/lipofuscin bodies. Moreover E + MP-treatment results in larger cytoplasmic volume and more sloughing of apical cell excrescences than following E treatment. It is suggested that, similar to that in the gallbladder, the action of progestin is paramount in favoring cytoplasmic morphological changes in the cystic duct which, along with the alteration of mucus, cell sloughing, decreased bile acids and motility could also participate in the gallstone nucleation process as they are brought into the gallbladder with the incoming bile flow.

Gallstones induced by sex steroids in the female Syrian hamster: duration effects.

Scanning (SEM), and transmission (TEM) electron microscopy were used and correlated to morphologically characterize changes induced in the gallbladder epithelium of female Syrian hamster in response to treatments with estradiol (E) alone, and estradiol with medroxyprogesterone (E + MP). Compared with control (C), the E- and E + MP-treated groups demonstrated alterations in the serum lipid profile as well as significantly decreased body weights. The liver with gallbladder weights, as well as the uterus weights, were significantly increased. Two-month E and E + MP treatment groups exhibited increased number of anionically charged apical granules, and luminal mucoid elements. Contrastingly, the three-month treatment groups demonstrated larger and more gallstone-like deposits as compared to the C and two-month E and E + MP groups. This report presents a comprehensive overview of our previous and current data, including that of other investigators in relation to the various factors and parameters involved in the cholelithiatic process.

Morphological aspects of female Syrian hamster gallbladder induced by one-month sex steroid treatment.

Light (LM), transmission (TEM), and scanning (SEM) electron microscopy were used to characterize morphological changes induced in the gallbladder epithelium of female Syrian hamsters in response to one-month estradiol alone (E) and estradiol with medroxyprogesterone (E + MP) treatments. TEM data were correlated with the SEM observations. Compared with control (C), E- and E + MP-treated hamsters showed significant decreases in body weight, while the liver and gallbladder, and uterus weights increased. Moreover, E treatment induced some subcellular changes (microvilli, nucleus, mitochondria, RER, glycogen, abundant apical granules). The E + MP treatment appeared to exacerbate these similar changes and, in addition, induced apical excrescences and cell shedding. Both E and E + MP gallbladders showed luminal micelles, cellular debris and crystalliths associated with mucus. Simultaneously, an increased acidification of the mucoid content of the apical granules was noticed.

Comparison of N-linked oligosaccharides of recombinant human tissue kallikrein produced by Chinese hamster ovary cells on microcarrier beads and in serum-free suspension culture.

Glycosylation heterogeneity in recombinant human tissue kallikrein (r-HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were observed in glycoform distribution and oligosaccharide heterogeneity. High-performance liquid chromatography with a pellicular anion-exchange column under low pH eluant conditions was used to characterize the number and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion-exchange conditions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, encompassing mono-, di-, tri-, and tetrasialylated complex type oligosaccharides.

Cytometric study of the female Syrian hamster gallbladder epithelium following sex steroid administration.

This report is a cytometric study of the female Syrian hamster gallbladder epithelium following 1-, 2-, and 3-month administration of female sex steroids. Nulliparous, multiparous, young, old and pregnant hamsters were used in this study. A 1 month treatment with estrogen alone significantly increases the nuclear volume of the gallbladder epithelial cells, while E + P treatment significantly affects the nuclear volume only after a 2 month treatment. On the other hand, E + P and P treatments significantly increase the cell volumes as compared to the E-treated groups, this effect is most striking following the 1 month period. Prolonged sex steroid treatment (2 and 3 month) does not appear to influence the gallbladder epithelial cell and nuclear volumes as dramatically as that observed following the 1 month treatment. The nulliparous, progesterone-treated hamsters appear to have a greater cytoplasmic volume than the multiparous group and this is substantiated by the bulging apices and the luminal cellular excrescences observed with scanning and transmission electron microscopy. These observations are similar to those reported in ovariectomized hamsters (Gilloteaux et al., 1992). Further, the gallbladder epithelial cells and nuclei of the older female hamsters demonstrate an accentuated response to a 1 month sex steroid treatment as compared to the younger hamsters for the same treatment duration. These results enable us to hypothesize that changes induced by a short term sex steroid treatment participate in the gallstone nucleation process, while longer duration of the treatments contribute to progressive enlargement and accumulation of gallbladder calculi.

Apical excrescences in the gallbladder epithelium of the female Syrian hamster in response to medroxyprogesterone.

All the intact female Syrian hamsters treated with medroxyprogesterone (MP) for a one-month period, without dietary manipulation, display gallbladder surface epithelial changes, and intraluminal deposits. These changes include excrescences in various stages, bulging, and extrusion of material from the epithelial cells. The most striking scanning electron microscopic observations are the dramatic events, comparable to apocrine-like secretory events observed in another related study using oophorectomized hamsters. Since the hamster gallbladder does not possess mucous goblet cells, it appears that this phenomenon could be a response to the MP treatment, thus providing a larger amount of mucous product than usual with cellular material, in addition to the possible alteration in the quality of the bile following this treatment. As a result of MP treatment, intraluminal deposits were also confirmed by using light and transmission electron microscopy. In control hamsters these events were not observed, however, small blebs outlining surface epithelial cells are seen. The results in this report complement the previous studies using the male and oophorectomized Syrian hamster model subjected to similar experimental conditions.

Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO cells.

The effect of cloned gene copy number on growth and product formation has been studied in sufficient detail using a Chinese hamster ovary (CHO) cell line producing recombinant hepatitis B surface antigen (HbsAg). Batch culture experiments were carried out in T flasks in order to characterize cell growth and HbsAg secretion in various clones carrying different numbers of HbsAg gene copies integrated into CHO cell chromosomes. Specific growth rates were found to decrease with increasing gene copy number. Secreted HbsAg concentration and specific HbsAg secretion rates were found to increase with increase in gene copy number. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (dhfr) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autoradiography and densitometry also gave the same trends. The flow cytometry experiments show that while parental cells were quite homogeneous with respect to intracellular dhfr content, the amplified clones exhibit a great deal of heterogeneity in dhfr content. Pulse-chase experiments show that the efficiency of HbsAg secretion (defined here as the fraction of initially labeled HbsAg that is secreted into the extracellular medium at the end of a 23.5-h chase) decreases and also that the intracellular HbsAg degradation increases with increasing gene copy number.

Female sex steroid induced epithelial changes in the gallbladder of the ovariectomized Syrian hamster.

Ovariectomized Syrian hamsters treated by female sex steroids during a 1-month period show gallbladder surface epithelial changes in the fundic area consistent with apical bulging and decapitations of the epithelial cells. These events were detected in the infundibulum and the fundic or body regions of estrogen- and estrogen+progesterone-treated hamsters. In control hamsters, these events were restricted to the region in the vicinity of the bile duct. Following steroid treatment, intraluminal deposits detected resembled Ca-bilirubinate deposits described in previous studies while decapitations are similar to endometrial epithelium changes associated with hormonal physiological changes or treatments. Moreover some small electron-dense deposits are comparable to those found in human cholesterol gallstones. This report indicates that, besides an alteration in bile composition, cell fragments originating from the surface epithelium of the bile duct and/or of the gallbladder mucosal epithelium could participate in gallstone nucleation.

Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver--conditioned medium.

We have identified a novel growth factor, stem cell factor (SCF), for primitive hematopoietic progenitors based on its activity on bone marrow cells derived from mice treated with 5-fluorouracil. The protein was isolated from the medium conditioned by Buffalo rat liver cells. It is heavily glycosylated, with both N-linked and O-linked carbohydrate. Amino acid sequence following removal of N-terminal pyroglutamate is presented. The protein has potent synergistic activities in semisolid bone marrow cultures in conjunction with colony-stimulating factors. It is also a growth factor for mast cells. In two companion papers, we present the sequences of partial SCF cDNAs, identify SCF as a c-kit ligand, and map the SCF gene to the Sl locus of the mouse.

Cultivation of hybridoma cells in continuous cultures: kinetics of growth and product formation.

Mouse hybridoma cells were grown in suspension in continuous stirred bioreactors. Cell growth, substrate utilization, and monoclonal antibody (MAb) production were studied using serum-free medium. Steady-state data were obtained at different dilution rates, between 0.012 and 0.039 h(-1) Viability was profoundly affected by dilution rate, particularly near the lower end of the dilution-rate range investigated. MAb concentration and productivity went through a maximum with respect to dilution rate. Lactate yield on glucose declined with increasing dilution rate. Experiments were carried out to study the effects of medium glucose concentration on cell growth, product formation, and lactate yield on glucose. Reduction of glucose concentration in the feed medium did not considerably affect cell density and MAb concentration in the culture, but lactate levels dropped sharply; lactate yield on glucose declined substantially, indicating alterations in cell metabolic path ways for energy metabolism. Optimization strategy for continuous cell culture is discussed.