Separation of two bone cell populations from fetal rat calvaria and a study of their responses to parathyroid hormone and calcitonin

1983 ◽  
Vol 99 (3) ◽  
pp. 387-399 ◽  
Author(s):  
I. P. Braidman ◽  
D. C. Anderson ◽  
C. J. P. Jones ◽  
J. B. Weiss
Keyword(s):  
Alkaline Phosphatase ◽  
Parathyroid Hormone ◽  
Acid Phosphatase ◽  
B Cells ◽  
Adenylate Cyclase ◽  
Culture Medium ◽  
Cell Populations ◽  
C Cells ◽  
Type B ◽  
Type C

Bone cells released from perinatal rat calvaria by digestion with clostridial peptidase were separated into two distinct populations (designated types B and C) by equilibrium density centrifugation on a two-step gradient of Percoll. They were extensively characterized by light and electron microscopy and for behaviour in culture, acid and alkaline phosphatase activity, collagen synthesis, collagenase secretion and adenylate cyclase response to parathyroid hormone (PTH) and calcitonin. Type C cells were predominantly large with up to seven nuclei and an unusual cytoplasmic appearance in cytocentrifuge preparations. They did not proliferate in culture and we have established culture conditions which prevented their overgrowth by contaminating proliferative cells. In culture these cells had low alkaline and high acid phosphatase and high aryl sulphatase activity, and synthesized little collagen. In contrast type B cells were mostly smaller and many had irregular cytoplasmic projections. In culture they became polygonal in shape, proliferated rapidly, and reached confluence in 4–5 days. These were low in aryl sulphatase and acid phosphatase, high in alkaline phosphatase activity, and synthesized labelled collagen actively with [3H]proline and ascorbic acid included in the culture medium. The two cell populations were found to differ in culture in two important further respects. First, the type C cells showed an adenylate cyclase response to calcitonin but not to PTH, while the converse was true for type B cells; this was so over at least a 20-fold range of isobutylmethyl xanthine concentration. Secondly, type C cells in culture secreted an active collagenolytic enzyme. Type B cells secreted much lower levels of a predominantly latent collagenase which required activation by mersalyl. Co-culture of type C and type B cells led to a marked reduction in the content of active collagenase in the culture medium.

Effects of physiological concentrations of parathyroid hormone on acid phosphatase activity in cultured rat bone cells

1986 ◽  
Vol 111 (1) ◽  
pp. 17-26 ◽  
Author(s):  
I. P. Braidman ◽  
J. G. St John ◽  
D. C. Anderson ◽  
W. R. Robertson
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
B Cells ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Bone Cells ◽  
Cell Populations ◽  
C Cells ◽  
C Cell ◽  
Type C

ABSTRACT The mechanism by which parathyroid hormone (PTH) induces osteoclastic bone resorption is still incompletely understood. Recent evidence suggests that the hormone exerts its effects indirectly, via the osteoblasts. Bone cells isolated from fetal rat calvaria by enzymatic digestion were used. Two heterogeneous cell populations were isolated by equilibrium density centrifugation on Percoll gradients and maintained by differential culture conditions. These two populations, which are morphologically distinguishable from one another by light and electron microscopy, have been characterized previously both biochemically and with regard to their hormonal (PTH and calcitonin) responses. We have called them type C cells (containing cells with some of the properties of osteoclasts) and type B cells (containing osteoblast-like cells, as well as fibroblasts, chondrocytes and other stromal cells). In the present study, we have further characterized the functional relationship between the two cell populations, with particular regard to the hormonal responses of type C cultures. Acid phosphatase, measured cytochemically in individual cells, was used as a marker for C cell responses. C cells had significantly higher levels of acid phosphatase activity than either B cells or spleen macrophages. Calcitonin (0–10 pg/ml) decreased C cell acid phosphatase activity but was without effect on B cells or spleen macrophages. Co-culture of C cells with B cells produced increased enzyme activity only in the former; this effect could be mimicked if fibroblasts replaced B cells and cell contact was essential for this response. PTH (0–10 pg/ml) raised enzyme activity further in C cells only when they were cultured with B cells. When C cells were cultured so that they shared medium, but were not in contact, with B cells, PTH (2 pg/ml) still increased enzyme activity in the former. Fibroblasts were ineffective in this system. Spleen macrophages were also unresponsive to PTH when substituted for C cells. Calcitonin (10 pg/ml) blocked the effects of PTH on C cells. These results indicate that macrophages are probably not a significant proportion of the C cell population, and that PTH may produce increased acid phosphatase activity in C cells via a humoral factor produced by cells present in B cell cultures. J. Endocr. (1986) 111, 17–26

Comparative transduction mechanisms of hair cells in the bullfrog utriculus. I. Responses to intracellular current

1994 ◽  
Vol 71 (2) ◽  
pp. 666-684 ◽  
Author(s):  
R. A. Baird
Keyword(s):  
B Cells ◽  
Hair Cell ◽  
Hair Cells ◽  
Hair Bundle ◽  
C Cells ◽  
Type B ◽  
Lateral Border ◽  
Utricular Macula ◽  
Type C ◽  
Current Steps

1. Hair cells in whole-mount in vitro preparations of the utricular macula of the bullfrog (Rana catesbeiana) were selected according to their macular location and hair bundle morphology. The voltage responses of selected hair cells to intracellular current steps and sinusoids in the frequency range of 0.5-200 Hz were studied with conventional intracellular recordings. 2. The utricular macula is divided into medial and lateral parts by the striola, a 75- to 100-microns zone that runs for nearly the entire length of the sensory macula near its lateral border. The striola is distinguished from flanking extrastriolar regions by the elevated height of its apical surface and the wider spacing of its hair cells. A line dividing hair cells of opposing polarities, located near the lateral border of the striola, separates it into medial and lateral parts. On average, the striola consists of five to seven medial and two to three lateral rows of hair cells. 3. Utricular hair cells were classified into four types on the basis of hair bundle morphology. Type B cells, the predominant hair cell type in the utricular macula, are small cells with short sterocilia and kinocilia 2-6 times as long as their longest stereocilia. These hair cells were found throughout the extrastriola and, more rarely, in the striolar region. Three other hair cell types were restricted to the striolar region. Type C cells, found primarily in the outer striolar rows, resemble enlarged versions of Type B hair cells. Type F cells have kinocilia approximately equal in length to their longest stereocilia and are restricted to the middle striolar rows. Type E cells, found only in the innermost striolar rows, have short kinocilia with prominent kinociliary bulbs. 4. The resting potential of 99 hair cells was -58.0 +/- 7.6 (SD) mV and did not vary significantly for hair cells in differing macular locations or with differing hair bundle morphology. The RN of hair cells, measured from the voltage response to current steps, varied from 200 to > 2,000 M omega and was not well correlated with cell size. On average, Type B cells had the highest RN, followed by Type F, Type E, and Type C cells. When normalized to their surface area, the membrane resistance of hair cells ranged from < 1,000 to > 10,000 k omega.cm2. The input capacitance of hair cells ranged from < 3 to > 15 pA, corresponding on average to a membrane capacitance of 0.8 +/- 0.2 pA/cm2.(ABSTRACT TRUNCATED AT 400 WORDS)

Loss of hamster Leydig cells during regression after exposure to a short photoperiod

2018 ◽  
Vol 30 (8) ◽  
pp. 1137 ◽  
Author(s):  
E. Beltrán-Frutos ◽  
V. Seco-Rovira ◽  
J. Martínez-Hernández ◽  
C. Ferrer ◽  
L. M. Pastor
Keyword(s):  
B Cells ◽  
Leydig Cells ◽  
Light And Electron Microscopy ◽  
Type A ◽  
Nuclear Antigen ◽  
Control Group ◽  
C Cells ◽  
Type B ◽  
A Cells ◽  
Type C

The aim of the present study was to evaluate the changes that occur in hamster Leydig cells during regression. Animals were divided into control, mild regression (MR), strong regression (SR) and total regression (TR) groups. Leydig cells were characterised by light and electron microscopy. Terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) antibodies were used to detect apoptosis and proliferation respectively. Three types of Leydig cells (A, B and C) could be differentiated. Type A cells were small in size compared with Leydig cells from animals exposed to a long photoperiod, which was a result of a decreased cytoplasm and nucleus. Type B cells were even smaller than Type A cells in regression groups. Type C exhibited cytoplasm vacuolisation. The percentage of Type C cells from the control group was much lower than in the MR, SR and TR groups. (P < 0.05). In the SR and TR groups, there was a significant decrease in the percentage of Type B cells compared with the control and MR groups (P < 0.05). The total number of Leydig cells decreased during testicular regression (P < 0.05). The total number of Type A and B cells was significantly lower in the MR, SR and TR groups compared with the control group (P < 0.05). There were no significant differences in the proliferation and apoptosis index in the groups studied. The findings of the present study indicate that there are three types of Leydig cells (A, B and C) in all hamsters studied and that regression causes an increase in the number of Type C cells, so that the reduction in the number Leydig cells during the phases of regression studied must be the result of necrosis and/or necroptosis.

scholarly journals Cortical cell populations from rabbit kidney isolated by free-flow electrophoresis: characterization by measurement of hormone-sensitive adenylate cyclase.

1982 ◽  
Vol 92 (2) ◽  
pp. 505-513 ◽  
Author(s):  
A Vandewalle ◽  
B Köpfer-Hobelsberger ◽  
H G Heidrich
Keyword(s):  
Alkaline Phosphatase ◽  
Parathyroid Hormone ◽  
Adenylate Cyclase ◽  
Fast Moving ◽  
Cortical Cell ◽  
Free Flow ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Cell Populations ◽  
Slow Moving

Free-flow electrophoresis allows the separation of different cell populations from a cell suspension isolated from rabbit kidney cortex after perfusion of the kidneys with a calcium-binder, followed by gentle mechanical treatment. After electrophoretic separation, analysis of the adenylate cyclase activities after stimulation by various hormones allows the precise determination of the origin of the cell populations with different electrophoretic mobilities. Adenylate cyclase from the slow-moving main cell population was only sensitive to parathyroid hormone. These cells had also high alkaline phosphatase content, further demonstrating their proximal origin. The various fast-moving cell populations had adenylate cyclase sensitive to isoproterenol and arginine vasopressin but were less sensitive to parathyroid hormone than the slow-moving cells. Their alkaline phosphatase content was also much lower. This indicates that these fast-moving cell populations originate from both the granulous segment of the distal tubule and from the collecting ducts. The adenylate cyclase activity and the cyclic AMP contents of isolated proximal cells maintained in culture medium were also investigated.

scholarly journals Analytical subcellular fractionation of alveolar macrophages from normal and BCG-vaccinated rabbits with particular reference to heterogeneity of hydrolase-containing granules

1979 ◽  
Vol 178 (3) ◽  
pp. 761-767 ◽  
Author(s):  
D B Lowrie ◽  
P W Andrew ◽  
T J Peters
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Type B ◽  
Type C ◽  
Pulmonary Granulomas ◽  
B Granules ◽  
Isopycnic Centrifugation

Macrophages were obtained by pulmonary lavage from normal rabbits or rabbits that had developed pulmonary granulomas after receiving intravenous BCG vaccine 2-3 weeks earlier. The cells were disrupted in iso-osmotic sucrose and a low-speed supernatant was fractionated by isopycnic centrifugation on a linear sucrose density gradient. Three populations of hydrolase-containing granules (putative lysosomes) were found in both normal and BCG-induced macrophages. They were distinguished by their different distributions in the gradient and different sensitivities to disruption by digitonin and were termed:type A, containing lysozyme; type B, containing N-acetyl-beta-glucosaminidase, beta-glactosidase, beta-glucuronidase and possibly some lysozyme; type C, containing cathepsin D. Acid phosphatase appeared to be about equally distributed between type B and C granules. Type A and B granules from BCG-induced macrophages showed markedly greater equilibrium density than did those from normal macrophages. Beta-glucuronidase and acid phosphatase had greater specific activity in the induced cells.

Physiological properties of rat ventral pallidal neurons recorded intracellularly in vivo

1996 ◽  
Vol 75 (4) ◽  
pp. 1432-1443 ◽  
Author(s):  
A. Lavin ◽  
A. A. Grace
Keyword(s):  
B Cells ◽  
Nucleus Accumbens ◽  
Action Potential ◽  
Type A ◽  
C Cells ◽  
Postsynaptic Potentials ◽  
A Cells ◽  
Type C ◽  
Stimulation Of

1. The physiology of ventral pallidal (VP) cells was investigated using in vivo intracellular recording and staining techniques in adult rats. Based on electrophysiological criteria, three different types of cells were found: type A cells, which fired phasic spikes that did not exhibit a substantial afterhyperpolarization (AHP), type B cells, which exhibited a slow ramplike depolarization that preceded the short-duration action potential; the spike was followed by a prominent AHP, and type C cells, which were the only cells that fired spikes in couplets or bursts, with the spikes in a burst exhibiting a progressive increase in duration and a decrease in amplitude. These cells also exhibited a rebound low threshold spikelike event. Furthermore, 18% of the VP cells recorded exhibited a slow subthreshold oscillation of the membrane potential (< 1 Hz). 2. The response of VP cells to stimulation of fibers arising from the prefrontal cortex, nucleus accumbens, and mediodorsal thalamic nucleus (MD) was examined. In contrast to our initial predictions, all cells responded to nucleus accumbens stimulation with excitation. Type A and B cells responded to nucleus accumbens stimulation with excitation and to MD stimulation with antidromic-like responses, orthodromic excitation, or evoked inhibitory postsynaptic potentials. Only type A cells responded to prefrontal cortical stimulation. Type C cells only responded to stimulation of the nucleus accumbens, which resulted in evoked excitatory postsynaptic potentials. 3. The cells in the VP therefore can be segregated into three physiologically defined groups according to action potential discharge patterns and their response to afferent fiber stimulation.

Initial Characterization of Cells Derived from Human Periodontia

1989 ◽  
Vol 68 (5) ◽  
pp. 761-767 ◽  
Author(s):  
J.E. Piche ◽  
D.L. Carnes ◽  
D.T. Graves
Keyword(s):  
Alkaline Phosphatase ◽  
Cyclic Amp ◽  
Periodontal Ligament ◽  
Culture Medium ◽  
Gingival Tissue ◽  
Cell Populations ◽  
Moderate Increase ◽  
Platelet Poor Plasma ◽  
Initial Characterization

The studies presented in this report describe an initial characterization of cell types derived from explants of human periodontia. Cell cultures were established from human periodontal ligament (PL4, PL7), gingival tissue (GF2), and alveolar bone (BP1) by means of explant techniques and monolayer culture. Cells were studied at passage numbers 2-4 and were characterized on the basis of morphological, biochemical, and proliferative parameters. Subconfluent cells did not have distinct morphologies useful in distinguishing them from one another. At confluence, PL4 and BP1 cells formed multilayered cultures of randomly oriented cells, while PL7 and GF2 cells grew in a monolayer of parallel cells. Biochemically, PL4 and BP1 cells exhibited characteristics consistent with an osteoblast-like phenotype. These included a significant increase in PTH-stimulated cyclic AMP and high basal levels of alkaline phosphatase activity, which were decreased on exposure to PTH and increased after stimulation by 1, 25 dihydroxyvitamin D3. In contrast, PL7 and GF2 cells exhibited basal alkaline phosphatase levels that were low, and cyclic AMP levels were not modulated by PTH stimulation. Cell populations PL7 and GF2 did not proliferate in culture medium supplemented with 3% platelet-poor plasma. After the addition of platelet-derived growth factor (PDGF) to this medium, the proliferation of these cell populations was equal to that in media supplemented with 10% fetal bovine serum. In contrast, PL4 and BP1 cells did proliferate in culture medium supplemented with 3% platelet-poor plasma. The addition of PDGF to the medium resulted in only a moderate increase in the proliferation of cell populations PL4 and BP1. These results establish that both osteoblast-like and fibroblast-like cells can be cultured from adult human periodontal ligament and suggest methods for studying the cells in vitro.

scholarly journals Single-Cell Profiling Reveals Distinct Tumor Subtypes and Their Associated T-Cell Environments in Follicular Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1577-1577
Author(s):  
Xuehai Wang ◽  
Deanne Gracias ◽  
Michael Nissen ◽  
Elizabeth Chavez ◽  
Gabriela Cristina Segat ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Single Cell ◽  
Research Funding ◽  
Type A ◽  
Cell Populations ◽  
Type B ◽  
Tumor Subtypes

Abstract Follicular lymphoma (FL) is an indolent, but incurable malignancy as most patients eventually experience progressive disease. We hypothesized that clonal heterogeneity and patient-specific immune responses would contribute to variable clinical outcomes and that understanding the complexity of the entire tumor "ecosystem" would allow us to better match patients with specific types of tumor- and immune-targeted therapies. In this study, we performed 38-dimensional single-cell phenotyping by mass cytometry (CyTOF) to simultaneously characterize both the substructure of malignant B cell populations as well as the T cell microenvironment in a cohort of 77 diagnostic patient FL biopsies and 35 benign reactive LN (rLN) biopsies. We first applied the t-distributed Stochastic Neighbour Embedding (t-SNE) algorithm to explore intra- and inter- tumoral heterogeneity among malignant B cell populations. t-SNE mapping of individual samples showed that more than a third of FL samples contain at least two phenotypically distinct tumor subpopulations, supporting the notion of multi-clonal tumor architectures presumably due to ongoing clonal evolution. Batched analysis combining all 77 FL cases together with 35 rLN samples revealed two distinct tumor subtypes comprising about 25% (type "A") and 10% (type "B") of total FL samples, respectively, with individual tumors within each subtype showing highly similar and partially overlapping phenotypes. Mapping the same data using Uniform Manifold Approximation and Projection (UMAP), a dimensional reduction algorithm similar to t-SNE but preserves global structure more accurately, revealed that type A tumors localized in close proximity to normal germinal center (GC) B cells, thus fulfilling conventional expectations as to the histogenesis of FL. In contrast, type B tumors localized more closely to pre-GC B cells, implying the existence of an alternate histogenic path in FL. Importantly, we also performed single-cell RNA-Seq on a subset of FL cases which independently confirmed the type A vs type B distinction in whole transcriptomic space. We next analyzed matching T cell data using a modified Statistical Scaffold algorithm in order to place distinct subsets in context with conventionally defined normal T cell populations. Clustering analysis using multi-layer phenograph performed on T cells from all FL and rLN samples combined yielded hundreds of small, but phenotypically distinct populations that were then annotated according to the nearest conventionally defined T cell subset. These imputed designations were used as features to perform hierarchical clustering of samples which revealed 3 major clusters. Cluster1 was characterized by mostly naive T cell populations and contained the majority of rLN samples. Cluster2 was characterized by more differentiated effector T cell populations and was dominated by FL samples. Samples within Cluster2 could be further divided into Tfh, Treg and Th1-rich subgroups. Cluster3 was characterized by a diverse T cell environment including naive, memory and differentiated effector subsets and contained a mixture of rLN and FL samples. Integrative analysis correlating B- and T- cell features revealed type B FL tumors were associated with a Tfh-rich immune landscape. Taken together, these data reveal pervasive phenotypic heterogeneity in both malignant and immune cell compartments of patient FL samples and suggest that defining tumoral subtypes as well as the status of the local immune response within individual samples will support more refined diagnostic classification and highlight functional interactions most amenable to therapeutic targeting. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Celgene: Consultancy, Honoraria; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding. Steidl:Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding; Roche: Consultancy.

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