Cell Cycle Regulation and Differentiation in the Human Podocyte Lineage

Mature podocytes are regarded as growth-arrested cells with characteristic phenotypic features that underlie their function. To determine the relationship between cell cycle regulation and differentiation, the spatiotemporal expression of cyclin A, cyclin B1, cyclin D1, the cyclin-dependent kinase inhibitors (CKIs) p27 and p57, and markers of differentiating podocytes in developing human kidneys was investigated by immunohistochemistry. In S-shaped body stage, Ki-67, a cell proliferation marker that labels the G1/S/G2/M phase, was expressed in the majority (more than 80%) of presumptive podocytes, along with cyclin A (~20% of the Ki-67-positive cells) and cyclin B1 (less than 5% of Ki-67-positive cells) expression. Among these cells, cyclin D1 and CKIs were markedly down-regulated. At the capillary-loop stage, by contrast, CKIs and cyclin D1 were intensely positive in podocytes, whereas no Ki-67, cyclin B1, or cyclin A expression was seen. Moreover, double-immunolabeling and serial-section analysis provided evidence that CKIs and markers specific for differentiating podocytes, namely PHM-5 (podocalyxin-like protein in humans), synaptopodin (a foot process-related protein), and C3b receptor, were co-expressed at the capillary-loop stage. Podocytes were the only cells within the glomeruli that expressed CKIs at immunohistochemically detectable levels. Furthermore, bcl-2 (an apoptosis inhibitory protein) showed a reciprocal expression pattern to that of CKI. These results suggest that 1) the cell cycle of podocytes is regulated by cyclin and CKIs, 2) CKIs may act to arrest the cell cycle in podocytes at the capillary-loop stage, and 3) the specific cell cycle system in podocytes may be closely correlated with their terminal differentiation in humans.     

Anti-CD45R antibody-treated normal lymphocytes respond to interleukin 2 (IL2) after stimulation by allergens

Interleukin 2 (IL2) responsiveness was specifically induced by Dermatophagoides farinae (Df) antigen in Df-sensitized lymphocytes from asthmatic children, but not in normal lymphocytes. Df-induced IL2 responsiveness was also observed in normal lymphocytes pretreated (Day 0) with anti-CD45R antibody, which recognize suppressor inducer subset among CD4+ T cells. However anti-CD45R antibody was no longer effective when the lymphocytes were cultured for more than one day with the antigen, suggesting its effect in the initial phase of the reaction. The intensity of the response induced in normal lymphocytes by the anti-CD45R was comparable to that of the patients sensitized to the nominal antigen. The response of the patients was no longer augmented by the anti-CD45R antibody. Taken together, these data suggest that even normal lymphocytes have potentiality to elicit Df-induced IL2 responsiveness and it is probably derepressed by inhibiting suppressor inducer subset with the anti-CD45R antibody. Also suggested is a defective suppressor inducer activity in the lymphocytes which may lead to hyperreactivity to allergens in asthmatic children.     

Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde.

The mechanism of DNA degradation and its clinical applications were examined. When purified lambda phage and extracted liver DNA were fixed in phosphate buffered formaldehyde, the DNA did not degrade, but there was incomplete digestion with endonuclease. Rat liver tissues were fixed under various conditions and DNA extracted. Immediate fixation with buffered formaldehyde at low temperature, or the addition of EDTA to buffered formaldehyde blocked the DNA degradation. Analysis of pulsed field gel electrophoresis also showed that DNA was degraded before extraction. These results suggest that tissue nuclease has an important role in DNA degradation in tissue. Furthermore, formaldehyde fixation at low temperature, which may take time and which decreases slightly the staining capacity, is useful for the extraction of intact DNA. For clinical application, the detection of provirus was examined. Genomic DNA was extracted from a necropsy sample of adult T cell leukaemia fixed in formaldehyde; human T cell leukaemia virus type-I (HTLV-I) provirus was successfully detected by Southern blotting.     

Different Ca2+ dynamics between isolated hippocampal pyramidal cells and dentate granule cells

The hippocampal formation contains a variety of neuronal types. The principal neurons are granule cells in the dentate gyrus and pyramidal cells in Ammon's horn. These two neuron types show distinct cell morphology and display a different vulnerability to ischemic injury or various neurotoxins. In order to illustrate the difference in the pathophysiological properties of these neurons, we established a method for separately culturing granule cells and pyramidal cells. They were prepared from the dentate gyrus and Ammon's horn of 3-day-old Wistar rat pups and maintained for 7–9 days in culture. After transient exposure to N-methyl-D-aspartate or glutamate, both the cultured neuron populations displayed somatic Ca²⁺ transients with similar amplitudes, but the subsequent recovery to baseline was about twice as fast in granule cells than in pyramidal cells. Similar results were obtained for K⁺ depolarization-induced Ca²⁺ elevation, suggesting that the relatively rapid Ca²⁺ clearance in granule cells is independent of Ca²⁺ influx pathways. The present study provides the first evidence for a difference in Ca²⁺ dynamics and homeostasis between granule and pyramidal cells and may represent a cellular basis for the differential vulnerability of hippocampal neurons.     

Role of the spleen in immunosuppression of gastric cancer: predominance of suppressor precursor and suppressor inducer T cells in the recirculating spleen cells.

In order to analyse the role of the spleen on immunosuppression of gastric cancer, T cell phenotypes in the spleen cells (SC) were investigated by two colour fluorescence flow cytometry, with reference to their suppressor cell activity. Suppressor T cell phenotypes of CD4+2H4+ cells (suppressor/inducer T cells) and CD8+CD11+ (suppressor T cells) were distributed predominantly in SCs from patients with gastric cancer, while they were distributed scarcely in those with liver cirrhosis. Moreover, CD4+2H4+ cells and CD8+CD11+ cells were found predominantly in SCs and splenic vein lymphocytes (SVL) respectively. Among SCs, a significantly higher proportion of CD4+2H4+ cells was found in the recirculating SCs, but fewer were found in the residual SCs. Higher activity of Concanavalin-A induced suppressor cells was found in the former and that of spontaneously activated suppressor cells was found in the latter. These results suggest the suppressor precursor and suppressor/inducer T cells might distribute predominantly in the cells recirculating from the spleen, and that suppressor cells might be matured during the migration from the spleen.     

Monocyte activating factor in sarcoidosis. I. Existence of the factor in sarcoidosis sera

Sarcoidosis sera were found to have the ability to induce normal human monocytes to spread. Gel filtration of sarcoidosis sera on Sephadex G-200 showed that the factor mainly responsible for this activity had a molecular weight of about 70,000. The spreading factor also possessed the ability to increase all cell size of normal human monocytes as well as to increase their phagocytosis and glucose consumption. Accordingly, the spreading factor seems to be considered as a monocyte activating factor. Sarcoidosis sera showed a macrophage migration inhibitory activity, as well. On Sephadex G-200 column chromatography of the sera, the most obvious inhibitory activity was eluted in the fraction with a molecular weight of about 45,000. The macrophage migration inhibitory factor had the ability neither to increase cell size of normal human monocytes nor to increase their phagocytosis and glucose consumption.     

Three-dimensional two-layer collagen matrix gel culture model for evaluating complex biological functions of monocyte-derived dendritic cells

Dendritic cell-like cells (Mo-DCs) generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used as tools to treat cancer patients (DC-vaccines). Because Mo-DCs have multiple antigen presentation-related functions, including phagocytosis, migration, cytokine production, and T cell stimulation, establishment of a method for simultaneously evaluating the various functions of Mo-DCs is important. We developed a new in vitro three-dimensional two-layer collagen matrix culture model that consists of a collagen gel containing Mo-DCs as the lower layer and a collagen gel containing necrotic GCTM-1 tumor cells and/or T cells as the upper layer. We used this system to observe simultaneously multiple functions of Mo-DCs by phase-contrast or fluorescence microscopy and to assess IL-12 secretion during more than 2 weeks of culture. We also observed interactions between Mo-DCs and necrotic GCTM-1 or T cells on an individual cell basis by time-lapse videomicroscopy. In addition, we collected Mo-DCs from the collagen gels by collagenase treatment and analyzed the expression of antigen presentation-related molecules such as HLA-DR, CD80, CD83, and CD86 on Mo-DCs. This model may be a useful tool for evaluation of the various functions of Mo-DCs used as DC vaccines and for studies of the complex behaviors of Mo-DCs in vivo.     

Harderian gland dependency of immunoglobulin A production in the lacrimal fluid of chicken

Involvement of the Harderian gland (HG) in the production of lacrimal immunoglobulin (especially IgA) was investigated. The lacrimal concentration of each immunoglobulin class was not affected by surgical bursectomy but was reduced by cyclophosphamide (CY) and testosterone (TP) treatments. Surgical removal of the Harderian gland caused a remarkable reduction of both the lacrimal concentration of each immunoglobulin class and the specific antibody titre, and and IgA was almost undetectable. The lacrimal concentration of each immunoglobulin class, as well as the specific antibody titre, was not affected by surgical removal of the Lacrimal gland (LG). The route of antigen administration produced no difference in the class of lacrimal immunoglobulin produced. The results indicate that the production of immunoglobulin in chicken tears may be dependent on the HG and that lacrimal immunoglobulin may be synthesized and secreted locally in the HG. Lymphocytes of the HG are of bursa of Fabricius origin and are seeded into the HG prior to hatching and its lymphocytes do not appear to be involved in systemic immunity.     

Rat strain differences in the early stage of porcine-serum-induced hepatic fibrosis.

Rat strain differences in the early development of porcine serum (PS)-induced hepatic fibrosis were histologically and immunohistochemically examined using Brown Norway (BN), Sprague Dawley (SD) and Wistar rats. They were injected i.p. with 0.5 ml sterile PS twice a week for 4 and 8 weeks. In addition, rats treated with physiological saline in the same way served as controls. At 4 weeks, hepatic fibrosis accompanying fibrous septa mainly composed of type III collagens developed in BN and SD rats but not in Wistar rats. In addition, the numbers of eosinophils, CD3-positive cells and ED-1-positive cells significantly increased in BN and SD rats, that of CD45RA-positive cells in BN rats, and that of alpha-smooth muscle actin (SMA)-positive cells in SD rats, respectively. Such differences in the number of inflammatory cells may be related with the absence of hepatic fibrosis in Wistar rats at 4 weeks. At 8 weeks, hepatic fibrosis with formation of many small-sized pseudolobules was observed in all strains at almost similar degree, and the numbers of infiltrating cells increased in all strains of rats with some exception. In addition, the main location of inflammatory cells was different, suggesting a different role of each inflammatory cell in the process of hepatic fibrosis.