L3T4 effector cells in multiple organ-localized autoimmune disease in nude mice grafted with embryonic rat thymus.

Rat thymic grafts reconstituted T cell functions of BALB/c nude (nu/nu) mice to a considerable degree, but multiple organ-localized autoimmune diseases such as oophoritis and thyroiditis generally developed. The effector cell population in this autoimmune model was studied by adoptive transfer of the lesions into syngeneic nude mice. The transfer activity was not diminished when spleen cells were incubated with antiserum against rat cell antigen and C, but the activity was completely vanished by incubation with anti-Thy-1.2 plus C, indicating that the effector cells are T cells of mouse origin. Elimination of the L3T4+ subset virtually abolished the transfer activity, whereas that of the Lyt-2+ subset did not, indicating that the effector cells are L3T4+. Positive selection experiments by FACS also demonstrated that L3T4+ cells, but not Lyt-2+ cells, were capable of inducing the lesion, confirming the results with depletion experiments described above.

Cancer-associated fibroblasts induce cancer cell apoptosis that regulates invasion mode of tumours.

In the process of cancer spreading, different modes of invasion exist. One is expansive invasion, in which a group of cancer cells gradually expands along with cancer cell proliferation. Invasion of cancer cells is also modified by their interaction with stromal cells including cancer-associated fibroblasts (CAFs). Cancer cells co-invade with CAFs, and invasion by CAFs frequently precede invasion by cancer cells, which indicates CAF-led cancer cell invasion. Here, we show that CAFs induce apoptosis in gastric cancer cells, which prevented expansive invasion by cancer cells and instead facilitated CAF-led invasion. Death receptor 4 and activation of caspase-8 in cancer cells mediated cancer cell apoptosis induced by CAFs, which was dependent on contact between cancer cells and CAFs. Apoptotic cancer cells in turn released apoptotic vesicles and stimulated invasion of CAFs. Accordingly, cancer cells followed the migrating CAFs. Treatment with a caspase inhibitor, ZVAD, or forced expression of a death domain fragment in cancer cells prevented cancer cell apoptosis induced by CAFs and increased expansive invasion by cancer cells in extracellular gel invasion assays, while the rate of cancer cell invasion led by CAFs was decreased. Death domain-fragment expression also prevented intramural invasion by gastric cancer cells in the stomach. Because CAF-led invasion is characterized by the movement of individual cancer cells away from the tumour, adequate cancer cell apoptosis may promote cancer dissemination.

Immunohistochemical demonstration of Fc receptors of human lymph node cells using peroxidase-antiperoxidase (IgG).

Human lymph node cells, prepared from regional lymph nodes excised from four patients with gastric cancer, were incubated with peroxidase-antiperoxidase (IgG) (PAPIgG). After being washed, they were reacted with diaminobenzidine tetrahydrochloride in the presence of H2O2. Light microscopic examination revealed that a certain proportion of lymph node cells (18.2-32.2%) were labeled on their cell surface with brown-colored reaction products and that the labeled cells were composed of small lymphocytes. Electron microscopic examination demonstrated electron-dense irregular-shaped aggregates of reaction products on the cell surface of lymphocytes. Characterization experiments confirmed that the immune complexes of PAPIgG bound specifically with Fc receptors. PAPIgG, therefore, can be used as a specific indicator for Fc receptor of human lymph node cells.

Immunohistochemical detection of Fc receptor. II. Electron microscopic demonstration of Fc receptor by using soluble immune complexes of ferritin-antiferritin immunoglobulin G.

The mouse mesenteric lymph node cells were incubated in the soluble immune complexes of ferritin-antiferritin immunoglobulin G at 37 degrees C for 20 min. After being washed, postfixed with OsO4 and dehydrated by degraded ethanol series, the lymph node cells were observed by electron microscope. Apprroximately 15% of the cells (mainly composed of small lymphocytes) bound ferritin particles to the cell surface. The distribution pattern of the binding of ferritin particles (ferritin-antiferritin immunoglobulin G) took the form of discrete patches of irregular distribution interspaced with unlabeled portions. The electron microscopic features of ferritin particles (ferritin-antiferritin immunoglobulin G) attached to the cell surface suggest that a structure of constant conformation (Fc receptor) situated in the cell membrane takes part in the binding of ferritin-antiferritin immunoglobulin G.

Immunohistochemical detection of Fc receptor. I. Light microscopic demonstration of Fc receptor by using soluble immune complexes of peroxidase-antiperoxidase immunoglobulin G.

The mouse mesenteric lymph node cells (in the cell suspension and frozen sections) were incubated in the soluble immune complexes of peroxidase-antiperoxidase immunoglobulin G. After being washed, they were reacted with diaminobenzidine tetrahydrochloride. Light microscopically brown-colored granules were observed on the cell surface of a proportion of small lymphocytes. In frozen sections, a proportion of small lymphocytes were stained dark brown on the cell surface. Characterization and control experiments suggest that the binding of peroxidase-antiperoxidase immunoglobulin G to the cell surface is mediated by Fc receptor. Peroxidase-antiperoxidase immunoglobulin G, therefore, can be used as in indicator of Fc receptor.

Detection of purine nucleoside phosphorylase in paraffin sections by the immunoperoxidase method.

A method is described for immunohistochemical demonstration of purine nucleoside phosphorylase (PNP: EC 2.4.2.1) in paraffin sections from routine surgical histology specimens. A peroxidase-antiperoxidase (PAP) method was employed, using specific rabbit antiserum against human PNP, which was purified from postmature human erythrocytes. In human lymph nodes, intensive staining for PNP was observed in the vast majority of small lymphocytes in paracortical areas, in many small lymphocytes in medullary cords, and in a few small-to medium-sized lymphocytes in germinal centers. Small lymphocytes in the primary follicles and those in the mantle zones of secondary follicles were negative for PNP staining. Tingible body macrophages, lymphatic sinus cells, and most of the large cells in germinal centers did not stain with anti-human PNP (hPNP) antibody. Endothelial cells of small vessels in the cortex and plasma cells did not show any constant pattern of PNP staining intensity. Histochemistry revealed that the distribution pattern of PNP activity was quite similar to that demonstrated on paraffin sections by the PAP method.

Double immunoenzymatic detection of surface phenotype of proliferating lymphocytes in situ with monoclonal antibodies against DNA polymerase alpha and lymphocyte membrane antigens.

To detect the proliferating cells in situ, a monoclonal antibody against human DNA polymerase alpha (pol alpha) was employed because this enzyme is known to be present in the nucleus of the cells in G1, S, and G2 phases. In addition, the surface phenotype of pol alpha-positive proliferating lymphocytes in diseased lymph nodes was determined by double staining consisting of immunoperoxidase and immunoalkaline phosphatase methods with various monoclonal antibodies against lymphocyte membrane antigens. In the paracortical area of lymph nodes with reactive changes, proliferating cells were 17% or less, and most of them were helper T-cells, although suppressor T-cells and B-cells also proliferate to a certain extent. In contrast, the proliferating cell population in malignant lymphomas was generally more than 40%, and it showed a single surface phenotype, indicating monoclonal proliferation. In addition, an unusual T-cell antigen phenotype of proliferating cells was observed in some cases of peripheral T-cell lymphomas. Thus, this double staining provided the authors with valuable information regarding the proportion, localization, and surface phenotype of proliferating cells, which should be useful for diagnosis of the diseases of lymphoid system.

Elevation of urinary trehalase in mercuric chloride-induced nephrotoxic rabbits: urinary trehalase as a specific indicator of renal brush border damage.

The origin of urinary trehalase in mercuric chloride-induced nephrotoxic rabbits was demonstrated with biochemical and immunochemical techniques. Urinary trehalase was dramatically increased with HgCl2-induced nephrotoxicity. The nephrotoxic kidney showed an extreme decrease in specific fluorescence with fluorescein isothiocyanate (FITC)-conjugated antibody technique. Moreover, trehalase activity in the membrane fraction was remarkably decreased in the nephrotic kidney compared with the control. Judging from the results of immunodiffusion, urinary trehalase and renal trehalase exhibit the same antigenicity. From the data of a time course analysis of nephrotoxicity, the excretion of urinary trehalase was earlier than that of urinary sugar. Previous results show that renal trehalase is localized in the renal tubular brush borders. From these results, it is suggested that urinary trehalase is originated in the renal brush borders. In consideration of the results described in previous papers and in this paper, it is proposed that urinary trehalase is a good indicator of renal brush border damage.

[An autopsy case of Ehlers-Danlos syndrome showing various types of cerebrovascular disease].

We report a 73-year-old woman with Ehlers-Danlos syndrome (EDS) and hypertension who had developed various types of cerebrovascular disease. She had suffered from cerebral hemorrhage of the left putamen at the age of 58, of the left parietal lobe at 64 and cerebral infarction of right internal capsule at 71. EDS type II or III was suggested by two times of skin biopsies. A brain CT at the age of 73 revealed a comparatively large cerebral aneurysm in the territory of the anterior cerebral artery. The patient was treated conservatively, but died due to rupture of the aneurysm. The wall of the aneurysm was made up thin collagen fibers without elastic fibers. There were other multiple small aneurysms in the cerebral arteries, but none in other organs. Deposition of acid mucopolysaccharides was noted in the media of the abdominal aorta. Finally, the present case was thought most likely to be of EDS type IV. It was suggested that one of the causes of the cerebral hemorrhage at the ages of 58 and 64 and the infarction at 71 was related to hypertension, since brain MR angiography at 71 showed no clear aneurysms. In cases of EDS, one should consider the possible formation or rupture of cerebral aneurysm even though the course is favorable.

[Treatment of solid tumor by a small amount of direct current].

Solid tumor treatment was given in our animal laboratory to determine the mechanism of tumor disappearance by direct electric current, and clinical trials were done on 9 far advanced recurrent rectal cancers. Solid tumors of Yoshida sarcoma in Donryu rat were treated by 1 mA of constant direct current for 1 hour a day, for 4 days. The tumors disappeared in 13/16 within 21 days. Positive results of DNA Nick-end labeling and ladder patterns in the gel electrophoresis of DNA were observed in the regressing tumor specimen. It is considered that apoptosis is the one of the mechanisms of a disappearing tumor. As for the clinical trial concern, in 9 cases of rectal cancer, one CR and 5 PR were seen.

[Electron microscopic and histochemical study of the rat adrenal cortex treated with aminoglutethimide (Elipten, CIBA) and its reparative process]. / Elektronenmikroskopische und elektronenmikroskopisch-histochemische Untersuchungen über Nebennierenrinde der Ratte mit Aminoglutäthimid (Elipten, CIBA) und über ihren Reparaturprozess

Aminoglutethimide blocks the conversion of free cholesterin to delta5-pregnenolone. It's effects on the fasciculata cells were studied electron microscopically in Wistar rats fed with this chemical for a maximum of seven days. The animals surviving after this treatment were also examined to follow the process of restoration from the damage. Four to seven days the initiation of the feeding, the mitochondria were enlarged, variously deformed and contained intramitochondrial vacuoles. The lipid droplets were increased in size. Three to four days after withdrawal of aminoglutethimide, so-called "intramitochondrial mitochondria" appeared. The picture suggesting division of the mitochondrial matrx was also occasionally encountered. These features suggested formation of new mitochondria from preexisting mitochondria. The mitochondria resumed their normal shape and appearance starting about ten days after withdrawal of aminoglutethimide. The electron microscopic histochemical method demonstrated tubular crystals of digitonin-cholesterol in the intramitochondrial vacoles as well as in the lipid droplets 4 days after the initiation of the feeding. The withdrawal of aminoglutethimide was followed with disappearance of these crystals from the sites of deposition.

Phenotyping of proliferating lymphocytes in angioimmunoblastic lymphadenopathy and related lesions by the double immunoenzymatic staining technique.

Biopsy specimens of lymph nodes with the histologic characteristics of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) were obtained from 9 cases (4 cases of AILD and 5 cases of AILD-like T lymphoma [AILD-T]) and histologically analyzed by the use of a double immunoenzymatic staining technique with the combination of a monoclonal antibody against lymphocyte membrane antigen and that against human DNA polymerase alpha (pol alpha), which is detectable in the nucleus of the cells in G1, S, and G2 phases. In all 9 cases, the pol alpha + proliferating cells had a peripheral T-cell phenotype with T11 and Leu-4 antigens, whereas proliferating B cells with B1 antigen were rarely observed. As for T-cell subset antigens, the proliferating T cells had T4+ helper/inducer phenotype in 7 cases, while T8+ suppressor/killer T cells proliferated in 2 cases, although a significant number of T4+ proliferating cells were also recognized. The study on malignant lymphomas that evolved in the 2 cases showed that the T-subset antigens on major proliferating tumor cells were the same as those found in the preceding AILD lesions, suggesting that lymphoma T cells originate from the AILD lesion. The results suggested that AILD without histologic manifestations of malignancy and AILD-T may be a neoplastic disease derived from either subset of peripheral T cells.

[An autopsied case of malignant lymphoma of the pancreas with an elevation of the serum Ca 19-9 level].

A 37-year-old male was admitted to hospital because of a right hypochondrial pain and icterus. His physical examination showed hepatosplenomegaly, and the laboratory findings demonstrated abnormal hepatic and pancreatic functions. A CT examination revealed a large mass of a low density in the pancreas head and tail. Further, an immunological study revealed that the patients serum Ca 19-9 level was elevated, but that the CEA and AFP levels were normal. Both the pathological and cytological examinations, however, did not indicate a malignancy of the pancreas. The patient subsequently developed uremia, a hemorrhagic tendency, and then died. An autopsy confirmed a pancreatic tumor which occupied the head of pancreas. Histologically, the tumor contained round cells with scanty cytoplasms and showed a sarcomatous pattern. An immunohistochemical study showed that the LCA, MB-1, and the LN-1 for B cell markers were positive, while the MT-1 for T cells was negative. The case illustrates a malignant lymphoma of the pancreas which demonstrated a serum Ca 19-9 elevation.

DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis.

Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end labeling method. Myocardial cells corresponding to those with nick end labeling method-stained nuclei, on the other hand, had mostly pyknotic and karyolytic nuclei and some unremarkable nuclei, even nuclear ghosts, and showed degenerated cytoplasm, including contraction band necrosis in H&E-stained preparations. The agarose gel electrophoresis of DNA extracted from the corresponding areas mentioned above showed the ladder pattern of internucleosomal cleavage characteristic of apoptosis. The present study revealed that infarcted myocardial cells with nuclear outlines, even nuclear ghosts, showed a distinct DNA fragmentation with the ladder pattern of internucleosomal cleavage. It is concluded from this study that the damaged myocardial cells of acute myocardial infarction represent a coagulation necrosis having the biochemical nature of apoptosis.

Ontogenic development of T and B cells and non-lymphoid cells in the white pulp of human spleen.

The ontogenic development of lymphoid and non-lymphoid cells in human splenic white pulp was studied histologically with immunoperoxidase technique, together with that of lymphoid cells from fetal liver, bone marrow and thymus by membrane immunofluorescence assay. The primitive white pulp, which appeared as small accumulations of lymphocytes around arterioles at 14 weeks of gestation (g.w.), was mainly composed of B1 antigen-positive B cells. After the appearance of follicular structure accompanied by follicular dendritic cells (FDC) stained with anti-DRC1 antibody at 26 g.w., these perivascular structures of B cells were located in the periphery of the white pulp areas. A large number of B cells composing the perivascular structure had surface IgM (sIgM) and IgD (sIgD) from the earliest stage (14 g.w.), although this type of B cell with mature phenotype was seldom observed in fetal liver or bone marrow at this stage. It was suggested that the spleen is an important site for B-cell maturation from sIg-negative B cells observed in 10-14 g.w. fetal liver, and that FDC are not involved in this development of B cells. The organization of 9.6 antigen-positive T cells around arterioles developed 4 weeks later than that of B cells, at 18 g.w., although 11 g.w. fetal thymocytes already showed a phenotype very similar to that of infants. Interdigitating reticulum cells (IDC) stained with anti-S-100 protein serum appeared from 14 g.w. before the T-cell organization, suggesting that IDC may play an essential role in the homing of T cells.

Cyclophosphamide-induced apoptosis induces phocomelia in the mouse.

Phocomelia (absence of upper fore and/or hind limbs) was induced in mouse fetuses using cyclophosphamide. On day 11 of gestation, pregnant mice were injected intraperitoneally with 10 ml/kg of saline containing cyclophosphamide (CP) at a dosage of 20 mg/kg body weight. On day 18, the fetuses were removed by Caesarean section from dams given CP on day 11 and were examined for external anomalies. Of 22 fetuses from CP-treated dams, 13 were dead or absorbed, but the surviving 9 fetuses were found to have phocomelia with various other external anomalies. In order to examine the direct cytotoxic effect of CP on fetal limb buds, fetuses were removed at 8, 16, 24, and 48 h after CP administration on day 11, revealing the presence of frequent pyknotic nuclei and apoptotic bodies in hematoxylin and eosin (H & E) preparations. Cell-nuclei and apoptotic bodies were frequently observed by nick end-labeling in limb buds. Transmission electron microscopy demonstrated the typical changes of apoptosis. DNA extracted from the fetal limb buds submitted to CP was analysed by agarose gel electrophoresis, showing the ladder pattern characteristic of internucleosomal cleavage. These findings suggest that cyclophosphamide causes apoptosis in mouse fetal limb buds and that this process induces the external anomalies of phocomelia.