Functional inhibition of isolated pancreatic cells, new technic for the detection of macrophage cytotoxicity]

It is usually accepted that macrophages "activated" by lymphokines may be found cytotoxic against tumoral target cells but show no detectable cytotoxicity in in vitro tests using normal non tumoral cells as target cells. These data have been obtained mainly with the chromium-release test. The present paper describes a new test using normal isolated pancreatic cells as target cells and evaluating the effect of activated or non-activated macrophages on the insulin secretion response to glucose stimulation. The results show a striking decrease in this response following an 18-hr incubation of pancreatic islet cells with activated macrophages, as compared to that of the same cells incubated with control macrophages. This is clear evidence that activated macrophages may alter normal cells and suggests that their cytotoxic properties are not restricted to tumoral target cells.     

Cytotoxicity of mouse peritoneal cells after alloimmunization]

CBA Mice were immunized by two intraperitoneal injections of 30 X 10(6) DBA/2 or C57BL/6 spleen cells at days--12 and--2. Peritoneal cell population was obtained at day zero by washing the peritoneal cavity of Mice. Adherent cells were then separated using a 2 hrs. incubation in "Falcon" plates followed by washing. This macrophage-rich peritoneal cell population was found nonspecifically cytotoxic against 51Cr labeled tumoral target cells: P815 X DBA/2 mastocytoma cells, EL4 X C57BL/L lymphoma cells and spontaneous lymphoma AKR cells (same H--2k as CBA). This adherent peritoneal cell cytoxicity was demonstrated after 24 hrs. incubation with the target cells. It was found in nonspecific combination as well as when using target cells syngeneic to the donor. These findings suggest that adherent peritoneal cell cytotoxicity could be at least partly due to macrophages and result from factor (s) released by sensitized lymphocytes in vivo in the same way as has been previously demonstrated in vitro.     

Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-₆ integrin and anti-CD71 antibodies. RA cells express high levels of ₆ integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed ₆ integrin and ₁ integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.Received 2 July 2004; received after revision 20 August 2004; accepted 10 September 2004     

Embryonic origins of human vascular smooth muscle cells: implications for in vitro modeling and clinical application

Vascular smooth muscle cells (SMCs) arise from multiple origins during development, raising the possibility that differences in embryological origins between SMCs could contribute to site-specific localization of vascular diseases. In this review, we first examine the developmental pathways and embryological origins of vascular SMCs and then discuss in vitro strategies for deriving SMCs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We then review in detail the potential for vascular disease modeling using iPSC-derived SMCs and consider the pathological implications of heterogeneous embryonic origins. Finally, we touch upon the role of human ESC-derived SMCs in therapeutic revascularization and the challenges remaining before regenerative medicine using ESC- or iPSC-derived cells comes of age.     

A combined histochemical and autoradiographic study of the distribution and maturation of peritoneal mast cells in the rat

Summary The relationship of mitotic activity and degree of cytological differentiation for mast cells of the peritoneal cavity of the rat was studied using combined autoradiographic and histochemical techniques. A mitotic pool of mast cells can be differentiated from a non-mitotic pool, the former containing cells with immature cytoplasmic differentiation and the latter with fully developed cytoplasmic histochemical properties.     

Resolution of small G1 and G2 populations of L1210 leukemia by flow microfluorometric analysis aided by velocity sedimentation

L1210 leukemic cells grown in vitro were subjected to kinetic analysis using a flow microfluorometer. A single broad peak was found for the DNA content distribution if unfractionated cells were used; prior fractionation using lg velocity sedimentation allowed the separation of small peaks with smaller (G1) and larger (G2) DNA contents from the dominant S phase peak with intermediate DNA content.     

Rat pancreatic islet cells in primary culture: occurrence of giant cells amenable to patch clamping

Neonatal and adult rat pancreatic islet cells were maintained in dissociated cell culture for up to three weeks. The unexpected occurrence of giant (40-50 micron) cells was noted, some of which reacted positively to an insulin antiserum, indicating the presence of insulin. The giant cells were amenable to study using the extracellular patch clamp technique, which was used to demonstrate a population of membrane channels gating outwardly directed current in these cells.     

Antigenic correlation between rat thymus and superior cervical ganglion.

By using rabbit anti-rat thymocyte and anti-rat superior cervical ganglion sera in cytotoxicity, immunofluorescence and absorption assays, it has been shown that surface membranes of rat thymocytes and cervical ganglion cells (i.e. peripheral nervous tissue cells) contain common antigenic determinants.     

Estimation of nonspecific lectin-mediated staining of glutaraldehyde-fixed cells

Summary Lectin-mediated stainings are widely used for the visualization of carbohydrate-carrying cellular components using the electron microscope. The use of glutarladehyde-fixed cells for these stainings introduces the possibility of low nonspecific lectin-trapping by the glutaraldehyde which coats the cells. This trapping was estimated by means of peroxidase-binding to human leukocytes,Tetrahymena pyriformis andEscherichia coli cells and was shown to be prevented by rinsing the glutaraldehyde-fixed cells in an amino acid solution before exposure to the lectin.     

Demonstration of catecholaminergic neurons in rat retina by fluorescence histochemistry on cryostat sections]

A fluorescence histochemical technique using glyoxylic acid and cryostat sections has been worked out to visualize the dopamine-containing neurons of the retina. It allows the demonstration of catecholaminergic junctional cells and alloganglion cells, classically described. Moreover, the observation of some fluorescent fibers in the external zone of the inner nuclear layer, in contact with the outer plexiform, suggests the presence of catecholamine-containing interplexiform cells, in the Rat retina.     

Stimulus-response function at several levels of background luminance, in the cat visual areas 17 and 18.

Stimulus-response curves of simple cells of the visual cortex were obtained by using 500-msec stationary stimuli. Background influence on single unit responses was studied. The contrast sensitivity of simple cells increases as a function of background luminance. The resolution power of these cortical cells for detecting differences in stimulus contrast decreases at background levels above 0.09 cd/m2.     

Anti-myosin stains chromaffin cells.

The presence in fixed chromaffin cells of antigenic sites for a myosin antibody was demonstrated using immunofluorescence techniques. Tests on viable cells showed that at least some of the antigenic sites seem to be localized on or close to the cell surface and explained the cell agglutination that occurred with the addition of the myosin antibody to cells isolated by a method described in this paper.     

PPD-induced blastogenesis is auto-regulated by suppressor cells generated in vitro.

Suppressor cell induction can be demonstrated during antigen specific blastogenesis by using the same methods which have shown induction of suppressor cells by Con A. Since suppressor cells are rapidly generated during antigen specific blastogenesis, they must regulate the final level of blastogenesis induced during the seven day in vitro incubation.     

Occurrence of immunoreactive thyroglobulin in the parafollicular cells of dogs.

When the canine thyroid gland was stained by immunofluorescence and immunoperoxidase methods using undiluted thyroglobulin antiserum, a considerably stronger immunoreactivity was revealed in the parafollicular cells than in the colloid droplets and follicular cells. After induced hypercalcemia and antithyroid drug treatment, the immunoreactivity of the parafollicular cells coinciding with the movement of secretory granules containing calcitonin was conspicuously decreased. An application of diluted serum (above 1:10) produced a strong reaction in the colloid.     

Histochemical localization of alkaline and acid phosphatase activities in the skin ofMystus (Mystus) vittatus Bl. (Siluriformes)

Summary An attempt has been made to localize alkaline and acid phosphatase activities in the skin ofMystus vittatus by using histochemical techniques. The alkaline phosphatase activity is found in metabolically active cells such as basal columnar cells, mucous cells and polygonal support cells. The acid phosphatase activity is intense in the outermost squamous support cells and in the basal columnar cells. These activities have been correlated with some physiological functions of the epidermis.Acknowledgment. We are thankful to P. Vishwanatham, Government College, Mhow, and Dr R.S. Shrivastava, Holkar Science College, Indore, for providing laboratory facilities and to the Council of Scientific and Industrial Research, New Delhi, for a fellowship for M.S.     

The inner ear structures of the echidna—An SEM study

Summary Surface structures of the echidna cochlea were investigated using a scanning electron microscope technique. It was found that unlike typical mammalian cochleas, the echidna cochlea possesses four rows of inner hair cells and 6–9 rows of outer hair cells, and that the arrangements of the stereocilia of the outer hair cells were not uniform throughout the length of the basilar membrane.We are grateful for the valuable advice given by Mr J. Nailon.     

In vitro neurogenesis: development and functional implications of iPSC technology

Neurogenesis is the developmental process regulating cell proliferation of neural stem cells, determining their differentiation into glial and neuronal cells, and orchestrating their organization into finely regulated functional networks. Can this complex process be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology? Can neurodevelopmental and neurodegenerative diseases be modeled using iPSCs? What is the potential of iPSC technology in neurobiology? What are the recent advances in the field of neurological diseases? Since the applications of iPSCs in neurobiology are based on the capacity to regulate in vitro differentiation of human iPSCs into different neuronal subtypes and glial cells, and the possibility of obtaining iPSC-derived neurons and glial cells is based on and hindered by our poor understanding of human embryonic development, we reviewed current knowledge on in vitro neural differentiation from a developmental and cellular biology perspective. We highlight the importance to further advance our understanding on the mechanisms controlling in vivo neurogenesis in order to efficiently guide neurogenesis in vitro for cell modeling and therapeutical applications of iPSCs technology.     

胃癌侧群细胞耐药性研究及干细胞相关基因检测

目的研究胃癌侧群（Side Population,sP）细胞对化疗药物5-Fu（氟尿嘧啶）的耐药性及可能机制,并检测干细胞相关基因Nanog、Musashi-1及cD44的表达情况。方法选择人胃癌细胞株sGc-7901,以荧光染料H0echst 33342染色,维拉帕米桔抗对照,应用流式细胞仪分选sP细胞和nonsP细胞。细胞耐药实验比较sP细胞与nonsP细胞对化疗药物5．Fu的耐药性差异;westem_h10t检测ABcG2和bcl-2蛋白表达情况;流式细胞仪分析细胞周期;荧光定量PcR检测两组细胞中干细胞相关基因Nanog、Musashi-1及cD44mRNA的表达差异。结果胃癌细胞株sGc．790l中sP细胞的比例为2．8％,sP细胞对5-Fu的耐药存活率明显高于non-sP细胞（P〈0．05）,与nonsP细胞相比,sP细胞高表达耐药蛋白ABcG2和抗凋亡蛋白bcl-2,有更多的细胞处于c0／G1期（P〈0．05）,并高表达干细胞相关基因Musashi-1和cD44。结论胃癌sGC_7901细胞株中sP细胞对化疗药物5．Fu的耐药性明显高于nonsP细胞,其耐药机制可能与sP细胞高表达耐药蛋白ABcG2和抗凋亡蛋白bcl-2,有更多细胞处于G0／Gl期有关;Musashi-1和cD44可能是相对特异性的胃癌干细胞标志物。     

Immigration of lymphoid precursor cells into the thymic rudiment inXenopus

Summary The origin of thymic lymphocytes was investigated, using a new reliable method to mark cells inXenopus. It was easily observed that extraneous cells immigrated into the thymic rudiment 4 days after fertilization and differentiated into a cell population identified as thymic lymphocytes in a fully developed thymus. Clearly, lymphoid precursor cells are of extrinsic origin.This work was supported in part by a grant from the Ministry of Education, Science and culture of Japan (No. 59770026).