Intermediate Stage Of Differentiation Research Articles

CD8 lethargy in the absence of CD4 help.

CD4 T cell help was proposed to have a pivotal role in orienting CD8 T cell responses to antigen stimulation. By activating antigen-presenting cells (APC), CD4 cells would induce their expression of costimulatory molecules, the "signal two" required to induce full CD8 activation, preventing CD8 tolerance. Recent data on this subject is contradictory, as the absence of help did not always result in CD8 tolerance. These differences were attributed either to the presence of residual CD4 help or, respectively to the type of antigen stimulation, the peptide affinity, the CTL frequencies, and/or the strength of the response. We therefore reassessed the role of CD4 help in CD8 responses using a system where CD4 cells are absent and APC not activated. This system can be manipulated to induce CD8 tolerance (at high antigen concentrations) or CD8 memory (at low antigen concentrations). We found that the presence of CD4 help did not prevent tolerance induction. On the other hand, the absence of CD4 help did not induce CD8 tolerance, but rather led to differentiation stage intermediate between naive/memory/tolerant cells that we call "lethargy". These findings indicate that role of CD4 help in CD8 responses does not follow a simple on-off rule, as previously suggested. They also reveal that the "tolerance versus memory" dichotomy fails to account for all possible states/properties of antigen-experienced CD8 cells. Depending on the priming conditions, other intermediate stages of differentiation may occur.

Human macrophage lectin specific for galactose/N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages.

We studied the expression of a human macrophage lectin specific for galactose/N-acetylgalactosamine (hMGL) during macrophage differentiation. The expression of hMGL during the in vitro differentiation induced by human serum was examined by immunostaining and Western blotting with a specific mAb, MLD-1, as well as with RT-PCR analysis. hMGL was detected on cells at an intermediate stage of differentiation. These cells were round, slightly larger in size (12.7 +/- 0.2 microm) than monocytes (9.8 +/- 0.1 microm) and expressed the macrophage marker CD14, but lacked the dendritic cell marker CD1a. The highest levels of expression occurred after 2-4 days of culture. At this time point, MLD-1 prominently stained 20-40% of the cells. Monocytes cultured for 16 h or fully differentiated monocyte-derived macrophages were negative or weak for hMGL expression. Similar transient expression was also observed during granulocyte macrophage colony stimulating factor- or macrophage colony stimulating factor-dependent macrophage differentiation. The lectin was characterized as a functional endocytic receptor for glycosylated macromolecules, since the uptake of carbohydrate polymers was partially inhibited by the addition of MLD-1. The distribution of hMGL(+) cells in normal human skin was found by immunostaining to be mainly in the upper dermis distant from vascular structures. More than 90% of the hMGL(+) cells were double stained with anti-CD68 mAb and constituted approximately 20% of the CD68(+) cells. We suggest that the dermal hMGL(+) cells are a subset of differentiated cells derived from monocytes and that hMGL is a unique marker for cells at an intermediate stage of macrophage differentiation.

Dynamics of T cell responses in HIV infection.

Cytotoxic CD8(+) T cells play a major role in the immune response against viruses. However, the dynamics of CD8(+) T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8(+) T cells, in relation to Ag-specific CD4(+) T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8(+) T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.

Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural killer cell differentiation from progenitor cells

To determine whether production of type 1 and type 2 cytokines defines discrete stages of natural killer (NK) cell differentiation, cytokine expression was analyzed in human NK cells generated in vitro in the presence of interleukin-15 (IL-15) and/or IL-2 from umbilical cord blood hematopoietic progenitors. Like peripheral NK cells, the CD161(+)/CD56(+) NK cells from these cultures contained a tumor necrosis factor alpha (TNF-alpha)(+)/granulocyte macrophage-colony-stimulating factor (GM-CSF)(+) subset, an interferon gamma (IFN-gamma)(+) subset, mostly included within the former, and very few IFN-gamma(-)/IL-13(+) cells. Instead, most immature CD161(+)/CD56(-) NK cells, detectable only in the cultures with IL-2, produced IL-13, TNF-alpha, and GM-CSF, but not IFN-gamma, and contained an IL-5(+) subset. In short-term cultures with IL-12 and feeder cells, a proportion of the immature cells acquired the ability to produce IFN-gamma. Part of these produced both IFN-gamma and IL-13, irrespective of induced CD56 expression. These in vitro data indicate that ability to produce the type 2 cytokines IL-13 and IL-5 defines CD161(+) NK cells at intermediate stages of differentiation, and is lost upon terminal functional differentiation, concomitant with acquired ability to produce IFN-gamma.

Characterization of T cell differentiation in the murine gut.

Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-ε, recombination activating gene (Rag)-1, and pre-Tα mRNAs expression at single cell level; (c) we characterized TCR-β, -γ, and -α locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Tα chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 ε/δ and pre-Tα deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.

Effects of estrogen on collagen synthesis by cultured human osteoblasts depend on the rate of cellular differentiation

Estrogen is known to act on osteoblasts according to their stage of differentiation and estrogen receptor (ER) isoform expression. The aim of this study was to determine when type I collagen (COL1) synthesis by cultured low-passage, human bone-derived osteoblasts (hOBs) is upregulated in response to estrogen. Cell lines from female donors aged 1 and 66 years were cultured for 11 days on collagen in growth medium supplemented with human serum, hydrocortisone, and beta-glycerophosphate. Young-donor hOBs grew more quickly than old-donor hOBs and did not mineralize. Old-donor hOBs formed mineralized nodules 5 days after reaching confluence. Changes in mRNA levels with time for ERs, type I collagen, and alkaline phosphatase reflected the faster differentiation of the old-donor cells. The ERbeta/ERalpha ratio fell threefold in young-donor hOBs but rose 300-fold in old-donor hOBs. Increased ERbeta/ERalpha ratios prevented ligand-dependent downregulation of ERalpha transcription, resulting in reduced proliferation in old-donor hOBs. Upregulation of COL1 mRNA expression in response to estrogen was confined to intermediate stages of differentiation, resulting in significant increases in COL1 mRNA by estradiol only in young-donor cells. Since the young and old-donor hOBs were cultured under identical conditions, our results indicate that the response of hOBs to estrogen is largely dependent on intracellular mechanisms that control the timing of cellular differentiation.

Phylogeography and genetic characteristics of a putative secondary-contact zone of the loggerhead shrike in central and eastern North America

The loggerhead shrike (Lanius ludovicianus) is found throughout North America in grassland and open scrub habitats. We previously described four loggerhead shrike management units found in central and eastern North America within the geographic ranges of three subspecies, L. l. migrans, L. l. ludovicianus, and L. l. excubitorides. A contact zone has been suggested to occur between L. l. migrans and L. l. excubitorides. The present study uses mitochondrial DNA sequence data to explore historical, ecological, and geographical factors that may have played a role in the genetic differentiation of these four management units, and examines the evidence for and characteristics of the migrans–excubitorides contact zone. Geographically discrete, monophyletic clades were not evident, but the distribution of haplotypes (especially those exceeding 10% overall frequency) indicated some phylogeographic structure that perhaps reflects an intermediate stage of differentiation between paraphyly and reciprocal monophyly. The contact zone between L. l. migrans and L. l. excubitorides is supported by the mixing of haplotypes unique to each of the hybridizing populations and intermediate frequencies of common haplotypes. We interpret these patterns as possibly reflecting the impact of both glacial refugial dynamics and anthropogenic changes to habitat in eastern North America.

Expression and regulation of golli products of myelin basic protein gene during in vitro development of oligodendrocytes

The myelin basic protein (MBP) gene produces two families of proteins, the classic MBPs, important for myelination of the CNS, and the golli proteins, whose biological role in oligodendrocytes (OLs) is still unknown. The goals of this work were to study the in vitro pattern of expression of the golli products during OL differentiation and to compare it with that of the classic MBP products of the gene. Mouse primary glial cultures were analyzed at the mRNA and protein levels with an array of techniques. We found that OLs express golli mRNA primarily during intermediate stages of differentiation, which was confirmed by immunocytochemical analysis. Golli expression was low in proliferating OL progenitors as well as in terminally mature OLs. Golli proteins were found associated with the OL cell soma and nuclei and, to a lesser extent, with the cellular processes. We also found that golli proteins are not targeted to myelin in vitro and in vivo, in contrast to the classic MBPs. Finally, we found that golli expression is regulated during OL development and can be manipulated by growth factors such as basic fibroblast growth factor, neurotrophin-3, and retinoic acid.

In vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice.

Intranasal inoculation of the i strain of the parvovirus minute virus of mice (MVMi) into immunodeficient SCID mice induces suppression of myeloid and erythroid progenitors in the bone marrow (BM) and lethal leukopenia. In the present study, we investigated whether the mouse megakaryocytic lineage was susceptible to MVMi. In vitro and in vivo infections with purified MVMi were conducted and their effects on the megakaryocytic lineage studied. In vitro infection of BM cells showed a multiplicity of infection-dependent inhibition in the colony-forming ability of megakaryocytic progenitors (colony-forming unit megakaryocyte [CFU-MK]). Neutralization or heat inactivation of the virus abrogated this inhibition. Expression of the MVMi nonstructural-1 protein was detected in the in vitro infected and cultured megakaryocytic cells. In vivo, intranasal inoculation of a lethal dose of virus was incapable of producing significant thrombocytopenia, although an increase in mean platelet volume was observed. Significantly, in the BM of these animals, a progressive decrease in CFU-MK was noted from day 14 postinfection, with survival rates less than 1% by day 35 postinfection. At day 35 postinfection, intermediate megakaryocytic differentiation stages showed maintenance of the proportion and ploidy of cells and a moderate decrease in the total number of these cells per femoral BM. The results demonstrate that MVMi is capable of inhibiting the proliferative capacity of megakaryocytic committed progenitors both in vitro and in vivo. Moreover, the in vivo data show that depletion of BM CFU-MK is compensated by the system, and platelet counts in the peripheral blood are maintained close to normal values.

The Effects of AICAR on Adipocyte Differentiation of 3T3-L1 Cells

The AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has been found to inhibit the differentiation of 3T3-L1 adipocytes, if added at an early phase of differentiation. AICAR blocks the expression of the late adipogenic markers, fatty acid synthase and acetyl-CoA carboxylase, and of the transcription factors, C/EBPα and PPARγ. It also inhibits early clonal expansion of pre-adipocytes, prevents the fall in C/EBPβ expression during the intermediate stage of differentiation and inhibits the late phase expression of CHOP-10, an antagonist of C/EBPβ. These data suggest a possible inhibitory role for AMPK in the process of adipose differentiation and suggest that AMPK might be a target to block adipogenesis.

Expression of cathepsin K messenger RNA in giant cells and their precursors in human osteoarthritic synovial tissues.

To investigate the expression of cathepsin K messenger RNA (mRNA) in the giant cells found in human osteoarthritic (OA) synovium and associated reparative connective tissues, and to compare this with mRNA expression of cathepsins B, L, and S, which are cysteine proteases known to be highly expressed by cells of the monocyte/macrophage lineage. Sections of human OA synovium were processed for in situ hybridization and probed for cathepsins K, B, L, and S. Serial sections were reacted for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, which are selective markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. At 3 sites of monocyte infiltration/giant cell formation (granulation tissue, the intimal and subintimal synovial layers, and deep stroma extending to the periphery of osteophytic tissue), both TRAP-positive mono- and multinucleated cells and TRAP-negative, NSE-positive mononuclear precursors were identified. Cells containing both enzyme activities were also found, potentially indicating an intermediate stage of differentiation. The TRAP-positive mononuclear/giant cells, and the occasional NSE-positive precursor, expressed an intense signal for cathepsin K mRNA, but did not express cathepsins B, L, and S. In contrast, the deep zone of phagocytic-like cells adjacent to sites of ossification expressed high levels of mRNA for cathepsins L, B, and S as well as cathepsin K mRNA. Giant cells that form within OA synovial tissue express high levels of cathepsin K mRNA. It appears that cathepsin K acts principally to digest the bone (and cartilage) fragments sheered from the joint surface during OA. The high TRAP activity and the undetectable expression of the macrophage-associated degradative proteases (cathepsins B, L, and S) by synovial giant cells strengthens the hypothesis that cathepsin K is the primary protease involved in bone degradation. At sites of synovial osteogenesis, a population of phagocytic-like cells expressed TRAP and cathepsins B, L, S, and K, and may represent blood-derived macrophages pushed toward an osteoclast phenotype.

Unicellular-Unilineage Erythropoietic Cultures: Molecular Analysis of Regulatory Gene Expression at Sibling Cell Level

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.

Epidermal Langerhans Cell Development and Differentiation

Epidermal Langerhans cells (LC) play a critical role in host defense. Still we know rather little about the development and functional specialization of these bone marrow-derived dendritic cells (DC) located in the most peripheral ectodermal tissue of the mammalian organism. How LC develop from their primitive progenitors in bone marrow and to what extent LC are related in their development to other lineages of the hemopoietic system is still under debate. There are currently 3 major areas of debate: 1) which are the signals required for LC development and differentiation to occur, 2) what are the (molecular) characteristics of the intermediate stages of LC differentiation, and 3) how are LC related in their development and/or function to other cells of the hemopoietic system? A better understanding of LC development and answers to these questions can be expected from recently developed technologies which allow the in vitro generation of DC with the typical molecular, morphological and functional features of LC from purified CD34 + progenitor cells under defined serum-free culture conditions. TGF-α1 was found to be an absolute requirement for in vitro LC development under serum-free conditions upon stimulation with the classical DC growth and differentiation factors GM-CSF, TNF-α and SCF. The recently identified cytokine FLT3 ligand further dramatically enhanced in vitro LC development and even allowed efficient in vitro generation of LC colonies from serum-free single cell cultures of CD34 + hemopoietic progenitor cells.

Changes in the Properties of Gap Junctions during Neuronal Differentiation of Hippocampal Progenitor Cells

The cellular mechanisms that regulate progenitor cell lineage elaboration and maturation during embryonic development of the mammalian brain are poorly understood. Conditionally immortalized mouse hippocampal multipotent progenitor cells (MK31 cells) were found to be strongly coupled by gap junctions comprising connexin 43 (Cx43) during early neuronal ontogeny; the presence of this Cx type was confirmed by electrophysiological, molecular biological, and immunocytochemical assays. However, as progenitor cells underwent intermediate stages of neuronal differentiation under the influence of interleukin 7 (IL-7) alone or terminal differentiation after composite exposure to basic fibroblast growth factor, IL-7, and transforming growth factor alpha, coupling strength and the level of Cx43 expression declined. An additional population of junctional channels with distinct properties was detected at an intermediate stage of neuronal differentiation. Reverse transcription-PCR assays detected mRNA encoding Cx40 in IL-7-treated cells and Cx33 after both treatment conditions. Because functional channels in exogenous expression systems are not formed by pairing Cx40 with Cx43 or by pairing Cx33 with itself or additional connexins, these experimental observations raise the possibility that the progressive loss of coupling during differentiation of neural progenitor cells may involve downregulation of Cx43 coupled with potentiation of expression of Cx33 and Cx40. Furthermore, continued expression of Cx43 in differentiating neuroblasts could mediate intercellular communication between neuronal precursor cells and astrocytes by direct signaling via homotypic gap junction channels.

Immortalization of immunologically committed Epstein-Barr virus-transformed human B-lymphoblastoid cell lines accompanied by a strong telomerase activity

The immunological characteristics and immortalization processes of three EBV-transformed human B-lymphoblastoid cell lines, N0003, N0005 and N6803, with strong telomerase and infinitively proliferating activities are described. The three cell lines were apparently immortalized: they developed a strong telomerase activity at the population doubling levels (PDLs) between 11 and 135, and continued proliferation over 250 PDLs. All the cell lines expressed CD22, CD19 and CD20 antigens. They were uniformly stained with IgM (N0005), IgG (N6803) or IgA (N0003) at early PDLs between 17 and 20, and they secreted the corresponding class of Ig into the medium; the N6803 and N0003 cell lines continued to secrete each class of Ig at decreased levels while the N0005 cell line expressed or secreted virtually no Ig after immortalization. Karyotype analysis of the immortalized cell lines showed that they were derived from a single cell because they shared a set of abnormal chromosomes within each cell population, and two of the cell lines attained clonal characteristics before they developed a strong telomerase activity. These results indicate that the three immortalized cell lines with a strong telomerase activity correspond to the intermediate stages of B-cell differentiation naturally committed to a specific Ig class, and suggest that they were derived from a B-lymphoblastoid cell committed to a specific class of Ig with poor telomerase activity, rather than from a strongly telomerase-positive B-lymphoblastoid cell either committed or multipotential.

Expression of ?v?3 and ?v?8 integrins during oligodendrocyte precursor differentiation in the presence and absence of axons

We have shown previously that switching of the alpha v-associated beta1 and beta5 integrin subunits during differentiation of myelin-forming oligodendrocytes may regulate important aspects of cell behaviour such as migration (Milner et al., 1996: J Neurosci 16:7240-7252). In this study we have examined the developmental regulation of other alpha v-associated beta subunits in oligodendroglial cell cultures and also the control of their expression by neurons, using xenocultures to distinguish glial and neuronal integrins. We have found that oligodendroglia express alpha vbeta8 in addition to the previously-described alpha vbeta1, alpha vbeta3, and alpha vbeta5. Beta8 and beta3 together comprise the 80kD band seen in alpha v immunoprecipitations that represents the most abundant alpha v-associated beta subunit and show reciprocal patterns of expression during development. Alpha vbeta8 is expressed at high levels on oligodendrocyte precursors and differentiated oligodendrocytes but diminishes during the intermediate stages of differentiation. Alpha vbeta3, in contrast, shows an opposite pattern of expression, with the highest levels seen at the intermediate stages of differentiation and little expression on either oligodendrocyte precursors and differentiated oligodendrocytes. The expression of alpha vbeta3 is not altered by coculture with neurons, unlike that of alpha vbeta8, in which the decrease seen at the intermediate stages of differentiation is less marked in the presence of neurones. Our results confirm that switching of alpha v-associated beta subunits is an important feature of oligodendrocyte differentiation and suggest that alpha vbeta8 and alpha vbeta3 have distinct functions during myelination.

Isolation and Purification of Primary Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system, and are avidly studied by investigators interested in neural cell lineages, myelination, multiple sclerosis, and oligodendroglioma. This unit presents a strategy for manipulating the division and differentiation of oligodendrocyte progenitors by isolating O-2A progenitors from optic nerve and then purifying and expanding them by culture in a defined medium supplemented with platelet-derived growth factor (PDGF) plus basic fibroblast growth factor (bFGF). Cells are then allowed to differentiate through removal of the growth factors. A protocol is also provided for monitoring the cell type composition of the cultures by immunohistochemistry. In addition to making it possible to generate large numbers of cells, this approach allows access to intermediate differentiation stages. Cells can thus be studied as they differentiate into oligodendrocytes, making the actual process of differentiation open to examination.

Cellular and Molecular Effects of Growth Hormone and Estrogen on Human Bone Cells

The aim of the present thesis is to examine some aspects of the biological effects of growth hormone (GH) and estrogen on bone cells in vitro. The first part of the thesis describes characterization of model systems to study normal human osteoblasts and osteoclasts in vitro. Three culture systems for human osteoblasts have been characterized, representing different stages of osteoblasts differentiation/maturation: (1) mature osteoblasts cultured from trabecular bone explants (trabecular osteoblasts), (2) less mature osteoblasts (stromal osteoblasts) cultured from bone marrow and (3) osteoblast precursor cells cultured also from bone marrow. This classification is based on quantitative and qualitative differences in the expression of osteoblast phenotypic markers by these cells. These systems are useful in studying the regulation of hormones and growth factors of different stages of osteoblast differentiation. Further characterization of the osteoblast differentiation pathway is still needed, especially the identification of surface markers that can definitively identify intermediate stages of osteoblast differentiation. Normal human osteoclasts were cultured from bone marrow mononuclear cells and exhibited the main characteristics of osteoclast phenotype: production of tartrate-resistant acid phosphatase, presence of a ruffled border and the ability to resorb mineralized matrix. This model is useful for studying factors regulating osteoclast commitment and differentiation. The second part of the thesis deals with the effects of GH on proliferation and differentiation of trabecular and stromal osteoblasts. Effects of GH on human osteoblasts were dependent on their degree of maturation. GH stimulated cell proliferation in both trabecular and stromal osteoblasts. While it increased the functional activity of trabecular osteoblasts, these effects were absent in stromal osteoblast cultures. Human trabecular osteoblasts produce mainly IGF-II, IGFBP-3 and minute quantities of IGF-I in culture. GH does not seem to regulate the local production of IGF-II or IGFBP-3. However, IGFs and their binding proteins may exert important regulatory effects on the biological effects of GH on human osteoblasts, and this role needs to be studied. Sincer GH exerted profound effects on the biological functions of human osteoblasts in vitro, the hypothesis that either decreased production of GH or decreased sensitivity of bone cells to its action leads to bone loss and osteoporosis was examined. No differences in the basal or GH-stimulated production of IGF-I, IGF-II or IGFBP-3 were found between osteoporotic patients and age-matched normals. Similarly, The response of bone cells to in vivo and in vitro stimulation by GH was similar in the two groups. These studies do not support the hypothesis of the presence of major defects in production of GH or the presence of resistance to its effects in bone cells in patients with osteoporosis. The last part of the thesis deals with the cellular mechanisms mediating estrogen actions on bone cells. Thus, the potential role of estrogen regulation of bone-resorbing cytokines in the pathogenesis of bone loss in postmenopausal women was examined. Estrogen was found to inhibit IL-6 production and gene expression in an immortalized human osteoblastic cell line expressing high levels of estrogen receptors (hFOB/ER9 cell line). To further examine the relationship between estrogen deficiency and early postmenopausal bone loss, levels of IL-6, IL-6sR, IL-1 alpha, IL-1 beta and IL-1ra were measured in bone marrow plasma and bone marrow cultures obtained from 40 postmenopausal women, half of whom were on estrogen replacement therapy. No difference was found between the groups in any of these parameters. This suggests that neither IL-1 nor IL-6 by itself is the major mediator for increased bone loss due to estrogen deficiency in the early postmenopausal period. (ABSTRACT TRUNCATED)

Visual field representation in striate and prestriate cortices of a prosimian primate (Galago garnetti).

Microelectrode mapping techniques were used to study the visuotopic organization of the first and second visual areas (V1 and V2, respectively) in anesthetized Galago garnetti, alorisiform prosimian primate. 1) V1 occupies approximately 200 mm2 of cortex, and is pear shaped, rather than elliptical as in simian primates. Neurons in V1 form a continuous (1st-order) representation of the visual field, with the vertical meridian forming most of its perimeter. The representation of the horizontal meridian divides V1 into nearly equal sectors representing the upper quadrant ventrally, and the lower quadrant dorsally. 2) The emphasis on representation of central vision is less marked in Galago than in simian primates, both diurnal and nocturnal. The decay of cortical magnification factor with increasing eccentricity is almost exactly counterbalanced by an increase in average receptive field size, such that a point anywhere in the visual field is represented by a compartment of similar diameter in V1. 3) Although most of the cortex surrounding V1 corresponds to V2, one-quarter of the perimeter of V1 is formed by agranular cortex within the rostral calcarine sulcus, including area prostriata. Although under our recording conditions virtually every recording site in V2 yielded visually responsive cells, only a minority of those in area prostriata revealed such responses. 4) V2 forms a cortical belt of variable width, being narrowest (approximately 1 mm) in the representation of the area centralis and widest (2.5-3 mm) in the representation of the midperiphery (>20 degrees eccentricity) of the visual field. V2 forms a second-order representation of the visual field, with the area centralis being represented laterally and the visual field periphery medially, near the calcarine sulcus. Unlike in simians, the line of field discontinuity in Galago V2 does not exactly coincide with the horizontal meridian: a portion of the lower quadrant immediately adjacent to the horizontal meridian is represented at the rostral border of ventral V2, instead of in dorsal V2. Despite the absence of cytochrome oxidase stripes, the visual field map in Galago V2 resembles the ones described in simians in that the magnification factor is anisotropic. 5) Receptive field progressions in cortex rostral to dorsal V2 suggest the presence of a homologue of the dorsomedial area, including representations of both quadrants of the visual field. These results indicate that many aspects of organization of V1 and V2 in simian primates are shared with lorisiform prosimians, and are therefore likely to have been present in the last common ancestor of living primates. However, some aspects of organization of the caudal visual areas in Galago are intermediate between nonprimates and simian primates, reflecting either an intermediate stage of differentiation or adaptations to a nocturnal niche. These include the shape and the small size of V1 and V2, the modest degree of emphasis on central visual field representation, and the relatively large area prostriata.

Effects of osteogenic protein-1 (OP-1, BMP-7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specific.

Bone morphogenetic proteins (BMPs) are a group of cytokines that are characterized by their ability to stimulate osteoblast differentiation and bone formation. However, the influence of BMPs on osteoblastic cells at different stages of differentiation is not known. Since bone matrix proteins are differentially regulated during bone formation we have studied the effects of recombinant human osteogenic protein-1 (rhOP-1; BMP-7) on the expression of these proteins by fetal rat calvarial cells (FRCCs) at discrete stages of osteoblast differentiation. Continuous administration of rhOP-1 to FRCCs, beginning at confluence (day 7), produced a dose-dependent increase in the number, size and mineralization of bone-like nodules formed in the presence of vitamin C and beta-glycerophosphate. Within 9 h of administration, rhOP-1 stimulated a 3-fold increase in OPN mRNA which was reflected in a comparable increase in the low phosphorylated, 55 kDa form of osteopontin. In contrast, changes in type 1 collagen, alkaline phosphatase and bone sialoprotein mRNAs followed the differentiation of preosteoblastic cells, and were increased 2-, 4- and 5-fold, respectively, after 8 days (day 15). When administered at intermediate stages of osteoblast differentiation (days 12, 15 and 18) BSP remained refractory to rhOP-1 whereas the ALP was increased almost 2-fold, independent of the constitutive levels of mRNA expression. To determine the effects on osteoblasts, FRCCs were first grown to the bone nodule-forming stage (day 21) before rhOP-1 was administered. Only modest, transient increases in the expression of ALP and OPN mRNAs were evident whereas OC expression was increased more than 3-fold. In contrast, collagen type 1 and BSP mRNA levels were not changed significantly. These results suggest that rhOP-1 increases bone formation by promoting osteoblastic differentiation, as indicated by the increased number of bone forming colonies and by increasing the number of osteoblastic cells in the colonies, but not by increasing matrix production by individual osteoblasts. It is also evident that the regulation of bone matrix proteins by rhOP-1 is dependent upon the differentiated state of the cell.