Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens.

Enzymes, either acid phosphatase or horseradish peroxidase, were conjugated to antibodies with bifunctional reagents. The conjugates, enzymatically and immunologically active, were employed in the immunohistochemical localization of tissue antigens utilizing the reaction product of the enzymatic reaction as the marker. Tissues reacted with acid phosphatase-labeled antibodies directed against basement membrane were stained for the enzyme with Gomori's method, and those reacted with peroxidase-labeled antibody were stained with Karnovsky's method. The reaction products of the enzymes localized in the basement membrane. Unlike the preparations of the fluorescent antibody technique, enzyme-labeled antibody preparations were permanent, could be observed with an ordinary microscope, and could be examined with the electron microscope. In the latter, specific localization of antibody occurred in the basement membrane and in the endoplasmic reticulum of cells known to synthesize basement membrane antigens. The method is sensitive because of the amplifying effect of the enzymatic activity. The ultrastructural preservation and localization were better with acid phosphatase-labeled antibody than with peroxidase-labeled antibody, but acid phosphatase conjugated antibody was unstable and difficult to prepare. Peroxidase-antibody conjugates were stable and could be stored for several months at 4 degrees C, or indefiniely in a frozen state.

Human intrinsic factor secretion: immunocytochemical demonstration of membrane-associated vesicular transport in parietal cells.

The human gastric parietal cell synthesizes and secretes intrinsic factor (IF) and acid. In contrast to the cellular mechanisms of acid secretion, little is known about the mechanisms of IF secretion. To elucidate these mechanisms we obtained gastric secretions and sequential fundic biopsies from three subjects before and after pentagastrin stimulation (6 microgram/Kg s.c.). IF was localized in the biopsies using an ultrastructural immunoperoxidase technique using a well-characterized, monospecific antibody to human IF. IF output was quantified using a specific radioimmunoassay in concurrently obtained gastric secretions. Before stimulation, IF was associated with tubulovesicles scattered throughout the cytoplasm and with some in rough endoplasmic reticulum (RER). The tubulovesicles associated with IF migrated to the periphery of the secretory canaliculi within 8 min of stimulation. IF was present on secretory microvilli between 8 and 30 min when IF output in gastric juice was at its maximum. The cessation of IF secretion coincided with the depletion of IF associated with tubulovesicles. IF appeared in the perinuclear space and RER as the IF associated with tubulovesicles was secreted. These observations indicate that IF secretion depends upon membrane-associated vesicular transport and provides support for a membrane translocation-fusion hypothesis to explain the morphologic changes that occur in the parietal cell during secretion.

Distribution of kinetochore (centromere) antigen in mammalian cell nuclei.

Antigens associated with mammalian centromeres were localized at the high and electron microscopic levels using the peroxidase-labeled antibody method. The antibody used was of a type naturally occurring in the sera of patients with scleroderma. At the light microscopic level, it reacts specifically with the centromere regions of chromosomes in a variety of mammalian species and strains in discrete foci in interphase nuclei. We find that the number of foci approximates the number of chromosomes present in the various cell types. At the ultrastructural level, the antigenic foci are confirmed to lie in the kinetochore regions of each chromosome. In interphase nuclei, the antigenic foci were usually associated either with the inner surfaces of the nuclear envelope or with the nucleoli. These observations indicate that the centromere regions of the chromosomes in interphase are not randomly distributed within the nucleus but are usually fixed either to the inner surface of the nuclear envelope or to nucleoli.

Cellular localization of leucine-binding protein from Escherichia coli.

Rabbit antibody against lecine-binding protein isolated from Escherichia coli K-12 has been prepared. This antibody has been used in conjunction with enzyme-labeled antibody to allow an immunocytochemical localizati of leucine-binding protein in the Escherichil coli cell. This protein appears to be present only in the envelope and not in the cytoplasm.

Ultrastructural analysis of the early development of teratocarcinomas.

Genital ridges of 12-day embryos of strain 129 mice were transplanted into the testes of adult mice of the same strain. Of the transplants that differentiated into fetal testes, 80% contained intratubular teratocarcinomas by the 7th day after transplantation. These teratocarcinomas were studied ultrastructurally, daily from the 7th-14th day following transplantation, to gain information about the histogenesis of the tumors and the mode of development of multipotent cells. The embryonal carcinoma cells, which are the stem cells of teratocarcinomas, so closely resembled primordial germ cells that the former appeared to be derived from the latter. The cytoplasm of these multipotential cells was characterized by large numbers of dispersed ribosomes and polysomes with few membranous organelles other than mitochondria. Among the earliest signs of differentiation was the development of rough-surfaced endoplasmic reticulum and Golgi complexes, followed by modification of the plasma membrane.

Ultrastructural comparison of differentiation of stem cells of murine adenocarcinomas of colon and breast with their normal counterparts.

Two rats with chemically induced transplantable adenocarcinomas of the colon were given pulses of [3H]thymidine, and autoradiography with electron microscopes was used to compare the degrees of differentiation of the stem cells of the tumor and colon. The best differentiated portions of the tumor had acini composed of vacuolated, mucous, and argentaffin cells in various stages of differentiation. Vacuolated and mucous cells incorporated [3H]thymidine and corresponded in degree of differentiation to that of their labeled normal counterparts in the normal colon. An exceedingly undifferentiated labeled cell, hitherto undescribed, was identified in the tumor and crypts of the colon; this may be an undifferentiated colon stem cell that differentiates into vacuolated and mucous stem cells and/or into argentaffin cells. Normal stem cells of the breast and malignant stem cells of spontaneous adenocarcinomas of the breast of C3H mice had comparable degrees of differentiation. Since normal stem cells in these tissues were as undifferentiated as the least differentiated stem cells of the tumors, there is now no need to postulate dedifferentiation as a mechanism to explain the undifferentiated appearance of tumors.

A peek at the future through histological preparations.

When histologists and pathologists examine histological preparations, they can often predict the future of tissues that have not been excised from bodies. This is possible because, unlike chemical and physical matter, living organisms travel in time on a genetically mandated fixed path. To recognize a portion of the path that tissues have already traveled, histological methods such as histochemistry and immunohistochemistry are effective. In this regard the development of the peroxidase-labeled antibody method (Nakane and Pierce, J. Histochem. Cytochem. 14:929-931, 1966) contributed immensely. For the portion of path which cells and tissues were traveling when they were removed from bodies, the method of choice is localization of mRNA by in situ hybridization. Specific methods designed to predict the future path of tissues are still at the drawing board phase. However, by discerning the past and current portions of the path and by referring to the paths that other tissues have traveled, one may deduce the future path of the tissues in question. For some time now, it has been my dream to develop methods enabling us to peek at the future path of tissues more concretely. To accomplish this one requires new procedural approaches. Thus, we would like to introduce in situ nick translation and oligonucleotide histochemistry.

Fas and Fas ligand interaction is necessary for human osteoblast apoptosis.

We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin-2 followed by 12-o-tetradecanoylphorbol 13-acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast-like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast-like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast-like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast-like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane-type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.

Ontogenesis of adrenocorticotropin-related peptide determinants in the hypothalamus and pituitary gland of the rat.

The time of appearance of the antigenic determinants of ACTH-related peptides (16K fragment, ACTH, gamma-lipoprotein, and beta-endorphin) was determined immunohistochemically in the brain and pituitary gland of the fetal rat. Major antigenic determinants of the pro-ACTH/endorphin precursor first appear in basally located cells of the hypothalamus on day 12 of gestation. Long axonal fibers are evident at day 13. Laterally located cells give rise to lateral, superficial fibers, whereas medially located cells give rise to periventricular fibers. The antigenic determinants appear simultaneously in the anterior lobe of the pituitary gland on day 16 and in the intermediate lobe on day 17. The intensity of immunostaining with anti-16K fragment antiserum is stronger than with the other antisera in the brain but is equal with other antisera in the pituitary. Neuronal perikarya always stain less intensely than pituitary cells. It is concluded that 1) ACTH-related antigenic determinants are produced within the central nervous system, and 2) cells containing ACTH-related antigenic determinants in pituitary gland and brain develop independently of one another.

Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia.

Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma-pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.

Role of apoptosis of thyrocytes in a rat model of goiter. A possible involvement of Fas system.

Apoptosis, a physiological process of cell death, may modulate the mass of the thyroid gland. We investigated the role of apoptosis and the possible involvement of Fas/Fas ligand (FasL) system in apoptosis during goiter formation and involution in a rat model of goiter. Rats were fed a low iodine diet and a goitrogen, 6-propyl-2-thiouracil, to induce goiter. Rats with goiter were then fed a high iodine diet to study the phase of involution. We examined the presence of apoptosis by electron microscopy (EM) and terminal deoxy-UTP nick end labeling (TUNEL). We also investigated the association between Fas and FasL expression and thyrocyte apoptosis using immunohistochemistry and Western blotting. To evaluate the proliferation of thyrocytes, proliferating cell nuclear antigen was examined immunohistochemically. The number of apoptotic cells increased during goiter formation and the early stage of involution, which were also associated with increased number of Fas-positive thyrocytes, and some of these cells contained TUNEL-positive nuclei. However, the expression of FasL was almost constant throughout the experiment. Proliferating cell nuclear antigen/TUNEL ratio markedly increased during goiter formation but decreased particularly during the late stage of goiter involution. Our results indicate that apoptosis of thyrocytes is a main factor of cell loss during goiter formation and involution and suggest that the Fas/FasL system is involved in the induction of apoptosis of these cells. Moreover, the delicate balance between apoptosis and cell proliferation may play an important role in the control of thyroid gland mass.

Modern histochemical methods using enzymes as markers.

In the 25 years since the idea that histochemically detectable enzymes could serve as a marker of ligands, the world of histochemistry has undergone a dramatic change. Today slides of immunostained tissue sections may be filed together with hematoxylin- and eosin-stained slides and may be examined at one's leisure. Antigens are now localized at the ultrastructural level as well as at the light microscopic level permitting one to wonder about the intricacies and beauties of living creatures. Genes which mandate our body since the day of conception to the day of death can now easily be probed with enzyme markers. Even with all these success stories, some dreams which I had hoped to accomplish with the method are not yet realized. Unlike the chemical and physical properties of inert objects, living organisms are provided with a fixed genetic program. By recognizing the point in the program which is being executed, one can deduce the potential FUTURE activities of the cells and tissues. I hypothesize that this is not an impossible task since (1) histochemistry and immunohistochemistry already provide us information on the PAST activities of cells and tissues, (2) in situ hybridization describes PRESENT gene activities, and (3) newer methods allow for the determination of potential gene function. Thus it is possible to peek at the future.

Isolation and characterization of mouse C1q.

C1q was isolated from mouse serum and ascites fluid by absorption onto human IgG-coated latex beads followed by separation on 3-10% exponential gradient sodium dodecyl sulfate (SDS) polyacrylamide gels. Mouse C1q was also purified by low ionic strength precipitation of mouse serum. The purified C1q was heat-labile (56 degrees C, 30 min) both structurally and functionally, contained 4.3% hydroxyproline, 1.38% hydroxylysine, and 18.5% glycine, had an apparent molecular weight of 380,000 daltons, and reconstituted the hemolytic complement activity of C1q-depleted mouse serum. The negatively stained ultrastructural appearance of this purified material consisted of 6 globular units connected by strands. These data demonstrate that mouse C1q structurally and functionally is similar to human and rabbit C1q. A portion of polyacrylamide gel containing mouse C1q was injected into rabbits resulting in the production of monospecific antisera against mouse C1q. Thus, this procedure is a new, rapid and simple method to obtain monospecific antisera against mouse C1q.

The covalent coupling of proteins to periodate-oxidized sephadex: a new approach to immunoadsorbent preparation.

Proteins were covalently coupled in good yield to periodate-oxidized Sephadex. The reaction was studied in detail with respect to Sephadex type, sodium m-periodate concentration, protein (albumin or immunoglobulin) concentration, time course, temperature dependence, and pH optimum. The complexes were stable and were used successfully for the immunoadsorption of sheep anti-rabbit immunoglobulin antibodies.

Distribution of oncodevelopmental markers in neoplastic cells: therapeutic implications.

When the subcellular distribution of secretory component (SC) and carcinoembryonic antigen (CEA) were determined immunoelectronmicroscopically, SC was found on the baso-lateral surface and CEA on the apical surface of the normal gastrointestinal epithelium. In contrast, on the neoplastic cells SC and/or CEA were found all around the cell surface. Taking the change in the distribution of CEA on the neoplastic cells as an advantage, an attempt was made to develop an immunotherapeutic method for adenocarcinoma. The method was based upon an assumption that intravenously injected anti-CEA is not accessible to normal epithelial cells, since the tight junction will act as a barrier for the diffusion of antibodies from the interstitium to the apical cell surface, but the anti-CEA will form immunecomplexes with the CEA on the baso-lateral surface of neoplastic cells. Specifically, CEA-producing human gall bladder carcinoma were transplanted into nude mice. To the tumor-bearing mice, glucose oxidase-labeled anti-CEA was intravenously injected. As a control, glucose oxidase-labeled normal rabbit IgG was injected. This was followed with an injection of NaI. It was found that in those mice injected with the labeled anti-CEA, the size of tumor was reduced as much as 30% within three days. In the controls, the tumor continued to grow. In those injected with the labeled anti-CEA, CEA-anti-CEA immunecomplexes were deposited on the glomerular basement membrane, consequently a search for an insoluble apical antigen is currently made.

In situ localization of ribosomal RNAs is a reliable reference for hybridizable RNA in tissue sections.

Assessments of RNA integrity and its hybridizability are essential for successful implementation of in situ hybridization (ISH) for mRNA or viral RNA, particularly when paraffin-embedded specimens from surgical, biopsy, and autopsy cases are used. In this study, we examined the suitability of ISH of 28S ribosomal RNA (rRNA) for this purpose. Oligo-DNA with nucleotide sequences complementary to a well-conserved segment of 28S rRNA with auxiliary adenine-thymine-thymine (ATT) repeats at the 3' and 5' ends was synthesized. The oligo-DNA was made antigenic by converting the adjacent thymines to T-T dimers by UV irradiation and was used as a probe for ISH of 28S rRNA. The T-T dimers were detected by enzyme immunohistochemistry. When the results of ISH rRNA staining and that of total RNA staining by methyl green/pyronin Y were compared for various types of sections prepared from rat and human tissues, the staining intensities of total RNA did not always match those of ISH rRNA staining. In paraffin sections of formalin-fixed tissues, the degree of proteinase digestion influenced the ISH rRNA staining intensity, whereas it had no effect on the total RNA staining intensity. The intensities of ISH rRNA staining agreed well with those of various types of mRNA staining by ISH in 10 cases of paraffin-embedded pathological specimens. We therefore believe that ISH rRNA staining is a convenient parameter for evaluation of levels of hybridizable RNAs in tissue sections.

Localization of cyclic adenosine 3',5'-monophosphate-responsive element (CRE)-binding proteins by southwestern histochemistry.

Regulation of gene transcription requires an interaction between specific segments of nuclear DNA and specific proteins. We describe a method to localize the specific DNA-binding proteins using haptenized double-stranded (ds) DNAs. To demonstrate this method, an oligodeoxynucleotide (oligo-DNA) with a consensus base sequence of cyclic adenosine monophosphate-responsive element (CRE) (TGACGTCA) with three TTA repeats at the 5' end was synthesized. Since the CRE sequence is palindromic, the oligo-DNA was allowed to self-anneal and form ds DNA with three TTA repeats at both ends. The CRE ds-oligo-DNA was irradiated with UV light to form haptenic thymine-thymine (T-T) dimers. The haptenized CRE ds-oligo-DNA reacted by Southwestern analysis with a distinct set of proteins, previously identified as CRE-binding proteins, ranging from 40-90 KD. When the haptenized CRE ds-oligo-DNA reacted with frozen sections fixed with 4% paraformaldehyde in PBS (pH 7.4) followed by enzyme immunohistochemical localization of the T-T dimers, nuclei of intestinal epithelial cells and brain cells were heavily stained. Nuclear staining was blocked when the sections were reacted with the haptenized CRE ds-oligo-DNA in the presence of an excess amount of non-haptenized CRE ds-oligo-DNA. This method, henceforth referred as Southwestern histochemistry, should be a useful tool to localize proteins that bind to a specific DNA sequence and regulate the transcriptional activity of genes.

Hepatitis B. Cytologic localization of virus antigens and the role of the immune response.

Antigens of the hepatitis B virus have been localized within liver tissue by various immunologic, histochemical, and electron microscopic methods. The abundance and distribution of these virus antigens are in part determined by the host immune response. This interaction of immune mechanisms with the hepatitis B virus may be related to the pathogenesis and natural history of human hepatitis B virus infection.

Immunohistochemical examination of tumor-suppressor gene p53 product and pyrimidine dimer in solar keratosis.

In order to find biomarkers to measure the effects of UV irradiation, we examined the accumulation of p53 protein and pyrimidine dimers in 18 solar keratosis specimens. Frozen or AMeX-fixed solar keratosis specimens were immunohistochemically stained by anti-p53 mouse monoclonal antibody, pAb1801 and polyclonal anti-(pyrimidine dimer) antibody. Nuclear accumulation of p53 protein was found in 5/18 (28%) solar keratosis lesions. The percentage of cases showing nuclear p53 protein varied according to the histological type; in the bowenoid type it was 4/7 (57%); in the atrophic type it was 1/7 (14%). Nuclear pyrimidine dimers were not stained in solar keratosis, although the skin of UV-irradiated nude mice was positive. Accumulation of p53 protein is a good marker for early precancerous change caused by UV exposure.

Frequency of apoptosis relates inversely to invasiveness and metastatic activity in human colorectal cancer.

The frequency of apoptosis was determined in 102 cases of human colorectal cancer. The results were correlated with the frequency of cell proliferation and with clinicopathological characteristics such as degree of differentiation, invasiveness and metastasis. As a marker of apoptosis, intranuclear DNA strand breaks were localized with in situ nick translation (ISNT). As a marker of proliferation, proliferating cell nuclear antigen (PCNA) was localized immunohistochemically. The numbers of nuclei positive with ISNT and for PCNA per 1,000 nuclei on tissue sections were obtained. The labelling indices were compared with clinicopathological characteristics for each tumour. The ISNT labelling index of well differentiated colon carcinomas was higher than that of poorly differentiated carcinomas. Among similarly differentiated cancers, ISNT L.I. of colon carcinomas classified as Dukes A was higher than Dukes B/C, and L.I. of carcinomas which did not metastasize to lymph node or liver was higher than that of carcinomas which metastasized. The PCNA labelling index did not correlate with any of the clinicopathological characteristics or with the ISNT labelling index. The data suggest that apoptosis indices severe as a marker of tumour progression.