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Background: The phenotypical identification of diagnostic cells is crucial for the diagnosis of Hodgkin's lymphoma (HL) on fine-needle aspiration cytology (FNAC). The aim of this study is to evaluate the immunocytochemical (ICC) expression of CD30, CD15, and PAX5 in Hodgkin's cells (HC) and Reed-Sternberg cells (RSC) on smears and cell-blocks (CB) of HL and to compare the performance of each antibody on smears and CB., Methods: In 21 FNAC cases of histologically confirmed classical HL, ICC identification of HC and RSC was performed using CD15, CD30, and PAX5 on smears and CB, respectively., Results: CD30 was positive in 19/21 cases (90.5%; 11/11 smears and 8/10 CB), CD15 was positive in 14/21 cases (66.7%; 5/11 smears and 9/10 CB), and PAX5 was positive in 13/21 cases (61.9%; 9/11 smears and 4/10CB)., Conclusions: CD15, CD30, and PAX5 are useful to the FNAC identification of HC and RSC. CD30 is the most sensitive, followed by CD15 and PAX5, which are more effective on CB and smears, respectively., (© 2019 Wiley Periodicals, Inc.)

Studies published in recent years have changed the outlook on sterility and germ cell development by producing gametes from stem cells. In present study, a novel approach on differentiation of bone marrow-derived stage-specific embryonic antigen 1 positive (SSEA-1 + ) pluripotent stem cells into male germ cells has been addressed. SSEA-1 + stem cells were separated from murine bone marrow using magnetic-activated cell sorting (MACS) system and propagated on a feeder layer cells. To evaluate the pluripotency characteristic of the purified cells, they were differentiated toward cells of three germ layers. Later the SSEA-1 + stem cells were induced to differentiate along male germ cell lineage with retinoic acid. Flowcytometric analysis of SSEA-1 + stem cells revealed purity of about 62% which increased to 91% after cultivation over feeder cells. Expression of specific transcripts of Oct4, SSEA-1, Nanog, Dppa3, fragilis, Rex-1, SOX-2, and alkaline-phosphatase and immunofluorescence evaluation of Oct4 and SSEA-1 expression showed the differentiation of purified stem cells toward the cells of three germ layers. Differentiation potential of purified cells was positively evidenced by expression markers specific for primordial germ cells, spermatogonial stem cells and spermatogonia including Mvh, fragilis, Dppa3, Stra8, DAZL, Piwil2, β1, and α6-integrins as well as meiotic-specific marker SYCP3. Our results showed that SSEA-1 + pluripotent stem cells are able to differentiate into male germ cells. The results of the present study are encouraging enough to merit further investigation, provide a new hope for those suffering from infertility and introduce a novel platform for research on germ cell development., (© 2016 Wiley Periodicals, Inc.)

We studied the retinal cone bipolar cells of Carollia perspicillata, a microchiropteran bat of the phyllostomid family. Microchiroptera are strongly nocturnal, with small eyes and rod-dominated retinae. However, they also possess a significant cone population (2-4%) comprising two spectral types, which are hence the basis for daylight and color vision. We used antibodies against the calcium-binding protein recoverin and the carbohydrate epitope 15 (CD15) as reliable markers for certain cone bipolar cells. Dye injections of recoverin- or CD15-prelabeled cone bipolar cells in vertical slices revealed the morphology of the axon terminal system of individual bipolar cells. Seven distinct cone bipolar cell types were identified. They differed in the morphology and stratification level of their axon terminal system in the inner plexiform layer and in immunoreactivity for recoverin and/or CD15. Additional immunocytochemical markers were used to assess the functional ON/OFF subdivision of the inner plexiform layer. In line with the extended thickness of the ON sublayer of the inner plexiform layer in the microbat retina, more ON than OFF cone bipolar cell types were found, namely, four versus three. Most likely, in the bats' predominantly dark environment, ON signals have greater importance for contrast perception. We conclude that the microbat retina conforms to the general mammalian blueprint, in which light signals of intensities above rod sensitivity are detected by cones and transmitted to various types of ON and OFF cone bipolar cells., (© 2014 Wiley Periodicals, Inc.)

Background: Anaplastic large cell lymphoma (ALCL) of the breast capsule is a rare lymphoma involving capsular tissues and/or effusions associated with breast implants. While several studies have detailed the histological and immunohistochemical (IHC) features of these tumors, no study has yet described flow cytometry features of the neoplastic cells of this entity. Here, we report two cases from our institution in which multi-parametric flow cytometry was performed., Methods: The immunophenotype of ALCL in association with breast implant was evaluated by flow cytometry., Results: We show that much like CD30+ tumor cells of classical Hodgkin lymphoma (CHL) and ALCL of non-breast implant tumors, the neoplastic cells of this entity can be readily identified by flow cytometry. The neoplastic cells of both cases were largely devoid of T-cell antigens, but had expression of weak CD15, strong CD30, and expression of CD40. These results are correlated with routine morphologic and IHC analysis, supporting the flow cytometry immunophenotype., Conclusions: Flow cytometry can aid in the diagnostic evaluation of effusions or tissue samples in association with breast implant/prostheses., (© 2014 Clinical Cytometry Society.)

Lewis Y (LeY) is a carbohydrate tumor-associated antigen. The majority of cancer cells derived from epithelial tissues express LeY type difucosylated oligosaccharides. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharides. In a previous study we reported that FUT4 is associated with cell proliferation; however, despite the important role of FUT4 in cancer proliferation and apoptosis, little is known about the mechanisms underlying the regulation of FUT4 transcription. In the current study we investigated the regulation of FUT4 transcription in human breast cancer. We compared the regulation of human FUT4 gene transcription in human breast cancer cells (MCF-7 and MDA-MB-231) using promoter/luciferase analyses. Using a series of promoter deletion constructs, we identified a potential regulatory site located between 0.8 and 1.6 kb of the FUT4 promoter. As shown by EMSA and ChIP analyses, heat-shock factor 1 (HSF1) and Sp1are required for FUT4 promoter activity. In addition, we explored the role of HSF1 and Sp1 on cell proliferation, and found that the ERK1/2 MAPK and PI3K/Akt signaling pathways regulate the expression of FUT4, which play a role in cell proliferation via HSF1 and Sp1. These results suggest that FUT4 is a target gene for HSF1 and Sp1 that is required for cell cycle progression in breast cancer epithelial cells., (© 2013 Wiley Periodicals, Inc.)

The cells surrounding the central canal of the spinal cord are a source of stem/precursor cells that may give rise to neurons, astrocytes, or oligodendrocytes. However, they are a heterogeneous population that remains poorly understood. Here we describe a subpopulation characterized by their strong expression of the CB(1) cannabinoid receptor, oval/round soma, apical nucleus, a variable number of cilia (0, 1, or 2), and the presence of a single short and occasionally ramified basal process. These cells are mainly located in the lateral and dorsal central canal throughout the spinal cord. These CB(1)(HIGH) cells are closely related to the basal lamina labyrinths or fractones derived from subependymal microglia. In addition, CB(1)(HIGH) cells express some stem/precursor cell markers, including vimentin, nestin, Sox2, Sox9, and GLAST, but not others such as CD15 or GFAP. In addition, this cell population does not proliferate in the intact adult spinal cord, although up to 50% of these cells express the proliferation marker Ki67 in newly born rats or after a spinal cord contusion. The present findings contribute to our understanding of the spinal cord central canal structure and reveal the targets for endocannabinoids inside this neurogenic niche., (Copyright © 2012 Wiley Periodicals, Inc.)

Both cancer-related inflammation and tumor-induced immune suppression are associated with expansion of myeloid cell subsets including myeloid-derived suppressor cells. However, little known regarding characteristics of myeloid cells in patients with bladder cancer. In this study, we analyzed myeloid cells from peripheral blood (PBMC) and tumor tissue that were collected from patients with superficial noninvasive and invasive urothelial carcinomas. Our results demonstrate that PBMC from bladder cancer patients contain two major CD11b myeloid cell subsets: granulocyte-type CD15(high) CD33(low) cells and monocyte-type CD15(low) CD33(high) cells. The number of circulating granulocytic but not monocytic myeloid cells in cancer patients was markedly increased when compared to healthy individuals. Both myeloid cell subsets from cancer patients were highly activated and produced substantial amounts of proinflammatory chemokines/cytokines including CCL2, CCL3, CCL4, G-CSF, IL-8 and IL-6. Granulocytic myeloid cells were able to inhibit in vitro T cell proliferation through induction of CD4(+) Foxp3(+) T regulatory cells. Analysis of bladder cancer tissues revealed that tumors were infiltrated with monocyte-macrophage CD11b(+) HLA-DR(+) and granulocytic CD11b(+) CD15(+) HLA-DR(-) myeloid cells. Collectively, this study identifies myeloid cell subsets in patients with bladder cancer. We demonstrate that these highly activated inflammatory myeloid cells represent a source of multiple chemokines/cytokines and may contribute to inflammation and immune dysfunction in bladder cancer., (Copyright © 2011 UICC.)

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins β3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain., (Copyright © 2011 International Society for Advancement of Cytometry.)

Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of difucosylated oligosaccharide LeY which is overexpressed in the cancers derived from the epithelial tissues. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation through the MAPK and PI3K/Akt signaling pathways, but the relationship between FUT4 and apoptosis remained unclear. Here, we investigated the effect of FUT4 overexpression on cyclophosphamide (CPA)-induced apoptosis in A431 cells. Western blot analysis showed that FUT4 overexpression decreased expression of Bax, Caspase 3, and PARP proteins, and increased anti-apoptotic Bcl-2 protein in A431 cells. The anti-apoptosis effect of FUT4 was confirmed both by Annexin-V/PI and JC-1 assays. The results showed that FUT4 overexpression up-regulated phosphorylation of ERK1/2 and Akt which was inhibited by CPA in dose-dependent manner. By blocking the ERK/MAPK and PI3K/Akt pathways with specific inhibitors, we demonstrated that these two pathways were required in mediating the anti-apoptosis effect of FUT4. We concluded that FUT4 inhibited cell apoptosis induced by CPA through decreasing the expression of apoptotic proteins Bax, Caspase 3, and PARP and increasing the expression of anti-apoptotic protein Bcl-2 via the ERK/MAPK and PI3K/Akt signaling pathways in A431 cells., (Copyright © 2011 Wiley-Liss, Inc.)

Studies have shown that male germline stem cells (mGSCs), which are responsible for maintaining spermatogenesis in the male, could be obtained from mouse and human testis. However, the traditional cultural methods were mostly dependent on serum and feeder, and the initial mGSCs were either obtained from neonatal mice or the detailed description of its potency and origin was not provided. Here we reported a novel (retinol (RE) serum-free and feeder-free) system for the successful culture of adult germline stem cells from adult Kunming mice (8-24 weeks) testis. The isolated mGSCs cultured in RE serum-free and feeder-free medium maintained the typical morphology of undifferentiated embryonic stem cells (ESCs), and they proliferated well in RE medium analyzed by proliferation assay, RT-PCR, microarray, and Western blotting. These cells also showed typical properties of ESCs (alkaline phosphatase (AP) positive, expressions of Oct4, Sox2, Nanog, and SSEA1, with the capacity to form teratomas and differentiate into various types of cells within three germ layers). Taken together, we conclude that RE promotes the self-renewal of mGSCs and maintains the pluripotency of mGSCs, the RE serum-free and feeder-free system may be useful for the culture of pluripotent stem cell lines from adult testis tissues, which provides a new resource for tissue engineering and therapy for infertility., (Copyright © 2011 Wiley-Liss, Inc.)

Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone-dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin-positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S-cone selective (bb) bipolar cell. The calcium-binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate., (Copyright © 2010 Wiley-Liss, Inc.)

Lewis Y (LeY) is a carbohydrate tumor-asssociated antigen. The majority of cancer cells derived from epithelial tissue express LeY type difucosylated oligosaccharide. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharide. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation, but the mechanism is still largely unknown. Herein, we investigated the role of the mitogen-activated protein kinases (MAPKs) and phosphoinositide-3 kinase (PI3K)/Akt signaling pathways on FUT4-induced cell proliferation. Results show that overexpression of FUT4 increases the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt. Inhibitors of PI3K (LY294002 and Wortmannin) prevented the phosphorylation of ERK1/2, p38 MAPK, and Akt PI3K). Moreover, phosphorylation of Akt is abolished by inhibitors of ERK1/2 (PD98059) and p38 MAPK (SB203580). These data suggested that FUT4 not only activates MAPK and PI3K/Akt signals, but also promotes the crosstalk among these signaling pathways. In addition, FUT4-induced stimulation of cell proliferation correlates with increased cell cycle progression by promoting cells into S-phase. The mechanism involves in increased expression of cyclin D1, cyclin E, CDK 2, CDK 4, and pRb, and decreased level of cyclin-dependent kinases inhibitors p21 and p27, which are blocked by the inhibitors of upstream signal molecules, MAPK and PI3K/Akt. In conclusion, these studies suggest that FUT4 regulates A431 cell growth through controlling cell cycle progression via MAPK and PI3K/Akt signaling pathways., ((c) 2010 Wiley-Liss, Inc.)

Mesenchymal stem cells (MSCs) are self-renewing cells with the ability to differentiate into various mesodermal-derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL-MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL-MSCs contain a subpopulation of frizzled-9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA-1, and SSEA-4. They are additionally positive for nanog and Oct-4; two critical transcription factors directing self-renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP-10. The presence of nanog, Oct-4, SSEA-1, and SSEA-4 suggests that PDL-MSCs are less differentiated than bone marrow-derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled-9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells., ((c) 2010 Wiley-Liss, Inc.)

Purkinje cell protein 2 (PCP2), a member of the family of guanine dissociation inhibitors and a strong interactor with the G-protein subunit G alpha(o), localizes to retinal ON bipolar cells. The retina-specific splice variant of PCP2, Ret-PCP2, accelerates the light response of rod bipolar cells by modulating the mGluR6 transduction cascade. All ON cone bipolar cells express mGluR6 and G alpha(o), but only a subset expresses Ret-PCP2. Here we test the hypothesis that Ret-PCP2 contributes to shaping the various temporal bandwidths of ON cone bipolar cells in monkey retina. We found that the retinal splice variants in monkey and mouse are similar and longer than the cerebellar variants. Ret-PCP2 is strongly expressed by diffuse cone bipolar type 4 cells (DB4; marked with anti-PKCalpha) and weakly expressed by midget bipolar dendrites (labeled by antibodies against G alpha(o), G gamma 13, or mGluR6). Ret-PCP2 is absent from diffuse cone bipolar type 6 (DB6; marked with anti-CD15) and blue cone bipolar cells (marked with anti-CCK precursor). Thus, cone bipolar cells that terminate in stratum 3 of the inner plexiform layer (DB4) express more Ret-PCP2 than those that terminate in strata 3 + 4 (midget bipolar cells), and these in turn express more than those that terminate in stratum 5 (DB6 and blue cone bipolar cells). This expression pattern approximates the arborization of ganglion cells (GC) with different temporal bandwidths: parasol GCs stratifying near stratum 3 are faster than midget GCs stratifying in strata 3 + 4, and these are probably faster than the sluggish GCs that arborize in stratum 5., ((c) 2009 Wiley-Liss, Inc.)

Recently, we purified a population of CXCR4+/Oct-4+/SSEA-1+/Sca-1+/Lin(-)/CD45(-) very small embryonic-like stem cells (VSELs) from adult murine bone marrow (BM). After using flow cytometry, ImageStream analysis, confocal microscopy, and real time RT-PCR, we report that similar cells could be also identified and isolated from several organs in adult mice. The highest total numbers of Oct-4+ VSELs were found in the brain, kidneys, muscles, pancreas, and BM. These observations support our hypothesis that a population of very primitive cells expressing germ line/epiblast markers (Oct-4, SSEA-1) is deposited early during embryogenesis in various organs and survives into adulthood. Further studies are needed to determine whether these cells, after being isolated from various adult human organs similarly to their murine BM-derived counterparts, are endowed with pluripotent stem cell properties.

The organization of the adult human medial preoptic nucleus was studied by using chemoarchitectonic markers for acetylcholinesterase, nonphosphorylated neurofilament protein (SMI-32), calbindin-D28k, neuropeptide Y (NPY), melanin-concentrating hormone (MCH), cocaine- and amphetamine-regulated transcript (CART), and 3-fucosyl-N-acetyl-lactosamine (CD15) to establish human homologs to the subnuclei making up MPO in the rat, where their connections and functional significance are better understood. The human MPO comprises three subnuclei, the medial MPO, the lateral MPO, and the dorsomedially positioned uncinate subnucleus. As in the rat, the human medial MPO is magnocellular and contains numerous NPY- and CART-immunoreactive fibers and terminals as well as calbindin-positive neurons. The human lateral MPO, like its homolog in the rat, distinctively features numerous MCH-positive fibers and terminals as well as SMI-32-immunoreactive fibers. The uncinate subnucleus is wedged between the lateral surface of the paraventricular nucleus and the medial MPO and is characterized by variable NPY- and CART-immunoreactive fibers and terminals, also seen in the rat central MPO, suggesting that the subnuclei are homologues. The intermediate nucleus was distinguished by CD15-positive neuronal staining, whereas the majority of its neurons also contained acetylcholinesterase. The human intermediate nucleus is positioned on the lateral surface of MPO and by its chemo- and cytoarchitecture constitutes a distinct nucleus of the preoptic area characterized by close structural association with the MPO complex. These findings demonstrate that the human MPO is organized similarly to the rat MPO, in chemo- and cytoarchitectonically distinct subnuclei, which implies differences in their functional specialization, as seen in the rat.

The mammalian retina contains as many as 50-60 unique cell types, many of which have been identified using various neurochemical markers. Retinal neurons express N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and kainic acid (KA) receptor subunits in various mixtures, densities, and spatial distributions. Ionotropic glutamatergic drive in retinal neurons can be mapped using a cation channel permeant guanidinium analog called agmatine (1-amino-4-guanidobutane; AGB). This alternative approach to physiologically characterize neurons in the retina was introduced by Marc (1999, J Comp Neurol 407:47-64, 407:65-76), and allows the simultaneous mapping of responses of glutamate receptor-gated channels from an entire population of neurons. Unlike previous AGB studies, we colocalized AGB with various macromolecular markers using direct and indirect immunofluorescence to characterize the glutamate agonist sensitivities of specific cell types. Activation with NMDA, AMPA, and KA resulted in AGB entry into neurons in a dose-dependent manner and was consistent with previous receptor subunit localization studies. Consistent with the various morphological phenotypes encompassed by the calbindin and calretinin immunoreactive cells, we observed various functional phenotypes revealed by AGB labeling. Not all calbindin or calretinin immunoreactive cells showed ligand-evoked AGB permeation. A small proportion either did not possess functional glutamate receptors, required higher activation thresholds, or express functional channels impermeable to AGB. AMPA and KA activation of bipolar cells resulted in AGB permeation into the hyperpolarizing variety only. We also studied the glutamate ligand-gating properties of 3[alpha1-3]-fucosyl-N-acetyl-lactosamine (CD15) immunoreactive cells and show functional responses consistent with receptor subunit gene expression patterns. CD15-immunoreactive bipolar cells only responded to AMPA but not KA. The CD15 immunoreactive amacrine cells demonstrated an identical selectivity to AMPA activation, but were also responsive to NMDA. Finally, localization of AGB secondary to glutamate receptor activation was visualized with a permanent reaction product., (J. Comp. Neurol. 494:686-703, 2006. @ 2005 Wiley-Liss, Inc.)

Previously, we showed that radial glia-like (RG) cells differentiated from embryonic stem (ES) cells after retinoic acid induction (Liour and Yu, 2003: Glia 42:109-117). In the present study, we demonstrate that the production of RG cells from ES cells is independent of the neural differentiation protocol used. These ES cell-derived RG (ES-RG) cells are similar in morphology to RG cells in vivo and express several characteristic RG cell markers. The processes of these ES-RG cells are organized into radial arrays similar to the RG scaffold in developing CNS. Expression of Pax6, along with other circumstantial data, suggests that at least some of these ES-RG cells are neural progenitors. The progression of neurogenesis into gliogenesis during the in vitro neural differentiation of ES cells recapitulates the in vivo developmental process. The identification of two cell surface markers, SSEA-1 and GM1, on both the native embryonic RG cells and ES-RG cells, may facilitate purification of radial glial cells for future studies and cell therapy. Overall, our study suggests that differentiation of radial glial cells is a common pathway during the neural differentiation of ES cells., ((c) 2005 Wiley-Liss, Inc.)

The major inhibitory neurotransmitter glycine is used by about half of the amacrine cells in the retina. Amacrine cells provide synaptic output to bipolar, ganglion, and other amacrine cells. The present study investigated whether different bipolar and amacrine cell types in the primate retina differ with respect to the expression of glycine receptor (GlyR) subtypes. Antibodies specific for the alpha1, alpha2, and alpha3 subunits of the GlyR were combined with immunohistochemical markers for bipolar and amacrine cells and applied to vertical sections of macaque (Macaca fascicularis) and marmoset (Callithrix jacchus) retinae. For all subunits, punctate immunoreactivity was expressed in the inner plexiform layer. The GlyRalpha2 immunoreactive (IR) puncta occur at the highest density, followed by GlyR(alpha)3 and GlyR(alpha)1 IR puncta. Postembedding electron microscopy showed the postsynaptic location of all subunits. Double immunofluorescence demonstrated that the three alpha subunits are clustered at different postsynaptic sites. Two OFF cone bipolar cell types (flat midget and diffuse bipolar DB3), are predominantly associated with the alpha1 subunit. Two ON bipolar cell types, the DB6 and the rod bipolar cell, are predominantly associated with the alpha2 subunit. The glycinergic AII amacrine cell is presynaptic to the alpha1 subunit in the OFF-sublamina, and postsynaptic to the alpha2 subunit in the ON-sublamina. Another putative glycinergic cell, the vesicular glutamate transporter 3 cell, is predominantly presynaptic to the alpha2 subunit. The dopaminergic amacrine cell expresses the alpha3 subunit at a low density., ((c) 2005 Wiley-Liss, Inc.)

Diffuse bipolar cells in primate retina receive synaptic input from multiple cones and provide output to ganglion cells. Diffuse bipolar cells can be subdivided into six types (DB1-DB6) according to the stratification of their axon terminals in the inner plexiform layer, but their synaptic connectivity in the inner plexiform layer is not well understood. Here the stratification and synaptic connectivity of DB6 axon terminals were studied in the retinae of New World (marmoset) and Old World (macaque) monkeys. Immunohistochemical markers were applied to retinal sections. The sections were analyzed by confocal and deconvolution light microscopy as well as electron microscopy. The DB6 cells were identified with antibodies against CD15; rod bipolar cells were identified with antibodies against protein kinase Calpha (PKCalpha); and AII amacrine cells were identified with antibodies against calretinin. The axons of DB6 and rod bipolar cells occupy distinct regions in stratum 5 of the inner plexiform layer. The distal processes of calretinin-labeled AII cells are usually closely associated with rod bipolar axons but sometimes also with DB6 axons. Pre-embedding immunoelectron microscopy showed that the vast majority (over 86%) of the synaptic output of DB6 cells is onto amacrine cell processes, whereas less than 14% goes to ganglion cell processes. In double-labeled preparations DB6 axons occasionally made output onto calretinin-labeled amacrine processes. Thus it is possible that AII cells receive some input from DB6 cells., (Copyright 2004 Wiley-Liss, Inc.)

The epithelial versus mesenchymal phenotypes of embryonic ectoderm and mesoderm cells of the prestreak stage pig embryos were examined by electron microscopy and molecular marker analysis. During this period the embryonic disc remained flat or slightly convex while becoming oval or pyriform in shape. Mesenchyme cells expressing vimentin were present between the embryonic disc and the underlying visceral endoderm before a primitive streak (or groove) was apparent. The migration of mesenchyme appeared to occur in lateral and posterior directions from a mass of quiescent cells located in the pointed end of the pyriform embryonic disc that expressed Brachyury; these cells are proposed to be the precursors of the primitive streak and/or form the equivalent of the mouse early gastrula organizer (EGO). Cells with the TEC-1 (or SSEA-1) epitope, the marker most frequently used to characterize pluripotent cells, were initially distributed randomly in the embryonic ectoderm and then were found to localize in an anterior crescent which may contain the precursor cells of ectoderm and neurectoderm. As mitotic figures were found only in the anterior crescent, it is proposed that at least some of these proliferating cells migrate toward the EGO. While cytokeratins were barely detectable in the embryonic ectoderm cells, vimentin expression was supposed to be associated with the migratory capacity of these cells. These findings indicate that the early step of gastrulation, migration of extraembryonic mesoderm, occurs at a prestreak stage during which the embryonic disc becomes polarized. genesis 38:13-25, 2004., (Copyright 2003 Wiley-Liss, Inc.)

Diagnosis of Hodgkin lymphoma (HL) by fine-needle aspiration (FNA) is often hampered by aspirated blood that camouflage scattered Hodgkin cells and Reed-Sternberg (HRS) cells, and the absence of HRS cells in the smears submitted for immunophenotyping. The objective of this study was to develop a simple protocol to overcome these problems. The visibility of HRS cells in Diff-Quik, traditional, and Ultrafast Papanicolaou (UFP) stains in FNA smears were compared in 73 cases of HL. HRS cells were found to be most visible in UFP because of the hemolysis of aspirated blood and the highlighting of HRS cells by the red-staining nucleoli. UFP-stained smears containing HRS cells were subsequently immunophenotyped via the cell-transfer technique. UFP staining was found to have no deleterious effect on the immunoreactivity of cellular CD15 and CD30 antigens of HRS cells. This simple protocol enhances the cytologic diagnosis of HL, feasible even with a single smear., (Copyright 2002 Wiley-Liss, Inc.)

A critical phase of nervous system development is the formation of connections between axons and their synaptic targets. Intermediate targets play important roles in axon pathfinding by supplying growing axons with long- and short- range guidance cues at decision points along their trajectory. We recently identified Vema as a novel membrane-associated protein that is expressed at the ventral midline of the developing vertebrate central nervous system (CNS). We report that Vema is expressed in the floor plate, an intermediate target for pathfinding commissural axons located at the ventral midline of the developing mouse spinal cord. Interestingly, Vema expression overlaps with the position of an unique population of neurons situated at the midline of the ventral diencephalon and that function as intermediate targets for pathfinding retinal ganglion cell axons. The distribution of Vema in the developing spinal cord and optic chiasm resembles the expression patterns of a variety of molecules known to play important roles in axon guidance, including Robo2, Neuropilin2, and SSEA. The expression of Vema at two key choice points for pathfinding axons suggests an important role for this protein in regulating axon guidance at the midline of the developing mouse central nervous system., (Copyright 2002 Wiley-Liss, Inc.)

The organization of the human hypothalamus was studied in 33 brains aged from 9 weeks of gestation (w.g.) to newborn, using immunohistochemistry for parvalbumin, calbindin, calretinin, neuropeptide Y, neurophysin, growth-associated protein (GAP)-43, synaptophysin, and the glycoconjugate 3-fucosyl- N-acetyl-lactosamine. Developmental stages are described in relation to obstetric trimesters. The first trimester (morphogenetic periods 9-10 w.g. and 11-14 w.g.) is characterized by differentiating structures of the lateral hypothalamic zone, which give rise to the lateral hypothalamus (LH) and posterior hypothalamus. The PeF differentiates at 18 w.g. from LH neurons, which remain anchored in the perifornical position, whereas most of the LH cells are displaced laterally. A transient supramamillary nucleus was apparent at 14 w.g. but not after 16 w.g. As the ventromedial nucleus differentiated at 13-16 w.g., three principal parts, the ventrolateral part, the dorsomedial part, and the shell, were revealed by distribution of calbindin, calretinin, and GAP43 immunoreactivity. The second trimester (morphogenetic periods 15-17 w.g., 18-23 w.g., and 24-33 w.g.) is characterized by differentiation of the hypothalamic core, in which calbindin- positive neurons revealed the medial preoptic nucleus at 16 w.g. abutted laterally by the intermediate nucleus. The dorsomedial nucleus was clearly defined at 10 w.g. and consisted of compact and diffuse parts, an organization that was lost after 15 w.g. Differentiation of the medial mamillary body into lateral and medial was seen at 13-16 w.g. Late second trimester was marked by differentiation of periventricular zone structures, including suprachiasmatic, arcuate, and paraventricular nuclei. The subnuclear differentiation of these nuclei extends into the third trimester. The use of chemoarchitecture in the human fetus permitted the identification of interspecies nuclei homologies, which otherwise remain concealed in the cytoarchitecture., (Copyright 2002 Wiley-Liss, Inc.)

Fibroblasts demonstrate different phenotypes and functions according to the tissue of origin and its physiopathologic state. We previously showed that fibroblasts isolated in culture from myelometaplasic (MM) spleen differed phenotypically from fibroblasts from normal bone marrow (BM). We compared the influence of each type of fibroblasts on the behavior of CD34+ stem cells. Expansion of nucleated cells was observed when blood CD34+ cells were co-cultured for 3 weeks with MM spleen-derived fibroblasts in monolayers. Myeloid cell differentiation was also observed as indicated by a decline in CD34+ cells and increases in CD14+, CD15+ and CD41+ cells. This myeloid differentiation was enhanced in the presence of MM spleen compared with normal BM-derived fibroblasts. Similarly, proliferation and differentiation of BM CD34+ cells was better in the presence of BM rather than MM spleen-derived fibroblasts. In addition, fibroblasts from MM spleen also induced a differentiation of CD56+ natural killer (NK) cells whereas BM-derived fibroblasts did not. Overall, the data indicate that cultured fibroblasts from diseased tissue have distinct growth and differentiation regulatory characteristics. They also suggest a role for these cells in hematopoietic disorders.

Glycoconjugates bearing the epitope 3-fucosyl-N-acetyllactosamine (CD15) are believed to be involved in cell-cell interactions and are temporally and spatially regulated in the brain. In the rat postnatal cerebellum, CD15 is predominantly expressed in the molecular layer by Bergmann glial cells, but little CD15 expression is seen in other astroglia, and the basis for this restricted expression is not known. Adenoviral vectors were shown to efficiently deliver transgenes to cerebellar glial cells and were used to determine whether manipulation of glycosyltransferase activities could enhance the expression of CD15 in these cells. In dissociated cerebellar cell cultures, few glial cells normally express CD15. However, transduction of these cells with an adenoviral vector (AdGFPCMVFucT) that expressed both green fluorescent protein (GFP) and FLAG-tagged rat alpha 1, 3-fucosyltransferase IV (rFuc-TIV) resulted in high CD15 expression on the surface of all transduced glial cells. Likewise, infection of cerebellar slice cultures caused the appearance of CD15-positive transduced cells of glial cell morphology in the internal granule cell layer. Thus, enhancement of Fuc-T activity caused robust CD15 expression in cerebellar glial cells that normally show little expression of CD15, suggesting a role for Fuc-T levels in regulating CD15 expression in this cell type. The manipulation of levels of glycosyltransferases using adenoviral vectors may prove a useful tool to investigate questions of glycoconjugate regulation in glial cells in the developing rodent cerebellum., (Copyright 2000 Wiley-Liss, Inc.)

Embryonic germ (EG) cell lines established from primordial germ cells (PGCs) are undifferentiated and pluripotent stem cells. To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem (ES) cells. We isolated PGCs from 5.5-day-old (stage 28) chicken embryonic gonads and established a putative chicken EG cell line with EG culture medium supplemented with stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), interleukin-11 (IL-11), and insulin-like growth factor-I (IGF-I). These cells grew continuously for ten passages (4 months) on a feeder layer of mitotically active chicken embryonic fibroblasts. After several passages, these cells were characterized by screening with the periodic acid-Schiff reaction, anti-SSEA-1 antibody, and a proliferation assay. The chicken EG cells maintained characteristics of gonadal PGCs and undifferentiated stem cells. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types. The chicken EG cells were injected into stage X blastodermal layer and produced chimeric chickens with various differentiated tissues derived from the EG cells. Chicken EG cells will be useful for the production of transgenic chickens and for studies of germ cell differentiation and genomic imprinting.

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated growth, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic fibroblast cell line expressing recombinant, membrane-bound porcine stem cell factor (SCF). Fifteen lines proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages 15 and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism., (Copyright 1999 Wiley-Liss, Inc.)

Sialyl Lewis a and x antigens are well-known tumor-associated antigens expressed in many cancer tissues. The expression of the genes encoding 5 alpha1,3fucosyltransferases, which are able to synthesize the sialyl Lewis antigens, was examined in normal and cancerous lung tissues of patients with non-small cell lung carcinoma. In all 20 cases examined, the transcripts only for the Lewis gene, encoding the Lewis enzyme (alpha1,3/4fucosyltransferase, Fuc-TIII), were abundantly expressed in lung tissue, and interestingly they were markedly up-regulated in the lung cancer tissues of all 20 cases in comparison with normal lung tissues. Myeloid-type alpha1,3fucosyltransferase (Fuc-TIV) was expressed at an intermediate level but was not up-regulated in lung cancer tissues. The transcripts for plasma-type alpha1,3fucosyltransferase (Fuc-TVI) gene were detected at a very low level but were apparently up-regulated in cancer tissues. Fuc-TVI was found to exhibit stronger relative activity for sialyl Lewis x synthesis (almost 6. 4-fold that of Fuc-TIII). The amount of sialyl Lewis x antigen on mucins in the lung cancer tissues was found to be determined by both enzymes, the Lewis enzyme (Fuc-TIII) and Fuc-TVI. However, the amount of the sialyl Lewis a antigens was not determined by any of the alpha1,3-fucosyltransferases, although the expression of sialyl Lewis a antigens definitely required the Lewis enzyme., (Copyright 1999 Wiley-Liss, Inc.)

The expression of the terminal saccharide determinant CD15 (3[a1-3]-fucosyl-N-acetyl-lactosamine) was evaluated in the central auditory system of the human developing brain by using monoclonal antibodies against this epitope. CD15 immunoreactivity was first observed in the ventral cochlear nucleus at 10 weeks of gestation, whereas the dorsal cochlear nucleus became positive from 13 weeks of gestation. In both nuclei, the intensity of immunoreactivity increased until 16 weeks of gestation and lasted until 25 weeks of gestation. In the inferior colliculi, CD15 was poorly expressed in the central nucleus from 13 to 23 weeks of gestation and later with moderate levels until birth. Within the medial geniculate nucleus, a biphasic pattern of expression was observed with peaks around 14-17 and 21-24 weeks of gestation. Heterogeneous expression in the medial geniculate nucleus, which was associated either with neurons or the neuropil, allowed distinction of subnuclei. In many of the auditory pathway structures (e.g., ventral cochlear nucleus and central nucleus of the inferior colliculus), a heterogeneous pattern of CD15 expression in the form of repeating parallel bands, possibly related to tonotopic organization, became transiently apparent around 23 weeks of gestation, whereas in the magnocellular part of the medial geniculate nucleus, a striking modular or compartmental arrangement of immunoreactive structures (which could also be associated with tonotopic organization) was also noted at about 23 weeks of gestation. We propose that the initiation of CD15 expression in each nucleus heralds the appearance of functional contacts and that high levels of neuropil labeling are related to the formation of nonstabilized synaptic contacts. Thus, transient CD15 expression in the central auditory system is possibly correlated with phases of functional plasticity in this pathway.

Overlapping morphologic patterns that may be observed in goiter, follicular adenoma, and papillary carcinoma can limit the cytologic evaluation of the thyroid gland. In an attempt to develop a useful adjunctive test, the immunocytochemical reactivity of HBME-1, carcinoma antigen 19-9 (CA 19-9), and CD-15 (Leu-M1) was tested on 59 cell block preparations from fine-needle aspirations of the thyroid gland. HBME-1 monoclonal antibody was reactive in all 21 papillary carcinomas, in 4 of 18 adenomas, and in 5 of 20 goiters. CA 19-9 was identified in 13 of 21 carcinomas, 1 goiter, but none of the adenomas. CD-15 was present in 15 of 21 carcinomas, 1 goiter, and 1 adenoma. We conclude that HBME-1 is a sensitive marker of papillary thyroid carcinoma. CD-15 and CA 19-9 are less sensitive but more specific. This panel can be useful to help classify morphologically equivocal lesions. As with all immunocytochemical testing, caution must be exercised in the interpretation of results, and correlation made with morphologic and clinical data.

Sialyl Lewis Tn(STN), sialyl Lewis a(CA-9-9), sialyl Lewis a-b-(DU-PAN-2), and sialyl Lewis x(SLX) antigens were immunohistochemically examined in the primary tumor and metastatic lymph nodes in 35 patients with advanced gastric cancer. STN, CA-19-9, DU-PAN-2, and SLX were expressed in 91%, 60%, 31%, and 60% in the primary lesion, and 77%, 54%, 22%, and 51% in the metastatic lesion, respectively. In only four cases, (11%) were all four antigens expressed in both the primary and metastatic lesions. Three antigens were expressed in 49% of primary lesions and in 20% of metastatic lesions. Compared with expression in primary lesions, increased, unchanged and decreased expressions in metastatic lesions were noted in 23%, 37%, and 40% for STN, 20%, 40%, and 40% for CA-19-9, 17%, 57%, and 26% for DU-PAN-2, and 26%, 31%, and 43% for SLX, respectively. These results indicate that the tumor in the primary and metastatic lesions has a heterogeneous expression of sialyl-related antigens. However, metastases cannot be predicted based upon the expression of these antigens.

Poorly differentiated gastric adenocarcinoma with medullary features (poor medullary) is distinguished by a propensity for hepatic metastasis. To classify it antigenically, we compared it to poorly differentiated adenocarcinoma with scirrhous growth pattern (poor scirrhous), well and moderately differentiated adenocarcinoma (differentiated adenocarcinoma), and normal gastric mucosa (foveolar and deep epithelium) using immunohistochemistry with antibodies against CEA, AFP, NSE, and Lewis-type antigens. Lewis(a) antigen was significantly associated with differentiated adenocarcinoma and foveolar epithelium, although Lewis(x) antigen was significantly expressed in poor medullary, poor scirrhous, and deep gland epithelium. From the viewpoint of expression of Lewis(a), there was no significant differentiation between poor medullary and differentiated adenocarcinoma, but it was definite between poor scirrhous and differentiated adenocarcinoma. Therefore, we conclude that in antigenic expression, poor medullary carcinoma is allied with differentiated adenocarcinoma rather than poorly differentiated scirrhous carcinoma.

The rate of polarized secretion of a putative adhesion ligand, sialosyl Lewis(a) (19-9), by SW1116 colorectal carcinoma cells is stimulated at least 20-fold after pre-incubation with, and the incorporation of, retinoic acid (RA). In order to investigate the possible involvement of fatty acylation in the export of the epitope, purified ligands from carcinoma-cell membranes, membrane subfractions and media were analyzed during RA-induced secretion. Incorporation of radioactivity from (3H)palmitate into membrane subfractions and purified sialosyl Lewis(a) antigenic molecular species of M(r) > 150,000 (SiaLeams) was stimulated by RA treatment. Most of the intracellular lipid radioactivity which bound to solid-phase 19-9 antibody behaved chromatographically, either like ganglioside or like NH2 OH-labile acyl groups, but most of the (3H) bound to SiaLeams of post-incubation media behaved like base-labile fatty acyl groups, or free fatty acid. Release of base-labile lipid radioactivity after 3 hr (associated with antigen) was almost exclusively into the apical media of membrane inserts. Gas-liquid chromatography/mass spec. analyses of purified Sialeams revealed the presence of palmitate (16:0), as well as stearate (18:0) and oleate (18:1) fatty acyl groups. Our results suggest that fatty acylation of SiaLeams may be co-ordinated with alterations in glycosylation and participate in directing these molecules to the apical surface. Lipid analyses were consistent with ganglioside chaperonage of SiaLeams to the apical surface, where N-fatty-acylated gangliosides remain for the most part integrated into the bilayer, but some oxyester or thioester bonds may be cleaved to permit release of SiaLeams to the apical medium.

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examined the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3-6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo-derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial-like cells were isolated on REF and colonies of ES-like cells on the RUCs. The ES-like cells were positive for expression of alkaline phosphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat.

Dimethyl sulfoxide (DMSO) exerts a number of biological effects including the promotion of cell differentiation in cultured cells. In this study, we examined the effect of DMSO on the adhesion of tumor cells to endothelial cells. In vitro treatment of human gastric adenocarcinoma (NUGC4) cells with DMSO resulted in increased adhesion to interleukin-I (IL-I)-activated human endothelial cells compared with DMSO-untreated NUGC4 cells. In flow cytometry, treating NUGC4 cells with DMSO enhanced the expression of sialyl Lewis x (sialyl Le(x)) and sialyl dimeric Le(x) antigens on their surface. Also, the binding of Limulus polyphemus agglutinin (LPA), which specifically binds to cell-surface sialic acids, was increased by DMSO. The adhesion of DMSO-treated NUGC4 cells to activated endothelial cells was blocked by neuraminidase pre-treatment of tumor cells or by antibody against either endothelial leukocyte adhesion molecule-I (ELAM-I) or sialyl Le(x). Thus, it is suggested that enhanced adhesion following DMSO treatment is mediated by the interaction of sialyl Le(x) expressed on NUGC4 cells with ELAM-I of endothelial cells. The modulation of sialyl Le(x) antigen by DMSO provides a useful system for studying the regulatory mechanism of Lewis-related carbohydrate antigens and also for understanding the metastatic properties of cancer cells.