Cells In Serum-free Medium Research Articles

Mechanism of ethanol enhancement of apoptosis and caspase activation in serum-deprived PC12 cells

Neuronal death is one of the most prominent consequences of alcohol exposure during development. Ethanol-induced neuronal death appears to involve apoptosis. The objective of the present study was to characterize the effect of ethanol on neuronal cell viability and to determine the mechanism by which ethanol enhances apoptosis in neural cells. For these studies the rat pheochromocytoma (PC12) cells were used. PC12 cells were incubated for 24 h in the presence or absence of 100 mM ethanol. Apoptosis was induced by serum withdrawal. Ethanol in the presence of serum-containing media did not alter cell viability, while incubation of PC12 cells in serum-free media resulted in a significant increase in cell death that was further significantly increased by 35% in cells exposed to ethanol. The temporal response of the PC12 cells to serum withdrawal was studied over a period of 22 h. At least 18 h of ethanol exposure was necessary to observe a significant increase in death for cells incubated in serum-free media. An increase in the caspase-3 activity in PC12 cells deprived of serum was observed that was further increased by ethanol exposure. This increase of caspase-3 activity was correlated with an enhancement of caspase-9 activity. Ethanol exposure increased the amount of cytosolic cytochrome c in PC12 cells incubated in serum-free media but did not alter the level of cytochrome c in cells incubated in serum. Finally, a 26% increase was observed in the number of cells with depolarized mitochondria due to ethanol treatment. The present study implicates an increase in the mitochondrial outer membrane permeability as a potential mechanism of enhancement of apoptosis in serum-deprived PC12 cells by ethanol.

Expression and Function of Fibroblast Growth Factor 10 and Its Receptor, Fibroblast Growth Factor Receptor 2B, in Bovine Follicles1

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

H2S, Endoplasmic Reticulum Stress, and Apoptosis of Insulin-secreting Beta Cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.

Tumor Engineering: Orthotopic Cancer Models in Mice Using Cell-Loaded, Injectable, Cross-Linked Hyaluronan-Derived Hydrogels

Current cancer xenograft models used to evaluate new anticancer therapies are limited to "good take" cell lines, fail to mimic normal human disease, and poorly predict clinical outcomes. We now describe the use of an injectable, in situ cross-linkable synthetic extracellular matrix (sECM) to deliver and grow cancer cells in vivo. The hyaluronan (HA)-derived sECMs were seeded with breast, colon, and ovarian cancer cells prior to gelation, and then injected subcutaneously into mammary fat pads, subserosally in colons, and intracapsularly in ovaries, respectively. Two cell lines were used for each type of cancer, and results were compared with orthotopic injection of cells in serum-free medium. At 4 weeks postinjection, four parameters were measured: (i) incidence and size of cancer at the injection site, (ii) vascularization or necrosis of new cancer tissue, (iii) cancer seeding in adjacent tissues, and (iv) metastasis to lymph nodes and other vital organs. In addition, the activation of the phosphoinositide 3-kinase (PI 3-K) signaling pathway was analyzed immunohistochemically. Overall, orthotopic delivery of cancer cells in sECM hydrogels showed clear advantages: (i) increased incidence of cancer formation and reduced variability in tumor size, (ii) enhanced growth of organ-specific cancers with good tumor-tissue integration, (iii) improved vascularization and reduced necrosis within the tumor, (iv) reduced cancer seeding on adjacent tissues, and (v) better general health of animals. Thus, engineered tumors represent an improved approach to traditional tumor xenografts, and facilitate studies in cancer biology, invasion and metastasis, as well as the investigation of new therapeutic and diagnostic protocols.

Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells

Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5 × 10 11 infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.

Angiotensin-TGF-β1 Crosstalk in Human Idiopathic Pulmonary Fibrosis:Autocrine Mechanisms in Myofibroblasts and Macrophages

Angiotensin II (ANGII) has been identified as a proapoptotic and profibrotic factor in experimental lung fibrosis models, and patients with the ID/DD polymorphism of ANG converting enzyme (ACE), which confers higher levels of ACE, are predisposed to lung fibrosis (Hum. Pathol. 32:521-528, 2001). Previous work from this laboratory has shown that human lung myofibroblasts isolated from patients with Idiopathic Pulmonary Fibrosis (IPF) synthesize the ANGII precursor angiotensinogen (AGT) constitutively. In attempts to understand the mechanisms and consequences of constitutive AGT synthesis by myofibroblasts, we studied myofibroblast-rich primary cultures of lung fibroblasts from patients with IPF (HIPF isolates), primary fibroblasts from normal human lung (NLFs), the IMR90 and WI38 human lung fibroblasts cell lines, and paraffin sections of lung biopsies from patients with IPF. Compared to the normal NLF isolates, HIPF primary fibroblast isolates constitutively synthesized more AGT and TGF-beta1 mRNA, and released more AGT protein, ANGII and active TGF-beta1 protein into serum-free conditioned media (both p<0.01). Incubation of HIPF fibrotic isolates with the ANGII receptor antagonist saralasin reduced both TGF-beta1 mRNA and active protein, suggesting that the constitutive expression of AGT drives the higher expression of TGF-beta1 by the HIPF cells. Consistent with this premise, treatment of either the primary NLFs or the WI38 cell line with 10(-7) M ANGII increased both TGF-beta1 mRNA and soluble active TGF-beta1 protein. Moreover, induction of the myofibroblast transition in the IMR90 cell line with 2 ng/ml TGF-beta1 increased steady state AGT mRNA levels by realtime PCR (8-fold, p<0.01) and induced expression of an AGT promoter-luciferase reporter construct by over 10-fold (p<0.001). Antisense oligonucleotides against TGF-beta1 mRNA or TGF-beta neutralizing antibodies, when applied to the fibrotic HIPF cells in serum-free medium, significantly reduced AGT expression. In lung sections from IPF patient biopsies, immunoreactive AGT/ANGI proteins were detected in myofibroblasts, epithelial cells and presumptive alveolar macrophages. Together, these data support the existence of an angiotensin/TGF-beta1 "autocrine loop" in human lung myofibroblasts and also suggest ANG peptide expression by epithelia and macrophages in the IPF lung. These findings may explain the ability of ACE inhibitors and ANG receptor antagonists to block experimental lung fibrosis in animals, and support the need for evaluation of these agents for potential treatment of human IPF. This manuscript discusses the data described above and their implications regarding IPF pathogenesis.

Chitosan nanoparticles are compatible with respiratory epithelial cells in vitro

The aim of this work was to evaluate the biocompatibility of novel respirable powder formulations of nanoparticles (NP) entrapped in mannitol microspheres using human respiratory epithelial cell lines. Microspheres formulated at NP:mannitol ratios of 10:90, 20:80 and 40:60 were evaluated using the Calu-3 and A549 cell lines. The MTT cell viability assay revealed an absence of overt toxicity to Calu-3 or A549 cells following exposure to the formulations containing <1.3 mg NP/ml (equivalent to 0.87 mg NP/cm 2) for up to 48 h. Transepithelial electrical resistance (TER) and solute permeability in Calu-3 cell layers were determined following exposure of the cells to the NP:mannitol 20:80 formulation. After administration of the formulation dissolved in serum-free cell culture medium (1.3 mg/ml NP suspension) to the cells, neither TER nor permeability were altered compared to untreated cell layers. Confocal microscopy did not reveal any NP internalisation under the conditions used in this study, although evidence of mucoadhesion was observed. All the data presented are encouraging with respect to the development of chitosan NP-containing microspheres for the pulmonary administration of therapeutic macromolecules. Not only do the formulations possess suitable aerodynamic characteristics and the capacity to encapsulate proteins as shown previously; they have now been shown to exhibit in vitro biocompatibility.

Possibility of insulin-producing cells derived from mouse embryonic stem cells for diabetes treatment

Insulin injection therapy is the principal current treatment of type 1 diabetes. Patients, however, suffer from various complications generated by insufficient control of blood glucose levels over a long period. Therefore, a method which can infuse insulin in response to changes of blood glucose levels is eagerly desired. Transplantation of insulin releasing cells derived from embryonic stem (ES) cells has been expected to be one of promising approaches to realize this requirement. In this study, ES cell progeny which were derived in culture media with/without fetal calf serum contained two distinct kinds of cells immunostained by anti-insulin and anti-C-peptide antibodies. The cytoplasm and nuclei of one type of cell were immunoreactive against antibodies for insulin, while the other kind of cell only had the cytoplasm stained by the anti-insulin antibody. The first cell type was the major population of insulin-positive cells in serum-free medium, while the latter kind of cells was the major population in medium containing serum. Interestingly, the latter insulin-positive cells could be also immunostained by anti-C-peptide antibodies, and was observed even after nine subcultures in medium containing serum. Although there still remain many issues to be addressed in order to definitely demonstrate that insulin-positive cells derived from ES cells to be truly beta cells in the islets, these properties of the obtained cells are believed to promising cells for treatment of type 1 diabetes.

Serum-free cell culture system supplemented with lipid-rich albumin for hepatitis C virus (strain O of genotype 1b) replication

HuH-7 is a highly differentiated hepatoma cell line and the only cell line that supports robust RNA replication of the hepatitis C virus (HCV). HuH-7 cells cause cell death in serum-free culture condition. However, the effect is reversed by supplementation with selenium. Serum-free cell cultures are advantageous for vaccine development and experimental reproducibility. However, HCV RNA replication in HuH-7 cells in serum-free medium had not yet been achieved. Therefore, we tried to develop a system for robust HCV RNA replication in a serum-free cell culture. Although HuH-7 cells grew in serum-free medium in the presence of selenium, HuH-7 cells under these conditions did not support HCV RNA replication in long-term culture. Among the supplements tested, serum-free medium with lipid-rich albumin (LRA) was found to yield robust HCV RNA replication. HCV proteins were detected for more than 9 months in serum-free medium supplemented with LRA. This is the first report to demonstrate a long-term, serum-free cell culture that successfully maintained robust HCV RNA replication. This cell culture system is expected to be a useful tool for vaccine development, as well as for further investigation of cellular factors that are essential for HCV RNA replication.

Specific High Affinity Interactions of Monomeric Endotoxin·Protein Complexes with Toll-like Receptor 4 Ectodomain

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin.protein complexes of high specific radioactivity. The binding of endotoxin.MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD+/-MD-2. These interactions are specific, of high affinity (KD<300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr approximately 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin.MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin.protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.

Directed differentiation of human embryonic stem cells into functional hepatic cells†

The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A1, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low-density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition, we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests.

Development of standardized cell culture conditions for tumor cells with potential clinical application

Tumor cell lines have enormous value for the study of different aspects of cancer biology and have also recently gained great importance in autologous cell-based anti-tumor therapies. However, the use of these cells is still limited because in vitro growth is hampered by suboptimal culture conditions and current media contain fetal bovine serum (FBS), which poses serious safety concerns regarding clinical application. To address this drawback, we aimed to develop a strategy for optimization of the culture medium for human medullary thyroid carcinoma (MTC) cell lines as a model system. We combined the general cell screening system (GCSS), which continuously measured the growth behavior of cells in a 96-well plate format, with statistically based experimental designs. The results obtained clearly demonstrated that, just by changing the composition of the basal medium, a significantly enhanced growth rate could be observed, and by subsequent addition of several substances a serum-free cell culture medium could be developed. This medium allowed the propagation of two MTC cell lines comparable with conventionally used serum-supplemented medium. We present a fast and easy way to screen for substances that are essential for tumor cell growth in vitro. Furthermore, these tumor cells can be adapted to culture conditions that allow the use of the cells in safe cell-based therapies. This is of utmost importance because of increasing regulatory requirements.

VDR‐mediated gene expression patterns in resting human coronary artery smooth muscle cells

Vitamin D analogs such as paricalcitol and calcitriol that activate the vitamin D receptor (VDR) provide survival benefit for Stage 5 chronic kidney disease (CKD) patients, possibly associated with a decrease in cardiovascular (CV)-related incidents. Phenotypic changes of smooth muscle cells play an important role in CV disease. The role of vitamin D analogs in modulating gene expression in smooth muscle cells is still not well understood. In this study, DNA microarray analysis of approximately 22,000 different human genes was used to characterize the VDR-mediated gene expression profile in human coronary artery smooth muscle cells (CASMC) at rest. Cells in serum free medium were treated with 0.1 microM calcitriol (1alpha,25-dihydroxyvitamin D(3)) or paricalcitol (19-nor-1alpha,25-(OH)(2)D(2)) for 30 h. A total of 181 target genes were identified, with 103 genes upregulated and 78 downregulated (>two fold changes in either drug treatment group with P < 0.01). No significant difference was observed between calcitriol and paricalcitol. Target genes fell into various categories with the top five in cellular process, cell communication, signal transduction, development, and morphogenesis. Twenty-two selected genes linked to the CV system were also impacted. Real-time RT-PCR and/or Western blotting analysis were employed to confirm the expression patterns of selected genes such as 25-hydroxyvitamin D-24-hydroxylase, Wilms' tumor gene 1, transforming growth factorbeta3, plasminogen activator inhibitor-1, thrombospondin-1 (THBS1), and thrombomodulin (TM). This study provides insight into understanding the role of VDR in regulating gene expression in resting smooth muscle cells.

GATA-1 Protein Levels Are Not Altered and Are Correlated to the Jak-2 Mutation Status in Patients with Idiopathic Myelofibrosis (IMF).

GATA-1 is important in megakaryocytopoiesis, and the fact that GATA-1low mice eventually develop myelofibrosis indicates that GATA-1 is important in the pathogenesis of idiopathic myelofibrosis (IMF). Previous study has shown that GATA-1 levels were lower in protein content in CD61+ cells of patients with IMF (Vannucci et al., Am J Pathol 167:849 2005). But the study was done with pooled protein from only three patients. We used ELISA assay (GATA-1 ELISA kit, Panomics, CA) to assay the protein content from nuclear extracts of blood hematopoietic progenitor cells (CD34+cells) and megakaryocytes (CD61+ cells) from patients with IMF and compared the results with those of patients with other myeloproliferative disorders and with controls. CD 61+ cells were obtained from 10-day cultures of blood CD34+ cells in serum-free culture medium with thrombopoietin and stem cell growth factor as described. The results are shown in Table 1. No statistical difference was found in the GATA-1 content in the nuclear extracts in CD34+ or CD61+ cells. We further analyze the GATA-1 levels in relation to the JAK2 status and significant correlation between the levels of GATA-1 levels and positive JAK2 nutation were found in blood CD34+ cells (p=0.01) and subsignificant in CD61+cells (p=0.08) (Table2) .These preliminary results suggest that GATA-1 protein contents are not altered and higher GATA-1 levels are seen in JAK-2 mutation positive patients with IMF.Table 1GATA-1 Levels in Patients with IMF and othersIMFMPDControlsCD34+ cells27.58±3.2 (n=9)30.57±5.4 (n=4)28.06±2.9 (n=7)CD61+ cells58.99±11.53 (n=8)38.64±18.2(n=4)41.03±11.3 (n=6)GATA-1 Levels: mean± SE of ng/2μg protein; n: numbers of patientsTable 2Correlation between GATA-1 Levels and JAK2 Status in Patients with IMFJAK2 mutation PositiveJAK2 mutation NegativeP ValueCD34 + cells35.6±3.4 (n=4)21.1±2.7(n=5)0.01CD61+ cells73.7±13.0(n=4)43.8±13.7(n=5)0.08GATA-1 Levels: mean±SE(ng/2μg protein): n: numbers of patients

Megakaryocytes Cultured from Patients with IMF Produced Significantly Higher mRNA but Not Protein Levels of Fibrosing Grown Factors.

Currently, the prevailing concept concerning the etiology of bone marrow fibrosis in patients with idiopathic myelofibrosis (IMF) is that it results from excessive production of fibrosing growth factors including transforming growth factor beta (TGF-B1), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) from megakaryocytes and monocytes. Since megakaryocytes are difficult to isolate from bone marrow in IMF patients, this concept remains speculative. We obtained megakaryocytes (CD41+ cells) from 10-day in vitro culture of blood CD34+ cells in serum-free medium with thrombopoietin and stem cell factors as described and cultured monocytes from isolating blood CD14+ cells. Then quantitative analyses of fibrosing growth factors at the mRNA and protein levels were obtained. mRNA levels were obtained from real-time RT-PCR technique, and protein levels were obtained from ELISA analysis of the supernatant of CD41+ cells cultured 4 h in serum-free medium. The results showed 1) mRNA levels of TGF-B1, PDGF, and FGF produced by the megakaryocytes were significantly elevated in agnogenic myeloid metaplasia (AMM) compared with those in normal controls (p<0.05). While these growth factors were elevated several-fold in AMM compared with other myeloproliferative disorders (MPD) including essential thrombocythemia and polycythemia vera, they were not statistically significant. 2) mRNA levels of TGF-B1 were higher than levels of PDGF or FGF. 3) The mRNA levels of these growth factors produced from CD14+ cells were not significantly elevated in AMM compared with other MPDs or controls; the AMM mRNA levels were significantly elevated only in some patients. 4) The correlation of mRNA levels of these growth factors with the degree of myelofibrosis in AMM was significant with megakaryocytes (r=0.73) but not with monocytes (r=0.23). 5) ELISA analysis of the growth factors from the cultured megakaryocytes showed that, in most of the patients with AMM and other MPDs and in volunteer controls, the growth factors were undetectable, and only a few patients with AMM had significantly elevated protein levels of these growth factors. We conclude thatin IMF, megakaryocytes but not monocytes are the predominant cells producing fibrosing growth factors, andthe failure of finding increased protein levels of these growth factors in the in vitro system suggest that other factors are necessary to initiate translation of these growth factors in the megakaryoctes, and neutrophil emperipolesis with releasing factors may be important in this process.

HIV gp120-induced Interaction between CD4 and CCR5 Requires Cholesterol-rich Microenvironments Revealed by Live Cell Fluorescence Resonance Energy Transfer Imaging

Binding of the human immunodeficiency virus (HIV) envelope gp120 glycoprotein to CD4 and CCR5 receptors on the plasma membrane initiates the viral entry process. Although plasma membrane cholesterol plays an important role in HIV entry, its modulating effect on the viral entry process is unclear. Using fluorescence resonance energy transfer imaging, we have provided evidence here that CD4 and CCR5 localize in different microenvironments on the surface of resting cells. Binding of the third variable region V3-containing gp120 core to CD4 and CCR5 induced association between these receptors, which could be directly monitored by fluorescence resonance energy transfer on the plasma membrane of live cells. Depletion of cholesterol from the plasma membrane abolished the gp120 core-induced associations between CD4 and CCR5, and reloading cholesterol restored the associations in live cells. Our studies suggest that, during the first step of the HIV entry process, gp120 binding alters the microenvironments of unbound CD4 and CCR5, with plasma membrane cholesterol required for the formation of the HIV entry complex.

Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium

Objectives: Development of an ELISA for human insulin that utilizes monoclonal antibodies (mAbs) produced in serum-free medium. Design and methods: Insulin mAbs were produced in vitro by hybridomas in serum-free medium. A two-step ELISA was developed to replace bovine insulin (standard) and bovine serum albumin (assay buffer) with non-animal reagents. Results: The sensitivity of the insulin ELISA was 0.73 uU/mL with a dynamic range of 2–200 uU/mL. No cross-reactivity with either human C-peptide or human proinsulin was observed. The intra- and inter-assay CVs were < 7%. The mean recovery of insulin added to plasma samples was between 102.2% and 105.7%. The mean linearity of dilution was between 93% and 110% of undiluted plasma samples. The animal serum-free (ASF) insulin ELISA showed no marked degradation of any kit component when stored at 37°C for up to 7 days. Significantly higher fasting insulin levels were observed in overweight or obese subjects ( n = 12) compared to lean subjects ( n = 10, p < 0.05). Feeding markedly increased fasting insulin levels in both lean ( p < 0.02) and overweight or obese ( p < 0.005) subjects. Excellent correlation was observed between insulin levels measured by ASF insulin ELISA and another CE marked insulin ELISA ( y = 1.06 x − 0.44, r = 1.00, n = 44). Conclusions: This novel insulin ELISA provides precision and reliability equal to methods currently used in clinical research and serves as a guide for the development of other serum-free immunoassays.

Secretion of Vascular Endothelial Growth Factor by Primary Human Fibroblasts at Senescence

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. Nonetheless, the senescence response is thought to be antagonistically pleiotropic and thus contribute to aging phenotypes, including, ironically, late life cancers. The cancer-promoting activity of senescent cells is likely due to secreted molecules, the identity of which remains largely unknown. Here, we have shown that senescent fibroblasts, much more than presenescent fibroblasts, stimulate tumor vascularization in mice. Weakly malignant epithelial cells co-injected with senescent fibroblasts had larger and greater numbers of blood vessels compared with controls. Accordingly, increased vascular endothelial growth factor (VEGF) expression was a frequent characteristic of senescent human and mouse fibroblasts in culture. Importantly, conditioned medium from senescent fibroblasts, more than medium from presenescent cells, stimulates cultured human umbilical vein endothelial cells to invade a basement membrane, a hallmark of angiogenesis. Increased VEGF expression was specific to the senescent phenotype and increased whether senescence was induced by replicative exhaustion, overexpression of p16(Ink4a), or overexpression of oncogenic RAS. The senescence-dependent increase in VEGF production was accompanied by very little increase in hypoxic-inducible (transcription) factor 1 alpha protein levels, and hypoxia further induced VEGF in senescent cells. This result suggests the rise in VEGF expression at senescence is not a hypoxic response. Our findings may in part explain why senescent cells stimulate tumorigenesis in vivo and support the idea that senescent cells may facilitate age-associated cancer development by secreting factors that promote malignant progression.

Regeneration of Megakaryocytopoiesis and ThrombopoiesisIn Vitrofrom X-Irradiated Human Hematopoietic Stem Cells

In the present study, we investigated whether X-irradiated hematopoietic stem cells can be induced to undergo megakaryocytopoiesis and thrombopoiesis in vitro using cytokine combinations that have been demonstrated to be effective for conferring increased survival on irradiated human CD34(+) megakaryocytic progenitor cells (colony-forming unit megakaryocytes; CFU-Meg), such as thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor and FLT3 ligand. Culture of nonirradiated CD34(+) cells in serum-free medium supplemented with multiple cytokine combinations led to an approximately 200- to 600-fold increase in the total cell numbers by day 14 of culture. In contrast, the growth of X-irradiated cells was observed to be one-sixth to one-tenth that of the nonirradiated cultures. Similarly, total megakaryocytes were increased by 50- to 130-fold, while culture of X-irradiated cells yielded one-fourth to one-eighth of the control numbers. At this time, CD41(+) particles, which appeared to be platelets, were produced in the medium harvested from nonirradiated and irradiated cultures. Although radiation suppressed cell growth and megakaryocytopoiesis, there were no significant differences in thrombopoiesis between the two types of culture. These results suggest that X-irradiated CD34(+) cells can be induced to undergo nearly normal terminal maturation through megakaryocytopoiesis and thrombopoiesis by stimulation with appropriate cytokine combinations.

Generation and characterization of a single‐gene mouse–human chimeric antibody against hepatitis B surface antigen

Antibody against hepatitis B surface antigen (HBsAg) is used for passive immunotherapy in certain cases of hepatitis B infection. The authors have earlier reported a high-affinity mouse monoclonal (5S) against HBsAg. However, this mouse antibody cannot be used for therapeutic purposes because it may elicit antimouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. A single-chain variable fragment (scFv), derived from the mouse monoclonal 5S, was fused with the fragment crystallisable (Fc) fragment of human IgG1. The scFv region is expected to bind to the antigen, whereas the Fc fragment can provide the effector functions required for virus neutralization. This chimeric molecule was expressed in Chinese hamster ovary (CHO) cells in serum-free medium. It was purified by affinity chromatography and characterized by in vitro binding studies. Purification and characterization indicated that this chimeric scFv-Fc fusion protein is secreted as a disulfide-linked, glycosylated, homodimeric molecule. The yield of the purified chimeric antibody was approximately 4.6 mg/L. In vitro analyses confirmed that this chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. Because it is a single-gene product, this chimeric scFv-Fc has the advantage of stable expression. Being chimeric and bivalent, it is expected to be less immunogenic and therefore suitable for further in vivo studies on virus neutralization.