Generation of antigen-specific CD4+ T cell lines from naive precursors

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4⁺ T cells from previously unprimed individuals. Freshly isolated CD4⁺ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp 160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (MΦ). In short-term (< 8 days) cultures, CD4⁺ T cells or their CD4⁺, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4⁺, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or MΦ. KLH- and SWM-specific CD4⁺ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and MΦ in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4⁺ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.     

Hematopoietic turnover index in reactive and neoplastic bone marrow lesions: quantification by apoptosis and PCNA labeling

 In order to determine the dynamics of hematopoietic cell turnover, proliferative activity and incidence of apoptosis (programmed cell death) were evaluated in bone marrow trephine biopsies. Selection of patients (20 in each group) included in addition to a control group, idiopathic thrombocytopenia (ITP), reactive thrombocytosis (TH), secondary polycythemia-smokers' polyglobuly (PG), primary (essential-hemorrhagic) thrombocythemia (PTH), polycythemia vera (PV), and finally acute myeloid leukemia (AML). Apoptosis was demonstrated by the in situ end-labeling technique (ISEL) and proliferative activity by applying the monoclonal antibody PC10 raised against proliferating cell nuclear antigen (PCNA). To assess dynamic features of hematopoiesis, an index was calculated consisting of the ratio between PCNA-positive nuclei and the apoptotic cell fraction. This factor was termed the hematopoietic turnover index (HTI). Morphometric analysis revealed that the HTI was significantly increased in AML and PV. According to cell culture studies both disorders are characterized by either a prevalent proliferation of the myeloid or erythroid cell mass. On the other hand, PG, PTH, and TH showed no relevant enhancement of this index in comparison to the control specimen. In vitro experiment results are in keeping with the finding that PG and PTH are not associated with a significant expansion of the erythroid lineage (CFU-E). Similar to ITP and TH, in PTH megakaryocyte proliferation (CFU-MEG) is the predominant feature of cell turnover. Differences between PTH and TH are in line with the reduced in vitro formation of CFU-MEG in the latter disorder. In conclusion, our in situ study on turnover rates of the bone marrow in various neoplastic and reactive lesions extends previous experimental data on hematopoietic cell kinetics. Received: 10 March 1997 / Accepted: 18 May 1997     

Ontogeny and thymus-dependence of T cell surface antigens in Xenopus : flow cytometric studies on monoclonal antibody-stained thymus and spleen

Recently generated anti-Xenopus T cell monoclonal antibodies (mAbs) to the 120 kDA XTLA-1 determinant and against the putative CD5 and CD8 homologues, together with anti-IgM and anti-MHC class II mAbs, are used in dual colour flow cytometric experiments to characterize cell surface antigenic expression on lymphocytes in thymus and spleen of Xenopus laevis during larval and early adult life and also in metamorphosis-inhibited animals. Histological confirmation of T cell emergence early in larval ontogeny is supplied by cryostat sections stained for CD8. Five-day thymectomy i.e. prior to T-lineage cell differentiation in the thymus, abolishes T cell marker expression in the spleen for up to 1 year. Moreover, late larval (20 days) or early adult (3 months) thymectomy (i.e. removal after peripheralization of T cells has occurred) also leads to severe depletion of mAb-defined T cells in the spleen.     

Mast cell activation involves plasma membrane potential- and thapsigargin-sensitive intracellular calcium pools

Summary— The regulation and role of the intracellular Ca²⁺ pools were studied in rat peritoneal mast cells. Cytosolic free calcium concentration ([Ca²⁺]i) was monitored in fura-2 loaded mast cells. In the presence of Ca²⁺ and K+, compound 48/80 induced a biphasic increase in [Ca²⁺]i composed of a fast transient phase and an apparent sustained phase. The sustained phase was partially inhibited by the addition of Mn²⁺. DTPA, a cell-impermeant chelator of Mn²⁺, reversed this inhibition, suggesting that a quenching of fura-2 fluorescence occurs in the extracellular medium. In the absence of extracellular Ca²⁺, the transient phase, but not the sustained one, could be preserved, provided that mast cells were depolarized. The transient phase was completely abolished by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor. Maximum histamine release induced by either compound 48/80 or antigen was obtained in the absence of added Ca²⁺ only when mast cells were depolarized. These histamine releases were inhibited by low doses (< 30 nM) of thapsigargin. Thapsigargin at higher doses induced histamine release which was unaffected by changing the plasma membrane potential, but was completely dependent on extracellular Ca²⁺, showing that a Ca²⁺ influx is required for thapsigargin-induced exocytosis. Together, these results suggest that the mobilization of Ca²⁺ from thapsigargin sensitive-intracellular pools induced by compound 48/80 or antigen is sufficient to trigger histamine release. The modulation of these pools by the plasma membrane potential suggest their localization is close to the plasma membrane.     

The long-term effect of pinealectomy on the crypts of the rat gastrointestinal tract

Abstract: Previously it has been found that rat small bowel crypt cell hyperplasia occurred several weeks after pinealectomy. To determine if this effect was longer-lasting (because of the possible role of the pineal in bowel malignancy) the crypt cell proliferation rate was determined in rat small bowel and colon 6 months after pinealectomy, using a stathmokinetic technique. Although the hyperproliferative effect of pinealectomy was well maintained in the small bowel crypts after 6 months, the hyper proliferative effect in the colonic crypts was much less marked. There is no obvious explanation for these findings, although it is possible that regional differences in levels of gut neuropeptides or melatonin are involved. The mechanism of the effect of pinealectomy on the crypts remains unexplained—in particular, why the effect is so prolonged.     

Differential Distribution of MAP1a and Aldolase c in Adult Mouse Cerebellum

MAP1a is a microtubule-associated protein with an apparent molecular weight of 360 kDa that is found in the axonal and dendritic processes of neurons. Two monoclonal anti-MAP1a antibodies, anti-A and anti-BW6, revealed different epitope distributions in the adult mouse cerebellum. Anti-A stained Purkinje and granule cells uniformly throughout the cerebellum. In contrast, anti-BW6 selectively stained the dendrites of a subset of Purkinje cells, revealing parasagittal bands of immunoreactivity in the molecular layer. The compartmentation of the BW6 epitope was compared to the Purkinje cells as revealed by immunostaining with anti-zebrin II, a well known antigen expressed selectively by bands of Purkinje cells. The anti-BW6 staining pattern was complementary to the zebrin II bands, the zebrin II^- Purkinje cells having BW6⁺ dendrites. These results demonstrate that MAP1a is present in two forms in the mouse cerebellum, one of which is segregated into parasagittal bands. This may indicate a unique MAP1a isoform or may reflect differences in the metabolic states of Purkinje cell classes, and regional differences in their functions.     

重症肌无力患者胸腺肥大细胞的形态数量和超微结构研究

为探讨研究重症肌无力（ＭＧ）患者胸腺内肥大细胞的形态数量和其超微结构变化与ＭＧ发病的关系。方法对２８例ＭＧ患者作胸腺肥大细胞的形态半定量分析和光镜观察，其中１６例的胸腺进行电镜检查。结果ＭＧ胸腺不仅表现组织学异常，且肥大细胞数量明显增多，尤其是在非增生胸腺内出现大量肥大细胞，而在增生的髓质和生发中心区几乎见不到。电镜观察提示肥大细胞与上皮细胞、Ｔ淋巴细胞和毛细血管关系密切，且含多种形态的分泌颗粒。结论肥大细胞对胸腺细胞的分化成熟、胸腺屏障和胸腺功能，以及ＭＧ发病均起到一定的重要作用。     

Frequency of T-Cell Progenitors in Nude Mice

The hypothesis that prothymocytes are distinct from and regulated independently of multilineage hemopoietic progenitors was tested by enumeration of these two cell populations in normal versus congenitally athymic (nude) mice. The absence of a thymus and of peripheral T cells in nude mice had no effect on the frequency of either multilineage progenitors (day 12 CFU-S) or prothymocytes (CFU-T), suggesting that there is no feedback regulation of CFU-T frequency. Thymus seeding from the bone marrow is therefore likely to be regulated by the availability of niches for prothymocyte maturation, rather than by feedback control of prothymocyte production.     

The mitogenic effect of KGF and the expression of its cell surface receptor on cultured normal and malignant human oral keratinocytes and on contiguous fibroblasts

This study examined the mitogenic response to keratinocyte growth factor (KGF) of normal and tumour-derived human oral keratinocytes in which the degree of cellular differentiation was known and in contiguous fibroblast cultures derived from the malignant epithelial cultures. Keratinocytes, but not fibroblasts, were stimulated by KGF. There by demonstrating epithelial target cell specificity of the ligand. KGF-induced stimulation of the tumour-derived keratinocytes cultured in the absence of the 3T3 fibroblast support broadly correlated with the degree of cellular differentiation; well-differentiated keratinocytes were stimulated more by KGF than their less differentiated counterparts. Malignant oral keratinocytes expressed KGF cell surface receptors (K_D 451-709 pM; receptors/cell 2306-413645), but KGF receptor mRNA did not correlate with either KGF-induced mitogenesis or the degree of epithelial cell differentiation. When the tumour-derived keratinocytes were cultured in the presence of 3T3 fibroblasts, the mitogenic response to KGF was comparable to normal epithelial cells. The results suggest that KGF-mediated growth stimulation may not be significant in providing a selective advantage for the growth of malignant keratinocytes.