Normal Human Lymph Node Cells: An Electron Microscopic Study

Lymph nodes, from 15 patients undergoing surgery for conditions not related to lymphoid tissue disease, have been examined with the electron microscope. The human lymph node cell types—including lymphocytic, reticularand plasma cells—have been described at low and medium electron microscopic magnifications, and the criteria for their identification are discussed. Thecharacteristic features outlined for identification of these cell types provide abasis for comparison with pathologically altered lymph node cells.

Submitted on May 4, 1965 Accepted on September 25, 1965     

Brief Report: Nuclear Bodies of Normal and Pathological Human Lymph Node Cells: An Electron Microscopic Study

Nuclear bodies in normal and pathologic human lymph node cells have beenexamined with the electron microscope and their structure has been illustratedand described. In normal lymph node cells, nuclear bodies are 0.3-0.5 micronsin diameter, are slightly less electron dense than the nucleolus, and consist ofperipheral fibrillar material with centrally located, dense granules, 200-400 Åin diameter. Morphologically abnormal nuclear bodies have been observedin a case of Hodgkin’s disease. The appearance of these atypical bodies wouldsuggest either contact and fusion of two or more atypical bodies, or possiblythe existence of single, large, irregular bodies.

Submitted on June 24, 1966 Accepted on August 26, 1966     

Response of Human Insulinoma Cells to Extracellular Calcium Is Different from Normal B Cells

The preoperative determination of thelocalization of a small insulinoma is sometimesdifficult using routine imaging techniques. We have usedthe selective arterial calcium injection (SACI) test todetermine the location of the tumor preoperatively. Thepathophysiologic basis of the SACI test is based on theresponsiveness of insulinomas to calcium injected intothe feeding artery. In this study, we demonstrated the in vitro response of the insulinoma cellsto the extracellular calcium challenge by usingprimary-cultured insulinoma cells. Human insulinomacells were obtained from three patients. MIN6 cells(normal pancreatic B cells) were used as a control;their insulin response to various stimuli resembles thatof normal B cells. The insulin secretory dynamics inresponse to extracellular calcium were observed using a perfusion system. Second, the change ofthe concentration of cytosolic free calcium([Ca²⁺]_i) was monitored byfluorometry using fura-2/AM. When the concentration ofextracellular calcium ([Ca²⁺]_o) was changed from 2.54 mM to 10 mM, insulinsecretion from the insulinoma cells was markedlyincreased within 6 min (10- to 18-fold at maximum), andrapidly returned to the basal level; at the same time, [Ca²⁺]_i was immediatelyelevated and reached a peak within 1 min. In contrast,in the MIN6 cells, the insulin secretion and [Ca2+]iwere not significantly changed when[Ca²⁺]_o was switched to 10 mM. The results of these in vitro experiments agreedwith the clinical results of the SACI test. The positiveresponse of the insulinoma to the SACI test is probablydue to the different response of insulinoma cells to the extracellular calcium challengecompared with normal B cells. The role of[Ca²⁺]_i may be important in themechanism underlying the SACI test.     

日本血吸虫感染小鼠肠系膜淋巴结Th17细胞的免疫应答

目的观察C57BL/6小鼠感染日本血吸虫后肠系膜淋巴结Th17细胞的免疫应答。方法 20只C57BL/6小鼠随机分为感染组和对照组,每组10只,感染组小鼠经腹部皮肤感染日本血吸虫尾蚴,每鼠(40±5)条。感染后5~6周分离小鼠肠系膜淋巴结的淋巴细胞,分别用抗小鼠CD3单克隆抗体(anti-CD3,1μg/ml)和抗小鼠CD28单克隆抗体(anti-CD28,1μg/ml)刺激,培养4 h后收集细胞,RT-PCR检测小鼠肠系膜淋巴结淋巴细胞中白细胞介素17(IL-17)和维甲酸相关孤独受体(ROR-γt)mRNA的转录水平;培养72 h后,ELISA检测细胞培养上清液IL-17和γ干扰素(IFN-γ)的含量。同时用佛波酯(PMA,10 ng/ml)和离子霉素(1μg/ml)刺激淋巴细胞5 h后,胞内细胞因子染色,流式细胞术检测Th17细胞的含量和其他细胞因子的产生。结果 ELISA检测结果显示,感染小鼠肠系膜淋巴结培养物上清液中IFN-γ[(214.3±62.6)pg/ml]和IL-17[(176.8±62.1)pg/ml]的含量明显高于健康小鼠[(46.7±13.9)和0 pg/ml](P<0.05)。RT-PCR检测结果显示,IL-17和ROR-γt mRNA转录水平也明显高于健康小鼠。感染小鼠肠系膜淋巴细胞CD4+T细胞中,Th17细胞的比例为(0.55±0.03)%,明显高于健康小鼠[(0.16±0.01)%](P<0.05)。在CD4+T细胞中,IL-17+IL-4+细胞占0.06%,IL-17+IFN-γ+和IL-17+IL-5+细胞各占0.02%,IL-17+IL-9+细胞占0.01%,未检测到IL-17+IL-10+和IL-17+Foxp3+细胞。结论日本血吸虫感染C57BL/6小鼠的肠系膜淋巴结能诱导Th17细胞产生。Th17细胞能分泌IL-4,及少量的IFN-γ、IL-5和IL-9,不分泌IL-10,也不表达Foxp3。     

Possible Species Differences in RNA Metabolism of Lymph Node Cells

Differences in the rates of incorporation of tritiated uridine U-H³ oflymph node cells of different species have been found. Plasma cells fromhumans, guinea pigs, mice, and rabbits have a higher rate of incorporationof U-H³ than do plasma cells from rats, whereas human blast cells have alower rate of U-H³ incorporation than blast cells from the various rodents.

Submitted on July 31, 1964 Accepted on October 12, 1964     

胃腺癌局部淋巴结中树突状细胞对转移和预后的影响

目的:研究胃腺癌局部淋巴结中树突状细胞(DC)对转移和预后的影响。方法:将S-100蛋白作为DC特异性标记物,应用S-P免疫组化方法检测胃腺癌局部淋巴结中DC的数量和分布。结果:转移组淋巴结中DC较非转移组明显减少。在57例转移组中,DC显著者18例,5年生存率55.56％;不明显者39例,5年生存率23.08％。30例非转移组中,DC显著者19例,5年生存率73.68％;不明显者11例,5年生存率36.36％。经x~2检验,在上述两组中,DC显著者的转移和5年生存率与不明显者的差异均有显著性意义(P<0.05)。结论:胃腺癌局部淋巴结中DC程度同转移和预后密切相关。     

Outflow Paths of Cells from the Lymph Node Parenchyma to the Efferent Lymphatics

Observations in the mouse lymph nodes using thin section histology made the authors conclude that the outflow of lymphocytes to the efferent lymphatics is probably a passive desquamation of cells. In the medullary sinuses this outflow seems to be restricted to circumscribed desquamation zones. The authors describe a specific type of structure, ad interim named ‘mud streams’, which probably represents a concentration of outflowing lymphocytes in extended desquamation zones, reaching deep into the parenchyma as temporary first segments of the efferent sinus system. These structures, which are difficult to recognize in routine, thick sections are very spectacular in 1 μ sections, together with the high endothelium venules they decide the specific morphologic pattern of the paracortical area of lymph nodes.     

Effects of Long-time Exposure to Lipopolysaccharide on Intestinal Lymph Node Immune Cells and Antibodies Level in Mice

Background: Endotoxin, widely present in the living environment of humans and animals, leads to endotoxemia during a short period. However, the long-term effects of endotoxin on immune function are unclear. Objective: To determine the importance of long-term endotoxin treatment on function of immune system. Methods: The mice were treated with different doses of lipopolysaccharide (LPS) for a month; the collected samples were then analyzed in terms of value changes in hematological parameters, lymphocyte subtypes, and immunoglobulins level. Results: The number of monocytes (MONO) and neutrophils (NEU) in the three treatment groups was significantly lower than the control after 30 days. However, the proportion of CD8+ T lymphocytes showed a rising trend in the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) while the CD4+ T cell was reduced. At the same time, a decrease was observed in the percentage of CD19+CD38+ B lymphocytes. Interestingly, the change of lymphocytes in PPs was more significant than that in MLNs, suggesting that immune response in the PPs occurred before the MLNs. Consistent with the changes in B cells, the content of IgA and IgG showed a downward trend. Conclusion: Long-term exposure to low-dose endotoxin had little or no effect on the immune function of the body, suggesting that the endotoxin can be rapidly eliminated by the immune system. Nonetheless, the number of immune cells was reduced in the high-dose group. T- and B-lymphocytes were significantly reduced, resulting in a decrease in immunoglobulin level, and showing a significant immune suppression state.     

一种简便的人正常胃黏膜上皮细胞原代培养方法

背景：原代培养的人正常胃黏膜上皮细胞与体内姊妹细胞相似，是进行胃生理和病理研究的理想工具，但我国尚未见简便、可靠的人正常胃黏膜上皮细胞原代培养方法的报道。目的：探讨简便的人正常胃黏膜上皮细胞原代培养方法。方法：以胶原酶Ⅱ和分散酶磁性搅拌分离人正常胃黏膜上皮细胞。以甲基噻唑基四唑（M1Tr）比色法检测细胞活力，以观察细胞大体生长过程；以溴脱氧尿苷（BrdU）标记法检测S期细胞数量，以观察细胞微观增殖能力。以PAS染色鉴定胃黏膜上皮细胞黏原颗粒；以细胞角蛋白（CK）-18免疫荧光染色鉴定上皮细胞。以光学显微镜和透射电子显微镜观察细胞形态结构。初步观察传代细胞的贴壁生长情况。结果：培养第2～3d．胃黏膜上皮细胞活力增幅最大．第4d达最高点，之后逐渐下降；BrdU孵育18h后，33．3％的细胞处于和曾经处于S期。接种密度高时，细胞PAS染色均呈紫红色，可见细胞核旁黏原颗粒；接种密度低时，仅成团细胞PAS染色呈阳性；培养第3d，绝大部分细胞CK-18阳性。接种第2d，胃黏膜上皮细胞成团簇状生长，增殖迅速，第4d后逐渐停止生长．第5d开始出现漂浮死亡细胞，最终完全被成纤维细胞取代；透射电子显微镜可见微绒毛、分泌颗粒、连接复合体等上皮细胞特征。传代胃黏膜上皮细胞增殖、铺展能力较原代细胞降低。结论：磁性搅拌酶分离培养可得到数量多、纯度高的人正常胃黏膜上皮细胞。     

Colony Formation by Normal and Leukemic Human Marrow Cells in Culture: Effect of Conditioned Medium From Human Leukocytes

"Conditioned medium" obtained fromcultures of human peripheral leukocytespromoted the growth of human marrowcells in cell culture. This material alsopermitted the growth of small coloniesfrom the marrow of patients with acutemyelogenous leukemia in relapse; in itsabsence, only occasional colonies wereobserved.

Submitted on June 30, 1970 Accepted on July 24, 1970     

Cell Migration Inhibition in Human Lymphomas Using Lymph Node and Cell Line Antigens

In an attempt to demonstrate in vitroreactions against autologous and allogenic lymph node (LN) extracts inlymphoma patients, 15 cases werestudied using as controls two normalLN extracts and leukocytes from 17blood donors. The techniques appliedwere migration inhibition tests carriedout directly on peripheral leukocytesand indirectly using lymphocyte culture supernatants on guinea pig peritoneal macrophages. The antigens(Ag) used were partially purified salineextracts of neoplastic LN, culturedneoplastic cells of lymphosarcoma(LS) origin, and normal LN. It wasobserved that autologous LN extractselicited migration inhibition in the fourcases of LS studied and in four out ofsix cases of Hodgkin’s disease (HD),while normal LN extracts gave negative results in these patients. Positivereactions were observed in four out ofsix LS cases using allogenic LS extracts, either from LN or from cell lines.No cross-reactions could be demonstrated in HD. All Ag gave negativeresults with leukocytes from normalblood donors. The positive autologousreaction suggests the presence of atumor Ag in neoplastic LN, while theimmunologic cross-reaction betweensome of the LS patients would pointto a common Ag, persisting in at leastone of the LS cell lines and perhapsrelated to a virus.

Submitted on July 15, 1971 Accepted on September 12, 1971     

Rates of Proliferation and Interrelationships of Cells in the Mesenteric Lymph Node of the Rat

Single and multiple injections of tritiated thymidine were combined withradioautography to study the rates of proliferation and interrelationships ofthe various cell lines in the mesenteric lymph node of the rat. The appearanceand labeling patterns of the different cells are described from studies of bothsmears and tissue sections. Reticular cells exhibit wide variations in labelingintensity, phagocytize labeled lymphocytes, and become labeled in highpercentages only when TTH is administered over a period of many days.Other slowly proliferating cell types include small lymphocytes, fat cells,endothelial cells and mast cells.

Rapidly proliferating cell lines include plasmablasts, hemohistioblasts, proplasmacytes and large lymphocytes. The generation time of plasmablasts andhemohistioblasts was determined to be approximately 9 and 12 hours respectively. Mature plasma cells constitute a non-dividing population which isrenewed in lymph node in not more than 5 days.

Evidence is presented that the most primitive cells in the lymphocyte andplasma cell lines are the hemohistioblasts and plasmablasts respectively.Reticular cells most probably are not stem cells. No evidence is found tosupport previous reports that plasma cells derive from lymphocytes.

Submitted on April 26, 1963 Accepted on July 5, 1963     

Development of Cellular Immunity in the Human Fetus: Dichotomy of Proliferative and Cytotoxic Responses of Lymphoid Cells to Phytohemagglutinin

The reactivity of cells in vitro was investigated with specimens from various lymphoid organs of seven human fetuses. Thymocytes responded to stimulation by phytohemagglutinin with significant increases in synthesis of DNA, but failed to produce destruction of xenogeneic target cells. In cells from bone marrow, precisely the converse pattern of reactivity to the mitogen was detected. Lymphocytes from spleen and peripheral blood demonstrated both phytohemagglutinin-dependent functions, while hepatic cells did not respond to phytohemagglutinin. Based on the striking dichotomy of phytohemagglutinin-dependent responses in fetal thymocytes and bone-marrow lymphoid cells, we conclude that phytohemagglutinin-dependent cytotoxicity and DNA synthesis are functions of different populations of lymphoid cells during human embryonic development.     

Human Fetal Endothelial Cells in Culture

Human endothelial cells were isolated from the umbilical cord vein by collagenase treatment and cultured for periods up to 6 weeks. The cultured cells were identified as endothelium by cell morphology and growth pattern, the presence of Weibel-Palade bodies, and their ability to stimulate allogeneic lymphocytes (Hirschberg et al 1974). Cultured fibroblast-like cells derived from the umbilical cord were clearly different in all three respects. Approximately one third of the primary endothelial cultures showed clear evidence of proliferation during the first 3–4 days in culture as judged by cell counting. Replicating ability in a culture was correlated with cell density at the time of seeding. Autoradiography of endothelial cells after exposure to ³H-thymidine showed a 30-fold increase in nuclear labelling from day 1 to day 3 in culture. The endothelial cells have so far been subcultured three times.     

Response to Prednisolone of Cultured Immunoglobulin-producing Human Lymphoid Cells

The survival of asynchronously growinglymphoid cells (T₁ cells) in vitro decreasedto 50% viability after 1 hr treatment withconcentrations of prednisolone above 10µg/ml. Treatment with prednisolone for24 hr produced a decrease in cell survivalto a plateau of 30% viability for concentrations above 10 µg/ml. After 48 hrtreatment, 10 µ/ml prednisolone reduced viability to 12%. Synchronouslygrowing T₁ cells were least sensitive toprednisolone treatment (50 µg/ml) during S phase (DNA synthesis period) andmost sensitive during G₁ (pre-DNA-synthesis period). This cell cycle specificity ispostulated to be an explanation for thepresence of prednisolone-resistant cells inasynchronously growing populations. Immunofluorescence studies on the effect ofprednisolone (100 µg/ml) on immunoglobulin production indicated there wasno correlation between the length of drugtreatment and reduction in the percentageof immunoglobulin-producing cells. Theseresults suggest that the mechanism ofprednisolone-induced immunosuppressionin lymphoid cells is primarily lymphopenia.

Submitted on November 30, 1973 Revised on January 23, 1974 Accepted on January 28, 1974     

DNA Polymerases in Normal and Leukemic Human Hematopoietic Cells

DNA polymerase activities were assayedin bone marrow cells and peripheral leukocytes from normal people and patientswith acute myelogenous, chronic lymphocytic, and chronic myelogenous leukemia.Extracts of subcellular components werefractionated by velocity sedimentationthrough sucrose density gradients andassayed using activated DNA as template.Two major DNA-dependent DNA polymerases were found in human cells withmolecular weights of approximately50,000 and 200,000 daltons, respectively.The DNA polymerase of high molecularweight is located in the soluble cytoplasmic fraction and is inhibited byN-ethylmaleimide. The low molecularweight polymerase is detected in extractsof nuclei and in the soluble fraction. It isresistant to inhibition by N-ethylmaleimide. In all cell types tested, total DNApolymerase activities were much higher incytoplasmic than in nuclear extracts.Lymphocytes purified from normal peripheral blood had three to four times asmuch of both the high and low molecularweight polymerase activities per cell aspurified granulocytes. Leukemic myeloblasts had 10 to 20 times as much cytoplasmic DNA polymerase activity as moremature leukocytes from normal peripheralblood. In general, immature granulopoietic cells contained higher total DNA polymerase activities than more maturegranulocytes, and the major increases inpolymerase activities were in the highand low molecular weight cytoplasmicenzymes rather than in the nuclearenzyme.

Submitted on October 9, 1973 Revised on January 23, 1974 Accepted on January 28, 1974