Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium - Serum-free growth of IGF-I producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

HUMAN CHOLESTERYL ESTER TRANSFER PROTEIN EXPRESSION ENHANCES THE MOUSE SURVIVAL RATE IN AN EXPERIMENTAL SYSTEMIC INFLAMMATION MODEL: A NOVEL ROLE FOR CETP

Mice expressing human cholesteryl ester transfer protein (huCETP) are more resistant to Escherichia coli bacterial wall LIPS because death rates 5 days after intraperitoneal inoculation of LIPS were higher in wild-type than in huCETP(+/-) mice, whereas all huCETP(+/+) mice remained alive. After LIPS inoculation, plasma concentrations of TNF-alpha and IL-6 increased less in huCETP(+/+) than in wild-type mice. LPS in vitro elicited lower TNF-alpha production by CETP expressing than by wild-type macrophages. In addition, TNF-alpha production by RAW 264.7 murine macrophages increased on incubation with LPS but decreased in a dose-dependent manner when human CETP was added to the medium. Human CETP in vitro enhanced the LIPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused C-14-LPS from Salmonella typhimurium was greater in huCETP(+/+) than in wild-type mice. Present data indicate for the first time that CETP is an endogenous component involved in the first line of defense against an exacerbated production of proinflammatory mediators.

Influence of estrogen deficiency and its treatment with alendronate and estrogen on bone density around osseointegrated implants: radiographic study in female rats

Objective. This study evaluated the influence of estrogen deficiency and its treatment on bone density around integrated implants. Study design. Implants were placed in female rat tibiae. The animals were assigned to 5 groups: control, sham, ovariectomy, estrogen, and alendronate. The control group was humanely killed to confirm integration of the implant. The others were submitted to ovariectomy or sham surgery. Bone density was measured by digital radiographs at 6 points on sides of the implant. Results. The analysis of radiographic bone density revealed estrogen privation had a negative impact only in the cancellous bone. The estrogen group differed significantly ( P <.05) from the ovariectomy and alendronate groups. The alendronate group presented the highest density for all evaluated regions. Conclusion. Ovariectomy caused a decrease in the radiographic bone density in the cancellous region. Estrogen replacement therapy and alendronate were effective treatments in preventing bone mass loss around integrated implants.

Bone mineral apparent density in juvenile dermatomyositis: the role of lean body mass and glucocorticoid use

Objective: To analyse bone mineral density (BMD) in juvenile dermatomyositis (JDM) and its possible association with body composition, disease activity, duration of disease, glucocorticoid (GC) use, and biochemical bone parameters, including osteoprotegerin (OPG) and receptor activator of nuclear factor B (RANKL). Methods: Twenty girls with JDM and 20 controls matched for gender and age were selected. Body composition and BMD were analysed by dual-energy X-ray absorptiometry (DXA) and bone mineral apparent density (BMAD) was calculated. Duration of disease, cumulative GC, and GC pulse therapy use were determined from medical records. Disease activity and muscle strength were measured by the Disease Activity Score (DAS), the Childhood Myositis Assessment Scale (CMAS), and the Manual Muscle Test (MMT). Inflammatory and bone metabolism parameters were also analysed. OPG and RANKL were measured in patients and controls using an enzyme-linked immunosorbent assay (ELISA). Results: A lower BMAD in the femoral neck (p< 0.001), total femur (p< 0.001), and whole body (p=0.005) was observed in JDM patients compared to controls. Body composition analysis showed a lower lean mass in JDM compared to controls (p=0.015), but no difference was observed with regard to fat mass. A trend of lower serum calcium was observed in JDM (p=0.05), whereas all other parameters analysed, including OPG and RANKL, were similar. Multiple linear regression analysis revealed that, in JDM, lean mass (p< 0.01) and GC pulse therapy use (p< 0.05) were independent factors for BMAD in the hip region. Conclusions: This study has identified low lean mass and GC pulse therapy use as the major factors for low hip BMAD in JDM patients.

Effect of Severe Dietary Magnesium Deficiency on Systemic Bone Density and Removal Torque of Osseointegrated Implants

Purpose This study evaluated the effect of severe magnesium (Mg) dietary deficiency on systemic bone density and biomechanical resistance of bone tissue to the removal torque of osseointegrated implants Materials and Methods The sample consisted of 45 rats, each received a titanium implant in their tibial metaphysis After 60 days, the animals were divided into three groups (n = 15) according to their dietary Mg the control group received the recommended content of Mg, group Mg1 received a 75% reduction in dietary Mg content, and group Mg2 was fed a diet with a 90% reduction in Mg con tent Animals were sacrificed 150 days after implant placement Serum concentrations of Mg were measured and the effect of Mg deficiency on systemic bone density was evaluated by densitometry of the lumbar vertebrae and femur Biomechanical characteristics were measured by resistance of the bone tissue to removal of the implants Results Lower Mg serum concentrations were found for the Mg1 and Mg2 groups, however, densitometric analysis and torque evaluations showed a statistically significant difference only in the Mg2 group (P < 05) There was a statistically significant difference in removal torque between the Mg2 group and the control group Conclusions This study showed that a severe deficiency of Mg decreased the systemic bone density and removal torque of osseointegrated implants INT J ORAL MAXILLOFAC IMPLANTS 2010 25 1125-1130

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Characterization of high density lipoprotein particles in familial apolipoprotein A-I deficiency

Our aim was to characterize HDL subspecies and fat-soluble vitamin levels in a kindred with familial apolipoprotein A-I (apoA-I) deficiency. Sequencing of the APOA1 gene revealed a nonsense mutation at codon 22, Q[22] X, with two documented homozygotes, eight heterozygotes, and two normal subjects in the kindred. Homozygotes presented markedly decreased HDL cholesterol levels, undetectable plasma apoA-1, tuboeruptive and planar xanthomas, mild corneal arcus and opacification, and severe premature coronary artery disease. In both homozygotes, analysis of HDL particles by two-dimensional gel electrophoresis revealed undetectable apoA-I, decreased amounts of small a-3 migrating apoA-II particles, and only modestly decreased normal amounts of slow a migrating apoA-IV- and apoE-containing HDL, while in the eight heterozygotes, there was loss of large alpha-1 HDL particles. There were no significant decreases in plasma fat-soluble vitamin levels noted in either homozygotes or heterozygotes compared with normal control subjects. Our data indicate that isolated apoA-I deficiency results in marked HDL deficiency with very low apoA-II alpha-3 HDL particles, modest reductions in the separate and distinct plasma apoA-IV and apoE HDL particles, tuboeruptive xanthomas, premature coronary atherosclerosis, and no evidence of fat malabsorption.

A methodology for current density calculations in high-frequency RF resonators

As nuclear magnetic resonance imaging and spectroscopy move inexorably toward higher field-strength magnets in search of improved signal-to-noise ratio, spectral resolution, and spatial resolution, the way in which radiofrequency (RF) probes are designed changes. At higher frequencies, resonant cavities become the favored RF ''coil'' type and may be built using streamline elements to reduce the inductance of the system. In modeling such systems, the quasi-static approach of assuming that current flows evenly in all conductor cross sections and that adjacent conductors do not affect each other becomes less reasonable. The proximity of RF conductors in resonators typically causes RF eddy currents to flow, whereby the current density in each rung is altered by the RF fields generated by nearby conductors. The proper understanding and prediction of how resonators will perform require a model of the current densities flowing in conducting sections, including all RF eddy current effects. Very few models of this type have been presented in the literature. This article presents an overview of one such model and of how it may be applied to a variety of resonators, both shielded and unshielded, circular, and elliptical, in cross section. Results are presented from a shielded head coil operating at 2 tesla. (C) 1997 John Wiley & Sons, Inc.

Human leukemia inhibitory factor upregulates LDL receptors on liver cells and decreases serum cholesterol in the cholesterol-fed rabbit

In a previous study, we found that the cytokine (human) leukemia inhibitory factor (hLIF) significantly reduced plasma cholesterol levels and the accumulation of lipid in aortic tissues of cholesterol-fed rabbits after 4 weeks of treatment. The mechanisms by which this occurs were investigated in the present study. This involved examining the effect of hLIF on (1) the level of plasma cholesterol at different times throughout the 4-week treatment and diet period; (2) smooth muscle cell (SMC) and macrophage-derived foam cell formation in vitro; and (3) LDL receptor expression and uptake in the human hepatoma cell line HepG2. At time zero, an osmotic minipump (2-mL capacity; infusion rate, 2.5 mu L/h; 28 days) containing either hLIF (30 mu g.kg(-1).d(-1)) or saline was inserted into the peritoneal cavity of New Zealand White rabbits (N=24). Rabbits were divided into four groups of six animals each. Group 1 received a normal diet/saline; group 2, a normal diet/hLIF; group 3, a 1% cholesterol diet/saline; and group 4, a 1% cholesterol diet/hLIF. hLIF had no effect on the plasma lipids or artery wall of group 2 rabbits (normal diet). However, in group 4 rabbits, plasma cholesterol levels and the percent surface area of thoracic aorta covered by fatty streaks was decreased by approximate to 30% and 80%, respectively, throughout all stages of the 4-week treatment period. In vitro, hLIF failed to prevent lipoprotein uptake by either SMCs or macrophages (foam cell formation) when the cells were exposed to P-VLDL for 24 hours. In contrast, hLIF (100 ng/mL) added to cultured human hepatoma HepG2 cells induced a twofold or threefold increase in intracellular lipid accumulation in the medium containing 10% lipoprotein-deficient serum or 10% fetal calf serum, respectively. This was accompanied by a significant non-dose-dependent increase in LDL receptor expression in hLIF-treated HepG2 cells incubated with LDL (20 mu g/mL) when compared with controls (P

Microvascular Lymphatic Density Analysis in Cutaneous Regressive and Nonregressive Superficial Spreading Melanomas Using the Lymphatic Marker D2-40

Background: The prognostic significance of spontaneous regression in melanoma, especially thin lesions, has been a controversial issue for the past 20 years, although recent studies suggest that extensive and late regression may be related to worse prognosis. Many data suggest that lymphangiogenesis predicts metastatic spread in melanoma. Methods: We have quantified lymphatic microvascular density (LMVD) in thin (<= 1.0 mm) superficial spreading melanomas comparing regressive and nonregressive melanomas, regressive and nonregressive areas from the same tumor, and early and late histological stages of regression in the same tumor. In addition, we tried to correlate lymphangiogenesis and tumor growth phase. We conducted histological examinations and immunohistochemical analyses using monoclonal antibody D2-40 with subsequent quantification by image analysis of 37 melanomas, 16 regressive and 21 nonregressive (controls). Results: We found higher LMVD in the late stage of regression compared with nonregressive area (internal control) of regressive melanomas. Conclusions: Our study suggest that the late stage of spontaneous regression in thin melanomas may be related to worse prognosis as it showed higher LMVD, and evidence shows that this is related with increased risk of metastatic spread. But this supposition must be confirmed by a longer follow-up for detection of lymph node metastases.

Altered loading patterns and femoral bone mineral density in children with unilateral Legg-Calve-Perthes disease

Maximization of bone accrual during the growing years is thought to be an important factor in minimizing fracture risk in old age. Mechanical loading through physical activity has been recommended as a modality for the conservation of bone mineral in adults; however, few studies have evaluated the impact of different loading regimes in growing children. The purpose of this study was to compare bone mineral density (BMD) in weight-bearing and non-weight-bearing limbs in 17 children with unilateral Legg Calve Perthes Disease (LCPD). Children with this condition have an altered weight-bearing pattern whereby there is increased mechanical loading on the noninvolved normal hip and reduced loading on the involved painful hip. Thus, these children provide a unique opportunity to study the impact of differential mechanical loading on BMD during the growing years while controlling for genetic disposition. BMD at four regions of the proximal femur (trochanter, intertrochanter, femoral neck, total of the regions) was measured using dual energy x-ray absorptiometry (DXA), and the values were compared between the involved and noninvolved sides of the children with LCPD. The BMD of both sides also were compared with normative values based on both chronological and skeletal age data. A significantly higher BMD was found on the noninvolved side over the involved side for all regions (P

Metabolism of triglyceride-rich lipoproteins and lipid transfer to high-density lipoprotein in young obese and normal-weight patients with polycystic ovary syndrome

Objective: To clarify whether the metabolism of triglyceride-rich lipoproteins and lipid transfer to high-density lipoprotein (HDL) are altered in patients with polycystic ovary syndrome (PCOS). Design: Case control study. Setting: Endocrinology clinics. Patient(s): Eight normal-weight (NW) and 15 obese (013) patients with PCOS were compared with 10 NW and 10 Ob women without PCOS paired for age and body mass index. Intervention(s): Determination of triglyceride-rich lipoprotein metabolism and lipid transfer to HDL. Main Outcome Measure(s): Participants were injected triglyceride-rich emulsions labeled with (14)C-cholesteryl esters and (3)H-triglycerides and the fractional clearance rate (FCR, in min(-1)) of labels was determined. Lipid transfer from artificial nanoemulsions to HDL was performed by incubating radioactively labeled lipid nanoemulsions with plasma during 1 hour, followed by radioactive counting of HDL-containing supernatant after chemical precipitation. Result(s): Lipolysis estimated by triglyceride FCR was equal in PCOS groups (NW = 0.043 +/- 0.032, Ob = 0.033 +/- 0.009) and respective controls (NW = 0.039 +/- 0.015, Ob = 0.044 +/- 0.019). However, the remnant removal as estimated by cholesteryl ester FCR was reduced in both PCOS groups (NW = 0.005 +/- 0.006, Ob = 0.005 +/- 0.005) compared with controls (NW = 0.016 +/- 0.006, Ob = 0.011 +/- 0.072). Lipid transfer rates were not different among groups, but triglyceride transfer rates were positively correlated with homeostasis model assessment estimate of insulin resistance in PCOS. Conclusion(s): PCOS patients showed decreased removal of atherogenic remnants even when fasting glucose was <100 mg/dL. This reinforces the usefulness of the measures taken to prevent cardiovascular events in PCOS patients. (Fertil Steril (R) 2010;93:1948-56. (C)2010 by American Society for Reproductive Medicine.)

Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum

Objective: To analyze vascular density and immunolocalization of angiogenic vascular endothelial growth factor (VEGF) and its receptor Flk-1 in the proliferative and secretory eutopic human endometrium. and in three different sites of endometriosis: the ovary, bladder, and rectum. Design: Prospective study. Setting: University hospital. Patient(s): Thirty women with endometriosis (10 ovarian, 1.0 bladder, 10 rectal) and 32 control women (10 proliferative endometrium, 10 secretory endometrium, 4 normal ovary, 4 normal bladder, 4 normal rectum). Intervention(s): Normal endometrial samples were obtained from women during laparoscopic ablation of subserous myoma, and biopsy specimens of endometriosis were obtained from patients undergoing surgery for the diagnosis and treatment of endometriosis. Normal tissues of ovary, bladder, and rectum were obtained from these organs beside the lesions of endometriosis. Main Outcome Measure(S): Blood vessels were quantified according to the number of von Willebrand factor-positive endothelial cells. The VEGF and Flk-1 distribution were evaluated semiquantitatively by immunohistochemical staining. Result(s): More blood vessels were found in cases of endometriosis, particularly rectal endometriosis, compared with the respective control samples and with the eutopic endometrium, and they were localized in endometrial stroma around the glands. The VEGF and Flk-1 expression levels were also higher in cases of endometriosis, especially rectal endometriosis. Conclusion(s): Vascularization and VEGF and Flk-1 expression are significantly higher in deeply infiltrating endometriosis affecting the rectum, reinforcing the hypothesis that antiangiogenesis therapy may constitute a new modality of treatment, especially in cases of deep endometriosis involving the rectum.

Influence of LH and high-density lipoprotein cholesterol (HDL-C) on metformin response in women with polycystic ovary syndrome

Objective: To search for predictors of metformin response in women with polycystic ovary syndrome (PCOS) through a detailed analysis of clinical and laboratory parameters. Study design: We designed a prospective study to investigate clinical and laboratory parameters to search for predictors of metformin response in women with PCOS. A total of 53 PCOS patients were given metformin 850 mg twice a day for 6 months, after which patients were classified as responders or non-responders. Parameters analyzed for comparison between the two groups were: plasma fasting insulin glucose/insulin ratio; oral glucose tolerance test (OGTT) with insulin (120 min); HOMA and QUICKI tests; total cholesterol and fractions, triglycerides; LH, FSH, estradiol, progesterone, testosterone, androstenedione, 17-OH progesterone, and DHEAS. Results: From all patients, 30(56.6%) were responders and 23(43.3%) were non-responders. Multinomial analysis showed that the positive response to metformin was associated with higher levels of basal LH (p = 0.038) and lower levels of high-density lipoprotein cholesterol (HDL-C) (p = 0.015). Conclusion: In weight-matched PCOS subjects, laboratory markers might predict the metformin response. Higher levels of basal LH and lower levels of HDL-C are correlated with a positive response to metformin treatment in PCOS subjects. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Multiple endocrine neoplasia type 1 in Brazil: MEN1 founding mutation, clinical features, and bone mineral density profile

Objective: Only few large families with multiple endocrine neoplasia type 1 (MEN1) have been documented. Here, we aimed to investigate the clinical features of a seven-generation Brazilian pedigree. which included 715 at-risk family members. Design: Genealogical and geographic analysis was used to identify the MEN1 pedigree. Clinical and genetic approach was applied to characterize the phenotypic and genotypic features of the family members. Results: Our genetic data indicated that a founding mutation in the MEN1 gene has occurred in this extended Brazilian family. Fifty family members were diagnosed with MEN1. Very high frequencies of functioning and non-functioning MEN1-related tumors were documented and the prevalence of prolactinoma (29.6%) was similar to that previously described in prolactinoma-variant Burin (32%). In addition, bone mineral density analysis revealed severe osteoporosis (T,-2.87 +/- 0.32) of compact bone (distal radius) in hyperparathyroidism (HPT)/MEN1 patients. while marked bone mineral loss in the lumbar spine (T,-1.95 +/- 0.39). with most cancellous bone, and femoral neck (mixed composition: T,-1.48 +/- 0.27) were also present. Conclusions: In this study, we described clinically and genetically the fifth largest MEN1 family in the literature. Our data confirm previous findings suggesting that prevalence of MEN1-related tumors in large families may differ from reports combining cumulative data of small families. Furthermore. we were able to evaluate the bone status in HPT/MEN1 cases, a subject that has been incompletely approached in the literature. We discussed the bone loss pattern found in our MEN1 patients comparing with that of patients with sporadic primary HPT.