Jean-Paul Valet

Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities

SummaryThe two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 3 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and α-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.

The effect of dexamethasone on formation of a fibronectin extracellular matrix by rat hepatocytes in vitro

Abstract Monolayer cultures of newborn rat hepatocytes were initiated in the presence or absence of dexamethasone, and fibronectin was analysed by indirect immunofluorescence microscopy using a specific antiserum raised in rabbit. Dexamethasone-treated hepatocytes produce a well defined extracellular matrix of fibronectin that begins to form as early as 24 h after treatment. Non-treated hepatocytes exhibit very little immunofluorescence staining. Moreover, examinations by phase contrast microscopy reveal a very good preservation of the hepatocyte typical epithelial morphology and a drastic inhibition of fibroblast growth in the treated cultures.

Allergenicity and cross-reactivity of rye grass pollen extracts revealed by monoclonal antibodies☆

Using monoclonal antibodies the immunogenic and allergenic characteristics of rye Group I were redefined by SDS-PAGE analysis and immunoblotting. The purified rye Group I from NIH possesses a major component of approximately 34000 Da against which most of the sera from grass-sensitive patients and none from non-atopic volunteers contain specific IgE antibodies. Monoclonal antibodies to rye Group I were raised and used to purify the antigen and to verify the cross-reactivity between grass extracts. The 3 monoclonal antibodies studied recognized different components of timothy grass and 2 of them recognized kentucky june grass but none recognized components of ragweed extracts.

The relationship between cell volume, ploidy. and functional activity in differentiating hepatocytes.

Liver cells were isolated by collagenase perfusion from rats of 1 day, and 1, 3, 5, and 12 weeks of age, fractionated by velocity sedimentation at 1g (STAPUT), and the major cell types were identified in terms of specific functions. Alphafetoprotein and albumin were used as markers of differentiating hepatocytes and these functional activities were evaluated in a quantitative manner using a radio-immunoassay. The capacity of this cell type to store35S-BSP, an indicator of bile formation, was also evaluated. Sinusoidal cells and hematopoietic cells were identified on the basis of their ability to take up99mTC-colloid sulfur and to incorporate59Fe, respectively. The fractionation procedure allowed a good separation of sinusoidal cells from hepatocytes at all postnatal ages and also of erythroid cells still present during the first week after birth. With increasing age, alphafetoprotein-producing hepatocytes exhibited changes in sedimentation velocities that parallelled those of albumin-producers. In turn, the latter hepatocyte subpopulation underwent gradual shifts in modal peak velocities similar to those of bile-forming hepatocytes. The fractionated hepatocytes obtained at different ages were further analyzed in terms of cell volume and nuclear ploidy using a Coulter counter system. This quantitative analysis obtained at the cellular level demonstrated that during the age-related differentiation of hepatocytes, which occurs during the postnatal period and results in the gradual appearance of cells of higher ploidy levels, the extent of albumin production and bile formation can be correlated with the hepatocyte volume.

MORPHOLOGICAL DIFFERENCES BETWEEN EPITHELIAL AND FIBROBLAST CELLS IN RAT LIVER CULTURES, AND THE ROLES OF CELL SURFACE FIBRONECTIN AND CYTOSKELETAL ELEMENT ORGANIZATION IN CELL SHAPE*

Monolayer cultures set up from isolated rat liver cells contain a mixture of cell types whose composition can vary depending on the isolation procedure and also the time spent in culture.2 It is accepted now that liver cell preparations obtained by collagenase perfusion lead to primary cultures of hepatocytes that can survive in a quiescent state for a period up to 1-2 weeks.-5 The same cell preparations also contain small numbers of low differentiated epithelial cells with good proliferative capacity that may appear as small colonies during the 2-3 weeks in cultures, and eventually can give rise to cell lines.36 Some of these epithelial cells have pale translucent cytoplasms while others exhibit dense cytoplasms.R8g The relationship between these two liver epithelial cell types is still unknown, but it has been suggested that they could be precursor cells of hepatoc tes. Liver cell suspensions prepared by tissue digestion with trypsin, collagenaseR or Dispase I* are enriched in these low differentiated epithelial cells, mainly because of severe damages caused to hepatocytes during the isolation procedure. In addition to all these liver cells of epithelial morphology found in short or long-term monolayer cultures, proliferating fibroblasts are frequently resent. In fact, the latter can grow to an extent where they invade the whole culture. These various cell types have been so far distinguished mainly on the basis of their morphology as they appear under phase contrast microscopy. However, all these differences in cell morphology imply major differences in cell size, shape and spreading as the cells are cultured on solid substrata, and there are increasing evidences indicating that these phenomena are dependent on the degree of or ani zation of a specific cell surface protein and the cytoskeleton fibrillar network. A vast amount of work has been reported during the recent years on the detection and macromolecular organization of a major external glycoprotein, called cellular fibronectinI5 on the surface of a variety of adherent cells, mainly fibrob1a~ts.l~ This protein is thought to be involved in cell adhesiveness and thereby possibly in cell shape. Finally, the same protein exists in plasma or At least three kinds of fibers are known to constitute the cytoskeleton of cells, namely microtubules, microfilaments and intermediate filament^,^ and recent advances have been made on the arrangements of these fibrillar structures in fibroblastic cells by using indirect immunofluorescence with antibodies produced against protein constituents specific for the respective fibers, tubulin for microtubules,Y actin for microfilaments, and prekeratin for intermediate filaments.2 We report here on the characterization of hepatocytes, dense and clear epithelial 8

Restricted specialization of differentiating hepatocytes in terms of albumin and alpha-fetoprotein production

Hepatocytes were isolated from 1-day and 1,2,3 and 12-week old rat livers by collagenase perfusion and the relative numbers of albumin (ALB) and alpha-fetoprotein (AFP) producers were evaluated using the reverse hemolytic plaque assay. The percentage of ALB producers remained essentially constant to 35% over the 12-week period. In contrast, the percentage of AFP producers varied from 19% at 1 day up to 28% at 1 week and then down to 0.1% at 3 weeks. Moreover, a double identification of secreting hepatocytes, using an adaptation of the plaque assay, demonstrated that AFP producing hepatocytes were also ALB producers. These results are explained in terms of a restricted specialization of differentiating hepatocytes during normal development.

Development of a reverse enzymoallergosorbent test (REAST) to detect timothy-specific IgE antibodies comparison with RAST☆

Radioallergosorbent test (RAST) for the measurement of IgE antibodies has been introduced more than 15 years ago and a number of technical modifications have since improved its sensitivity and reproducibility. The test has been applied to the diagnosis of allergy and to determine changes in the levels of IgE antibodies following immunotherapy. However, specific IgG antibodies are raised during such a therapy and can interfere with the RAST. We have developed a reverse enzymoallergosorbent test (REAST) where microtiter plates are first coated with a purified polyclonal anti-IgE antibody, then with the serum to test and finally with peroxidase-labeled antigen. This assay is antigen specific as shown by the significant inhibition of binding of the labeled antigen in presence of unlabeled specific antigen (greater than 95%) and the absence of inhibition in presence of irrelevant antigens. The values found in atopic patients (85 subjects) were significantly higher than in the non-atopic donors (35 subjects) (1.14 U +/- 1.20 vs. 0.01 U +/- 0.02, P less than 0.0005) and there was a good correlation with the Pharmacia RAST (P less than 0.0005). The levels of specific IgE by both REAST and RAST correlated well with the clinical symptomatology.

Expression of gooseberry-proximal in the Drosophila developing nervous system responds to cues provided by segment polarity genes

SummarySegment polarity genes define the cell states that are required for proper organization of each metameric unit of the Drosophila embryo. Among these, the gooseberry locus has been shown to be composed of two closely related genes which are expressed in an overlapping single-segment periodicity. We have used specific antibodies raised against the protein product of the gooseberry proximal (gsb-p) gene to determine the spatial distribution of this antigen in wild type embryos, and to monitor the effects of segment polarity mutants on the pattern of the gsb-p protein distribution. We find that the gsb-p protein accumulates beneath each posterior axonal commissure in the progeny of neuroblasts deriving from the epidermal compartments of wingless (wg) and engrailed (en) expression. The results of this analysis support the idea that gsb-p has a specific role in the control of cell fates during neurogenesis, and indicate that en and wg provide critical positional cues to define the domain in which gsbp will be activated. Furthermore, these data suggest that, in order to be expressed in the embryonic CNS, gsb-p may preliminarily require activity of the gooseberry-distal gene in the epidermis.

Detection of immunoglobulin-secreting lymphocytes by the use of a hemolytic plaque assay in liquid phase

This paper describes a modification of the reverse hemolytic plaque assay allowing the enumeration of immunoglobulin (Ig)-secreting lymphocytes at rest or after in vitro stimulation. Ig-secreting cells are mixed with anti-Ig coated sheep red blood cells, developing anti-Ig antiserum and complement in liquid medium. This mixture is pipetted between two slides and the hemolytic plaques are numerated after incubation at 37 degrees C. The class of Ig secreted can be determined using a monospecific developing antiserum. This technique is easier to perform and more economical than the previously described method using agarose without loss of sensitivity, the number of plaques detected being about the same and the kinetic of their appearance comparable.

Biochemical and Immunocytochemical Localization of Heat‐Shock Proteins in Drosophila Cultured Cells a

Treatment of living cells at supraoptimal temperatures or with various chemical or physical aggressors induces the synthesis of a group of proteins known as heat-shock proteins (HSP)..2 The function of these ubiquitous proteins is unclear although a role in cellular protection has been ~uggested.~ In Drosophila cultured cells, biochemical fractionation of HS cells shows an enrichment of most HSPs (with the exception of HSP 82) in the nuclear pellet following HS.4, In the course of studies on the characterization of a major intermediate filament-like cytoskeletal protein of 46,000 in these we observed that the group of low molecular weight HSPs tended to copurify with a Triton-high salt insoluble cytoskeletal fraction (FIGURE 1). In order to investigate the significance of this finding and to elucidate the function of HSPs, we prepared polyclonal antibodies against HSP 82, 70, 68, and 23 and studied their intracellular distribution by immunofluorescence techniques following heat shock and during recovery. The antibodies were purified by affinity and their specificity checked by immunoblotting. The results are summarized in FIGURE 2. HSP 82 shows an exclusive cytoplasmic distribution with a reversible concentration near the plasma membrane during HS. The HSP 68-70 complex, which is present in the cytoplasm and perinuclear region of unshocked cells, is found both in the nuclear and the cytoplasmic compartments following HS. In the nucleus, they are found in nucleoli as well as in the nucleoplasm. HSP 23 is only present in stressed cells and concentrates in the nucleolus and perinuclear region upon heat shock. When stresses other than heat are used, the HSPs do not migrate in the nucleus. Thus immunofluorescent localization studies confirm and clarify previous cellular fractionation studies.