Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage

Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65–270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage) in mice.     

Human Epidermal Melanocyte and Keratinocyte Melanocortin Receptors: Visualization by Melanotropic Peptide Conjugated Microspheres (Latex Beads)

The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle⁴,D-Phe⁷]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromo-lecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) micro-spheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.     

New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.     

Natural and activated cytotoxic lymphocytes which act on autologous and allogeneic tumor cells

Summary A considerable proportion of human blood lymphocytes that express natural cytotoxic potential belong to the T subset. They are heterogenous with regard to their surface receptor characteristics. Results obtained with the exceptionally sensitive cell lines (such as K562, Molt-4) and freshly harvested cells as targets reveal different types of cytotoxicity. While certain cell lines may be killed without the involvement of antigen recognition, the freshly harvested cells are recognized and killed by lymphocytes that carry receptors for surface antigens on these targets. In accordance, such tests can reveal the cytotoxicity of T cells that carry receptors for histocompatibility antigens. In view of the high frequency of these cells in the lymphocyte population, enlargement of the clone is not always necessary and activation suffices.Cytotoxicity against autologous tumor cells derived from surgical specimens of sarcomas and carcinomas was demonstrated only in a few patients when the blood lymphocytes were used directly after collection. Cytotoxic cells could be generated in a proportion of cases when a stimulus was given, e.g., co-cultivation with allogeneic lymphocytes in the conventional MLC test. Co-cultivation of autologous lymphocytes and tumor biopsy cells was the most efficient measure for the generation of auto-tumor cytotoxicity. This condition allows the enlargement of the reactive clone. When this was inhibited, e.g., by the presence of interferon in the mixed culture no auto-tumor killing was generated.We would like to emphasize that tumor cells seem not to share the membrane properties of the in vitro model lines, which exhibit high sensitivity to the natural or activated killer cells without the involvement of antigen recognition.     

Studies on the recognition of xenogeneic cells by nonimmune macrophages. I. Destruction of chicken fibroblasts in vitro by murine macrophages.

Human cord blood lymphocytes were compared with adult lymphocytes with regard to proportions of cells with surface markers for surface immunoglobulin (Ig), receptors for C′3 and the Fc-portion of IgG, as well as two types of erythrocyte rosettes (rapid and late E-rosettes). A significant decrease (P < 0.02 ? 0.05) in both early and late E-rosettes was noted when cord cells were compared to adult lymphocytes. After 20 hr of incubation at 37 °C, proportions of cells bearing Fc receptors in cord blood samples showed striking increments (P < 0.001) when compared with adult lymphocytes. T cell enrichment studies and sequential depletion of cells bearing Fc receptors as well as E-rosette forming cells indicated that the precursors of cells generating Fc receptors in vitro did not arise from cells with Fc receptors or T cell markers.     

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[³H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[³H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[³H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.     

Mucosal immunoregulation: environmental lipopolysaccharide and GALT T lymphocytes regulate the IgA response

In this review, we have emphasized: 1) bacterial lipopolysaccharide (LPS) involvement in IgA responses to orally administered thymic-dependent (TD) antigens; 2) characterization of Peyer's patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti-LPS immunity to infection. Gut LPS, which interacts with PP lymphoreticular cells, is a major determinant for host responses to orally administered TD antigens. Bacteroides species are the principal microflora present in the gastrointestinal tract and our studies with phenol-water LPS extracts from Bacteroides fragilis indicate that both polysaccharide and lipid A activate lymphoreticular cells. The B. fragilis lipid A moiety, like that derived from E. coli and Salmonella LPS, induces B cell mitogenic responses in cultures from LPS responsive mice, but does not stimulate C3H/ H3J B cells. The inability of lipid A to stimulate gut-associated lymphoreticular tissue (GALT) cells of C3H/HeJ mice results in the induction of greater T helper cell activity in this tissue in response to orally administered TD antigens and ultimately results in an elevated IgA response pattern. Murine PP contain accessory cells (approximately 1% dendritic cells and 6-8% macrophages) and lymphocytes T (35-38%) and B (40-42%). Recent studies with antigen-specific T cell clones from C3H/ H3J PP have resulted in the isolation of IgA isotype-specific T helper cells (PP Th A cells). PP Th A cells are antigen-specific, bear Fc alpha receptors, and require H-2 histocompatibility with B cells for helper activity. PP Th A cells most effectively collaborate with surface IgA (sIgA)-bearing B cells (IgA committed B cells) for IgA isotype responses. Other studies have shown that PP dendritic cells and T cells form clusters when stimulated in vitro with sodium periodate and that these clusters promote polyclonal IgA responses in B cell cultures. Polyclonal IgA responses in cultures containing PP cell clusters from C3H/ H3J mice are considerably higher than those in identical cultures from LPS responsive mice. In other studies, the environmental influence on GALT B cells and their resultant commitment to IgA isotype is under investigation. CBA/N, X-linked immunodeficient (xid) mice possess an immature splenic B cell population which cannot respond to thymic-independent class-2 (TI-2) or certain TD antigens. However, GALT B cells of xid mice possess a mature Lyb-5+ B cell subpopulation capable of both TI-2 and TD responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Surfaces of murine lymphocyte subsets differ in sialylation states and antigen distribution of a major N-linked penultimate saccharide structure

Rat liver beta-galactoside alpha-2,6-sialyltransferase and Vibrio cholerae sialidase were used with cytidine-5'-monophospho-N-acetyl-[3H]neuraminic acid (CMP-[3H]NeuAc) to specifically probe the distribution and sialylation state of Gal beta 1-4GlcNAc residues on N-linked saccharides on the surfaces of murine lymphocytes. The relative extent of exogenous sialyltransferase-mediated sialylation (per cellular protein) was thymocytes greater than T-cells greater than T-cell lymphoma (EL-4) greater than B-cells greater than B-cell lymphoma (AKTB-1b) greater than splenocytes. Prior desialylation increased exogenous resialylation by 23.8-, 13.1-, 7.1-, 7.9-, 7.0-, and 5.3-fold for splenocytes, B-cells, T-cells, EL-4, AKTB-1b, and thymocytes, respectively. Though numerous glycoproteins were labeled, the majority of the Gal beta 1-4GlcNAc residues were detected on a relatively small number of cell surface proteins, many of which are well-defined lymphocyte antigens. Gal beta 1-4GlcNAc residues on thymocytes were found to exist in an undersialylated state on T200 but not on other antigens (e.g., Thy-1). T200 was found to be fully sialylated on mature cells (i.e., hydrocortisone-resistant thymocytes and splenic T-cells), suggesting that its sialylation state is developmentally regulated. These studies indicate that the number, sialylation state, and polypeptide distribution of the penultimate structure, Gal beta 1-4GlcNAc, differ on N-linked saccharides on the surfaces of different lymphocyte populations.     

T细胞IFN-g表达水平检测方法的建立及其在马传染性 贫血疫苗免疫机理研究中的应用

[目的]马传染性贫血病毒(EIAV)弱毒疫苗致弱机制和免疫保护机理的研究可以为慢病毒疫苗的研究提供重要的模型.为探讨IFN-γ表达水平与疫苗保护性免疫的关系,本研究旨在建立一种准确、有效地检测EIAV感染马不同T细胞亚型表达IFN-γ水平的方法.[方法]我们将分离的马传贫弱毒疫苗免疫马(FDDV)、强毒感染马(LV)和健康马的外周血单核细胞(PBMC),体外分别经病毒(FDDV)和PMA/Inomycin激活、 BFA 阻断蛋白分泌、荧光标记马的特异性表面抗体和IFN-γ抗体等过程后,进行流式检测.[结果]疫苗免疫马产生的特异性IFN-γ水平为CD4 1.7(0.9%/CD8 6.1(1.2%,而强毒组则为CD4 0.6(0.1%/CD8 2.4(0.9%.[结论]本研究建立的多荧光参数流式细胞术同时检测细胞内IFN-γ染色和淋巴细胞亚型的方法,具有良好的特异性,稳定性和重复性.为研究EIAV弱毒疫苗免疫保护机制奠定了基础.     

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification¹.A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types^2,3. However, no single ‘marker’ has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple ''markers''. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response⁵, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody⁶. For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells⁷. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.     

Immunoregulation in the rat: requirements for in vitro B cell responses to classical TI-1 and TI-2 antigens

The presence of suppressor cells and their mediators has made it difficult to induce B cell mitogenic or immune responses in rat spleen cell cultures. In the present study, we have defined culture conditions required for induction of in vitro thymic independent (TI) immune responses in the rat. Rat spleen cell cultures support low responses to various trinitrophenyl (TNP) haptenated antigens including TNP-Brucella abortus (TNP-BA), TNP-lipopolysaccharide [LPS; either phenol (Ph)- or butanol (Bu)-water extracted], TNP-Ficoll, and TNP-dextran. However, all of these antigens induced good splenic anti-TNP PFC responses when given at appropriate doses in vivo. When spleen cells were depleted of adherent cells and cultured with TI antigens in vitro, good anti-TNP PFC responses were seen with TNP-BA, whereas, lower responses were induced by TNP-LPS (Ph or Bu). No responses were observed in cultures incubated with either TNP-Ficoll or TNP-dextran. Purified splenic B cell cultures [prepared by panning on plates coated with anti-rat F (ab')2] supported good responses to TNP-LPS (Ph or Bu) and TNP-BA. The addition of irradiated splenic adherent cells (macrophages, M phi) to either M phi-depleted or purified B cell cultures completely abrogated in vitro responses to TNP-BA or TNP-LPS (Ph or Bu). Purified splenic B cell cultures generally responded poorly to TNP-Ficoll or TNP-dextran. Addition of indomethacin (IM) to spleen cell cultures abrogated suppression and allowed anti-TNP PFC responses to TNP-BA, TNP-LPS (Ph or Bu), TNP-Ficoll, and TNP-dextran. Furthermore, nude spleen cell cultures treated with IM, also allowed significant TNP-Ficoll and TNP-dextran immune responses; however, untreated cultures did not respond to these antigens. Our studies indicate that rat splenic B cell cultures are responsive to TI antigens, and highest responses occur with the murine TI-1 class, e.g., TNP-BA and TNP-LPS. Inhibition of suppression with IM restored splenic B cell responses to the murine TI-2 class, i.e., TNP-Ficoll and TNP-dextran.     

Signal transduction in T helper cells: CD4 coreceptors exert complex regulatory effects on T cell activation and function

The immune system provides a highly sophisticated surveillance mechanism to detect diverse antigens and to protect the host organism from invading pathogens and altered cells (e.g., virus-infected and tumor cells). Adaptive immune responses depend on the recognition of antigen by specific antigen receptors that are expressed on the surface of T and B lymphocytes. Helper T cells provide regulatory functions and direct the adaptive immune system to respond appropriately to a particular antigen (i.e., cytotoxic T cell responses against viral infections and tumor cells, humoral responses against extracellular bacteria and parasitic worms). Helper T cells express CD4 coreceptors, which recognize conserved domains on proteins expressed by the class II major histocompatibility complex, the same proteins that present antigen to the T cell receptor. Recent progress in T cell biology has identified multiple regulatory functions of CD4 during thymocyte development and antigen stimulation of mature T helper cells. Signaling pathways induced by engagement of CD4 independently of T cell receptor signaling mediate these regulatory functions. In this review, we discuss the regulation of T cell signaling and emphasize the functional consequences of proper and improper CD4 coreceptor signaling.     

Differentiation antigens in human T-cell activation: Evidence that anti-VH antibodies react with a membrane structure on human T Lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series

The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human V_H antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin V_H fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIal, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIal and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.     

Neuropeptide Y stimulates retinal neural cell proliferation--involvement of nitric oxide

Neuropeptide Y (NPY) is a 36 amino acid peptide widely present in the CNS, including the retina. Previous studies have demonstrated that NPY promotes cell proliferation of rat post-natal hippocampal and olfactory epithelium precursor cells. The aim of this work was to investigate the role of NPY on cell proliferation of rat retinal neural cells. For this purpose, primary retinal cell cultures expressing NPY, and NPY Y₁, Y₂, Y₄ and Y₅ receptors [Álvaro et al. , (2007) Neurochem. Int., 50, 757] were used. NPY (10–1000 nM) stimulated cell proliferation through the activation of NPY Y₁, Y₂ and Y₅ receptors. NPY also increased the number of proliferating neuronal progenitor cells (BrdU⁺/nestin⁺ cells). The intracellular mechanisms coupled to NPY receptors activation that mediate the increase in cell proliferation were also investigated. The stimulatory effect of NPY on cell proliferation was reduced by l -nitroarginine-methyl-esther ( l -NAME; 500 μM), a nitric oxide synthase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ; 20 μM), a soluble guanylyl cyclase inhibitor or U0126 (1 μM), an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK 1/2). In conclusion, NPY stimulates retinal neural cell proliferation, and this effect is mediated through nitric oxide–cyclic GMP and ERK 1/2 pathways.     

Heterokaryon-based reprogramming of human B lymphocytes for pluripotency requires Oct4 but not Sox2

Differentiated cells can be reprogrammed through the formation of heterokaryons and hybrid cells when fused with embryonic stem (ES) cells. Here, we provide evidence that conversion of human B-lymphocytes towards a multipotent state is initiated much more rapidly than previously thought, occurring in transient heterokaryons before nuclear fusion and cell division. Interestingly, reprogramming of human lymphocytes by mouse ES cells elicits the expression of a human ES-specific gene profile, in which markers of human ES cells are expressed (hSSEA4, hFGF receptors and ligands), but markers that are specific to mouse ES cells are not (e.g., Bmp4 and LIF receptor). Using genetically engineered mouse ES cells, we demonstrate that successful reprogramming of human lymphocytes is independent of Sox2, a factor thought to be required for induced pluripotent stem (iPS) cells. In contrast, there is a distinct requirement for Oct4 in the establishment but not the maintenance of the reprogrammed state. Experimental heterokaryons, therefore, offer a powerful approach to trace the contribution of individual factors to the reprogramming of human somatic cells towards a multipotent state.     

Fabrication of a biomimetic spinal cord tissue construct with heterogenous mechanical properties using intrascaffold cell assembly

In tissue engineering studies, scaffolds play a very important role in offering both physical and chemical cues for cell growth and tissue regeneration. However, in some cases, tissue regeneration requires scaffolds with high mechanical properties (e.g., bone and cartilage), while cells need a soft mechanical microenvironment. In this study, to mimic the heterogenous mechanical properties of a spinal cord tissue, a biomimetic rat tissue construct is fabricated. A collagen-coated poly(lactic-co-glycolic acid) scaffold is manufactured using thermally induced phase separation casting. Primary rat neural cells (P01 Wistar rat cortex) with soft hydrogels are later printed within the scaffold using an image-guided intrascaffold cell assembly technique. The scaffolds have unidirectional microporous structure with parallel axial macrochannels (260 ± 4 µm in diameter). Scaffolds showed mechanical properties similar to rat spine (ultimate tensile strength: 0.085 MPa, Young's modulus [stretch]: 0.31 MPa). The bioink composed of gelatin/alginate/fibrinogen is precisely printed into the macrochannels and showed mechanical properties suitable for neural cells (Young's modulus [compressive]: 3.814 kPa). Scaffold interface, cell viability, and immunostaining analyses show uniform distribution of stable, healthy, and elongated neural cells and neurites over 14 culture days in vitro. The results demonstrated that this method can serve as a valuable tool to aid manufacturing of tissue constructs requiring heterogenous mechanical properties for complex cell and/or biomolecule assembly.     

Measles virus-induced suppression of lymphocyte reactivity in vitro

Measles virus (Edmonston strain B), in various multiplicities of infection, was added to human lymphocytes which were cultured in medium containing fetal bovine serum. Live measles virus was found to cause an almost complete inhibition of [³H]-thymidine incorporation in lymphocytes cultured in the presence of phytohemagglutinin, pokeweed mitogen, tuberculin purified protein derivate (PPD), or allogeneic lymphocytes. Analysis of cell size in the lymphocyte cultures revealed that blast transformation was inhibited as well. Measles virus, inactivated by heat or ultraviolet irradiation, did not cause inhibition. The inhibitory effect of measles virus was only measurable in the initial stages of culture; when added later, i.e., 24 hr before measuring [³H]-thymidine incorporation, it had no effect. The diminished reactivity of measles virus-infected lymphocytes cannot be explained by cytopathologic effects or by altered kinetics of lymphocyte transformation. When lymphocytes were cultured at 39 °C the extent of virus-induced suppression was significantly reduced. Very small amounts of pooled normal human serum, as well as IgG, prepared from the serum of a patient with subacute sclerosing panencephalitis, were able to prevent the inhibitory effect of measles virus.     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

T3 monoclonal antibody activation of nonspecific cytolysis: a mechanism of CTL inhibition

The T3 antigen is expressed on all cytotoxic T lymphocytes (CTL). Monoclonal antibodies (MAb) to the T3 antigen previously have been shown to inhibit CTL-mediated killing of cells expressing the relevant target antigens. The mechanism of T3 MAb inhibition, however, remains undefined. In this report, we describe a novel effect of the T3 MAb: the stimulation of allospecific CTL clones to kill target cells that do not express the relevant HLA antigens. The stimulation of nonspecific killing was seen only with MAb to the T3 antigen; MAb to other function-associated antigens (e.g., LFA-1, LFA-2, LFA-3, T4, T8, HLA-A,B,C, and DR) had no effect. T3 MAb stimulated nonspecific killing by CTL clones expressing both the T4+ and T8+ phenotype and by CTL clones specific for both class I and class II HLA alloantigens. Target cell susceptibility to T3 MAb stimulated killing was variable. CTL clones lysed some target cell lines very efficiently (e.g., K562, Daudi, and M124.1) but lysed other cell lines much less efficiently (e.g., 23.1, Mann, and L cells). In CTL-mediated cytotoxicity assays with target cells expressing the relevant HLA antigens, T3 MAb demonstrated the expected inhibition of cytolysis. Thus, the ability of T3 MAb to stimulate and inhibit CTL-mediated cytolysis suggests that both effects may be the result of a common mechanism of activation.     

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.