High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

High-throughput organ-on-chip platform with integrated programmable fluid flow and real-time sensing for complex tissue models in drug development workflows.

Drug development suffers from a lack of predictive and human-relevant in vitro models. Organ-on-chip (OOC) technology provides advanced culture capabilities to generate physiologically appropriate, human-based tissue in vitro, therefore providing a route to a predictive in vitro model. However, OOC technologies are often created at the expense of throughput, industry-standard form factors, and compatibility with state-of-the-art data collection tools. Here we present an OOC platform with advanced culture capabilities supporting a variety of human tissue models including liver, vascular, gastrointestinal, and kidney. The platform has 96 devices per industry standard plate and compatibility with contemporary high-throughput data collection tools. Specifically, we demonstrate programmable flow control over two physiologically relevant flow regimes: perfusion flow that enhances hepatic tissue function and high-shear stress flow that aligns endothelial monolayers. In addition, we integrate electrical sensors, demonstrating quantification of barrier function of primary gut colon tissue in real-time. We utilize optical access to the tissues to directly quantify renal active transport and oxygen consumption via integrated oxygen sensors. Finally, we leverage the compatibility and throughput of the platform to screen all 96 devices using high content screening (HCS) and evaluate gene expression using RNA sequencing (RNA-seq). By combining these capabilities in one platform, physiologically-relevant tissues can be generated and measured, accelerating optimization of an in vitro model, and ultimately increasing predictive accuracy of in vitro drug screening.

Cardiotrophin-1 in choroid plexus and the cerebrospinal fluid circulatory system.

There is a growing recognition of choroid plexus functioning as a source of neuropeptides, cytokines and growth factors in cerebrospinal fluid (CSF) with diffusional access into brain parenchyma. In this study, choroid plexus and other components of the CSF circulatory system were investigated by Western blotting, reverse transcriptase polymerase chain reaction and immunohistochemistry for production of interleukin-6-related cytokines characterized by neuroactivity [cardiotrophin-1 (CT-1), ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M] and signaling through the gp130/leukemia inhibitory factor receptor-beta receptor heterodimer. Western blot analysis showed that CT-1 was the only cytokine family member detectable in adult rat choroid plexus, as in leptomeninges. The specificity of detection was verified with blots of the same tissues from CT-1-deficient mice. Levels of both CT-1 mRNA and protein were constitutively high in rat from birth through adulthood in choroid plexus, up-regulated postnatally in leptomeninges and undetectable in brain parenchyma. Using antigen retrieval, CT-1 immunolocalized to choroid epithelial cells in all choroid plexuses in addition to leptomeninges (arachnoid and pial-glial membranes). Ependymal cells lining the ventricular neuroaxis, unlike the central canal, were also CT-1-immunoreactive. Western blots indicated rat choroid epithelial cells express and release CT-1 immunoreactivity under defined culture conditions and also revealed the presence of a CT-1-like protein in human choroid plexus and CSF. Previously, CT-1 has been conceptualized to function as a target-derived factor for PNS neurons. Our study clearly demonstrates production of CT-1 in the postnatal and adult CNS, specifically by cell types comprising the blood-CSF barrier, and its accumulation in ventricular ependyma. This finding has broad implications for CT-1 functioning apart from other leukemia inhibitory factor receptor ligands as a CSF-borne signal of brain homeostasis, one possibly involving regulation of the barrier itself, the ependyma or target cells in the surrounding parenchyma, including the subventricular zone. A rationale for studies examining CT-1-deficient mice in these respects is provided by the data.

4-Hydroxynonenal, a lipid peroxidation byproduct of spinal cord injury, is cytotoxic for oligodendrocyte progenitors and inhibits their responsiveness to PDGF.

Oligodendroglial reactions to compression injury of spinal cord include apoptosis, secondary demyelination, and remyelination failure. Within hours after contusion, the membrane lipid peroxidation (MLP) byproduct, 4-hydroxynonenal (HNE), increases rapidly in gray matter and thereafter in white matter tracts beyond the initial lesion level. Considering that HNE is a mediator and marker of neuronal MLP toxicity in various neurodegenerative conditions, the present study examined its effect on the regeneration potential of oligodendrocyte progenitors, as defined by their capacity to survive, proliferate and migrate in primary culture. Treatment of oligodendroblasts with HNE evoked a time- and dose-dependent cytotoxicity resembling apoptosis at aldehyde concentrations known to be produced by neurons and achieved in tissue undergoing peroxidative injury. In addition, sublethal concentrations of HNE inhibited the mitogenic and chemotactic responses of more immature progenitors to platelet-derived growth factor. These effects appear to be mediated in part by the formation of HNE adducts with progenitor proteins located within the plasma membrane and cytoplasmic compartments. Our data are the first to show that HNE can have direct, deleterious effects on oligodendrocyte precursors. The present study also suggests a mechanism by which the striking accumulation of HNE in white matter tracts surrounding the site of spinal cord compression injury and in other ischemic-hypoxic insults associated with MLP could suppress the potential regenerative response of endogenous oligodendrocyte progenitor cells.

Synthesis, assembly, and turnover of alpha and beta-erythroid and nonerythroid spectrins in rat hippocampal neurons.

The synthesis and turnover of alpha-erythroid, beta-erythroid, alpha-nonerythroid and beta-nonerythroid spectrins was investigated in cultured rat hippocampal neurons. [35S]methionine and subunit specific antibodies were used to label and immunoprecipitate newly synthesized spectrins in 12- to 14-day-old cultures. Synthesis experiments, performed under normal resting conditions, showed that the ratio of newly synthesized alpha-erythroid/beta-erythroid and alpha-nonerythroid/beta-nonerythroid spectrins is 1/1 (mol/mol) both in the soluble and insoluble fractions. Soluble and insoluble alpha and beta erythroid spectrin turn over rapidly (half-life=16-24 min). Soluble nonerythroid alpha-spectrin (half-life=80 min) and beta spectrin (half-life=53 min) turn over more slowly than their insoluble counterparts (30-34 min). The nonerythroid alpha spectrin turnover was significantly different (p<0.05) from the other measurements except for nonerythroid beta spectrin, indicating that these subunits are protected from rapid proteolytic degradation until they are assembled in the membrane skeleton.

Glial cell-specific differences in response to alkylation damage.

Oligodendrocytes are preferentially sensitive to the toxic, carcinogenic, and teratogenic effects of methylnitrosourea (MNU). The mechanisms responsible for this enhanced sensitivity have not been fully elucidated. One of the most vulnerable cellular targets for this chemical is mitochondrial DNA (mtDNA). To determine if differences in mtDNA damage and repair capacity exist among the different CNS glial cell types, the effects of MNU exposure on oligodendroglia, astroglia, and microglia cultured separately from neonatal rat brain were compared. Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed that whereas no cell type-specific differences in initial mtDNA damage were detected, mtDNA repair in oligodendrocytes, oligodendrocyte progenitors, and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, >60% of N-methylpurines were removed from the mtDNA by 24 hr. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes, oligodendrocyte progenitors, and microglia during the same time period. Mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis. In the present study, apoptosis, as determined by DNA laddering and ultrastructural analysis, was clearly induced by MNU treatment of cultured oligodendrocyte progenitors and microglia, but not in astroglia. These data demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis. However, further experimentation is necessary to determine if a causal relationship exists and contributes to the vulnerability of oligodendroglia following exposure to N-nitroso compounds in the environment or in chemotherapeutic regimen.

In vitro death of jimpy oligodendrocytes: correlation with onset of DM-20/PLP expression and resistance to oligodendrogliotrophic factors.

Severe hypomyelination in the jimpy (jp) mouse mutation results from premature death of most oligodendrocytes (OCs). We have applied an immunopanning technique to successfully purify oligodendroblasts (OBs) directly from neonatal jp brainstem in order to determine if their death during differentiation into OCs is preventable in culture by diffusible oligodendrogliotrophic factors. No significant differences in the yield (0.9-1.1 x 10(5) cells/brainstem) or viability (approximately 90%) of OB populations from jp and wild-type (wt) littermates were observed, indicating that cell death occurs at a later stage in the mutant lineage. When cultured in a basally defined, insulin-containing medium, wt and jp OBs died 1-2 days later as their differentiation into GalC+ OCs began. Survival was not enhanced by known trophic factors (ciliary neurotrophic factor, leukemia inhibitory factor, neurotrophin-3) for differentiating rat OCs. In medium conditioned by neonatally derived rat or wt mouse astrocytes, however, wt OBs survived terminal OC differentiation, expressing first GalC, then DM-20/PLP on their surface 1-2 days later, before elaborating myelin-like membrane. By contrast, jp OBs in sister cultures survived differentiation initially as well as their normal counterparts did but rapidly died thereafter, beginning at the time when PLP/DM-20 immunoreactivity became detectable on premature wt GalC+ OCs. Additionally under these conditions, there survived a minor population (<5%) of jp cells, including mature OCs, which expressed stunted membranes and DM-20/PLP immunoreactivity in their cytoplasm, and undifferentiated progenitors. This model supports the concept that OC death in jp is effected by an intrinsic program, one mechanistically related to jp PLP/DM-20 gene expression and refractory to trophic cues in the environment.

In vitro oligodendrogliotrophic properties of cell adhesion molecules in the immunoglobulin superfamily: myelin-associated glycoprotein and N-CAM.

To determine if cell recognition molecules interact trophically with oligodendrocytes (OCs), their effect as growth substrates for differentiating oligodendroblasts was studied in primary culture. Oligodendroblasts purified from postnatal rat cerebrum by immunopanning were plated on substratum-bound cell adhesion molecules or extracellular matrix glycoproteins in chemically defined medium in which OCs terminally differentiate but survive poorly. Growth on myelin-associated glycoprotein (MAG) and neural cell adhesion molecule (N-CAM) selectively increased the number of viable cells per culture 2 weeks after plating as much as tenfold and sixfold, respectively, over background survival on an albumin substrate, whereas L1, tenascin-R, tenascin-C, fibronectin, and laminin were ineffective. Neither MAG nor N-CAM stimulated bromodeoxyuridine incorporation into cultures, indicating that enhanced proliferation did not contribute to better survival. Compared to growth on polyornithine alone, oligodendroblast differentiation in the added presence of MAG or N-CAM was qualitatively unchanged; > 90% of surviving cells developed into OCs that matured further by immunocytochemical and morphological criteria. A striking difference, however, was the quantitative effect of MAG and N-CAM substrates on oligodendrite outgrowth, increasing myelin-like membrane formation two- to threefold (> 8 x 10(3) microns2/cell). These findings support the concept that autotypic or heterotypic cell contact-mediated signaling by recognition molecules at the OC surface contributes trophic support of myelinogenesis.

Regulation of oligodendrocyte cell survival and differentiation by ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and interleukin-6.

The regulation and maintenance of developmental lineages by trophic factors, both cell-mediated and soluble, is a key aspect of cellular differentiation in the nervous system. In this review we focus on oligodendrocytes and their progenitors and how differentiation and survival are regulated by four neuropoietic cytokines: ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and interleukin-6 (IL-6). We discuss how these cytokines act as "broad spectrum" factors. That is, how, even within a specific cell lineage, a given cytokine may have different effects on the target cells at various stages of differentiation.

Astroglial control of oligodendrocyte survival mediated by PDGF and leukemia inhibitory factor-like protein.

Programmed death and the identification of growth factors delaying this process in the oligodendrocyte lineage suggest that other cell types provide oligodendrogliotrophins. To determine their source, primary cultures of oligodendroblasts immunopurified from postnatal rat cerebrum were used to screen other cultured neural and non-neural cell types for the release of survival factors into a defined insulin-containing medium. In non-conditioned medium, oligodendroblasts died 1-2 days after undergoing terminal differentiation into oligodendrocytes, as defined by the onset of expression of galactocerebroside. In medium conditioned by astrocytes, unlike the other tested cell types, differentiated oligodendrocytes survived for weeks in a mature myelinogenic state. Survival was partially reduced by immunoabsorption of the medium with antibodies to platelet-derived growth factor and abolished by immunoabsorption with antibodies to leukemia inhibitory factor. By the same criterion, survival activity was not attributed to other astrocytic products, ciliary neurotrophic factor and basic fibroblast growth factor. Membrane ultrafiltration analysis indicated the activity corresponded to heat-labile protein smaller (M(r) = 10(-30) x 10(3)) than native rat leukemia inhibitory factor (M(r) = 43 x 10(3)). The astrocytic stimulus was > 4-fold more efficacious than other known oligodendrogliotrophic cytokines, including ciliary neurotrophic factor, neurotrophin-3 and leukemia inhibitory factor itself, tested singly or in combination, and promoted survival additively with these agents. These findings suggest that astrocytes function as paracrine regulators of oligodendroblast and oligodendrocyte survival and that their effect is mediated initially by platelet-derived growth factor and thereafter by a powerful cytokine related to leukemia inhibitory factor.

Oligodendroblasts distinguished from O-2A glial progenitors by surface phenotype (O4+GalC-) and response to cytokines using signal transducer LIFR beta.

The developmental potential of progenitors at two final stages of the macroglial lineage giving rise to oligodendrocytes in postnatal rat brain was studied in response to defined and serum inducers of astrocyte gene expression. Cell immunoselection [with Gd3 ganglioside, O4 and galactocerebroside (GalC) antibodies] was used to isolate G+D3O4- and O4+GalC- phenotypes directly from premyelinating cerebrum. In a basal defined culture medium, G+D3O4- progenitors differentiated infrequently into oligodendrocytes on a growth substratum comprised of meningeal cell-derived extracellular matrix. Their conversion into astrocytes, as determined by immunofluorescence analysis of glial fibrillary acidic protein expression, was induced by oncostatin-M as well as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, but not interleukin-6, and required extracellular matrix. By comparison, O4+GalC- progenitors were refractory to astrocyte induction under these conditions, as in short-term cultures of optic nerve, and differentiated into myelinogenic oligodendrocytes instead. Only in response to an overriding stimulus in fetal bovine serum did O4+GalC- progenitors, like their immediate precursors, become astrocytic. These data functionally distinguish two classes of astrocyte-inducing agents to provide clear evidence of an oligodendroblast, a progenitor defined by surface phenotype (O4+GalC-) and an altered response of the oligodendrocyte lineage to cytokines using signal transducer LIFR beta.

Glial cell mitogens bFGF and PDGF differentially regulate development of O4+GalC- oligodendrocyte progenitors.

The oligodendrocyte lineage in cerebrum is characterized by the expression of immunologically identified surface antigens resulting in the sequential appearance of three distinct phenotypes, A2B5+O4-, O4+GalC-, and O4+GalC+. In the present study we have placed O4+GalC- progenitors immunopanned from premyelinating rat cerebrum into a basal, defined medium that by itself does not support well either their proliferation or survival. The response of these progenitor cells to platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) was then examined. The results demonstrate that both PDGF and bFGF stimulated proliferation and short-term survival of newly cultured cells, but that their effect on the course of O4+GalC- differentiation was strikingly different. PDGF delayed postmitotic development by transiently reverting (ED50 = 3 ng/ml) O4+GalC- progenitors to A2B5+O4- preprogenitor-like cells that subsequently differentiated even in the continued presence of PDGF. bFGF restored mitogenic activity of the O4+GalC- progenitors to a saturable level at low doses (ED50 = 1 ng/ml); doses of bFGF > or = 10 ng/ml impaired differentiation of the progenitors into GalC+ cells and were also mitogenic for newly differentiated GalC+ oligodendrocytes. These data imply that bFGF supplants PDGF as a mitogen during lineage progression from A2B5+O4- to O4+GalC- progenitors. Lineage reversion of O4+GalC- cells in response to PDGF is suggested as a mechanism for facilitating remyelination by triggering the proliferative expansion of O4+GalC- progenitor-like cells persisting into adulthood.

Two proliferative stages of the oligodendrocyte lineage (A2B5+O4- and O4+GalC-) under different mitogenic control.

Cell proliferation during successive stages of oligodendrocyte development was delineated in the rat brain and optic nerve. Surface antigens, A2B5, O4, and galactocerebroside (GalC) identified three cell populations emerging in sequence; the incorporation of bromodeoxyuridine into newly synthesized DNA identified the proliferative cells. In vivo, progenitor cells with phenotypes A2B5+O4- and A2B5+O4+GalC- were both proliferative, whereas differentiated GalC+ oligodendrocytes were not. Under basal conditions of culture, the proliferation of both progenitor cell types of the optic nerve was nearly abolished. Activity was restored for A2B5+O4- precursor cells with medium conditioned by either type-1 astrocytes, meningeal cells, or cerebellar interneurons. In contrast, intermediate O4+GalC- cells (proligodendrocytes) were refractory to the astroglial and meningeal signals, but remained as responsive as their precursor cells to the neuronal stimulus. These data further characterize the O4+GalC- proligodendrocyte as a distinct developmental stage, one that specifies a changing response of the cell to environmental mitogens.

Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development.

Three monoclonal antibodies that react with antigens on the surface of developing oligodendrocytes in a stage-specific manner, O1, O4 (Sommer and Schachner, 1981), and R-mAb (Ranscht et al., 1982), have been studied with respect to their specificities for a number of purified lipids. The observed specificities were consistent regardless of how the antigens were presented to the antibodies. O1 reacted with galactocerebroside, monogalactosyl-diglyceride, and psychosine and, in addition, labeled an unidentified species in rat brain extracts. R-mAb reacted with galactocerebroside, monogalactosyl-diglyceride, sulfatide, seminolipid, and psychosine; the reaction of R-mAb with sulfatide was nearly equal to that with galactocerebroside. O4 reacted with sulfatide, seminolipid, and to some extent with cholesterol. However, oligodendrocyte progenitor cells labeling with O4 that had not yet begun to express the O1 antigen failed to incorporate 35SO4 or [3H]galactose into sulfatide or seminolipid, the syntheses of which first appear in O1-positive cells. Therefore, O4 stains, in addition to sulfatide and seminolipid, and unidentified antigen that appears on the surface of oligodendrocyte progenitors prior to the expression of sulfatide and galactocerebroside. In primary cultures of rat brain, developing O4+ oligodendrocyte progenitors stained slightly earlier with R-mAb than with O1, and thus R-mAb transiently stained a larger population of oligodendrocytes than did O1. None of the three antibodies produced a detectable reaction on Western immunoblot after separation of brain proteins on reducing gels. In conclusion, the results show that O4, R-mAb, and O1 have multiple overlapping specificities, including previously unrecognized cross-reactions.

Oligodendrocyte progenitors isolated directly from developing telencephalon at a specific phenotypic stage: myelinogenic potential in a defined environment.

Oligodendroglia differentiate asynchronously in the developing central nervous system, passing through a series of stages identified by the sequential expression of specific differentiation antigens, culminating in the formation of the myelin sheath. In the work presented here, oligodendrocyte progenitors at a temporally narrow and well-defined phenotypic stage of development have been isolated in high purity and yield directly from postnatal rat telencephalon. This stage is identified by the expression of the O4 antigen, the earliest recognized surface marker specific for the oligodendroglial lineage, but the absence of the differentiation marker galactosylcerebroside (GalC). These O4+ GalC- progenitors first appear at birth (10(5)/telencephalon), 2-3 days before O4+ GalC+ oligodendrocytes. The work presented here demonstrates that a major subpopulation of O4+ GalC- progenitors (80%), which we have termed 'proligodendrocytes', is fully committed to terminal oligodendrocyte differentiation. A relatively small, maximal set of nutritional supplements are sufficient for proligodendrocytes to carry out the myelinogenic cascade of differentiated gene expression in a temporally normal manner, in quantitatively significant amounts, in normal ratios of myelin protein isoforms, and in a regulated relationship to the inclusion of myelin-specific products into myelin-like membrane sheets. An important corollary is that this step of myelinogenesis does not require contact with other cell types, in particular neurones and astrocytes, nor does it require unknown growth factors unique to these cell types. Additionally under these conditions, there exists a developmentally quiescent subpopulation (20%) of O4+ GalC- cells that may have significance for understanding the progenitors previously described in adult brain and suggested to be instrumental in remyelination under pathological conditions.

Stimulation of oligodendrocyte differentiation in culture by growth in the presence of a monoclonal antibody to sulfated glycolipid.

Perturbation of myelinogenesis by monoclonal antibodies against galactolipids is being used to study the role of these lipids in oligodendrocyte differentiation. We report here a marked stimulatory effect on oligodendrocyte differentiation when mixed primary cultures initiated from 19-21 day fetal rat telencephala are grown in the presence of a monoclonal antibody against sulfogalactolipids. When such cultures were grown in the presence of the IgM antibody 04 [Sommer and Schachner, Dev Biol 83:311-327 1981], the oligodendrocytes formed aggregates connected by fasciculated processes. Immunofluorescence microscopy and biochemical analyses of treated cultures demonstrated 2-3 fold increases in the fraction of 04-positive cells expressing myelin basic protein, and in the levels of myelin basic protein RNA, myelin basic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase activity, and 35SO4 incorporation into sulfatide. Greater than 90% of the cells positive for myelin basic protein in treated cultures were in aggregates. The specific activities of oligodendrocyte markers were unaffected in control cultures grown with nonspecific myeloma IgM. Since there was no increase in the total number of 04-positive cells in treated cultures, the increases in the specific activities of the myelin protein markers appears to be due to an increase in the fraction of cells expressing these markers. Time course studies demonstrated that both the rate and extent of oligodendrocyte differentiation were enhanced in treated cultures. These data are discussed with regard to possible mechanisms of the stimulation, considering not only potential direct effects of the antibody on the cell physiology, but also possible indirect effects due to antibody-induced aggregation.

Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes.

Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.

Myelin-specific domain on the plasmalemma of oligodendroglia: differential expression in the rat and hypomyelinating mouse mutants jimpy and quaking.

Monoclonal antibody, 1A9, prepared against bovine white matter, recognizes a proteinaceous, myelin-specific domain in the CNS that is restricted to the surface of oligodendroglia in primary dissociated cell cultures. The antigen is not detected in the PNS or non-neural tissues. Antibody binding is abolished by heating, exposure to SDS and delipidation, indicating that a conformationally sensitive epitope is recognized. The antigen is present in tracts of developing white matter in rat cerebellum beginning at 5 days postnatally. In developing cultures of fetal rat brain the period of rapid onset for the phenotypic expression of 1A9 antigen is similar to that of galactocerebroside, corresponding to 2-4 postnatal days of age. The 1A9 antigen is not observed in white matter or cultured oligodendroglia of the hypomyelinating jimpy mutant mouse, but its expression is qualitatively normal in the quaking mutant. The possibility is raised that 1A9 may be the primary target of the jimpy mutation.

A monoclonal antibody to the endothelium of rat brain microvessels.

A rat brain fraction enriched with microvessels was used as the immunogen to produce mouse hybridoma cell lines secreting monoclonal antibodies. One of these antibodies, selected from 156 supernatants by enzyme-linked immunosorbent and immunofluorescent assays, reacted only with the endothelium of microvessels in the brain. The endothelium-specific antibody labelled the cytoplasm of microvascular endothelial cells, their luminal membranes, and an extracellular layer, the endocapillary coat, which covered the luminal surface of these cells. In the kidney, the antibody specifically stained the brush border of the proximal tubuli, and in the liver, the antibody specifically stained bile canaliculi. This demonstrates that 3 morphological structures with important transport functions, cerebral microvascular endothelium, brush border of kidney proximal tubuli, and liver bile canaliculi, express the same epitope.

Extra-neural glial fibrillary acidic protein (GFAP) immunoreactivity in perisinusoidal stellate cells of rat liver.

The dendritic processes and perinuclear cytoplasm of stellate-shaped perisinusoidal cells in frozen sections of rat liver were specifically labeled with antisera raised independently to glial fibrillary acidic protein (GFAP), the major component of intermediate filaments in astrocytes. A liver protein co-migrating with authentic GFAP and immunoreactive with GFAP antisera was demonstrated with immunoblots of brain and liver extracts enriched in intermediate filament proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study presents yet another example of immunoreactivity to GFAP, or a highly similar protein localized outside the CNS, in cells of mesenchymal origin exhibiting some morphological features common to astroglia.