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Pit cells, which are recovered in low density fraction of lymphocytes derived from the liver (L-Fr.1,2), are recognized as perisinusoidal cells. In the present study, the accumulation of liver-derived low (L-Fr.1,2) and high (L-Fr.3,4) density 51Cr-labeled lymphocytes was assessed in the liver, spleen and peripheral blood of syngeneic BALB/c mice in comparison with those derived from the spleen (S-Fr.1,2 and S-Fr.3,4) and peripheral blood (P-Fr.1,2 and P-Fr.3,4). L-Fr.1,2, L-Fr.3,4 and P-Fr.1,2 accumulated selectively in the liver, whereas P-Fr.3,4 did not accumulate in the liver. L-Fr.3,4 was composed of two major subpopulations; Thy1+CD4+ and Thy1+CD8+. This suggested that L-Fr.3,4 were distinctive from P-Fr.3,4 or L-Fr.1,2 and inhabited the liver. P-Fr.1,2 and S-Fr.1,2 tended to accumulate in the liver and 51Cr labeled P-Fr.1,2 and S-Fr.1,2 were recovered mainly in L-Fr.1,2 L-Fr.3,4, S-Fr.1,2 and P-Fr.3,4 48 h after the transfer of these cells, suggesting that a part of P-Fr.1,2 and S-Fr.1,2 might be in dynamic equilibrium with those of L-Fr.1,2 and could be a part of the source of L-Fr.3,4. Results also suggested that some of L-Fr.1,2 of T cell lineage might change into L-Fr.3,4 in the liver and might be released into the systemic circulation. Thereafter, L-Fr.3,4 might behave as P-Fr.3,4 and no longer show organ-specific accumulation.

The fine structure of the hepatic sinusoids of 81 human embryos and fetuses and their development from 5 to 12 weeks gestation were studied. At 5 weeks gestation, sinusoid-like structures and Kupffer-like cells were observed between liver cell cords. Between 6 and 8 weeks gestation the sinusoids were completely developed. Definite Kupffer cells appear at this developmental stage, when the bone marrow has not yet formed. Floating macrophages form cell aggregates in the sinusoids which contact endothelial cells and settle as Kupffer cells. Erythroblastophagia is observed in Kupffer cells and macrophages. The endothelial linings are closed, with the attenuated cell processes and intercellular junctions between the adjoining endothelial cells. No transition was observed between Kupffer cells and endothelial cells. The findings suggest that Kupffer cells in the human embryo are extrahepatic in origin and that they reach the sinusoids via the circulatory system. Ito cells, which store fat, originate from mesenchymal cells in the septum transversum.

The effects of repeated d-galactosamine (GalN) administration - i.e. subacute GalN intoxication - on rat liver cell nucleoli were examined. After an initial intraperitoneal injection of 375 mg GalN/kg body weight the rats were treated with 250 mg GalN/kg body weight daily at intervals between 24 h and 30 days. The rats were sacrificed six h after the last injection by decapitation. Specimens of liver were studied by light and electron microscopy. According to the nucleolar size three stages were to be distinguished. Stage I: decreased nucleolar size after one injection, stage II: increased and maximal nucleolar size after two to five injections, stage III: slightly increased relatively stable nucleolar size after six and more injections. Fine structural observations suggest a nucleolar hyperfunction in subacute GalN intoxication as well as a minor disturbance of the transcription and the transfer of the nucleolar RNA.

One of the most sensitive and specific signs of the galactosamine effect upon the rat liver cell is the appearance of PAS-positive and diastase-resistant granules within the cytoplasm of hepatocytes. Light-microscopic, histochemical, biochemical, and electron-microscopic findings reveal that the appearance of these ADB (= atypical dense bodies) depends upon a working glycogen metabolism at the time of GalN treatment. The ADB are composed of particles resembling, due to shape and size, ribosomes and beta particles of glycogen. Most of them are surrounded by the rER, but they are never enclosed by a limiting membrane. Due to sequential changes they can be generally classified into three types; the early, the intermediate, and the late type. In seven experiments it can be shown, that the appearance of the ADB depends upon the time and dosage after GalN treatment. They occur even if an additional treatment with galactose or uridine prevents the liver from the features of a hepatitis, as also shown in the livers of newborn animals up to 3 weeks of age. The histochemical response against various glucosidases, hexosaminidases, pronase, and RNAse as well as against various fixatives indicates that ADB are composed of, at least, two different constituents, the former RNAse-sensitive and visible with routine light-microscopic staining procedures, the latter RNA-resistant, PAS-positive, and invisible after staining with H & E or toluidine blue. The latter is diastase-resistant, suggesting that this portion of ADB does not represent the usual glycoproteins but some abnormal metabolite of glycogen. The ADB can be detected with maximal accumulation in the cytoplasm of hepatocytes at that time when the glycogen content determined in the liver homogenate by biochemical methods is greatly reduced.

Liver tissues obtained from 78 human fetuses between 5 weeks and 10 months of gestation were observed with light and electron microscope. In the third month of gestation hepatic megakaryocytopoiesis reached the maximum level and then appeared to decline. At the maximum level immature to mature megakaryocytes were usually smaller than those from adult bone marrow. They were observed between growing liver cells, within hepatic sinusoids and in the mesenchyme surrounding the liver parenchyma in the given order of frequency. Immature megakaryocytes were characterized by the presence of both demarcation vesicles or tubules and a few small round specific granules. The nucleus was mononuclear but frequently indented. With cell maturation, the megakaryocytes were stained intensely by PAS-technique. The nuclei were multilobulated. Large cytoplasm was dominated by numerous specific granules and well-developed demarcation membrane system. Some megakaryocytes passed through the sinusoidal wall and possibly to hepatic sinusoids. These observations indicate that the main site of hepatic megakaryocytopoiesis may be extravascular and more likely between liver cells. Small megakaryocytes in fetal livers are discussed in relation to the occurrence of similar megakaryocytes in various blood dyscrasias.

A case of focal nodular hyperplasia of the liver was reported, in which various diagnostic imaging modalities were performed. Hepatic colloid scan (SPECT) in this case shows typical focal accumulation in the lesion. Ultrasonic image shows a sharply demarcated hypoechoic lesion in the left lobe. But no significant informations of this tumor were obtained in the plain CT, CE-CT and angiography. Echo-guided biopsy was performed and focal cirrhotic finding in the lesion without liver cirrhosis was obtained.

Mitochondria isolated from livers of rats which received D-galactosamine (375 mg/kg body wt., four times) demonstrated a marked decrease in respiratory control ratios, the ADP/O ratios, and state 3 respiration rates and an increase in state 4 respiration rates. The aberration was profound with site I being altered prior to sites II and III. Quantitation of phospholipids revealed a reduction of total phospholipids per mg protein with decreases in phosphatidylcholine and phosphatidylethanolamine contents. Caldiolipin was the only phospholipid which remained unaltered. Fatty acid composition was altered in these phospholipids; caldiolipin was altered most severely, showing reductions in linoleic and arachidonic acids, and an elevation in saturated fatty acids and in some other small components of fatty acids. In phosphatidylethanolamine, palmitic acid decreased, whereas stearic and docosahexenoic acids increased. These changes were smaller in phosphatidylcholine fatty acids. These mitochondria were also characterized by an altered composition in high molecular weight polypeptide components. By experiments with normal mitochondria in vitro, galactosamine, but not other aminohexoses, was proved to be an uncoupling agent of the oxidative phosphorylation system. Electron microscopic observation demonstrated that both in vivo and in vitro treatments with galactosamine induced marked disorganization of mitochondrial structures. These results suggest that mitochondrial damage is also included in galactosamine-induced hepatic lesion.

In rats hepatocellular cytoplasmic changes after daily repeated D-galactosamine (GalN) intoxication--i.e. subacute GalN intoxication--were studied by light and electron microscopy. The number of GalN injections--and thus the days of survival--was between one and 30. The rats were killed six hours after the last GalN injection. Less degenerative changes were found after repeated GalN injections. An increased formation of atypical dense bodies (ADB), a temporary pronounced lipid accumulation and changes of the rough and smooth endoplasmic reticulum were prominent features of subacute GalN intoxication. The implications with respect to a modified GalN action in subacute GalN intoxication are discussed with special reference to biochemical data obtained in the same experimental model (Schuchhardt et al., 1977).

A 30-year-old previously healthy housewife presented with bilateral pulmonary infiltrates. She was treated with various antibiotics but died with disseminated intravascular coagulation. Cultures of tracheal aspirates obtained prior to her death yielded Trichosporon beigelii. Postmortem examination revealed T. beigelii pneumonia with fungal dissemination in other organs. In addition, many organs were found to be infiltrated with atypical mononuclear cells which exhibited cytochemical properties of histiocytes. It was considered, therefore, that T. beigelii pneumonia developed on the basis of underlying malignant histiocytosis. T. beigelii in her lung sections was fluorescently stained with rabbit antiserum to the fungus isolated from the patient. This is the first report of Trichosporon beigelii pneumonia associated with malignant histiocytosis.

In the human lymphoreticular system, the alpha and beta subunits of S-100 protein are found in ordinary monocyte-macrophages and non-phagocytic histiocytes such as Langerhans cells and interdigitating reticulum cells, respectively. The beta subunit is also present in some CD8+ T cells. In the present study, we investigated the ontogeny of these histiocytes and lymphocytes in humans. Yolk sacs and 4 to 21-week fetuses were examined immunohistochemically for the presence of S-100 protein subunits using antisera monospecific to each subunit. S-100 alpha + macrophages were present in the yolk sacs and the hepatic sinusoids of the 4th week embryos prior to bone marrow hematopoiesis. These macrophages later appeared in other lymphoid organs when anlagen of these organs were formed. No S-100 beta + cells were found in the yolk sacs. S-100 beta+ histiocytes were first detected in the hepatic sinusoids of the 5th week embryo, and after the 8th week of gestation, they were distributed in other lymphoid organs. S-100 beta+ lymphocytes were not found in the liver. They were first detected in the thymus at the 12th week of gestation, and were subsequently distributed in other lymphoid organs. These results suggest that S-100 beta+ lymphocytes and histiocytes may belong to different cell lineages, and the former may not be the precursor of the latter.

Distribution and fine structure of macrophages were studied in 10 human embryos in the 6th and 7th week of gestation, 5.5 to 12 mm in crown-rump length. The yolk sac macrophages were found in the extravascular mesenchymal tissues and intravascular spaces long before the first appearance of bone marrow and lymphatic tissues in the embryos. In addition to the macrophages, the fibroblastic cells and the cells of erythropoietic series were also present in the extravascular space. The macrophages showed a variety of cellular structures suggesting transition from immature cell type with no heterophagolysosomes to mature cell type in phagocytosis. The mature macrophages avidly phagocytized the primitive erythroblasts and occasionally platelets. They were positively stained for lysosomal enzymes and were characterized by numerous pleomorphic heterophagolysosomes which exhibited various stages of digestion of phagocytized blood cells. The origin of intravascular macrophages may be in either migrated extravascular macrophages or phagocytic endothelial cells. The phagocytosis and degradation of erythroblasts appear to be one of the main functions of yolk sac macrophages. The presence of the macrophages in mitosis indicates their proliferation in situ.

When the mouse lymphoma (L-1210) cells are treated with methylnitrosourea (MNU) at 37 degrees C for 30 min and then cultured for 4 h in a normal medium nuclear structure and functions of the cells are changed. We have investigated the mechanism as to how nuclear structure and functions are changed by MNU. In MNU-treated cells euchromatin area diminishes and chromatin condensation occurs. [3H]thymidine and [3H]uridine uptakes of the MNU-treated cells decrease. In contrast, when the MNU-treated cells are cultured in the presence of 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) synthetase changes of nuclear structure do not appear. [3H]Thymidine and [3H]uridine uptakes are partially and almost completely recovered, respectively. Autoradiographs of the cells labelled with [3H]NAD, a substrate of poly(ADP-ribose) synthetase show that silver grains due to [3H]ADP-ribose are densely located only in the cells where chromatin condensation occurs. Chromatin-bound proteins of molecular masses 20-25 X 10(3) daltons are specifically poly(ADP-ribosyl)ated in the MNU-treated cells. These results suggest that MNU-induced chromatin condensation is caused by poly(ADP-ribosyl)ation of chromatinbound proteins.

Findings of perivascular mesenchymal cells and fibroblasts in mice receiving large doses of vitamin A were described. Liver, lung, intestine and skin were investigated by fluorescence and electron microscopy. Marked increase of fluorescence of vitamin A was observed in the sinusoidal wall of the liver, in the alveolar septa of the lungs, in the propria mucosa, submucosa and muscular layer of the intestine and in the dermis of the abdominal skin. Increased fluorescence of these organs corresponded, ultrastructually, to the appearance of numerous fat droplets in Ito cells of the liver, septal cells of the lung and fibroblasts of the intestine and of the skin. All of these cells showed the same morphological features and the same distribution in the tissue, namely in the interstitial connective tissue space. These findings indicate that vitamin A storing cells are distributed widely in the connective tissue of various organs and that perivascular vitamin A storing mesenchymal cells and interstitial fibroblasts are probably of common fibroblastic cell line.

The ultracytochemical acridine orange (AO) method has been employed to demonstrate DNA template activity within experimental brain tumors induced by transplacental administration ethylnitrosourea (ENU) in Sprague-Dawley rats. Ultrastructural examination revealed that acridine orange binds to DNA exclusively within the active extended euchromatin portion of the cell nucleus of brain tumor and of foci of atypical cell proliferation. No AO reaction products were visible in the non-neoplastic cell nuclei adjacent to the brain tumors. The percentages of AO positive cells in brain tumor cells in rats given an intraperitoneal injection of 3H-thymidine 1 hr before sacrifice were between 2.4% and 8.0%. The average number of AO chromatin reaction products per single section of a cell nucleus varied between 7 and 21. The ratios of areas of euchromatin to heterochromatin were found to be slightly larger in AO positive nuclei then in AO negative nuclei in each of the tumors. The present results suggested that rat brain tumors induced by transplacental administration ENU exhibit de-repression of DNA template normally repressed in the adult state. The usefulness of this method for study on the development of experimental brain tumors is briefly discussed.

Morphologic and radiologic studies were undertaken on 26 human embryos and fetuses to determine the stage and site of the earliest bone marrow formation. Up to the 10th week of gestation, primary bone marrow is not present anywhere although the intramembranous ossification occurs in the maxilla and mandible and also in the middle portion of the clavicle. At the 11th week of gestation, X-ray examination showed in two fetuses the bone formation in the clavicle, scapula, maxilla, mandible, and the diaphysis of the long bones. Serial sections of these fetuses revealed that the primary bone marrow occurs first in the middle portion of the clavicle. From a series of our embryological studies, the concept of the mononuclear phagocyte system which involves the bone-marrow-derived monocytic origin of tissue macrophages, is not accepted, at least, on the origin of Kupffer cells in human fetal livers.

Focusing on hematopoiesis, especially granulocytopoiesis, livers from ten human fetuses between 7 to 13 weeks of gestation were observed with light and electron microscope. In the hepatic mesenchymal tissue variable number of granulocytes in varying stage of maturation were found from 10 to 13 weeks of gestation. The granules of neutrophils in human fetal livers were classified into three distinct types. The first granules appeared in myeloblasts. The second granules, possibly formed in Golgi complex, were more larger and observed after the promyelocytic stage. The third granules, predominant in mature neutrophils, were pleomorphic. Other than granulocytic, erythrocytic and stromal cells, unclassifiable cells were occasionally encountered. The role of mesenchymal tissues in the granulocytopoiesis, the development of fetal neutrophils and the problem of hematopoietic stem cells were discussed.

The proliferative activity of Ito cells in acute liver injury induced by carbon tetrachloride (CCl4) was studied by light and electron microscopic autoradiography. At 48 hours after a single intraperitoneal injection of CCl4, the livers of the mice given vitamin A per os for preceding 9 days and those of the mice without vitamin A-pretreatment were removed. Small tissue blocks of each group were respectively incubated at 37 degrees C for 1 hour in culture medium containing 3H-thymidine. After CCl4 injection, perisinusoidal and sinusoidal cells adjacent to centrilobular necrotic liver cells increased in number and size. Some of them were labelled by 3H-thymidine. On the other hand, the perisinusoidal and sinusoidal cells in the peripheral zone in which liver cells are not markedly degenerated nor necrotic showed no noticeable increase in number. They contained very few or no silver grains after 3H-thymidine. In control mice the labelling of perisinusoidal cells was hardly observed. Electron microscopic autoradiography revealed that most of the labelled perisinusoidal cells in the centrilobular zone possess characteristics of Ito cells in their location and in the fine structures such as the presence of small fat droplets, well-developed rough endoplasmic reticulum, and Golgi complex in the cytoplasm. These findings indicate that Ito cells incorporate 3H-thymidine in DNA synthesis after hepatocellular necrosis resulting in cell proliferation.

An unusual case of solid hepatic hamartoma, which could not be correctly categorized under the WHO classification, is reported. The well defined, solid nodular lesion, arising in the anterior edge of the left hepatic lobe, was found in a 15-month-old female infant. The lesion was composed of irregularly shaped yellow islands of parenchyma and white-gray nodules of fibrous stroma of various sizes, which were intermingled revealing a mosaic pattern. Prominent bile ductal proliferation was observed histologically in the fibrous stroma, associated in parts with dense or loose periductal bundles and vascular proliferation. The stromal proliferation seemed to result in erosion of the islands of differentiated parenchymal cells with no regeneration. Alpha-fetoprotein was demonstrated immunohistologically in the parenchymal cells of the lesion. From these findings, the essential component of this solid hamartoma was thought to be markedly proliferating bile ducts associated with periductal fibrosis, resulting in a fibroadenomatous pattern.

An indirect immunoperoxidase method capable of identifying an opportunistic fungus, Trichosporon beigelii, in formalin-fixed, paraffin-embedded tissue has been developed. The authors studied autopsy materials from 19 patients with various fungal infections, including 3 patients with disseminated T. beigelii infection, 8 patients with localized or systemic candidiasis, 4 patients with invasive aspergillosis, 2 patients with pulmonary mucormycosis, and 2 patients with systemic cryptococcosis. T. beigelii could be successfully visualized in tissue sections from all three patients with this infection and a patient with systemic candidiasis who was found to have concomitant infection by T. beigelii. The capsule of Cryptococcus neoformans was also stained, indicating the presence of cross-reactive antigen between T. beigelii and C. neoformans. C. neoformans-absorbed antiserum was reactive with T. beigelii but not with other fungi, including C. neoformans.

Small tissue blocks of livers obtained from normal and CCl4-treated mice were incubated in culture medium supplemented with 3H-leucine for one hour to observe the protein synthesis of Ito cells in acute carbon tetrachloride liver injury by a light and electron microscopic autoradiography. In normal mouse liver very few Ito cells are labeled with 3H-leucine. On the contrary, in acutely damaged mouse liver by a single intraperitoneal injection of CCl4, the Ito cells in the centrilobular zones where liver cells show marked degeneration and necrosis are densely labeled. No labeling, however occurs over the Ito cells in the intermediate and peripheral zones of the same liver lobules, where liver cells showed no cellular degeneration. These findings show that the Ito cells are inactive in 3H-leucine incorporation under physiological conditions. After acute liver cell injury the Ito cells located in the necrotic areas of liver cells are markedly activated for protein synthesis.