Determination of autoantibodies to antigens of thymic epithelial cells of clean up team workers and patients who survived acute radiation sickness at distant periods after irradiation]

A group of patients, suffering from sequelae of acute radiation sickness (ARS), and liquidators was studied 5 years after exposure to a complex of factors resulting from the Chernobyl A.P.S. disaster. Studied were: the antibody titres to antigens of the cytoplasm of thymus epithelial reticulum cells and to Hassall's corpuscles the levels of serum immunoglobulins M, G, A; and the content of serum alpha 1-thymosin. Patients with ARS sequelae and liquidators showed a high level and incidence of autoantibodies to antigens of cytoplasm of thymus epithelial reticulum cells and to Hassall's corpuscles. There were no significant differences between the antibody levels in the blood of patients with ARS sequelae and liquidators. The antibodies were found to belong to IgM class; there was a correlation between the serum IgM titres and the rate of the indirect immunofluorescence reaction with autoantibodies to antigens of the cytoplasm of the thymus epithelial reticulum cells. To identify autoantibodies cryostat sections of human and mouse, (CBA x C57BL/6) F1, thymus as well as the epithelial and stromal cell culture of mouse thymus can equally be used.     

Melanocytes in Human Embryonic and Fetal Skin: A Review and New Findings

Melanocytes account for approximately 5–10% percent of the cells in adult epidermis. Unlike the ectodermally derived keratinocytes, they originate in the neural crest and migrate into the epidermis early in development. There has been an interest in melanocytes in developing human skin since the late 1800s, when concentrated pigmented cells were identified in the sacro-coccygeal skin of Japanese fetuses. This observation led to speculation and subsequent investigation about the racial nature of the melanocytes in this site (the Mongolian spot), the presence of melanocytes in fetuses of other races, the timing of appearance of these cells in both the dermis and epidermis, and their origin. The early investigators relied primarily on histochemical methods that stained either the premelanosome or the pigmented melanosome, or relied upon the activity of tyrosinase within the melanosome to effect the DOPA reaction. Studies by electron microscopy added further documentation to the presence of melanocytes in the skin by resolving the structure of the melanosome regardless of its state of pigmentation. All of these methods recognized, however, only differentiated melanocytes. The thorough investigations of melanocytes in the skin from a large number of black embryos and fetuses by Zimmerman and colleagues between 1948 and 1955 provided insight into the time of appearance of melanocytes in the dermis (10–11 weeks' menstrual age) and the epidermis (11–12 weeks) and revealed the density of these cells in both zones of the skin of several regions of the body. The precise localization of the melanocytes in the developing hair follicles was contributed by the studies of Mishima and Widlan (J Invest Dermatol 1966; 46:263–277). More recently, monoclonal antibodies have been developed that recognize common oncofetal or oncodifferentiation antigens on the surface or in the cytoplasm of melanoma cells and developing melanocytes (but not normal adult melanocytes). These antibodies recognize the cells irrespective of the presence or absence of melanosomes or their activity in the synthesis of pigment and therefore are valuable tools for re-examining the presence, density, and distribution patterns of melanocytes in developing human skin. Using one of these antibodies (HMB-45), it was found that dendritic melanocytes are present in the epidermis between 40 and 50 days estimated gestational age in a density comparable with that of newborn epidermis and are distributed in relatively non-random patterns. A number of questions about the influx of cells into the epidermis, potential reservoirs of melanoblasts retained within the dermis, division of epidermal melanocytes, and the interaction of melanocytes and keratinocytes during development remain unresolved. The tools now appear to be available, however, to begin to explore many of these questions.     

Specific antigen of granular cells of the human endometrium

Rabbits were immunized by homogenates of endometrium obtained from women during 10-12 weeks of gestation. A specific antiserum was obtained by absorption of the crude antiserum by blood cells and plasma proteins of men with different kinds of ABO and Rh antigens, till disappearance of positive reaction with men's serum protein in the Ouchterlony test. Such an adsorbed specific antiserum continued to react with the sera of pregnant women. Two antigens, numbers 1 and 2, respectively, were determined by the Ouchterlony test. Another group of rabbits was immunized by antigens detected in the precipitation test. A monospecific antidecidual antiserum (ADS 1092) was obtained against number 2 antigens. This antiserum revealed only one antigen in sera of women with gestation and did not react with sera of non-pregnant women. In the slides of endometrium of pregnant women of 10-12 weeks of gestation ADS 1092 had a strong positive reactive with the cytoplasm of one type of endometrium cells. The immunomorphological analysis by the non-direct Coons test and the PAP-test permits to identify cells with the positive reaction as granular cells. It is concluded that the granular cells may be a source of one of the serum antigens detected in women with gestation.     

The distribution of blood group antigens in rodent epithelia

Summary The pattern of distribution of antigens cross-reacting with antibodies to human blood group antigens A and B and two precursor molecules was examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice. Staining for blood group antigen A was negative. In all epithelia examined, blood group antigen B was present at the surface of basal and parabasal cells, and the H antigen at the surface of spinous cells. N-acetyllactosamine was present on the cell membranes in the upper spinous and granular cell layers of epidermis and forestomach epithelium and was not expressed in the oral epithelia except for a limited area in the dorsal tongue epithelium.Thus, the expression of antigen varies both regionally and, as earlier shown in human epithelium, with the stage of maturation of cells within a given epithelium. The observed sequence of expression of these antigens during maturation differs from that of human epithelia, but the present study provides a basis for further experimental studies of the role of cell surface antigens in epithelial homeostasis and maturation.     

Reaction of loose connective tissue fibroblasts after poly-4-vinylpyridine administration

Mice were injected with poly-4-vinylpyridine at a dose known to be effective for immunogenesis stimulation. Within 24 hours after the injection fibroblasts reacted by dendrit production, cytoplasm vacuolization and clasmocytosis. 2-3 days after the polymer injection the fibroblast cytoplasm was restored. The immunofluorescence employed rabbit serum natural antibodies to fibroblast antigens. The reaction observed may be used for the estimation of biological polymer activity.     

Immunocytochemical identification of bovine Langerhans cells by use of a monoclonal antibody directed against class II MHC antigens

We undertook a study to develop a reliable light microscopic technique for identifying Langerhans cells (LC) in bovine epidermis. Monoclonal antibodies (MCA) detecting bovine class II MHC antigens were used in conjunction with an avidin-biotin-peroxidase complex (ABC) immunocytochemical staining method. The specificity of the MCA for LC was confirmed ultrastructurally by use of gold-labeled second antibody. Epidermal sheets and epidermal single-cell suspensions examined by light microscopy confirm that bovine epidermal LC express class II antigens. Anti-bovine class II MCA is a dependable reagent for identification of LC in normal bovine epidermis.     

Localization of basal cell antigen in the epithelial tissues of animals and humans detected by means of natural antibodies

By means of the immunofluorescent method using rabbit serum that contains natural antibodies against the basal cell antigen of epidermis, the distribution of the antigen has been demonstrated in cells of the basal layer of all types of the stratified epithelium. The reaction is also noted in cytoplasm of the epithelial cells in the thymus and the tracheal mucous membrane. This demonstrates their histogenic affinity to stratified epithelii. The antigen studied is not species-specific, since it is revealed in the stratified epithelium of all species examined (human being, mouse, rat, guinea pig, rabbit). It is possible to use the basal cell antigen as a marker for immunomorphological reveal of epithelial cells in the thymus in the process of its physiological and pathological involution.     

Cell surface expression of invariant gamma-chain of class II histocompatibility antigens in human skin

A rat monoclonal antibody (McAb 21:9) reactive with the human invariant gamma-chain of class II major histocompatibility complex (MHC)-encoded antigens was isolated and was shown to react with the carbohydrate-carrying, COOH-terminal part of the gamma-chain. The McAb 21:9 binds to a molecule that is identified as the gamma-chain for the following reasons: it has an apparent m.w. of 33,000, similar to that of the gamma-chain; it has a two-dimensional gel migration pattern identical to that of the gamma-chain; and it associates with immature, but not processed class II antigens. When used for immunohistochemical staining on sections of normal human skin, only dendritic, class II MHC antigen, and anti-Leu-6 reactive Langerhans cells are labeled in the epidermis. HLA-DR-expressing keratinocytes present in the tuberculin reaction, cutaneous T cell lymphoma, and lichen planus, however, did not react with the anti-gamma-chain antibody, nor with a HLA-DQ-reactive antibody. Cell surface expression of the gamma-chain was observed on 1 to 3% of normal viable epidermal cells in suspension. By using double indirect immunofluorescence, it was possible to demonstrate the simultaneous binding of anti-gamma-chain, anti-HLA-DR, anti-Leu-10, and anti-Leu-6 antibodies, respectively, on the same cells, thus confirming their identity as Langerhans cells. The presence of the gamma-chain on the surface of the immunocompetent Langerhans cells may indicate that the cell surface, not the cytoplasm as has been suggested, is the site of the primary function of the gamma-chain.     

Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.     

Humoral response to Staphylococcus aureus antigens evaluated by the western blotting method]

Cellular antigens extracted from the cells of four Staphylococcus aureus strains from different kinds of infections (sepsis, osteomyelitis, furunculosis) were analysed by the western blotting technique. Antibiotic sensitivity pattern of the strains was compared. One isolate was found to be MRSA strain. Sera samples from patients of whom strains were isolated and four sera from blood donors (as a control) were used in the investigation. IgG levels for purified staphylococcal antigens (lipase, alpha-toxin and teichoic acid) were estimated. Interaction between extracted bacterial antigens and serum antibodies of IgG class were analysed in homologous and heterologous systems. The most strong immunological reaction of the investigated sera with staphylococcal antigens was observed in the case of homologous system. Serum from sepsis patient was found to be the most reactive serum with all staphylococcal antigens mixtures.     

The expression of desmosomal and corneodesmosomal antigens shows specific variations during the terminal differentiation of epidermis and hair follicle epithelia.

Using five monoclonal antibodies (MAb), we studied by indirect immunofluorescence the desmosomes and a junctional structure specific to cornified layers, the corneodesmosome, in normal and plantar epidermis and in the various sheaths of the anagen hair follicle. The monoclonal antibodies DP1&2.2-15, PG5.1, and DG3.10, specific for desmoplakins I/II, plakoglobin, and desmoglein I, respectively, were used to study the desmosome antigens, and G36-19 and G20-21 to study the corneodesmosome antigens. The distribution and sequence of expression of the five antigens allowed the nine epithelial differentiation pathways studied to be merged into four distinct families: non-plantar epidermis, characterized by the absence of desmosome and corneodesmosome antigens in the stratum corneum; the outer root sheath of the hair follicle, which behaves like the viable layers of the epidermis with regard to the desmosome antigens but does not express the corneodesmosome antigens; plantar epidermis and the three components of the inner root sheath in which the corneodesmosome antigens are present up to the desquamating layer; and the three components of the hair shaft, which are characterized by the absence of expression of both the desmosome and the corneodesmosome antigens in its mature portion.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Immunoelectron microscopic demonstration of tissue antigens with monoclonal antibodies

Two methods of demonstrating tissue antigens by ultrastructural enzyme immunohistochemistry were tested. The monoclonal antibodies Ki-M1 and Ki-M4 were chosen for testing the methods because Ki-M1 identifies a relatively stable, and Ki-M4 a very unstable antigen. The two antibodies react selectively with human macrophages and interdigitating reticulum cells or dendritic reticulum cells of lymphoid follicles. The Ki-M1 reaction product is confined to the surface membrane. Ki-M4 reactivity is located on the surface membrane and, less often and to a lesser extent, in the cytoplasm. The technical prerequisites for reliable conservation of the antigens identified by these two antibodies were standardized. The results indicated that prior fixation in 4% paraformaldehyde is preferable for optimum preservation of stable antigens. Application of the primary antibody prior to fixation was found to be the best procedure for demonstrating unstable antigens, although nonspecific reactions were seen more often with this method.     

Antibodies to a nucleolar protein are localized in the nucleolus after red blood cell-mediated microinjection

To determine whether red blood cell-mediated microinjection of antibodies can be used to study nuclear protein localization and function, we microinjected antibodies that have been shown to react specifically with nucleolar acidic phosphoprotein C23 into Walker 256 cells. The intracellular distribution of microinjected anti-C23 antibodies and preimmune immunoglobulins were determined by immunofluorescence. At 3 h after microinjection, affinity-purified anti- C23 antibodies were localized in the cytoplasm and nucleolus. At 17 h after microinjection, the affinity-purified antibody was localized to those nucleolar structures previously shown to contain protein C23. Furthermore, the antibody remained localized in the nucleolus for at least 36 h after microinjection. In contrast to the results obtained with specific antibodies, preimmune immunoglobulins remained in the cytoplasm 36 h after microinjection. These results indicate that red blood cell-mediated microinjection of antibodies can be used to study nucleolar and nuclear antigens.     

Histocompatibility antigens controlled by theI region of the murineH-2 complex

Spleen cells from A.TH mice, presensitized in vivo by skin grafting, were restimulated in vitro by A.TL lymphocytes, and A.TH anti-A.TL effector cells were generated. The effector cells lysed, in the CML assay, A.TL blasts. This reaction, which was againstI-region antigens, could be inhibited by the addition to the reaction mixture of anti-La sera directed against A.TL antigens. The inhibition was specific, since normal mouse serum, reciprocal antiserum (A.TL anti-A.TH), and anti-H-2 sera did not have a significant effect on the reaction. The Ia antibodies also specifically inhibited the reaction of A.TH anti-A.TL effector cells against CBA targets. Con A blasts were significantly poorer targets inI-region CML than LPS blasts. As CML targets, macrophages and cells of a mammary adenocarcinoma were as good as, if not better than, the LPS blasts. The experiments support the notion that Ia antigens are the targets in theI-region CML.     

Effect of pertussis antibodies on the induction of suppressor cells of humoral immunity in mice

Standard pertussis agglutinative serum as well as antibodies to total and purified protective pertussis antigens, isolated from the serum by immunoadsorption technique, were studied. The treatment of donors with pertussis antibodies affected the T-suppressor formation, induced by high doses of sheep red blood cells, as was shown on the model of syngeneic transfer. Immunomodulating effect of antibodies, complementary to immunogen fraction of pertussis cells, was decreased. It was suggested that the observed antilymphocyte activity of antipertussis antibodies could play a certain role in postvaccination complications after corpuscular pertussis vaccine administration.     

Production and characterization of DR xenoantisera: use for detection of serum DR antigens.

Immunoadsorbents consisting of rabbit anti-DR antibodies bound to Staphylococcus aureus were used to bind DR antigens from NP40 cellular extracts of cultured B lymphoid cells. When injected into rabbits, these DR antibody absorbents elicited the production of antibodies that were shown by both serologic and immunochemical techniques to react specifically with the DR antigen molecule. A rosette inhibition assay with B lymphoid cells and anti-DR xenoantisera was used to detect DR antigens in human serum. DR serum antigens could be found in an enriched preparation of serum lipoproteins obtained by centrifugation in KBr. In addition, sera from some patients with neoplastic diseases contained higher levels of DR serum antigens than those found in normal individuals.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.     

Indication of antigens and antibodies using the reaction of compound immune adsorption and reactivation of the phage.

A method of determining antibodies by their adsorption on large-pore or surface immunosorbents with subsequent treatment of the carrier with anti-immunoglobulin serum and antiphage serum isologous to the antibodies and then with the bacteriophage has been presented. The adsorbed virions are split off by means of papain-induced hydrolysis of the antibody complex. The antigens are determined by the reaction of phage fixing inhibition. The method permits to determine small amounts of antibodies to proteins, haptenes and cells with objective calculation of results.     

Simultaneous immunocytochemical visualization of bromodeoxyuridine and neural tissue antigens.

5'-Bromodeoxyuridine (BrdU) is a thymidine analogue which can be detected by monoclonal antibodies (MAb). We have developed a method for the simultaneous visualization of BrdU and a wide range of neural antigens in paraformaldehyde-fixed brain sections. Pregnant mice were injected intraperitoneally with a single pulse of BrdU. Young adult offspring were processed for immunocytochemistry following a double immunoperoxidase sequence. BrdU was detected using diaminobenzidine (DAB) intensified with nickel ammonium sulfate and neural antigen-containing elements were visualized with DAB alone. BrdU-positive nuclei and tissue antigen-immunoreactive cells were easily differentiated. Furthermore, double-labeled cells characterized by the presence of a black immunoreactive nucleus surrounded by a brown immunopositive cytoplasm were unambiguously recognized. Satisfactory results were obtained using either MAb or polyclonal antibodies against a variety of cell antigens, including neuropeptides, CA++ binding proteins, and cytoskeletal components of the glial cells. The method reported here permits analysis of the neurogenesis and proliferation of subsets of neurons and glial cells, identified by immunocytochemical markers.