Autologous rosette forming cells in patients with renal diseases

We studied the distribution of autologous rosette forming cells (ARFC) in the peripheral blood of 30 healthy adult donors, 30 patients with IgA nephropathy, 20 patients with primary glomerular diseases, eight patients with systemic diseases and 25 patients with other renal diseases. The mean percentages of ARFC were markedly reduced in the IgA nephropathy patients compared with the healthy adult donors. The values for ARFC were even more significantly reduced in IgA nephropathy patients compared with patients with primary glomerular diseases, systemic diseases and other renal diseases. This means that the immunoregulatory abberation in IgA nephropathy primarily involves T cells.     

The relationship between immune rosette forming cells and plaque forming cells in the lizard, Calotes versicolor

The specificity of immune rosette forming cells was assessed using the in vitro blocking of rosette formation with sonicated erythrocyte fragments. An appreciable degree of inhibition of rosette formation against appropriate erythrocytes was observed. The ability of immune rosette forming cells to form plaques was analyzed by separating immune rosettes on a fetal calf serum (FCS) gradient or by separating immune spleen cells on Ficoll-isopyenic gradient. The majority of rosette forming cells were found in a fraction different from that of plaque forming cells. We confirmed these results using a rosette-plaque assay. The data indicate that the majority of antigen-binding cells do not secrete antibody at the peak of immune response.     

Analysis of the immunoglobulin isotype expression by peripheral blood rosette forming cells in chickens

We have analysed the nature of rosette forming cells (RFC) in peripheral blood lymphocytes (PBL) from chickens which had been immunized with SRBC 7 days previously. Although none of the RFC secreted antibody immediately after harvest, a PFC response was detected after in vitro culture in the presence of SRBC. This response was of IgG isotype and was abolished by depleting RFC from cell suspensions prior to culture. In chickens with a partial immunodeficiency (produced by bursectomy 3 days post-hatching) RFC counts and IgG antibody levels were suppressed during the primary response but the in vitro memory response was unimpaired when tested 4 weeks later; thus, RFC appear to be the precursors of antibody secreting but not memory cells. The normal levels of IgM haemagglutinins found in bursectomized chickens suggested that peripheral blood RFC constitute part of the maturation pathway of IgG but not IgM antibody producing cells.

Incubation of primed PBL with anti-M1 (IgM) allotype sera inhibited rosette formation. With PBL from M1^a/M1^b heterozygous chickens, only 50% RFC inhibition was achieved by either anti-M1^a or anti-M1^b serum. This result, when interpreted in terms of allelic exclusion, implies that M1 receptors are endogenous cell products rather than passively acquired molecules. We conclude that RFC in chicken PBL represent B cells which are committed to IgG antibody synthesis and still express a high density of IgM antigen binding receptors at an advanced stage of maturation.

     

Rat thymocyte subpopulations: heterogeneity of the fetal calf serum dependent rosette forming cells

We demonstrate heterogeneity within a rat thymic cortical population, the fetal calf serum dependent rosette forming cells. Approximately half of these cells are guinea pig serum sensitive. Guinea pig serum sensitive and insensitive rosette forming cells show different distribution patterns on a continuous gradient of Percoll. They contain different amounts of TdT enzyme and regenerate with different time kinetics following sublethal irradiation. These data demonstrate new aspects of heterogeneity within the rat thymic cortical population.     

Origin of T lymphocyte colony-forming cells in cell populations depleted of sheep erythrocyte rosette forming cells.

Cell populations depleted of sheep erythrocytes (E) rosette-forming T cells (E-cells) contain cells capable of giving rise to T cell colonies. We have characterized the T cell colony-forming cell from human bone marrow, blood and tonsil E- cells using a T-cell colony assay. Depletion of CD2+, CD3+ or CD4+ cells from E- cells reduced colony formation by 70-100%. Removal of CD8+ cells did not reduce, but rather enhanced colony formation by 50% or more. The most effective reduction (100%) in colony formation was obtained with anti-CD4, indicating that CD4 is a marker of all colony-forming T cells. The CD4+ lymphocytes generated two types of colonies, types I and II, in the presence or absence, respectively, of CD8+ lymphocytes. Type I were small and compact, reached a peak on days 5-7, and contained CD4+ and CD8+ cells. Type II were large and diffuse, reached a peak on days 9-10 and contained CD4+ cells. In continuous culture of single type II colony cells, we observed a consistent increase of CD8+ cells. In one colony the combined percentage of CD4+ and CD8+ cells exceeded 100% (averaging 83% CD4+ and 72% CD8+), indicating the presence of dual markers on some cells. We suggest that colony forming T cells are CD2+ CD3+ CD4+, the CD4+ antigen being the most consistent marker of such precursor cells.     

Characterization of low dose induced suppressor cells in adjuvant arthritis in rats.

Characterization of suppressor cells in adjuvant arthritis was performed by using highly susceptible DA strain rats. The results showed that suppressor cells were induced after a single inoculation of subarthritogenic dose of mycobacterial adjuvant. Relatively long incubation period was required for the induction of suppressor cells. Such cells were predominated in the draining lymph node and, after fractionation, only sIg- population was effective in conferring unresponsiveness. In vivo irradiation or hydrocortisone treatment suggested that low dose induced suppressor cells were resistant against such immunosuppressive treatments. In addition, by using alkyldiamine as a non-mycobacterial arthritogenic adjuvant, it was suggested that unresponsiveness induced by low dose priming with mycobacterial adjuvant was antigen specific.     

Density gradient analysis of human active rosette forming T-lymphocytes

Human peripheral blood lymphocytes have been fractionated on a Percoll density gradient. We found an even distribution of active rosette-forming T-cells among lymphocyte subpopulations of different densities.     

Modulation of E rosette forming activity of human lymphocytes by rosette inhibiting factor (RIF) ann rosette restoring factor (RRF).

The mechanism of action of RIF and RRF on spontaneous rosette formation by human T lymphocytes with SRBC was studied on cells isolated from healthy donors and from patients with active pulmonary tuberculosis. E rosette formation by lymphocytes from healthy donor is inhibited in vitro by RIF. The effect of RIF is reversible since the primary activity of lymphocytes can be restored by contact with RRF. On the other hand, E rosette formation by lymphocytes from tuberculous patients by means of RRF can be depressed by RIF to the starting level. It was found that prostaglandin synthetase inhibitors (indomethacin and RO 20-5720) prevent inhibition or restoration of E rosette activity by RIF and RRF, respectively.     

A simple method for electronic counting of rosette forming human blood lymphocytes.

We investigated the possibility of using a standard electronic laboratory cell counter, of a type which permits manual settings of discriminator and sensitivity levels, for the quantitation of E rosette forming human blood lymphocytes. The electronic counting was performed on rosette specimens prepared from 10 healthy donors, nine lymphoma patients and eight patients with chronic lymphocytic leukaemia (CLL) of B cell type. The simple procedure which was earlier developed for the electronic counting of thymocyte rosettes in guinea-pigs was used and now supplemented with a method for rapid, graphical determination of rosette frequency. The low level of E rosettes in CLL patients was easily distinguished from the higher level seen in the other groups and there was a good agreement between electronic and visual determinations. In samples with a high rosette frequency, the cell counter initially gave 10-30% lower values than when recorded visually. This discrepancy was corrected, however, by a minor adjustment of discriminator level and the introduction of a correction procedure for the presence of double rosettes. We conclude that the electronic counter can well be used for the counting of human rosette forming blood lymphocytes and, due to a high precision and large capacity, this approach would prove to be useful when screening of a large number of samples is required.     

Cells involved in autologous rosette formation in mice. II. Identification of cells forming autologous rosettes in peripheral blood

In the study on mice, we showed that autologous rosette forming cells (ARFC) in peripheral blood belong to two categories. First, representing the bulk of ARFC are "null" cells, the other cells are Lyt 123+. The biological significance of the cells which form rosettes in the periphery remains still unknown.     

Characterization of bone forming cells in posttraumatic myositis ossificans by lectins.

Lectins were used to characterize bone forming cells in posttraumatic myositis ossificans. The lectins applied were as follows: Arachis hypogaea (PNA): specific for beta-D-galactose (1,3)N-acetyl-D-galactosamine (Gal-1,3 GalNac), Canavalia ensiformis (Con A): specific for alpha-D-glucose (D-Glc) and alpha-D-mannose (D-Man) and Wheat germ (WGA): specific for N-acetyl(1,4)D-glucosamine (Glc-NaC) and neuraminic acid. The development of myositis ossificans was characterized by the appearance of a WGA binding cell population. The lectin-binding sites appeared as a cluster in the supranuclear cytoplasm, corresponding to the Golgi-complex. However, the WGA lectin-binding sites disappeared in the mature form of myositis ossificans. We assume that these lectin binding cells may be the bone marrow derived precursors of myofibroblast-like cells which are responsible for bone formation within the damaged muscle.     

高糖作用的肾小球系膜细胞SnoN/Ski蛋白表达及泛素化降解

目的: 观察高糖作用的大鼠肾小球系膜细胞内核转录共抑制因子SnoN/Ski及泛素连接酶Arkadia的表达变化,初步探讨SnoN/Ski泛素化降解在糖尿病肾病中的作用。方法: 将体外培养的大鼠肾小球系膜细胞分别设正常对照组 、高糖甲组(培养基含20 mmol/L葡萄糖)、高糖乙组(培养基含30 mmol/L葡萄糖)、高糖+MG132组(30 mmol/L葡萄糖培养基中预先加入0.5 μmol/L特异性泛素蛋白酶体抑制剂MG132)和甘露醇组。用蛋白免疫印迹技术和免疫荧光染色-激光共聚焦显微镜检测各组细胞SnoN/Ski及Arkadia蛋白的表达。结果: (1)正常系膜细胞中SnoN/Ski蛋白大量表达,Arkadia蛋白表达较弱。(2)高糖作用后SnoN/Ski蛋白表达减弱,Arkadia蛋白表达增强(P<0.05)。(3)与高糖组比较,高糖加入MG132后SnoN/Ski蛋白表达增加,Arkadia蛋白表达减弱(P<0.01)。(4)甘露醇组与正常组比较SnoN/Ski及Arkadia蛋白表达均无明显差异(P>0.05)。结论: (1)高糖可通过泛素蛋白酶体途径降解SnoN/Ski蛋白致其低表达。(2)E3连接酶Arkadia参与了高糖诱导的SnoN/Ski的泛素化降解 。     

骨形态蛋白2体外诱导大鼠骨髓间充质干细胞分化为心肌样细胞

目的 应用骨形态蛋白2(BMP-2)体外诱导骨髓间充质干细胞(MSCs)向心肌样细胞分化,探索MSCs向心肌细胞分化的诱导方法.方法 取SD大鼠四肢骨骨髓,分离培养MSCs,应用BMP-2定向诱导,相差显微镜观察细胞形态学变化,应用免疫细胞化学、激光扫描共焦显微镜技术检测结蛋白(desmin)、α-横纹肌肌动蛋白(α-sareomeric actin)、心肌特异性肌钙蛋白(C-TnT)的表达,透射电镜鉴定.在诱导后7d、21d和28d 3个时间点以半定量RT-PCR方法检测细胞心肌早期转录因子(GATA4)和心肌特异性α-肌凝蛋白重链(α-MHC)的表达.结果 BMP-2诱导后的MSCs细胞伸出伪足,排列方向渐趋一致.MSCs体外经BMP-2诱导后分化的细胞结蛋白、α-横纹肌肌动蛋白、C-TnT均表达阳性,结蛋白、α-横级肌肌动蛋白阳性率较高,分别为37.28%和63.94%,而C-TnT阳性率较低为34.66%.透射电镜下可见到平行排列的肌丝,大量的粗面内质网和线粒体,富含糖原和核糖体.RT-PCR结果显示,GATA4于诱导后7d弱表达,21d表达增强,28d表达减弱.α-MHC在诱导后7d不表达,21d弱表达,28d表达明显.结论骨髓间充质干细胞在BMP-2诱导下可定向分化为心肌样细胞,是自体心肌细胞的一种良好供体来源.     

Feedback inhibition of IgM memory cells by high antigen dose

The ability of high and low antigen doses (SRBC) to recruit IgM memory cells has been compared in several strains of mice. In intact animals priming with low doses was more efficient than priming with high doses. If, however, mice of different strains were X-irradiated two days after priming and repopulated, regardless of whether syngeneic or allogeneic splenocytes were used, they fell into two categories--those in which more memory cells were found after low antigen priming and those in which the reverse was true (Swiss mice). Two possible explanations are offered to explain these interstrain differences--antibody mediated suppression, and generation of suppressor T cells. Our data favor the latter, and we assume that suppressor T cells appear at different intervals after priming in different strains of mice, and that these cells are radioresistant.