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At least 127 records · Page 7Linked to original sources
PubMed2003

Efficient tumor cell lysis by autologous, tumor-resident T lymphocytes in primary ovarian cancer samples by an EP-CAM-/CD3-bispecific antibody.

The epithelial cell adhesion molecule (Ep-CAM) is expressed on the surface of most human carcinomas, including ovarian, breast, lung, prostate and colorectal carcinoma. Ep-CAM was shown to be a valid target for monoclonal antibody-based therapies. We have investigated whether an Ep-CAM-/CD3-bispecific single-chain antibody called bscEp-CAM x CD3 is effective in tumor cell elimination within the cellular microenvironment of primary ovarian cancer tissue. The ex vivo elimination of ovarian cancer cells in tumor preparations from 21 patients was monitored by flow cytometry using Ep-CAM/CA-125 double-labeling or Ep-CAM single-labeling combined with propidium iodide uptake of cells. Methodology was established by the ovarian cancer cell line OvCAR. A total of 17 (81%) patient samples showed a dose-dependent tumor cell elimination by bscEp-CAM x CD3. High and specific tumor cell lysis was seen at bscEp-CAM x CD3 concentrations as low as 1 ng/ml, at very low effector:target ratios and in the absence of T cell costimulation. The high efficacy of the bispecific antibody may be due to the non-restricted activation of tumor-resident cytotoxic T lymphocytes. In clinical trials, the ex vivo data with the T cell-recruiting bispecific antibody bscEp-CAM x CD3 may translate into a high response rate and efficacy of tumor cell elimination.

Antibodies, Bispecific↗
PubMed1986

Influence of growth phase, nutrition and hypoxia on heterogeneity of cellular buoyant densities in in vitro tumor model systems.

EMT6/Ro multicellular spheroids were sequentially dissociated into outer, middle and inner layers, and the buoyant densities of these subpopulations were compared to aerobic or exponential or plateau-phase monolayers by isopycnic density centrifugation using Percoll as the gradient medium. Spheroids were more heterogeneous in buoyant density distributions than monolayer cultures. The outer-layer cells of spheroids and fed plateau monolayers peaked at a similar density; the majority of middle-layer cells banded close to unfed plateau and exponential hypoxic monolayers, while most inner-layer cells banded where fed or unfed plateau hypoxic monolayer peaked in density. Our data suggest that metabolic heterogeneity due to growth phase, together with altered cellular microenvironment brought about by differences in oxygenation and external nutrient supplies between spheroids and monolayers, can be manifested in one biophysical parameter such as buoyant density.

Animals↗
PubMed1988

Physicochemical characterization of deposits associated with HA ceramics implanted in nonosseous sites.

Pellets of well-characterized microporous hydroxyapatite (HA) ceramic were implanted in hamsters in two nonosseous sites: (1) in the fatty tissue of the gingival crease, far from bony tissue and (2) in intraperitoneal sites. The implants in site 1 were placed directly in contact with tissues, cells, and extracellular fluids while the implants in site 2 were placed in special chambers made of plexiglass cylinders covered in both ends with millipore filters, preventing contact with tissues and cells, but not with extracellular fluids. The hamsters were sacrificed and the implants recovered after 8, 16, 30, 150, and 365 days. The pellets were characterized using x-ray diffraction, infrared absorption, thermogravimetry, scanning and transmission electron microscopy, and calcium and phosphate analyses before and after implantation. Physicochemical analyses of HA ceramic implants before and after implantation demonstrated the formation of new material which was significantly different from the HA ceramic in terms of the following: (a) morphology (size of shape) of crystals; (b) intimate association of the inorganic phase of the new material with an organic phase similar to inorganic/organic association in bone; (c) the inorganic phase of the new material is a CO3-apatite, similar to that of bone, while the HA in ceramic is CO3-free; (d) electron diffraction of apatite of new material is similar to that of bone apatite. This study also demonstrated that the new material associated with the HA ceramics implanted in two different nonosseous sites were identical in spite of the differences in their microenvironment (cellular and acellular).

Animals↗
PubMed2007

Enhanced insulin-like growth factor-I (IGF-I) cell association at reduced pH is dependent on IGF binding protein-3 (IGFBP-3) interaction.

The cellular microenvironment impacts how signals are transduced by cells and plays a key role in tissue homeostasis. Although pH is generally well regulated, there are a number of situations where acidosis occurs and our work addresses how low pH impacts cell association of insulin-like growth factor-I (IGF-I) in the presence of IGF binding protein-3 (IGFBP-3). We have previously shown that IGF-I cell binding was enhanced in the presence of IGFBP-3 at low pH and now show that this binding is IGFBP-mediated as it is inhibited by Y60L-IGF-I, a mutant with reduced affinity for the IGF receptor (IGF-IR), and unaffected by insulin, which binds but not IGFBPs. Using surface plasmon resonance (SPR), we show that direct binding between IGF-I and IGFBP-3 is pH sensitive. Despite this, the key step in the process appears to be IGFBP-3 cell surface association as Long-R(3)-IGF-I, a mutant with reduced affinity for IGFBPs, shows a similar increase in cell association at pH 5.8 in the presence of IGFBP-3 but does not exhibit pH-dependent binding by SPR. Further, analysis indicates a large increase in low-affinity binding sites for IGF-I in the presence of IGFBP-3 and an elimination of IGF-I enhanced binding when a non-cell associating mutant of IGFBP-3 is added in place of IGFBP-3. That the IGFBP-3-mediated binding localizes IGF-I away from IGF-IR is suggested by triton-solubility testing and indicates additional complexities to IGF-I regulation by IGFBP-3. Identifying the pH-dependent binding partner(s) for IGFBP-3 is a necessary next step in deciphering this process.

Animals↗
PubMed2007

Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells.

Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.

Animals↗
PubMed1979

Solute compatibility with enzyme function and structure: rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates.

The major nitrogenous osmolytes present in the cells of marine invertebrates, notably the free amino acids glycine, alanine and proline, and trimethylamine oxide and betaine, are highly compatible with proper enzyme function and structure. These nitrogenous osmolytes display either non-perturbing or, in some cases, favorable effects on enzyme-substrate and enzyme-cofactor complex formation, catalytic velocity and protein structural stability. In contrast, inorganic salts (KCl and NaCl) and certain of the free amino acids which play only a minor osmotic role, e.g., arginine and lysine, have strongly perturbing effects on one or more of these enzymic parameters. The compatible nitrogenous solutes therefore are suitable for use at high (several tenths molar) concentrations and at widely varying concentrations in osmo-conforming species. Certain nitrogenous solutes, especially trimethylamine oxide, betaine and glutamate, offset some of the perturbing effects of inorganic ions on enzyme function. The selective accumulation of osmolytes thus involves not only the concentration of non-perturbing solutes, but also a balanced accumulation of solutes with opposing effects on enzymes. The selection of end-products of anaerobic metabolism also appears to be based, in part, on considerations of solute compatibility with enzyme function. Octopine is a non-perturbing solute, whereas arginine, which is condensed with pyruvate to form octopine, is very strongly perturbing. Succinate has marked stabilizing effects on protein structure. We conclude that the composition of the intracellular fluids of marine invertebrates reflects selection for osmolytes and end-products whose net effects create a cellular microenvironment which is conducive to optimal enzyme function and structure. The accumulation of compatible solutes may preclude the necessity for widespread changes in protein structure in adapting to concentrated or highly variable osmotic environments.

Amino Acids↗
PubMed2004

Astrocyte-specific overexpression of insulin-like growth factor-I promotes brain overgrowth and glial fibrillary acidic protein expression.

Insulin-like growth factor-I (IGF-I) is widely expressed in the central nervous system (CNS). Whereas during normal development IGF-I is expressed predominantly by neurons and to a much lesser degree by glial cells, its expression in astrocytes, and often in microglia, is increased during and/or after variety of CNS injuries. Recently we have generated a new line of IGF-I Tg mice, called IGF-I(Ast/Tet-Off) Tg mice, in which IGF-I transgene is expressed specifically in astrocytes and is tightly controlled by the tetracycline analog doxycycline. In this study we examined whether IGF-I derived from astrocytes is capable of promoting neural cell growth during development. When the IGF-I transgene is allowed to be expressed, IGF-I(Ast/Tet-Off) Tg mice exhibit markedly increases in 1) brain weight; 2) brain DNA and protein abundance; and 3) number of neurons, oligodendrocytes, and astrocytes, as well as myelination, findings similar to those observed in our other lines of Tg mice that express IGF-I transgene predominantly in neurons. Unlike Tg mice with neuron-specific IGF-I expression, which manifest marked increases in the concentrations of oligodendrocyte/myelin-specific proteins, however, IGF-I(Ast/Tet-Off) Tg mice exhibit an increase in the concentration of glial fibrillary acidic protein, an astrocyte-specific protein. Furthermore, when transgene expression is blunted, brain overgrowth in IGF-I(Ast/Tet-Off) Tg mice ceases. Our data indicate that astrocyte-derived IGF-I is capable of promoting neural cells growth in vivo. Our data also suggest that IGF-I's actions in CNS depend in part on the location of its expression and cellular microenvironment and that continuous presence of IGF-I expression is necessary for brain overgrowth.

Age Factors↗
PubMed2005

The human prion protein alpha2 helix: a thermodynamic study of its conformational preferences.

We have synthesized both free and terminally-blocked peptide corresponding to the second helical region of the globular domain of normal human prion protein, which has recently gained the attention of structural biologists because of a possible role in the nucleation process and fibrillization of prion protein. The profile of the circular dichroism spectrum of the free peptide was that typical of alpha-helix, but was converted to that of beta-structure in about 16 h. Instead, below 2.1 x 10(-5) M, the spectrum of the blocked peptide exhibited a single band centered at 200 nm, unequivocally associated to random conformations, which did not evolve even after 24 h. Conformational preferences of this last peptide have been investigated as a function of temperature, using trifluoroethanol or low-concentration sodium dodecyl sulfate as alpha- or beta-structure inducers, respectively. Extrapolation of free energy data to zero concentration of structuring agent highlighted that the peptide prefers alpha-helical to beta-type organization, in spite of results from prediction algorithms. However, the free energy difference between the two forms, as obtained by a thermodynamic cycle, is subtle (roughly 5-8 kJ mol(-1) at any temperature from 280 K to 350 K), suggesting conformational ambivalence. This result supports the view that, in the prion protein, the structural behavior of the peptide is governed by the cellular microenvironment.

Humans↗
PubMed1993

The effect of changes in iron redox state on the activity of enzymes sensitive to modification of SH groups.

Iron ions in micromolar concentrations induced a rapid and selective inhibition of the activity of skeletal muscle creatine kinase (CK), sarcoplasmic reticulum (SR) Ca(2+)-ATPase, and pyruvate kinase (PK). This effect of iron was dependent on the presence of adenine nucleotides and on the redox state of iron. Changing the redox state of the media created different Fe2+/Fe3+ ratios which selectively depressed different enzymes: depression of PK activity occurred when iron was predominantly in its reduced form and, consequently, when there was a high Fe2+/Fe3+ ratio; depression of SR Ca2+ uptake and SR Ca(2+)-ATPase activity occurred when the Fe2+/Fe3+ ratio was close to 1; depression of CK activity occurred when iron was predominantly in its oxidized form and the Fe2+/Fe3+ ratio was low. All iron-sensitive enzymes possessed sulfhydryl groups, accessible to N-ethylmaleimide (NEM), which were essential for their activity. The rate of inhibition of enzyme activity with NEM increased in the order PK < Ca(2+)-ATPase < CK. Iron-induced depression of CK and PK activities was reversible by dithiotreithol. Results suggest that changes in the redox state of cellular microenvironments, which inevitably occur during reperfusion of ischemic tissue or rapid increase in tissue oxygen consumption, may selectively depress the activity of several enzymes bearing SH groups that are sensitive to modifications and that are essential for their activity. Iron-induced depression of enzyme activity depends on the availability of iron bound to adenine nucleotides and possibly to other low molecular weight chelators and on the Fe2+/Fe3+ ratio generated by the induced redox change.

Animals↗
PubMed2007

Perfusion effects and hydrodynamics.

Biological processes within living systems are significantly influenced by the motion of the liquids and gases to which those tissues are exposed. Accordingly, tissue engineers must not only understand hydrodynamic phenomena, but also appreciate the vital role of those phenomena in cellular and physiologic processes both in vitro and in vivo. In particular, understanding the fundamental principles of fluid flow underlying perfusion effects in the organ-level internal environment and their relation to the cellular microenvironment is essential to successfully mimicking tissue behavior. In this work, the major principles of hemodynamic flow and transport are summarized, to provide readers with a physical understanding of these important issues. In particular, since quantifying hemodynamic events through experiments can require expensive and invasive techniques, the benefits that can be derived from the use of computational fluid dynamics (CFD) packages and neural networking (NN) models are stressed. A capstone illustration based on analysis of the hemodynamics of aortic aneurysms is presented as a representative example of this approach, to stress the importance of tissue responses to flow-induced events.

Animals↗
PubMed2006

Chronic spinal nerve ligation induces microvascular permeability disturbances, astrocytic reaction, and structural changes in the rat spinal cord.

The possibility that a chronic nerve ligation impairs the spinal cord cellular microenvironment was examined using leakage of endogenous albumin, reaction of astrocytes, and structural changes in a rat model. Rats subjected to 8 weeks of unilateral L4/L5 nerve ligation (a model of neuropathic pain) showed leakage of albumin, up-regulation of glial fibrillary acidic protein (GFAP) immunoreaction, and abnormal cell reaction. Distortion and loss of nerve cells as well as general sponginess of the gray matter was clearly evident. Cell changes were present in both dorsal and ventral horns and were most marked on the ipsilateral side compared to the contralateral cord. Nerve cell and glial cell changes are normally present in the regions showing intense albumin immunoreactivity, indicating disruption of the blood-spinal cord barrier (BSCB). Our observations indicate that a chronic nerve lesion has the capacity to induce selective breakdown of the BSCB that could be responsible for activation of astrocytes and abnormal cell reaction. These findings enhance our understanding of the pathophysiology of neuropathic pain and/or other spinal cord disorders.

Adaptation, Physiological↗
PubMed1988

Haemopoietic regulation and the role of the macrophage in erythropoietic gene expression.

The macrophage is considered as an "active" component of the haemopoietic cellular microenvironment with respect to erythropoietin (epo) production during embryonic, foetal and adult erythropoiesis. Emphasis is placed on steady-state rather than pathophysiological conditions. In addition, the signals capable of affecting the functional capacity of the macrophage with regard to colony stimulating factor and epo production are also taken into account. Evidence is given demonstrating that a subpopulation of resident macrophages in vitro and in the mouse bone marrow, under normal conditions, can express the epo gene. These results indicate that erythropoiesis can be regulated by short-range or cell-to-cell interactions within the bone marrow.

Animals↗
PubMed1990

Contrary effect of lactic acid on expression of neuron-specific enolase and glial fibrillary acidic protein in human glioma cells.

We examined the effect of lactic acid on cultured human glioma cell lines expressing glial fibrillary acidic protein (GFAP), vimentin and neuron-specific enolase (NSE). The growth of the cells was inhibited by the lactic acid in a dose-dependent manner. At 56 mM of lactic acid, the surviving cells of the KNS-42-c2 cell line developed slender processes and increasingly formed bizzar giant cells. In an immunofluorescence study of the lactic acid-resistant cells, the GFAP-positive cells prominently decreased in number, while the NSE-positive cells clearly increased. The vimentin was not affected throughout the experiment. After removing lactic acid from the medium, the GFAP-positive cells gradually increased in number. The method of dot immunoassay was useful for quantifying GFAP in cellular extracts. It indicated that the amount of GFAP decreased in the cells cultured with lactate-containing media and increased to the primary values after removing the lactic acid. These results may suggest that the morphological and immunochemical diversities of glioma cells are secondarily affected by cellular microenvironments such as lactic acid.

Cell Line↗
PubMed1987

Multicellular spheroids. A review on cellular aggregates in cancer research.

Cellular aggregates have been used in developmental biology and in experimental cancer research for several decades. Spherical aggregates of malignant cells, i.e. multicellular tumor spheroids, may serve as in vitro models of tumor microregions and of an early, avascular stage of tumor growth. The similarities between the original tumor and the respective spheroids include volume growth kinetics, cellular heterogeneity, e.g. the induction of proliferation gradients and quiescence, as well as differentiation characteristics, such as the development of specific histological structures or the expression of antigens. Research using cell aggregates has been focussed on mechanisms involved in the control of proliferation, invasion and metastasis. Immunological studies with spheroids have resulted in the characterization of defense cells which are responsible for specific host-versus-tumor reactions. The vast majority of investigations on spheroids concerns the simulation of therapy with regard to various treatment modalities, combination treatments and systematic analyses of using various endpoints in predictive assays. Only a few pathophysiological studies on the interrelationship among tumor-specific micromilieu, cellular metabolism, proliferative status, and cellular viability have been undertaken with the spheroid model up to now. Since these studies are indicative of a large influence of the cellular microenvironment on basic biological properties of cancer cells, investigations of these epigenetic mechanisms should be intensified in future research on cell aggregates. Similarly, the molecular basis of the biological peculiarities found in malignant cells grown as three-dimensional aggregates has to be investigated more intensively.

Animals↗
PubMed1983

Serum-independent modulation of hemicyst formation by dissolved oxygen in postconfluent epithelial monolayers.

Hemicyst formation is considered a manifestation of either transepithelial solute and fluid movement or secretory activity in culture. This study shows that hemicyst formation in postconfluent monolayers of rhesus monkey kidney (LLC-MK2) cells is modulated by the dissolved oxygen concentration (PO2) of the culture medium. Either daily replacement of serum-free medium or displacement of the gas phase with 18% vol/vol O2 (initial medium PO2 = 125 to 135 mm Hg) enhances formation of hemicysts. Use of 30% O2 (medium PO2 approximately equal to 175 mm Hg) does not further increase the incidence, but neither 10% O2 (medium PO2 = 90 to 95 mm Hg) nor 1% O2 (medium PO2 = 35 to 50 mm Hg), the approximate range of dissolved oxygen values in blood, supports hemicyst formation unless cultures are gently rocked to disrupt diffusion gradients. Phase photomicrography of living cultures shows that the surface of a turgid hemicyst is furrowed, and cinephotomicrography reveals that the walls vibrate subtly. When hypoxic conditions (0 to 1% O2) are introduced this vibration ceases within 2 to 3 h, whereas collapse and disappearance of turgid hemicysts requires 18 to 20 h, seems virtually synchronous, and is reversible. Hemicysts form in a broad osmotic range, and increased electrolyte concentration increases the incidence. Hemicysts persist in locally dense areas when cell-free strips are etched in the postconfluent monolayer; no DNA synthesis is detected under these conditions, but two-dimensional cell spreading into the denuded area is seen along the edge of the wound. We conclude that the dissolved oxygen supply in the cellular microenvironment modulates functional expression by differentiated kidney epithelial cells in culture and that increased electrolyte concentration also enhances expression of this phenotypic marker.

Animals↗
PubMed1990

Studies on human CFU-Mix microvolume culture by use of limiting dilution assay.

By use of limiting dilution assay we investigated the bipotent and pluripotent hemopoietic progenitor cells (CFU-Mix) from 21 normal adults, cultured for 32 times. The CFU-Mix counts were 35.7 colonies/10(6) bone marrow cells. According to different cellular elements they could be divided into 5 groups, namely GEMM, GE, GL, GMeg and GM phi. It was suggested that differentiation of the stem cell is of stochastic process, influenced by different hemopoietic growth factors and cellular microenvironment. Our model seems to be suitable for studying differentiation of the stem cells. The rates of 3H-TdR and 55 + 59Fe incorporation were determined, and the 2nd day of culture was found to be the delayed stage of cell growth.

Adolescent↗
PubMed1996

The vitronectin receptor (alpha V beta 3) as an example for the role of integrins in T lymphocyte stimulation.

Integrins are a family of cell surface receptors which mediate the adhesion of cells to each other or to extracellular matrix (ECM) proteins. The interaction of integrins with their ligands or counter-receptors was initially considered to be a one-way process in that cells actively regulate the interaction of integrins with their ligands ('inside-out signal'). In contrast, it was not obvious that cells would receive a signal from the outside via the integrin heterodimers following ligand binding ('outside-in signal'). Recent evidence increasingly supports the active role of integrins in cell activation and proliferation. Many reports describe the effects of integrin-mediated signaling in lymphoid cells. Our studies of gamma/delta T cells, expressing the beta 3 integrin vitronectin receptor (VNR), reflect some of the consequences this active interaction between lymphocytes and the ECM could have for T cell activation and differentiation. The VNR has been described as a T cell costimulatory molecule. We recently reported that the VNR has the potential to stimulate cytokine secretion in T cell hybridomas without involvement of T cell receptor-mediated signals. Further studies demonstrated tyrosine phosphorylation of proteins following VNR cross-linking and the interaction of the VNR with protein kinases. Intensive research focuses on the signal transduction mechanisms of integrins and their interaction with other costimulatory or activation molecules. This knowledge is important to better understand the role of adhesion molecules, the ECM, and the cellular microenvironment for lymphocyte activation and differentiation.

Animals↗
PubMed2005

Do myoepithelial cells hold the key for breast tumor progression?

Mammary myoepithelial cells have been a neglected facet of breast cancer biology, largely ignored since they have been considered to be less important for tumorigenesis than luminal epithelial cells from which most of breast carcinomas are thought to arise. In recent years as our knowledge of stem cell biology and the cellular microenvironment has been increasing, myoepithelial cells are slowly starting to gain more attention. Emerging data raise the hypothesis whether myoepithelial cells play a key role in breast tumor progression by regulating the in situ to invasive carcinoma transition and that myoepithelial cells are part of the mammary stem cell niche. Paracrine interactions between myoepithelial and luminal epithelial cells are known to be important for regulation of cell cycle progression, establishing epithelial cell polarity, and inhibiting cell migration and invasion. Based on these functions, normal mammary myoepithelial cells have been called "natural tumor suppressors." However, during tumor progression myoepithelial cells seem to loose these properties, and eventually this cell population diminishes as tumors become invasive. Better understanding of myoepithelial cell function and their role in tumor progression may lead to their exploitation for cancer therapeutic and preventative measures.

Breast Neoplasms↗