Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.

Liver cells isolated from newborn rats and seeded on a non-adherent plastic substratum were found to spontaneously re-aggregate and to form, within a few days, spheroidal aggregates that eventually reached a plateaued diameter of 150-175 micron. Analyses on frozen sections from these spheroids by immunofluorescence microscopy using antibodies to various cytoskeletal elements and extracellular matrix components revealed a sorting out and a histotypic reorganization of three major cell types. A first type consisted of cells that segregated out on the aggregate surface forming a monolayer cell lining; a second type was identified as hepatocytes that regrouped in small islands often defining a central lumen; and a third group of cells reorganized into bile duct-like structures. This intercellular organization in the aggregates was paralleled by the accumulation of extracellular matrix components (laminin, fibronectin, and collagen) and their deposition following a specific pattern around each cell population structure. Determinations of albumin secretion and tyrosine aminotransferase induction by dexamethasone and glucagon at various times after the initiation of the cultures revealed a maintenance of the hepatocyte-differentiated functions for at least up to 2 mo at the levels measured at 3-5 d. It is concluded that cells dispersed as single cells from newborn rat liver conserve in part the necessary information to reconstruct a proper three-dimensional cyto-architecture and that the microenvironment so generated most likely represents a basic requirement for the optimal functioning of these differentiated cells.

New generation high performance in situ polarized 3He system for time-of-flight beam at spallation sources.

Modern spallation neutron sources generate high intensity neutron beams with a broad wavelength band applied to exploring new nano- and meso-scale materials from a few atomic monolayers thick to complicated prototype device-like systems with multiple buried interfaces. The availability of high performance neutron polarizers and analyzers in neutron scattering experiments is vital for understanding magnetism in systems with novel functionalities. We report the development of a new generation of the in situ polarized 3He neutron polarization analyzer for the Magnetism Reflectometer at the Spallation Neutron Source at Oak Ridge National Laboratory. With a new optical layout and laser system, the 3He polarization reached and maintained 84% as compared to 76% in the first-generation system. The polarization improvement allows achieving the transmission function varying from 50% to 15% for the polarized neutron beam with the wavelength band of 2-9 Angstroms. This achievement brings a new class of experiments with optimal performance in sensitivity to very small magnetic moments in nano systems and opens up the horizon for its applications.

Differential cytokeratin and alpha-fetoprotein expression in morphologically distinct epithelial cells emerging at the early stage of rat hepatocarcinogenesis.

The various liver cell populations emerging during the transitory reappearance of alpha-fetoprotein (AFP) in serum of 3'-methyl-4-dimethylaminoazobenzene-ingesting rats were analyzed in situ and in vitro on isolated cell preparations in terms of their cytokeratin and AFP expression using single and double indirect immunofluorescence microscopy. A polyclonal guinea pig antibody raised against cow hoof prekeratin, which recognized a Mr 52,000 cytokeratin, was found to react with bile ductular epithelial cells and oval cells but not with hepatocytes. A monoclonal antibody against a Mr 55,000 cytokeratin reacted not only with bile ductular and oval cells but also with hepatocytes. In contrast, a polyclonal antibody against porcine eye lens vimentin reacted with sinusoidal cells and stroma cells. To assess further the heterogeneity of the emerging cell populations, liver cells were isolated after 4 weeks of treatment and fractionated according to cell size and ploidy level into 4 fractions (I to IV) by velocity sedimentation at 1 X g. A cell-type analysis using AFP and albumin as functional markers revealed the presence of AFP-producing cells in Fraction IV at a mean velocity equivalent to that of newborn diploid rat hepatocytes, whereas most of the albumin-producing cells were distributed in Fractions I to III at velocities similar to those of adult tetraploid rat hepatocytes. A similar analysis based on the differential expression of Mr 52,000 and Mr 55,000 cytokeratins and vimentin in bile ductular and other diploid epithelial cells, hepatocytes, and mesenchymal cells showed that large cells in Fractions I to III were tetraploid hepatocytes, whereas viable cells present in Fraction IV were diploid epithelial cells and mesenchymal cells in proportion of 62 and 38%, respectively. These cell populations could be resolved further by changing the sedimentation time. A subsequent examination of the Mr 55,000 cytokeratin-containing diploid epithelial cells in Fraction IV using double immunofluorescence microscopy resolved three cell populations with respect to Mr 52,000 cytokeratin and AFP expression, namely, two cell populations expressing either protein marker and a third one containing both markers. These results suggest a ductular origin of oval cells and a possible relation to immature hepatocytes.

An "abnormal" lymphoid proliferation simulating Hodgkin's disease in a burn patient.

A particularly exuberant and unusual lymphoid proliferation in the spleen of a burn patient is presented. Many Reed-Sternberg cell variants and occasional diagnostic Reed-Sternberg cells strongly suggested a diagnosis of Hodgkin's disease. The pattern of distribution and cytology of this lymphoid infiltrate, however, are incompatible with Hodgkin's disease or a non-Hodgkin lymphoma. Morphologic features in this case suggest that the Reed-Sternberg cell is a B lymphocyte. The most probable etiologies of this unusual lymphoid proliferation are reviewed.

Metastatic neuroblastoma in bone marrow aspirate smears.

The authors observed rosette formation and partially fibrillar, blue-grey, extracellular material in Wright-Giemsa-stained bone marrow aspirate smears in five preparations from three cases of neuroblastoma metastatic to bone marrow. These features are little publicized in the literature and textbooks. These findings in neuroblastoma metastatic to bone marrow should prove helpful in differentiating that entity from acute leukemia and from other metastatic small-cell neoplasms in bone marrow.

Kondo physics in non-local metallic spin transport devices.

The non-local spin-valve is pivotal in spintronics, enabling separation of charge and spin currents, disruptive potential applications and the study of pressing problems in the physics of spin injection and relaxation. Primary among these problems is the perplexing non-monotonicity in the temperature-dependent spin accumulation in non-local ferromagnetic/non-magnetic metal structures, where the spin signal decreases at low temperatures. Here we show that this effect is strongly correlated with the ability of the ferromagnetic to form dilute local magnetic moments in the NM. This we achieve by studying a significantly expanded range of ferromagnetic/non-magnetic combinations. We argue that local moments, formed by ferromagnetic/non-magnetic interdiffusion, suppress the injected spin polarization and diffusion length via a manifestation of the Kondo effect, thus explaining all observations. We further show that this suppression can be completely quenched, even at interfaces that are highly susceptible to the effect, by insertion of a thin non-moment-supporting interlayer.

In situ polarized 3He system for the Magnetism Reflectometer at the Spallation Neutron Source.

We report on the in situ polarized (3)He neutron polarization analyzer developed for the time-of-flight Magnetism Reflectometer at the Spallation Neutron Source at Oak Ridge National Laboratory. Using the spin exchange optical pumping method, we achieved a (3)He polarization of 76% ± 1% and maintained it for the entire three-day duration of the test experiment. Based on transmission measurements with unpolarized neutrons, we show that the average analyzing efficiency of the (3)He system is 98% for the neutron wavelength band of 2-5 Å. Using a highly polarized incident neutron beam produced by a supermirror bender polarizer, we obtained a flipping ratio of >100 with a transmission of 25% for polarized neutrons, averaged over the wavelength band of 2-5 Å. After the cell was depolarized for transmission measurements, it was reproducibly polarized and this performance was maintained for three weeks. A high quality polarization analysis experiment was performed on a reference sample of Fe/Cr multilayer with strong spin-flip off-specular scattering. Using a combination of the position sensitive detector, time-of-flight method, and the excellent parameters of the (3)He cell, the polarization analysis of the two-dimensional maps of reflected, refracted, and off-specular scattered intensity above and below the horizon were obtained, simultaneously.

Differential effects of calcium deprivation on the cytoskeleton of non-tumorigenic and tumorigenic rat liver cells in culture.

Normal rat liver T51B epithelial cells and Morris no. 7795 hepatoma cells growing exponentially were exposed for 24 h to standard medium containing low (0.02 mM) calcium, a concentration which drastically reduces the proliferation of normal but not tumour cells. Cell surface morphology was examined by scanning electron microscopy (SEM); and the distribution and organization of microtubules, cytokeratin and vimentin filaments, and microfilaments were analysed by indirect immunofluorescence microscopy using specific antibodies. Calcium deprivation caused the loss of intercellular cohesion in both cell types and the appearance of some microvilli and blebs, particularly on tumour cells. However, marked differential (normal vs tumour cells) effects on the organizational integrity of the cytoskeleton fibrillar network were observed. Extracellular calcium deprivation led to a particular rearrangement of microtubules, and a perinuclear accumulation of cytokeratin and vimentin filaments in normal, but not in tumour cells. A massive concentration of actin-containing microfilaments was observed in the cell periphery and blebs of hepatoma cells. In the light of the possible involvement of calcium in controlling cytoskeleton assembly, the differing cytoskeletal changes of the two cell types may be linked to their different proliferative capabilities in low-calcium medium.

Flow cytometric analysis of human breast tumors and assessment of in vitro chemosensitivity by clonogenic assays.

We report a double-agar clonogenic system adapted to human breast cancer. We optimized the conditions for cell growth and clonogenicity with respect to hormones (insulin, estradiol, progesterone) and components of the extracellular matrix (collagen, laminin and fibronectin). Using our experimental improvements, 67% of the breast tumor samples received were grown successfully. Tests on 21 tumors with three agents: Doxorubicin, Methotrexate and 5-Fluorouracil permit objective discrimination of the in vitro pharmacosensitivity of human breast tumors. Flow cytometric analysis reveal that 64% of the tumors were diploid and 36% were aneuploid. The aneuploid tumors grew better in the double agar layer system used for the clonogenic assay. The diploid tumors were especially rich in estrogen (ER+) and progesterone (PR+) receptors whereas the aneuploid tumors were mostly estrogen and progesterone receptors negative (ER-/PR-). Finally, we noted no difference in drug responsiveness depending on the tumor ploidy and steroid receptor content.

Hormonally induced formation of extracellular biomatrix in cultured normal and neoplastic liver cells. Effect of dexamethasone.

We have examined by scanning electron microscopy the cell surface of rat hepatocytes (HEP), propagable liver epithelial (CE) cells, Morris # 7777 and # 7795 hepatoma (MH) cells at different times after seeding onto fibronectin-precoated substratum in presence of a defined medium. Upon seeding, HEP exhibited numerous microvilli and at later times, the cells flattened and lost most of these structures. When HEP were cultured in presence of dexamethasone (DEX), numerous microfibrils were observed extending into the substratum and on the cells and formed elaborate extracellular matrices. By indirect immunofluorescence we have demonstrated that these extracellular matrices are composed of fibronectin and collagens (type I, III). In contrast to HEP, sparse or confluent CE cells showed few microvilli and only very low amount of extracellular matrices were observed at confluency, even in presence of DEX. Growing or confluent MH # 7777 cells exhibited some microvilli while MH # 7795 cells showed a lower number of these surface structures at confluency. These hepatoma cell lines produced little extracellular matrices in absence or presence of DEX. Tyrosine aminotransferase (TAT), a liver specific intracellular protein, was inducible in HEP and MH but not in CE cells. Thus, these data demonstrate that the hormonal induction of a liver specific function is accompanied by the production of extracellular matrix only in the case of normal hepatocytes (HEP), but not in the case of cancerous (MH) or epithelial (CE) cells.

Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression.

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (Mr) 55,000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of Mr 52,000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55,000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Juvenile hemangioendothelioma: report of a case and review of the literature.

A solitary juvenile hemangioendothelioma on the maxillary gingiva of a newborn white male has been presented. No recurrence or evidence of systemic involvement has been noted in the 18 months following excision. The literature was reviewed with emphasis on the varying location and behavior of the lesion and the methods of treatment. Simple excision of the isolated, histologically benign neoplasm is recommended provided adequate follow-up is assured.

Selective increase in cytokeratin synthesis in cultured rat hepatocytes in response to hormonal stimulation.

Addition of a combination of insulin, dexamethasone and EGF at seeding time to cultured rat hepatocytes in serum-free medium caused a selective increase in the biosynthesis of particular cytokeratin components. This increase was prominent during the first day in culture. No significant increases were detected in the absence of hormones or in the presence of either hormones added alone or in pairs, except in the case of insulin plus dexamethasone, which yielded an effect close to that obtained with the three factors. Interestingly, the latter condition also maintained a high level of albumin production over a 6-day period in culture.