Phenotypic Analysis of the Chicken Thymic Microenvironment During Ontogenic Development

The development of monoclonal antibodies (mAb) reactive with the thymic microenvironment has identified distinct subpopulations within the stromal component, but the function of these subregions in intrathymic T-cell differentiation remains essentially an enigma. In this study, we have used such a panel of mAb to examine the chicken thymus during ontogenic development to gain insight into the contributions of these thymic regions to the distinct phases of T-cell development and to further characterize the development of this organ. Our reagents have demonstrated the complex differentiation of the primitive endodermal epithelium into more specialized structures and the development of other thymic stromal components from mesectodermal cells. We also describe molecules localized to the subcapsular and perivascular regions, which have an ontogenic expression corresponding to the early localization and stimulation of thymic precursors and another molecule on the medullary vasculature expressed corresponding to the exit of mature cells from the thymus. In addition, two markers of distinct medullary epithelial clusters are initially expressed corresponding to the appearance of T-cell receptor-1 (TcR-1) and TcR-2 positive cells in the medulla, respectively. These mAb potentially represent excellent reagents for further definition of the thymic modulation of T-cell differentiation.     

Thymic epithelium tolerizes chickens to embryonic graft of quail bursa of Fabricius

We demonstrated previously that isotopic and isochronic grafts of the quail bursa of Fabricius rudiment performed at 5 days of incubation (E5) into chick embryos resulted in the development of a chimeric bursa whose chick host B lymphocytes and accessory cells differentiated in a foreign, quail epithelial environment. Such animals reject their grafted bursa by the age of 2-3 weeks post-hatching (1,2). Isotopic embryonic grafts of the thymus epitheliomesenchymal anlagen from the quail donor of the bursal rudiment were carried out at E4.5 (before their colonization by hemopoietic precursor cells), following partial or complete host thymectomy. The quail thymic epithelial stroma was accepted and invaded by chick hemopoietic precursor cells that further differentiated into lymphocytes and dendritic cells. Tolerance of the foreign bursa was induced in such thymobursal chimeras. This demonstrates that the thymic epithelium has the capacity to induce tolerance of xenogeneic rudiments when both grafts are implanted at early stages of embryonic development. We also report on the production of two birds in which removal of the chick host thymus was complete thus generating chimeras in which host T and B lymphocytes differentiated in a completely xenogeneic epithelial environment.     

Stromal Elements for Tumor Diagnosis: A Brief Review of Diagnostic Electron Microscopic Features

Tumor diagnosis mainly depends on the appearance of the tumor cells in recapitulating the appearance of primordial cells from which they arise. However, certain tumors may present with specific stromal changes that may assist/enhance the diagnosis. In this presentation, diagnostic stromal features have been reviewed. The cytoplasm is enclosed by a unit membrane, which serves as a barrier to, as well as an interface with, surrounding structures. Epithelial cells usually show characteristic basal–apical orientation. In mesenchymal tissue, different types of interface can be found in different types of mesenchymal tissue. External lamina can be defined as an anatomic structure, which encloses anatomic functional units. In epithelial tissue, cells in a functional unit are enclosed within a well-defined external lamina (EL). In malignant epithelial tumors, EL can become increasingly indistinct as tumors become less differentiated, and one has to look for it diligently. Within the external lamina, epithelial cells are closely packed with closely apposed cell membranes and cell attachment junctions. In contrast to epithelial tissue, mesenchymal tissue is usually characterized by the stromal elements they produce. Individual cells are embedded in the stroma, and individual mesenchymal cells represent the functional unit. Vascular endothelial cells are an exception since their relationship to stroma resembles to that of epithelial cells. Thus, tumors deriving from mesenchymal cells known to have external lamina such as muscle cells and Schwann cells tend to show total enclosure of cells by external lamina. In malignant muscle tumors, external lamina production can be focally present and found only by diligent search. In Schwann cell tumors, the presence of EL is prominent in low-grade tumors and more irregular and variable in malignant tumors. In the latter, stromal aggregation of scrolls of external lamina can be characteristic. Similar features are seen in ossifying fibromyxoid tumors. Fibronexus junctions (composed of extracellular fibronectin fillements linking intracellular 5-nm filaments) is claimed to be typical of myofbroblasts. Finding them in spindle cell tumors justifies a diagnosis of myofibroblastomas. There have been several stromal changes diagnostic for certain tumors found only by electron microscopy. Fibrous long-spaced collagen (known as Luse bodies) is diagnostic for peripheral nerve sheath tumors, but they can rarely be found in other tumors. Luse bodies usually appear as focally as crystallized aggregates apart from the regular collagenous interstitial stroma. They should be distinguished from other nonspecific long-spaced collagen changes. The changes are diffusely stromal in contrast to Luse bodies. Spiny collagen and amianthoid fibers are interesting collagen fibrils and their diagnostic value is questionable. Skeinoid fibers (SF) are short-spaced collagen of 41- to 45-nm banding so-named because of their peculiar appearance by electron microscopy simulating skeins of yarn. They were originally described in neurogenic tumors and small intestinal stromal tumors with features of gastrointestinal autonomic nerve tumors (GANT). Although there have been a few sporadic case reports of the presence of skeinoid fibers in nonneurogenic tumors, the frequent presence of SF in spindle cell tumors signifies their neurogenic nature in this authors’ experience. An exception to this is that SF can be a constant element of rare ciliary body tumors known as ciliary mesectodermal leiomyomas, in which tumor cells show some resemblance to smooth muscle as well as Schwann cells. In addition to SF, several other types of peculiar crystallized collagen were observed in GANT tumors, particularly those with multiple tumor syndromes such as neurofibromatosis and Carney's triad. They simulate the appearance of railroad tracks or centrosomes. The reason for this is not known. The authors speculate that such collagen crystallization may be caused by genetic alterations involving collagenosis. Further studies will be necessary to clarify their pathogenesis. Another peculiar stromal change is electron-dense stromal filamentous aggregates with extra-long banding of > 250-nm periodicity previously described in Ewing sarcomas. This stromal change simulating a tiger skin pattern is also seen in primitive neuroectodermal tumors and malignant melanomas. In view of continually new discoveries of stromal changes that can be used for the differential diagnosis of tumors, the importance of close evaluation of stromal elements of tumors, and diligent application of electron microscopy in tumor diagnosis cannot be overemphasized.     

Phenotypic Mapping of The Chicken Embryonic Thymic Microenvironment Developing Within an Organ Culture System

The chicken thymic microenvironment, as it developed in an embryonic thymus organ culture system, was phenotypically mapped using a panel of mAb defining both epithelial and nonepithelial stromal cell antigens. We have previously reported that thymocyte proliferation and differentiation will proceed for up to 6–8 days in thymus organ culture, hence demonstrating the functional integrity of the thymic microenvironment in vitro. During this time, the stromal component reflected that of the normal embryo with cortical and medullary epithelial areas readily identifiable by both morphology and surface-antigen expression. An abundance of subcapsular and cortical epithelial antigens was detected in the cultured thymus, particularly those normally expressed by the epithelium lining the capsule, trabeculae, and vascular regions (type epithelium) in the adult and embryonic thymus. Medullary epithelial antigens developed in organ culture, although were present in lower frequency than observed in the age-matched embryonic thymus. MHC class II expression by both epithelial and nonepithelial cells was maintained at high levels throughout the culture period. With increasing time in culture, the ratio of epithelial to nonepithelial cells decreased, concurrent with a decrease in thymocyte frequency and suggestive of a bidirectional interaction between these two cell types. Thus, a functionally intact thymic microenvironment appears to be maintained in embryonic thymus organ culture, a model that is currently being exploited to assess the role of stromal antigens, as defined by our mAb, in the process of thymopoiesis.     

抗鸡免疫球蛋白(IgM、IgG)单克隆抗体的制备与鉴定

本文报道将SRBC免疫鸡的血清经33％硫酸铵沉淀,再经Sephadex G-200凝胶柱两次层析,收集第Ⅰ,Ⅱ蛋白峰作为免疫原免疫BALB/c小鼠。建立了2株(3A7、1C4)分泌抗鸡IgG和4株(6AS、5E2、6C7、6B1)抗鸡IgM特异性单克隆抗体的杂交瘤细胞系;琼脂扩散试验表明6株细胞系分泌的单克隆抗体均为IgG1亚类。所制腹水的ELISA滴度达10~5～10~6。用免疫印迹技术(IB,Immunobllotting)对其中2株(3A7和6A5)的特异性作了进一步鉴定。     

4种抗支原体单克隆抗体免疫学特性的研究

为了有效地控制细胞质量、我们从1987年开始、相继建立了抗口腔支原体(M.orale),精氨酸支原体(M.arginini),猪鼻支原体(M.hyorhinis).和莱氏无胆甾原体(A.laidlawii)的杂交瘤细胞系共20株。并用IFA法、BA法或APAAP法对单克隆抗体进行了检测和鉴定。单克隆抗体(McAb)亚类鉴定结果:8株为IgG1,2株为IgG2b,其余10株为IgM。抗体效价测定大多数在4000～8 000之间。利用支原体纯培养的菌落进行了IFA染色试验,封闭试验,生长抑制试验和免疫电镜检查,均证实了它们的特异性,并获得2株对同属支原体有抗原交叉的McAb(3D1和3A11)。利用上述McAb检测实验室常用细胞的支原体污染,较常规法和DNA荧光染色法简便,快速,特异性较高。     

抗人乳腺癌单克隆抗体HD1的特性

HD1是由1株抗人乳腺癌杂交瘤腹水扩增,亲和层析纯化得到的单克隆抗体,分泌抗体(IgG)的蛋白含量:上清为10μg/ml,腹水为1.5mg/ml。用ABC法检测,HD1与3株人乳腺癌细胞系呈阳性反应,而与胃癌、结肠癌等细胞和正常成纤维细胞、淋巴细胞呈阴性反应。组织化学染色显示对部分乳腺癌石蜡切片为阳性,而对多种正常组织为阴性。HD1对3株乳腺癌细胞系的亲和力在1.3×10~(-8)～2.7×10~(-8)mol/L之间。免疫印迹实验表明,HD1可使乳腺癌细胞MCF-7和475-A细胞膜Mr为60000的抗原组分染色,使另一株乳腺癌细胞BCaP-37 Mr为56000和94000的组分着色。对3株乳腺癌细胞有抗体介导的细胞毒效应(18％～27％)。以上的研究有助于对HD1性质及其相对应抗原的了解,为抗乳腺癌单抗的应用提供了可靠的依据     

Phenotypic characteristics of a population of normal killer cells of autologous erythrocytes

Laboratory of Biophysics, Institute of Biophysics, Siberian Branch, Academy of Sciences of the USSR, Krasnoyarsk. (Presented by Academician of the Academy of Medical Sciences of the USSR I. A. Terskov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 1, pp. 62–64, January, 1989.     

Thymic Medulla Epithelial Cells Acquire Specific Markers by Post-Mitotic Maturation

The development of thymocyte subsets and of the thymic epithelium in SCID and RAG-2^-/– mice was monitored after normal bone-marrow-cell transfer. The kinetics of thymic reconstitution and their relationships with cell proliferation were investigated by using bromodeoxyuridine to detect DNA-synthesizing cells among lymphoid cells by 3-color flow cytometry, and in epithelial compartments by staining frozen sections. Thymocytes started to express CD8 and CD4 10 days after transfer, simultaneously with extensive proliferation. The first mature CD4⁺ single-positive cells were generated, from resting CD4⁺CD8⁺ cells after day 15. During this day 10–15 period, many epithelial cells positive for cortexspecific or panepithelial markers were labeled with BrdUrd after pulse-injection. Organized medullary epithelium also developed after day,15, that is, synchronously with the appearance of mature thymocytes, but medullary cells were never found BrdUrd⁺. These results suggest that, in these models, the reconstitution of the thymic epithelial network proceeds through expansion of preexisting cortical or undifferentiated cells and by later maturation (acquisition of specific markers) of medullary cells. This last process is dependent of the presence of mature thymocytes.     

Characterization of murine complement receptor type 2 and its immunological cross-reactivity with type 1 receptor.

We have previously demonstrated that some mAbs prepared against mouse complement receptor type 1 (CR1) bind a 150,000 Mr protein in addition to the 190,000 Mr CR1 protein. We now identify the 150,000 Mr murine protein as complement receptor type 2 (CR2), since: (i) one of the monoclonal antibodies that bind this protein inhibits rosette formation between mouse B cells and C3d-bearing sheep erythrocytes; (ii) as is known for human CR2, this protein is present on B lymphocytes but not T lymphocytes; and (iii) this protein must have affinity for C3b, since it has weak factor I cofactor activity. In addition, this protein resembles the 145,000 Mr human CR2 molecule in size. Since four of the five mAbs that were produced by immunization with CR1 also bound CR2, and they bind to different CR1 epitopes, it seems that murine CR1 and CR2 share multiple epitopes. Injection of mice with one of the CR1-CR2 cross-reactive mAbs almost eliminated both CR1 and CR2 expression, but did not decrease B cell numbers or the expression of B cell IgM, Ia, or B220 antigens. In contrast, injection of mice with a non-cross-reactive anti-CR1 antibody only modulated CR1 expression. These antibodies should thus provide useful tools for the study of the in vivo roles of B cell complement receptors.     

In Vitro and In Situ Characterization of Fish Thymic Nurse Cells

We present an enzyme- and immuno-cytochemical, and ultrastructural characterization of trout thymic nurse cells (TNCs). Our data suggest that isolated trout thymic multicellular complexes are epithelial cells with acidic compartments that may be involved in the processing of antigens and in the generation of the MHC-II proteins that these cell express, and also that isolated TNCs are the In Vitro equivalent of the pale and intermediate electronlucent epithelial cells located in the inner zone of the trout thymus, constituting indirect evidence of the phylogenetical relationships of the inner zone of the teleost thymus with the thymic cortex of higher vertebrates.     

抗人IgD单克隆抗体的制备及其特性鉴定

本文采用Ultrogel AcA34凝胶过滤,DEAE离子交换及亲和层析法,从骨髓瘤病人血清中分离高纯度的IgG,以此为抗原免疫小鼠,利用常规杂交瘤技术制备了3株分泌抗人IgD单克隆抗体的杂交瘤细胞株,分别命名为ZD2、ZD61和ZD94,此3株杂交瘤细胞分泌的抗体与IgD有特异性反应,而与人其它类别的免疫球蛋白重、轻链无交叉反应,均属IgG1亚类。免疫转印结果显示,均能识别分子量为67kD的IgD重链条带。用ELISA法测定3株抗体分别识别两种不同的抗原决定簇。     

大鼠抗人血红蛋白单克隆抗体的制备及其特性定鉴

将人血红蛋白(Hb)免疫LOU/M(IgK-1a)大鼠,取脾细胞与IR983F大鼠骨髓瘤细胞融合,制备了3株大鼠抗Hb单克隆抗体(McAb)2A10、9B5,4F12杂交瘤细胞株。3种McAb腹水效价分别为1:16×10~4、1:3.2×10~4、1:323×10~4。培养上清效价分别为1:256、1:16、1:8。亚类测定分别为IgM、IgG2_a、IgG2_4、染色体记数2A10为60±5,9B5为72±6。3种McAb的亲和常数分别为1.28×10~6M~(-1)、6.35×10~7M~(-1),3.64×10~8M~(-1)。特异性检测表明3种McAb与人Hb及人红细胞的亲和力明显高于其它种类动物的红细胞,与无关蛋白无结合活性。用这些McAb检测Hb最低可测到1υg水平,检测人红细胞可检测到4～8个RBC/ml(相当于90～190ngHb/ml)。大大高于目前临床常用的测定潜血方法。     

Specific Topo-Optical Reactions of Connective Tissue Elements and Their Ultrastructural Interpretation

A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)_n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin.     

抗rHuIL-8单克隆抗体的制备和初步鉴定

本文以单核细胞衍生的基因重组人IL-8(MIL-8)为免疫原,经常规免疫、融合、克隆化制备了6株抗MIL-8的单克隆抗体(2G2、4E1、4C3、4D7、5C9、5A4)。其中5株(除4D7)对MIL-8的嗜中性粒细胞趋化活性具有中和作用,6株单抗均与内皮细胞衍生的重组人IL-8(EIL-8)具有交叉反应,但与表达IL-8的载体菌裂解液均无交叉反应。     

抗重组人肿瘤坏死因子-α(rHTNF-α)单克隆抗体特性及功能的研究

应用杂交瘤技木制备抗重组人肿瘤坏死因子-α单克隆抗体对于r-HTNF-α的纯化和TNF-α分子的抗原性及功能的研究将是一种主要工具。本实验对一组抗rHTNF-α单克隆抗体的特性和功能进行了研究。Western blotting结果表明Z_4、Z_8、Z_(12)、Z_ (20)、Z_(21)、B_3和E_6 抗体特异性地识别分子量为17000道尔顿的TNF-α它们与rIL-1、rIL-2、rIFNγ、rIFNα、和E coli菌裂解液无交叉反应。竞争性ELISA结果证实7种抗体分别识别TNF-α分子上5个不同的抗原决定簇。其中4个抗体可以识别TNF-α活性中心部位。两个抗体还可以识别天然TNF-α分子。