Localization of tissue antigens with the fluorescent antibody technique: application to human anterior pituitary hormones

Antisera have been prepared to adrenocorticotrophic hormone, thyrotrophic hormone and growth hormone extracted from human pituitaries and to gonadotrophin, and thyrotrophic hormone extracted from human urine.Precipitin, absorption-precipitin and gel-diffusion tests have been performed with these antisera.No conclusive evidence was obtained of the presence of a hormone-specific antibody in any of the antisera.Cross-reactions occurred indicating the presence of an antigen common to those extracts prepared from the pituitary and a further antigen, or antigens, common to those extracts prepared from the urine.Despite the absence of hormone-specific antibodies fluorescein-labelled samples of the antisera gave staining in the cytoplasm of cells in the anterior pituitary and did not stain other tissues.The implication of these results in the interpretation of tissue localization studies with labelled antisera is discussed.     

Amyloid Deposits in Pituitaries and Pituitary Adenomas: Immunohistochemistry and In Situ Hybridization

The patterns of deposition and immunoreactivity of interstitial amyloid were studied in 11 pituitary glands obtained at autopsy and 9 surgically resected pituitary adenomas using Congo red staining and a panel of antisera directed against 5 major amyloid fibril proteins and all pituitary hormones. The deposition pattern of amyloid in pituitary glands differed from that in adenomas but all amyloid deposits showed an immunostaining with anti-amyloid λ-light chain. The remaining antisera were immunonegative.In situ hybridization using an oligodeoxyribonucleotide-probe complementary to the mRNA coding for the constant region of human λ-light chain yielded no hybridization signals in the pituitaries or pituitary adenomas, excluding local synthesis and secretion of immunoglobulins. Since no case studied suffered from generalized Aλ-amyloidosis and adsorption of immunoglobulins to the unknown amyloid fribril protein of the pituitary seems to be unlikely, crossreaction of the polyclonal antisera with an undefined antigen is probable. The similar immunostaining properties of amyloid deposits in “normal” pituitaries and pituitary adenomas suggest they both originate from the same precursor protein.     

Antibodies to spermatozoa. III. Responses in rabbits and guinea-pigs to immunization with guinea-pig sperm cells

The antigens of guinea-pig sperm cells, of both the epididymal and ejaculated (or seminal) types, have been studied, using rabbit and guinea-pig antisera. Several antigens could be revealed by gel diffusion studies, using well-washed but non-ruptured sperm cells, indicating that intentional cell breaking is not essential for demonstrating the antigens. This release of soluble antigen was followed as a function of time and temperature, both as total protein in supernatants and in increasing strength of precipitation. With rabbit antiserum, epididymal sperm showed two antigens, that were also demonstrated in epididymal and testicular extract and in seminal sperm. These other materials revealed additional antigens with these antisera. Immunofluorescent staining was limited to the acrosomes. With guinea-pig antibodies, no precipitating antigen that was characteristic of sperm could be seen. These antisera showed immunofluorescent staining of the acrosomes. The staining could be distinguished, in terms of thermostability, from the staining produced by normal serum. No evidence was found for the occurrence of any sperm-coating antigens in the guinea-pig, especially since both antiseminal plasma and antivesicular fluid antisera failed to give immunofluorescent staining of the sperm cells.     

Characterization of rabbit cells by monoclonal and polyclonal antibodies.

Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a T-cell antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed.     

The occurrence of antigen reacting with antibody to porcine neurophysin

1. Antiserum was raised in rabbits to neurophysin prepared from posterior pituitaries of pigs. The presence of antibody reacting with porcine neurophysin was demonstrated in precipitation, gel diffusion and immunoelectrophoretic tests.

2. The antibody was species specific and did not react with protein from rat, bovine or guinea-pig neurohypophyses.

3. The occurrence of antigen reacting with anti-neurophysin serum was demonstrated in acetic acid extracts of porcine kidney but there was no evidence for the presence of antigen in extracts of liver or spleen prepared in the same way.

4. Protein fractions (`N-fractions') from various organs and tissues of pigs were obtained by the same methods used to prepare neurophysin from posterior pituitaries and were tested for antigenicity in reaction with neurophysin antibody. N-fractions from kidney, uterus, mammary gland and serum contained antigen while fractions from liver, spleen, brain and skeletal muscle did not react with the anti-neurophysin serum.

     

Immunohistological localization of TGFα, EGF,IGF-I,and TGFβ in the normal human pituitary gland

In previous studies, we demonstrated several growth factors, including epidermal growth factor (EGF), transforming growth factorβ (TGFβ), insulin-like growth factor-I (IGF-I, somatomedin C), and TGFe in extracts of the human pituitary. Using immunohistochemistry, reactivity for TGFα, EGF, and TGFβ was localized in sections of 12 of 12 autopsy-derived human pituitary glands. IGF-I reactivity was demonstrated in 5 of 5 pituitary glands. Sections staining for TGFα and IGF-I were double-stained for the full spectrum of anterior pituitary hormones (i.e., GH, PRL, ACTH, LH, FSH, TSH, and a-subunit). Intracellular EGF immunostaining was demonstrated within the posterior pituitary and in some squamous cell nests of the pars tuberalis. Occasional extracellular EGF reactivity was also noted within connective tissue of the anterior pituitary. Polyclonal antibody to TGFβ showed extracellular reactivity within connective tissue surrounding the gland as well as that separating cords of secretory cells. TGFβ staining was also noted in the mediae of small vessels. Monoclonal anti-TGFα antibody labeled scattered secretory cells throughout the anterior pituitary, including ones engaged in follicle formation, and some cells lining Rathke’s cleft remnants. TGFα reactivity in secretory cells was expressed in cells also staining for PRL, α-subunit, LH, FSH, and TSH. No TGFα reactivity was noticed in cells staining for either GH or ACTH. IGF-I reactivity was observed in occasional secretory cells within the anterior pituitary and in neuronal processes of the posterior pituitary. Anterior pituitary cells reactive for hormones were nonreactive for IGF-I. The presence of EGF and TGFβ reactivity within extracellular matrix suggests that it represents a possible storage site of these growth factors within the anterior pituitary. The presence of EGF and IGF-I within the axons and terminations of posterior pituitary implies either local or possibly hypothalamic production of EGF. Finally, our results indicate that TGFα and IGF-I, as well as perhaps EGF, are manufactured by specific pituitary cells and that they may have a role in the endocrine function of the anterior and posterior pituitary, respectively.     

Pituitary adenomas. An immunohistochemical study of hormone production and chromogranin localization.

Tumors from 42 surgically resected pituitaries and from 13 autopsy cases were studied immunohistochemically with polyclonal antisera to 7 anterior pituitary hormones and with a newly developed monoclonal antibody directed against human chromogranin for evaluation of the distribution of chromogranin in normal and neoplastic pituitaries. In addition, a prospective study was done for assessment of the prevalence, morphology, and endocrine cell types of pituitary tumors in 100 autopsy subjects. When these 55 pituitary adenomas were examined with monoclonal antibody (LK2H10) directed against human chromogranin, selective staining of normal adenohypophyseal cell types and pituitary tumors was observed. Most null-cell adenomas (12/14) were positive for chromogranin, whereas all prolactin (PRL)-producing adenomas (19/19) were negative. Growth hormone (GH) adenomas were focally positive (9/9). All oncocytomas (2/2), 1 thyrotropin (TSH) adenoma, and a follicle-stimulating hormone/luteinizing hormone adenoma were positive for chromogranin. One or more adenomas were present in 14% of the autopsy cases. The tumors occurred most frequently in patients in the fifth through the seventh decades of life. Immunohistochemical staining of 13 adenomas revealed 1 TSH, 1 ACTH, and 4 PRL-producing tumors, whereas 7 other tumors, which were null-cell or undifferentiated adenomas, failed to stain for any of the seven principle pituitary hormones. These results indicate that antibody LK2H10 to human chromogranin is useful in the immunohistochemical characterization of pituitary adenomas. Incidental pituitary microadenomas from autopsy-derived pituitaries most commonly produce PRL, or they belong to the null-cell or undifferentiated tumor group.     

Immunological Studies on Adrenal Glands: II. Immunization with Adrenals of the Same Species

Thirty-six guinea pigs were injected with a suspension of pooled guinea-pig adrenals incorporated into Freund adjuvants; twenty-two rabbits were immunized in a similar manner with rabbit adrenal suspensions.

Sera of twenty-six guinea pigs and twelve rabbits reacted in complement fixation and tanned cell haemagglutination tests with adrenal extracts but not with extracts of other organs. Potent antisera also produced one line reaction in double diffusion gel precipitation test with adrenal extracts.

The adrenal-specific antigen was thermolabile, possibly of protein character. Reactions of guinea-pig antisera were limited to guinea-pig adrenal; rabbit antisera reacted with adrenal of all tested mammalian species.

The serological reactions exhibited by antigens prepared from the antibody producer's own adrenal were as strong as those produced by adrenals of other animals of the same species. Additional evidence for auto-specificity of the antibody under investigation was obtained from the experiment in which a guinea pig injected with its own gland removed by left adrenalectomy formed adrenal antibodies.

Some but not all animals developed histological lesions in the adrenal gland. The interpretation of this observation was complicated by occasional positive histological findings in the adrenals of control animals.

     

Immunofluorescent studies on human spermatozoa. II. Characterization of spermatozoal antigens and their occurrence in spermatozoa from the male partners of infertile couples

The object of this study was to elucidate properties, organ specificity, and the occurrence in individual spermatozoa of the four antigens which can be identified in methanol-fixed human spermatozoa by the indirect immunofluorescence technique using human antisera, i.e. the antigen in the front part of the acrosome, in the equatorial segment, in the postnuclear region, and in the main tail piece.

With unfixed spermatozoa the reactions were weak and often uncharacteristic. After fixation with ethanol and acetone all four antigens were preserved, while after fixation with formalin the postnuclear antigen was not demonstrable. The antigens in the equatorial segment and in the postnuclear region were destroyed at 60°C and those in the front part of the acrosome and in the tail at 80°C. After the spermatozoa had been treated with trypsin the reaction with the three antigens in the head disappeared. However, with rabbit antiserum to human spermatozoa fluorescence could be induced even after heating the spermatozoa to 100°C or treating them with trypsin.

Absorption of the human antisera with seminal plasma, human milk, liver, kidney, and adrenal extract disclosed an antigenic relationship between the postnuclear antigen and seminal plasma as well as between the antigen in the front part of the acrosome and adrenal extract.

By means of known antisera the occurrence of acrosome antigens in the individual spermatozoa was investigated in ejaculates from seventy-six male partners of infertile couples. In ejaculates with a normal concentration of spermatozoa the antigens were generally demonstrable in at least 50% of the spermatozoa, whereas samples with a low concentration showed a marked variation, the staining percentages ranging from 0 to 90%.

     

Antibody production in vitro: The effects of anti-allotypic serum on the secondary response of rabbit spleen cell suspensions to sheep erythrocytes SRC

Rabbit anti-allotypic sera directed against rabbit immunoglobulins stimulated an increase in the number of direct PFCs in suspensions of cells from the spleens of rabbits killed after a boost of SRC. This effect occurred within the first 24 hours of incubation of these cells with antiserum in the absence of antigen in the culture medium. High levels of these antisera were much less stimulatory than low during the early period of culture, but more prolonged incubation with antiserum in high concentrations resulted in increased numbers of PFC in these cultures. Amounts of antisera which were stimulatory in the absence of SRC in the culture medium were found to inhibit the stimulatory effect of antigen in PFC during the first 1–2 days of culture. Like high concentrations of anti-allotypic sera alone, this early inhibition was followed by a stimulatory increase of PFC on more prolonged culture in the presence of both SRC and anti-allotypic serum.

Anti-allotypic sera affected the levels of PFC in these studies even in the absence of any change in the rate of DNA synthesis, as measured by the incorporation of [³H]thymidine. Further to this, it was found that concentrations of antisera which stimulated increased uptake of [³H]thymidine during 24–48 hours of culture, actually depressed the levels of PFC in these cultures. It was, therefore, concluded that the action of anti-allotypic sera on PFC in these cultures was by means of a direct effect on the process of production of antibodies by these cells and was not dependent on the stimulation of cell division in the antibody-producing population. This stimulatory effect of anti-allotypic sera was prevented by Actinomycin-D and the inhibitory effects of complement on PFC in cultures incubated with anti-allotypic sera indicated that antibodies had combined with receptor molecules on these cells. The effects of the combination of SRC and anti-allotypic sera on the levels of PFC would suggest that the same cell population in these cultures was being affected by both these agents and that the receptor molecule for them was specific antibody.

The effect of anti-allotypic sera of the PFC in these rabbit spleen cell suspensions was shown to have some degree of non-specificity. Since normal rabbit serum as well as rabbit antiserum against a Proteus vulgaris OX19 did not have any effect it was considered possible that this non-specific stimulation of PFCs was also due to antibodies directed against antigenic determinants in rabbit immunoglobulins.

     

Isolation and characterization of a rabbit non-tissue specific autoantigen which is shared by kidney, heart, adrenal and liver

An antigen that can be derived from rabbit kidney can be identified by use of suitable rabbit antisera as being non-tissue specific in addition to being an autoantigen, since it had been found to be distributed in kidney, heart, adrenal and liver. The purification of this autoantigen has been accomplished by successive use of four different methods. These were the salting out with ammonium sulphate, gel filtration on Sephadex G-200, dialysis against distilled water, and chromatography on DEAE-cellulose. The heterogeneity of the products derived from the procedure at each step was analysed by means of analytical electrophoresis and ultracentrifugation, and also by gel diffusion precipitation.

The final product was a homogeneous component, according to the criteria that were used. It had a sedimentation coefficient of 4.1S, suggesting a mol. wt of approximately 60,000. This autoantigen is of similar molecular size to several others that are, in contrast, adrenal specific or kidney specific.

     

In Vitro: The Adsorption of Antigen by Spleen Cells previously treated with Antiserum*

Rabbit spleen cells, which have been treated in vitro with certain rabbit or guinea-pig antisera (e.g. against human serum albumin) and washed, are capable of specifically adsorbing radio-isotope labelled antigen. The antibody responsible for this effect appears to be distinct from the main precipitating antibody in the serum and the term `cytophilic antibody' has been suggested. The activity of the cytophilic antibody is not destroyed by heating at 56° C. for half an hour.

The cytophilic antibody is not adsorbed by red cells. Spleen cells which have been treated with methanol before treatment with antiserum take up less antigen than non-methanol-treated cells tested in the same way. However, the addition of fresh normal spleen cells to the methanol-antibody-treated cells before the addition of antigen increased the uptake of the latter.

     

Immunological studies on adrenal glands: VI. The multiple nature of the rabbit adrenal autoantigens

Antisera prepared in rabbits by immunization with rabbit adrenal homogenate were shown to contain as many as three or four distinct antibodies to adrenal extract. This was generally demonstrated by concentrating the antisera. In some of the studies even the untreated antiserum revealed two or more antibodies. Three of the precipitation lines were specific for adrenal extract, while a fourth line reacted with extracts of many tissues, corresponding apparently to an antigen commonly distributed in the body. The possibility that serum groups might be involved in some of these responses was ruled out by a number of negative results on various serum and globulin samples tested as possible antigens. Further studies showed that at least three of the antibodies, and possibly all four of them, were in fact autoantibodies. The distribution of the corresponding autoantigens was evaluated by comparison of extracts of bovine adrenal cortex and medulla with those of whole rabbit adrenal. The medulla contained two of these antigens, and the cortex contained one of the others.     

A new differentiation antigen of plasma cells

A cell-surface differentiation antigen of mouse Ig-secreting cells, MSPCA (Mouse-specific plasma cell antigen), is identified by rabbit antiserum to mouse myeloma MOPC-104E (which secretes λμ) or myeloma MPC-67 (which secretes Kα); these two antisera contained little or no anti-Ig antibody. Although a11 5 mouse myelomas tested were MSPCA⁺, rabbit antisera to one of them, myeloma MOPC-7OA, contained anti-Ig antibody and little or no anti-MSPCA. This myeloma differs from myelomas MOPC-104E and MPC-67 in that the Ig components it makes, Kγ1, are readily demonstrable on the cell surface. These results suggest that rabbits immunized with myeloma cells may respond by producing preferentially either anti-MSPCA or anti-Ig antibody, depending on the surface Ig-phenotype of the myeIoma used for immunization.     

Analysis of mammosomatotropic cells in normal and neoplastic human pituitaries

The reverse hemolytic plaque assay (RHPA) was used to analyze hormone secretion in normal and neoplastic human pituitary cells. Immunocytochemical (ICC) staining for PRL and GH with the peroxidase method and ultrastructural ICC with colloidal gold labeling were used along with the RHPA to analyze for mammosomatotropic (MS) cells in these dissociated and cultured pituitary cells. MS cells were identified in both normal and neoplastic pituitary tissues. Quantitation of plaque areas, showed that PRL cells from normal pituitaries had significantly larger plaque areas than PRL cells from PRL-producing adenomas or from mixed PRL-GH producing adenomas.     

. II. : Fowl Antibody • The Composition of Specific Precipitates Formed by Antisera to Serum Albumin, Haemoglobin and Myoglobin and some Properties of Non-precipitating Antibody

Specific precipitates formed in 0.9 per cent and 8 per cent NaCl and the precipitates formed by raising the salt concentration of `0.9 per cent' supernatants to 8 per cent were measured quantitatively. With antisera to haemoglobin and myoglobin the antigen in the precipitates was also measured. Except for some very high antibody/antigen ratios found in some cases in antibody excess, these ratios were the same as those found with rabbit antibody, and did not depend on salt concentration.

Non-precipitating antibody, prepared by serial absorption of antiserum with small portions of antigen, did not precipitate with antigen even in 8 per cent NaCl; it co-precipitated with homologous rabbit antiserum and delayed its flocculation, but produced no permanent inhibition.

Rabbit antiserum to washed specific precipitates made from fowl antisera was used to confirm the presence of two globulins, one a macroglobulin, in the precipitates, and to study their different properties when free in whole serum and when combined with antigen.

     

Lymphocyte-transforming activity of homologous and heterologous antisera to rabbit leucocytes

Two out of nine rabbits receiving a course of immunization with homologous leucocytes produced antisera which caused blast formation and stimulated DNA synthesis of donor lymphocytes in vitro. Of the seven rabbits which failed to produce active antisera three had received only a short immunization course. The active antisera `transformed' lymphocytes from some rabbits but not those of others. This stimulatory effect was not related to the red cell types or the γ-globulin allotypes of the rabbits.

Heterologous antisera raised to rabbit leucocytes in rats and guinea-pigs were cytotoxic to rabbit leucocytes in vitro and also caused leucoagglutination but never stimulated DNA synthesis and blast formation.

     

分泌颗粒素Ⅱ和促肾上腺皮质激素在脑损伤大鼠垂体腺细胞共存(英文)

采用荧光双标记技术研究了脑损伤时大鼠垂体前叶促肾上腺皮质激素和分泌颗粒素 的定位。垂体前叶细胞呈现分泌颗粒素 染色强阳性 ,阳性物质位于胞浆中。免疫荧光双标记技术用于鉴定分泌颗粒素 和促肾上腺皮质激素共存的细胞 ,共聚焦显微镜观察显示促肾上腺皮质激素和分泌颗粒素 共存于同一细胞。结果表明 :分泌颗粒素 可在大鼠垂体前叶促肾上腺皮质激素阳性细胞表达 ,其生理作用可能是调节分泌泡的 p H值以利于促肾上腺皮质激素从其前体裂解。