Need for caution when interpreting Xpert® MTB/RIF results for rifampin resistance among children.

BACKGROUND: Recommended by the World Health Organization as an initial diagnostic test for TB in children, Xpert® MTB/RIF is widely implemented in many countries, including Kenya.METHODS: Three hundred HIV-positive and negative children (<5 years) were enrolled in Kisumu County, Kenya, from October 2013 to August 2015. Multiple specimen types were collected from each child and tested using Xpert, liquid culture, and phenotypic drug susceptibility testing (DST). Samples positive for rifampin (RIF) resistance on Xpert were tested using line-probe assay and sequencing.RESULTS: Of 32 children with bacteriologically confirmed TB, 27 had positive Xpert results. Of these, 3/27 (11%, 95% CI 4-28) had RIF resistance detected on Xpert, but not by phenotypic DST, line-probe assay, or sequencing. For these three children, five Xpert tests showed RIF resistance; all five tests had semi-quantitative "very low" results and delay or absence of probe D signal, whereas no Xpert results with higher semi-quantitative results showed RIF resistance. All three children responded well to standard TB treatment.CONCLUSIONS: False RIF resistance may be detected in pediatric specimens. Further study is needed to determine if false RIF resistance is associated with low bacterial load.

Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.

A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.

Monitoring the performance of mycobacteriology laboratories: a proposal for standardized indicators.

SETTING: Thailand Tuberculosis (TB) Active Surveillance Network: Bangkok, Chiang Rai, Phuket, Tak and Ubon-Ratchathani, Thailand. BACKGROUND: Mycobacteriology laboratories in resource-limited, high TB burden settings are expanding to perform conventional solid media culture and broth-based mycobacteriology culture. Indicators that measure how well a laboratory performs sputum microscopy have been developed and broadly implemented. Routine monitoring of sputum culture performance, however, is not as common. DESIGN: We implemented indicators for monitoring the quality of laboratory services in five province-level mycobacteriology culture facilities in Thailand. These indicators were derived from literature review, consultation with subject matter experts and our program experience. CONCLUSIONS: We believe that an international consensus document providing monitoring guidelines for mycobacteriology laboratories is urgently needed.

Control of gap-junctional communication in astrocytic networks.

Astrocytes, which constitute the most abundant cell type in mammalian brain, are extensively coupled to one another through gap junctions composed mainly of connexin43. In regions exhibiting high levels of connexin43 expression, tens of astrocytes are labeled following single-cell intracellular injection. Importantly, both the expression and the permeability of gap junctions are tightly regulated. Such long- and short-term regulations indicate that astrocytic networks might be subject to remodeling and to some plasticity. Since evidence for neuro-glial interaction exists, the degree of coupling between astrocytes could participate to set the tone of neuronal activity and to determine the sphere of influenced neurons. Research in this area is still at its early stages and significant progress requires a transition from the understanding of basic properties to the study of function.

Astrocytic neurotransmitter receptors in situ and in vivo.

In the brain, astrocytes are associated intimately with neurons and surround synapses. Due to their close proximity to synaptic clefts, astrocytes are in a prime location for receiving synaptic information from released neurotransmitters. Cultured astrocytes express a wide range of neurotransmitter receptors, but do astrocytes in vivo also express neurotransmitter receptors and, if so, are the receptors activated by synaptically released neurotransmitters? In recent years, considerable efforts has gone into addressing these issues. The experimental results of this effort have been compiled and are presented in this review. Although there are many different receptors which have not been identified on astrocytes in situ, it is clear that astrocytes in situ express a number of different receptors. There is evidence of glutamatergic, GABAergic, adrenergic, purinergic, serotonergic, muscarinic, and peptidergic receptors on protoplasmic, fibrous, or specialized (Bergmann glia, pituicytes, Müller glia) astrocytes in situ and in vivo. These receptors are functionally coupled to changes in membrane potential or to intracellular signaling pathways such as activation of phospholipase C or adenylate cyclase. The expression of neurotransmitter receptors by astrocytes in situ exhibits regional and intraregional heterogeneity and changes during development and in response to injury. There is also evidence that receptors on astrocytes in situ can be activated by neurotransmitter(s) released from synaptic terminals. Given the evidence of extra-synaptic signaling and the expression of neurotransmitter receptors by astrocytes in situ, direct communication between neurons and astrocytes via neurotransmitters could be a widespread form of communication in the brain which may affect many different aspects of brain function, such as glutamate uptake and the modulation of extracellular space.

Tuberculosis screening outcomes for newly diagnosed persons living with HIV, Nyanza Province, Kenya, 2009.

SETTING: Fifteen human immunodeficiency virus (HIV) clinics in Nyanza Region, Western Kenya. OBJECTIVE: To describe routine tuberculosis (TB) screening and diagnostic practices among newly enrolled people living with HIV (PLHIV) prior to the implementation of World Health Organization recommended TB intensified case finding. DESIGN: Retrospective chart abstraction of PLHIV aged ⩾7 years who were newly enrolled in HIV care in July and August 2009, and who had not received antiretroviral treatment in the preceding 2 years or been diagnosed with TB in the previous year. Factors associated with evidence of TB diagnostic evaluation among symptomatic PLHIV were assessed. RESULTS: Of 1020 patients included in the analysis, 995 (98%) were screened for TB at enrolment and 613 (62%) reported TB symptoms. Ninety-six (16%) patients with symptoms had evidence of referral for TB diagnostic evaluation, including patients at large clinics, those with advanced HIV disease and those reporting multiple TB symptoms. Among the 43 (45%) with documented evaluation results, 26 (60%) were diagnosed with TB. CONCLUSION: Although most PLHIV were screened for TB, very few underwent an evaluation, and the proportion diagnosed with TB was very low. Efforts to improve TB screening should focus on standardizing the intensified case finding algorithm and linkage to, and adequate infrastructure for, TB diagnostic evaluation.

Retroviral inhibition of cAMP-dependent protein kinase inhibits myelination but not Schwann cell mitosis stimulated by interaction with neurons.

Schwann cells are the myelinating glia of the peripheral nervous system. Neuron-Schwann cell contact profoundly affects several aspects of Schwann cell phenotype, including stimulation of mitosis and myelin formation. Many reports suggest that neuronal contact exerts this influence on Schwann cells by elevating Schwann cell cAMP and activating cAMP-dependent protein kinase A (PKA). To elucidate the importance of Schwann cell PKA in neuronal stimulation of Schwann cell mitosis and myelination, the gene encoding the PKA inhibitory protein RIalphaAB or PKIEGFP was delivered to Schwann cells using retroviral vectors. PKA inhibitory retroviral vectors effectively blocked forskolin-stimulated Schwann cell mitosis and morphological change, demonstrating the ability of the vectors to inhibit PKA in infected Schwann cells. Treatment of dorsal root ganglia neuron-Schwann cell cocultures with H-89 (10 microm) or KT5720 (1-10 microm), chemical inhibitors selective for PKA, significantly inhibited neuronal stimulation of Schwann cell mitosis. In contrast, retrovirus-mediated inhibition of Schwann cell PKA had no effect on the ability of neurons to stimulate Schwann cell mitosis. However, markedly fewer myelin segments were formed by Schwann cells expressing PKA inhibitory proteins compared with controls. These results suggest that activation of Schwann cell PKA is required for myelin formation but not for Schwann cell mitosis stimulated by interaction with neurons.

Receptor-mediated calcium signals in astroglia: multiple receptors, common stores and all-or-nothing responses.

Calcium signals following activation of P2Y purinergic, alpha 1 adrenergic, and muscarinic cholinergic receptors were examined in individual astroglial cells. ATP, phenylephrine and carbachol, each increased intracellular calcium levels ([Ca2+]i) to similar amplitudes in the presence or absence of extracellular Ca2+. The dose-response relationship showed that less than an order of magnitude increase in ligand concentration led to maximal increase in [Ca2+]i from basal levels. Simultaneous application of multiple ligands did not produce additive effects on [Ca2+]i. These data suggested that different ligands released Ca2+ from common stores and that each of the ligands could cause maximal release. Application of a second ligand immediately after the first ligand produced an additional Ca2+ rise, suggesting that the Ca2+ stores were rapidly refilled and that receptor desensitization rather than Ca2+ depletion accounted for the rapid decline of the Ca2+ peak. Caged IP3 produced Ca2+ signals similar to those produced by ligands. For a given cell, both caged IP3 and ligands sometimes produced only one level of partial Ca2+ increases, suggesting the presence of a pool of high IP3-sensitive stores. Together, our results indicate that neuroligands tend to generate an all-or-nothing Ca2+ release from IP3 sensitive stores. The interactions between different receptor systems most likely occur at the level of IP3 accumulation.

Schwann cells influence the expression of ganglioside GD3 by rat dorsal root ganglion neurons.

Cultured rat dorsal root ganglion neurons expressed ganglioside GD3 when grown in the absence of non-neuronal cells. Among the non-neuronal cells, fibroblasts, but not Schwann cells, also stained for ganglioside GD3 during the first few days in culture. When neurons were combined with non-neuronal cells the intensity of the GD3 immunoreactive neuronal processes was diminished at sites contacted by Schwann cells. This contact-mediated effect was specific for ganglioside GD3 since no difference was seen with A2B5 or JONES antibodies, which recognize different gangliosides.

Regulation of astroglial responsiveness to neuroligands in primary culture.

Previous studies from this laboratory indicate that type-1 astroglia in primary culture are pharmacologically heterogeneous. Two competing hypotheses were proposed to explain the development of glial heterogeneity. First, that the heterogeneity may reflect stable subclasses of astroglia that express a set of receptor-signalling systems. Second, that astroglia can undergo qualitative changes in their expression of receptor-signalling systems with time in vitro. To distinguish between these two hypotheses, experiments were designed to examine neuroligand-evoked calcium responses within clones of type-1 astroglia. If stable and distinct subsets of astroglia were present, a clone derived from a single cell would exhibit uniform responses to a given set of neuroligands. Alternatively, if the pharmacological properties of astroglia underwent qualitative changes, astroglial clones should contain pharmacologically distinct cells. A video-based imaging system and the Ca2+ indicator dye Fura-2 were used to monitor receptor-mediated increases in Cai2+ upon receptor activation. Interestingly, only a fraction of the cells within a given clone responded to carbachol or histamine with an increase in Cai2+, whereas treatment with a P2Y purinergic receptor agonist generally increased Cai2+ in 100% of the cells within the clone. To examine the stability of the receptor signalling over time, individual astroglia within a number of clones were tested on different days for their ability to respond to neuroligands. The results of these experiments indicated that individual astroglial cells tended to lose their responsiveness to certain ligands such as carbachol and histamine as they developed responsiveness to others such as norepinephrine. Our data indicate that during development neurotransmitter receptors on astroglial cells are regulated by both internal and external mechanisms. Glial proliferation produces a variety of pharmacologically distinct astroglial cells. Exposure to neurotransmitters can qualitatively turn off some, but not all, astroglial receptor systems.

Pharmacologically-distinct subsets of astroglia can be identified by their calcium response to neuroligands.

Type 1 astroglia have been reported to exhibit in excess of 20 different neuroligand receptors in vitro. The aim of this study was to determine if astroglia, like neurons, are heterogeneous with respect to their ability to respond to different neuroligand receptor agonists. Type 1 astroglia were loaded with the calcium indicator dye fura-2 and the influence of six different neuroligands on their intracellular calcium levels was examined using a video-based imaging system. Each of the six different neuroligands tested increased calcium levels in a subpopulation of glial fibrillary acidic protein immunopositive cells (astroglia). The percentage of cerebral cortical type 1 astroglia responding to a given neuroligand varied with the agonist and generally followed the order: 2-methylthio-ATP greater than phenylephrine greater than carbachol = serotonin greater than glutamate = histamine. The results also indicate that a single astroglial cell can respond to one agonist with a sustained rise in calcium levels and to an alternate agonist with oscillations in calcium levels. The pharmacological heterogeneity evident among astroglia does not appear to depend on cell proliferation or association with neurons. The results of our studies indicate that cultures of cerebral cortical type 1 astroglia are composed of distinct subsets of cells that can be distinguished by qualitative differences in their ability to respond to specific neuroligands with a rise in intracellular calcium.

Characterization of antisera to glutamate and aspartate.

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.

A dicistronic retroviral vector and culture model for analysis of neuron-Schwann cell interactions.

A dicistronic retroviral gene delivery system and tissue culture model has been developed for studies of neuron-Schwann cell interactions at the single cell level. The dicistronic retroviral vector contains a multiple cloning site followed by the encephalomyocarditis virus internal ribosomal entry site (EMCV-IRES) and a green fluorescent protein gene. This design allows for 5'-cap dependent translation of any gene of interest and 5'-cap independent translation of green fluorescent protein (GFP) from a single dicistronic RNA. The culture model consists of dorsal root ganglia (DRG) explants grown in defined medium. Under these conditions the Schwann cell population is selectively expanded and infected by the retroviral vector, allowing for rapid transfer of genes of interest selectively to a large percentage of Schwann cells in coculture with neurons. Infected cells are subsequently identified in living cultures by their expression of GFP. Infected (GFP expressing) Schwann cells in contact with neurites continued to exhibit: (1) increased mitotic activity, (2) increased sensitivity to elevate intracellular calcium in response to extracellular application of ATP, and (3) myelination. This viral construct has the added advantage that it allows identification of cells expressing transgenes among a heterogeneous population by fluorescence microscopy, FACS, or flow cytometry.

Immunocytochemically defined astroglia from fetal, newborn and young adult rats express beta-adrenergic receptors in vitro.

Autoradiography of radioligand binding was used to assess the expression of beta-adrenergic receptors (beta-AR) by immunocytochemically identified astroglia cultured from the cerebral cortices of rats 16 days in gestation through 28 days postnatal (DPN). Polygonal astroglia isolated from animals at each age examined were found to exhibit large numbers of beta-AR. In contrast, only low levels of beta-AR could be detected on process-bearing astroglia and fibroblasts. Quantitative analysis showed that there was an increase in the density of beta-AR on polygonal astroglia between 16 days in gestation and 1 DPN. This increase in beta-AR receptor density was present whether the cells were grown for long periods of time in culture (8-22 days) or for short periods of time in culture (1-5 days). The results also suggest that differences in the level of receptor expression between cells grown in short-term and long-term culture may be due in part to culture methodology.

Autoradiographic quantitation of beta-adrenergic receptors on neural cells in primary cultures. I. Pharmacological studies of [125I] pindolol binding of individual astroglial cells.

As part of an investigation into the expression of neurotransmitter receptors on astroglia, we have developed a method to label beta-adrenergic receptors on immunocytochemically stained cultured cells using autoradiography of 125I-labelled beta-adrenergic receptor ligands. The current investigation was undertaken to determine whether the binding of [125I]pindolol (*IPIN) to immunocytochemically stained cultured cells, as measured by quantitative autoradiography, would fulfill the usual pharmacological criteria for specific beta-adrenergic receptor binding. *IPIN binding experiments were carried out on individual astroglia obtained from neonatal rat cerebral cortex and grown as primary cultures on polylysine-coated glass slides. Autoradiographic silver grains on cells which stained for the intracellular astroglial marker, glial fibrillary acidic protein (GFAP), were quantified by a microcomputer-based video digitizing system. We determined that competition for *IPIN binding by propranolol and isoproterenol was stereospecific, that specific binding of *IPIN was saturable, and that *IPIN binding sites were lost after isoproterenol-induced desensitization. These results provide convincing evidence that we can quantitatively examine beta-adrenergic receptors on single cells. The use of computerized video methods greatly facilitates the rapid quantitation of autoradiographic grains associated with immunocytochemically identified cells. This study is a unique demonstration of receptor binding parameters derived from single cells in a known population, and represents a novel approach to the problem of assessing cell-type specific receptors on neural cells in mixed primary cultures.

Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: a new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates.

A new method has been developed for the preparation of essentially pure primary cultures of neurons and non-neuronal cells from 11-day embryonic chick sympathetic ganglia. This method utilizes (1) differences in cell-to-substrate adhesiveness between neurons and non-neuronal cells and (2) the capacity of neurons to form homotypic aggragates. The maximum difference in adhesiveness between neuronal and non-neuronal cells occurred when the ganglia were dissociated with trypsin following collection in a salt solution lacking divalent cations. This difference allowed the preparation of highly purified non-neuronal cultures and 85-90% pure neuronal cultures. Intermittent agitation during the period of cell separation markedly increased the purity of the neuronal cultures by (1) inhibiting neuronal but not non-neuronal cell attachment and (2) facilitating the formation of homotypic neuronal aggregates in the supernatant. Neuronal and non-neuronal cultures prepared under these conditions were more than 99% pure on the basis of both morphological and biochemical analyses. Both cell types exhibited attachment efficiencies greater than 95% and have been maintained for several weeks in vitro. Thus, completely isolated neuronal and non-neuronal cultures can be prepared and maintained for prolonged periods in the absence of cells of the other type.

Neuronal stimulation of (3H)thymidine incorporation by primary cultures of highly purified non-neuronal cells.

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-3H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [3H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [3H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [3H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [3H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [3H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.

Neuron-associated astroglial cells express beta- and alpha 1-adrenergic receptors in vitro.

In vivo, astroglial cells are closely interrelated with neurons. The present studies were undertaken to determine if certain astroglial properties are influenced when maintained in a heterogeneous cellular environment. Astrocytes and neurons were co-cultured from hippocampal tissue and the parameters examined included astroglial morphology and expression of beta- and alpha 1-adrenergic receptors (AR). Astroglial cells exhibit an extremely elongated morphology when growing in direct contact with neurons. Astroglia growing under the same culture conditions, but not in direct contact with neurons, exhibited a polygonal morphology. One hundred percent of the elongated astroglia expressed the beta-AR with an average density of 1320 binding sites/1000 microns 2. In contrast, only 40-50% of these elongated astroglia expressed alpha 1-ARs. Our results indicate that astroglia, maintained in the presence of neurons, continue to express beta- and alpha 1-ARs. These results suggest that under in vivo conditions, where astroglia normally exist in close contact with neurons, astrocytes may express surface receptors which enable them to sense neuronal activity and to selectively respond to such activity. The elongated astroglia described here may exhibit morphological features more reminiscent to that which exists in vivo.

Autoradiographic quantitation of beta-adrenergic receptors on neural cells in primary cultures. II. Comparison of receptors on various types of immunocytochemically identified cells.

We have developed a microcomputer-based video method to quantify neurotransmitter receptors on single, immunocytochemically labeled cultured cells. This method has been applied to determine whether beta-adrenergic receptors are more numerous on neurons, astroglia, oligodendroglia or fibroblasts in primary neural cell cultures, and to assess the heterogeneity of receptor expression within a single cell type. Dissociated cells from perinatal rat cerebral cortex were grown in very sparse cultures on polylysine-coated glass slides. The cultured cells were fixed and permeated, then stained with fluorescently labeled immunocytochemical markers for astroglia (glial fibrillary acidic protein), fibroblasts (fibronectin), oligodendroglia (galactocerebroside) or neurons (A2B5). beta-Adrenergic receptors were labeled with [125I]pindolol or [125I]cyanopindolol, and dry-mount autoradiography was carried out on the fixed cells. Cells were identified according to their morphology and cell-type specific staining, then autoradiographic grains associated with the defined cells were visualized by reflected polarized light microscopy and counted with a microcomputer-based video digitizing system. Using this technique, we have determined that fibroblasts have less than 15% of the number of beta-adrenergic receptors expressed by polygonal astroglia, whereas oligodendroglia and neurons had no detectable binding of 125I-labelled ligands. This suggests that in these mixed neural cell cultures, the great majority of beta-adrenergic receptors are associated with astroglia. Furthermore, we determined that process-bearing astroglia have less than 5% of the number of beta-adrenergic receptors expressed by polygonal astroglia. Since process-bearing astroglia are thought to be derived from polygonal astroglia, these results suggest that the beta-adrenergic receptor is lost from this population of astroglia during development.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-ganglioside antibodies reveal subsets of cultured rat dorsal root ganglion neurons.

Subsets of cultured dorsal root ganglion neurons were identified by using the anti-ganglioside monoclonal antibodies A2B5, D1.1, R24 and JONES. A2B5 and D1.1 labelled a population of cells that was relatively stable between 2 and 20 days in vitro, while the population of cells labeled with both R24 and JONES decreased with time, suggesting that the gangliosides recognized by Jones and R24 are developmentally regulated. Given the observation that the relative proportions of ganglioside species changes with time in culture, it is very important to carefully define the stability of ganglioside antigens before using them as cell markers.