Mobilization of iron and iron-related proteins in rat spleen after intravenous injection of lipopolysaccharides (LPS).

We used lipopolysaccharide (LPS) to provoke immune responses and observed the changes in the localization of iron and iron-related proteins, such as transferrin receptor, ferritin and hemosiderin in the rat spleen. After intravenous injection of 250 micrograms LPS (salmonella minnesota R595), spleen weight and serum IgM levels increased, cells incorporating 5-bromo-2'-deoxyuridine (BrdU), and transferrin receptor positive cells increased in the peripheral portion of the periarterial lymphoid sheath (PALS), the marginal zone (MZ) and the follicles. Ferritin positive cells increased markedly in the white pulp and stainable iron increased in the marginal metallophils (MM) and in the macrophages in the MZ and the outer PALS. Even in iron deficient rats, a similar change was observed for the localization of iron and iron-related proteins after injection of LPS. After injection of 0.4 mg keyhole limpet hemocyanin (KLH), changes similar to but less pronounced than that in the LPS injected rats were observed for serum IgM levels and for the localization of iron and iron-related proteins. These results showed that the iron in the MM and the macrophages in the white pulp have a dynamic response to immunological challenges and suggested that they play some role in immune responses.

Effects of nicotinamide and its isomers on iron-induced renal damage.

The effects of three isomers of pyridinecarboxamide (picolinamide (2-pyridinecarboxamide), nicotinamide (3-pyridinecarboxamide) and isonicotinamide (4-pyridinecarboxamide)) on iron-induced renal damage were studied. Pyridinecarboxamide (250 mg/kg body weight, ip) was administered 10 min before injection of ferric nitrilotriacetate (Fe(III)-NTA) (7.5 mgFe/kg body weight, ip). In picolinamide-treated rats, the renal tubular necrosis induced by Fe(III)-NTA was attenuated and serum creatinine did not increase. Picolinamide most efficiently suppressed renal lipid peroxidation in vivo-induced by Fe(III)-NTA. Non-heme iron levels in the kidneys after Fe(III)-NTA injection did not differ in groups to which pyridinecarboxamides were administered. To elucidate the protective effects of picolinamide, we studied the action of pyridinecarboxamides on lipid peroxidation and DNA damage in vitro. These isomers inhibited iron-induced lipid peroxidation of linolenic acid. Picolinamide had no effect on DNA damage, but nicotinamide and isonicotinamide promoted DNA damage by iron, especially when ascorbate was used as a reductant. None of these pyridinecarboxamide isomers changed the chlelate structure of Fe(III)-NTA as shown by electronic absorption spectra. Among the three isomers, picolinamide most effectively protected the kidneys against iron-induced renal damage, since it not only inhibited iron-induced lipid peroxidation, but also had little enhancing action on DNA damage by iron.

Transferrin receptors and selective iron deposition in pancreatic B cells of iron-overloaded rats.

Iron overload was produced in Wistar rats by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe(3+)-NTA) for one to six months. Pancreatic tissues from these iron-overloaded rats and untreated controls were examined for insulin (for B cells), glucagon (for A cells), transferrin receptor (TfR), transferrin (Tf) and ferritin (Ft) using immunohistochemical methods, and for iron by histochemical Berlin blue staining. In the islets of iron-overloaded rats, increased Ft staining appeared prior to deposition of Berlin blue-stainable iron, and the staining intensity of Ft and iron was stronger in B cells than in A cells. In the islets of untreated control rats, the staining intensity of TfR was stronger in B cells than in A cells. TfR staining of the islets was weaker in iron-overloaded rats than in the controls. These findings suggest that 1) iron uptake by islet cells in vivo is regulated and mediated by TfR, 2) intracytoplasmic Ft transforms into stainable iron in iron-overloaded rats, and 3) predominance of TfR expression in B cells may result in selective deposition of iron and predispose B cells to damage and diabetes mellitus in iron-overloaded rats.

Glial fibrillary acidic protein immunoreactivity of chondrocytes in immature and mature teratomas.

The immunoreactivity of chondrocytes for glial fibrillary acidic protein (GFAP), other intermediate filament proteins and S-100 protein was studied in formalin-fixed paraffin-embedded sections. A total of 95 cartilage specimens were examined from five immature teratomas, 12 mature teratomas, and a teratocarcinoma. GFAP-immunoreactive chondrocytes were abundant in immature cartilages, and as the cartilages maturated, these chondrocytes decreased and became distributed peripherally. Elastic cartilage had more GFAP-immunoreactive chondrocytes than non-elastic cartilage. GFAP-immunoreactive cartilage was often located close to central nervous tissue. Immunostaining for vimentin and S-100 protein revealed extensive distribution of immunoreactive chondrocytes in immature and mature cartilages, but in mature cartilage, chondrocytes at the center had less vimentin immunoreactivity. GFAP-immunoreactive chondrocytes also showed apparent immunostaining for vimentin. There was no difference in immunohistochemical staining for the alpha and beta subunits of S-100 protein. The immunoreactivities of teratoma cartilage specimens were quite similar to those of respiratory tract cartilage.

Macrophage heterogeneity in bone marrow culture in vitro.

Macrophages in mouse bone marrow cultures were investigated with macrophage-specific monoclonal antibody F4/80 and anti-Forssman glycosphingolipid (GSL) antibody, which was specific for macrophages in hematopoietic foci. Antibody F4/80 stained two types of cells, small macrophages and large flat macrophages associated with hematopoietic cells. The cytochemical and phagocytotic characteristics were similar between these two types of cells, but Forssman GSL was positive only for the large flat macrophages associated with hematopoietic cells. The data suggest that Forssman GSL positive macrophages, derived from resident bone marrow macrophages, play an important role in hematopoiesis and are clearly distinguished from small macrophages in vitro.

Rapid in vitro transformation system for liver epithelial cells by iron chelate, Fe-NTA.

We have previously found toxic effects of iron chelate, Fe-NTA on cultured normal rat liver epithelial cells (RL34). In the present study, when RL34 cells were exposed to 50 micrograms/ml iron of Fe-NTA for 15 days, besides the expected cytolytic effects in most cells, the appearance of resistant cells was observed. The resistant cells showed drastic morphological transformation, grew in soft agar, and induced hepatocellular carcinomas when transplanted into syngeneic newborn rats in a short period of time. Since DNA instability in the transformed cells was ascertained by differential AO staining, it is suggested that DNA damage by Fe-NTA is of a critical importance for extremely rapid neoplastic transformation of normal epithelial cells.

Intestinal absorption of cationic and anionic ferric colloids and complexes: biochemical, histochemical and morphological observations in rats.

This experiment was undertaken to study the possible difference in the intestinal iron absorption efficiency among iron compounds with different electric charges. Observation of rats given oral administration of 59Fe-labeled cationic cacodylate ferric (59Fe-Cac) colloid, anionic citrate ferric (59Fe-Cit) colloid, cationic 59Fe-Cac complex and anionic 59Fe-Cit complex revealed that iron absorption was more efficient in the 59Fe-Cac colloid, moderate in the 59Fe-Cac complex, low in the 59Fe-Cit colloid, and lowest in the 59Fe- cases given 59Fe-Cac colloid and 59Fe-Cac complex, a very high ratio activity was found in the liver and in the erythrocyte or hemoglobin in circulating blood, while the blood plasma, bone marrow, and spleen were low in activity. Histochemical observations of rat jejunal mucosa exposed independently for 10 min to the Fe-Cac colloid, anionic Fe-Cit colloid, and Fe-Cac and Fe-Cit complexes revealed that the cationic Fe-Cac colloid and Fe-Cac complex adhered to the luminal surface of the mucosa covering the apical area of villi with some ferric iron in the capillaries, while the anionic Fe-Cit colloid and complex did not adhere to the epithelial cells and were found free in the jejunal lumen. Electron microscopy revealed that Fe-Cac colloid particles were taken into epithelial cells by pinocytosis at the webs of microvilli, moved to the Golgi area, exocytosed to the intercellular spaces, and then translocated into the basement membrane toward blood capillaries.

Transferrin receptor expression in normal, iron-deficient and iron-overloaded rats.

The distribution of transferrin receptor (TfR)-positive cells and their staining intensity were examined in the liver, duodenum, pancreas, spleen, kidney and brain of iron-deficient, iron-overloaded and normal Wistar rats to elucidate the regulatory effects of iron on TfR expression in vivo. Iron deficiency was produced by an iron-deficient food and water regimen, and iron overload by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe3(+)- NTA) for 12 weeks. In iron-deficient rats, levels of hemoglobin (Hb = 5.9 +/- 0.7) and serum iron (SI = 29.9 +/- 4.4) were lower, and total iron-binding capacity (TIBC = 624.4 +/- 72.7) was higher than in normal rats (Hb = 15.6 +/- 0.9, SI = 206.5 +/- 20.5, TIBC = 416.0 +/- 56.0), and vice versa for SI (217.7 +/- 15.5) and TIBC (307.1 +/- 45.4) in iron-overloaded rats. In normal rats, TfR-positive granules were observed in hepatocytes and Kupffer cells of the liver, absorptive epithelium of the duodenum, acinar and Langerhans islet cells of the pancreas, macrophages and red pulp erythroblast of the spleen, and distal convoluted tubular epithelium of the kidney. Although the tissue distribution pattern of TfR-positive cells was similar in normal, iron-deficient and iron-overloaded rats, the staining intensity and number of TfR-positive cells were obviously higher in iron-deficient, and lower in iron-overloaded rats. We conclude that TfR expression is negatively regulated by the tissue concentration of iron.

Efficacy of traditional Chinese herbs on squamous cell carcinoma of the esophagus: histopathologic analysis of 240 cases.

Three types of traditional Chinese herb medicine were used to treat 98 patients with advanced esophageal squamous cell carcinoma prior to surgical treatment. Forty-two patients with the same diagnosis were treated with these herbs plus cyclophosphamide (endoxan). One hundred similar patients received surgical treatment without herbs or endoxan treatment as controls. Histologic examinations of surgical specimens were made on all of these patients. Stromal lymphoid-cell infiltration and cancer tissue degeneration were more prominent in Menispernum dehuricum DC- or Chelidonium majus L-treated patients, and were less clear in patients treated with herbs plus endoxan and the controls. The antitumor action of herbs is thought to be brought about by the activation of an immunological rejection mechanism. Herbs plus endoxan may result in the masking of the immunological response of hosts without obviously damaging cancer tissues.

Stimulation of glutathione synthesis in iron-loaded mice.

We have previously reported that the iron-loading of mice, by feeding them carbonyl iron, caused an elevation of hepatic glutathione concentration and an increase in glutathione excretion from the liver (Kawabata, T., Ogino, T. and Awai, M. (1989) Biochim. Biophys. Acta 1004, 89-94). To elucidate the mechanism of glutathione elevation, hepatic cysteine concentration and gamma-glutamylcysteine synthetase (L-glutamate: L-cysteine gamma-ligase (ADP-forming), EC 6.3.2.2) activity were measured and possible changes in cysteine metabolism were also compared between iron-loaded and control mice. Hepatic cysteine concentration was higher in iron-loaded mice (185 +/- 12 nmol/g wet wt.) than in the controls (164 +/- 8 nmol/g wet wt.), and gamma-glutamylcysteine synthetase activity was also elevated in iron-loaded mice (34.3 +/- 3.2 nmol/mg protein per min) compared with the controls (28.6 +/- 3.8 nmol/mg protein per min). A comparison of the metabolic pathways with intravenously injected [35S]cysteine showed that organ distribution of the isotope was not significantly different, and also the rate of [35S]cysteine uptake into the hepatic glutathione fraction exhibited no difference between the two groups of mice. This shows that hepatic cysteine turnover may not be different between the two groups of mice. Since hepatic cysteine concentration was higher in iron-loaded mice, the apparently equal turnover of hepatic cysteine suggests that GSH synthesis may be elevated in iron-loaded mice. The high gamma-glutamylcysteine synthetase activity is suggested to stimulate GSH synthesis in iron-loaded mice.

Capillary growth of rat aortic segments cultured in collagen gel without serum.

The process of angiogenesis was studied under serum-free conditions using rat aortic segments in three-dimensional collagen gel. In serum-free and growth-factor free conditions, the capillaries formed networks and tube-like structures, and the endothelial cells produced von Willebrand factor, laminin and type IV collagen, but the number of capillaries was lower and their growth was slower than in medium containing 20% fetal calf serum (FCS). Incorporation of bromodeoxyuridine (BrdU) and inhibition of growth by hydroxyurea suggested that capillary growth depended mainly on cell proliferation and not on migration. Capillary growth in PRMI 1640 or DMEM was similar and more efficient than in MEM. Only slight growth was seen in Medium 199 and HAM-F12. The addition of serum to the medium accelerated capillary growth in proportion to the amount added. In serum-free conditions, ITS(+) and fibroblast growth factor (FGF) promoted capillary growth, but not to a significant extent. There ware no differences in capillary growth among the gel matrices used (type I collagen, type I + II collagen, type I + IV collagen, fibrin and plasma clot).

[Pathogenesis and mechanism of iron overload: ferric nitrilotriacetate, hemosiderin, active oxygen, and carcinogenesis].

Iron overload is found clinically in such conditions as hemochromatosis and sideroblastic anemia, and after long term repeated transfusion in aplastic anemia. An animal model of iron overload was successfully developed in rats and rabbits by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe3+-NTA). This procedure induced a diabetic state with hyperglycemia, ketonemia, glycosuria and ketonuria. Blood venesection on these rats reduced the iron load in the liver and pancreas, and ameliorated the general diabetic symptoms. A single injection of Fe3+-NTA in rats induced a temporary elevation in plasma iron concentration, lipid peroxidation in the perfused liver homogenate expressed by malondialdehyde (MDA) formation, blood GOT, GPT, ALP and gamma-GTP sequentially. Fe3+-NTA uptake in the liver caused membrane lipid peroxidation, and subsequently produced a transit liberation of liver cell enzymes, although the incorporated liver Fe3+-NTA was only 1% of the injected dosage (7.5 mg iron/kg BW) at 3 hr after injection. The direct toxic effect of Fe3+-NTA to living cells was examined using cultured normal rat liver parenchymal cells (RL-34). Marked cytolysis was found in cells exposed to more than 25 micrograms of iron through Fe3+-NTA/ml. At 50 micrograms iron of Fe3+-NTA/ml, most cells were lethally injured and the remaining cells were piled up and aggregated at 15 days. They grew on soft agar culture, and when inoculated subcutaneously to five newly born rats a subcutaneous tumor developed in all animals within three weeks. Lung metastases were found in three of five inoculated rats. A spin trapping technique with electron spin resonance (ESR) on Fe3+-NTA employing 5, 5-dimethyl-l-pyrroline-N-oxide (DMPO) yielded a spin adduct with three doublets (DMPO-Z) which corresponded to singlet oxygen. By ESR in the presence of H2O2, the Fe3+-NTA solution strongly generated hydroxyl radical. The production of active oxygen species by Fe3+-NTA solution may explain the toxicity and carcinogenicity of Fe3+-NTA. The majority of stainable iron in the iron overloaded tissue was hemosiderin (Hs). We tried to purify the Hs from multi-transfused human spleen by the method of Weir et al. The purified Hs did not show a DMPO-OH adducts in the presence of H2O2 and DMPO on ESR measurement. The Hs iron was solubilized with several biological ligands in an acidic state in the presence of a reducing reagent like glutathione. Solubilized Hs iron produced iron chelate complexes which resulted in OH radicals production in the presence of H2O2 in acidic conditions below pH 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of capillary networks from bone marrow cultured in collagen gel.

The growth of capillaries from mouse bone marrow was studied in collagen gel. When the culture contained sufficient bone marrow cells (more than 1 X 10(6) cells) and cell clusters, short capillaries with lumina appeared about one to two weeks after inoculation, following the proliferation of fibroblastic cells and hemopoietic cells. Four weeks after inoculation, these capillaries formed a network among hemopoietic cells and adipocytes. Electron microscopic observations revealed that these capillaries had thin walls and poorly developed basement membranes, similar to the sinusoids of bone marrow. These capillaries did not appear when the amount of inoculated bone marrow was reduced or dispersed to the point that the marrow cell clusters disappeared. The quantity of the inoculum and the clustering of cells, therefore, seems to play important roles in the appearance of the capillaries.

Protective effects of glutathione against lipid peroxidation in chronically iron-loaded mice.

To elucidate the protective effects of glutathione against iron-induced peroxidative injury, changes in the hepatic glutathione metabolism were studied in chronically iron-loaded mice. When the diets of the mice were supplemented with carbonyl iron, iron deposition occurred primarily in the parenchymal cells of the liver. In addition, expiratory ethane production was elevated, suggesting an enhancement in lipid peroxidation. In iron-loaded mice, the total hepatic glutathione contents were higher (6.21 +/- 0.53 mumol/g wet wt.) than in control mice (4.61 +/- 0.31 mumol/g wet wt.), primarily due to an increase in the reduced glutathione contents. The value of oxidized glutathione was also higher (98.5 +/- 8.1 nmol/g wet wt.) than in the controls (60.8 +/- 9.5 nmol/g wet wt.), and the ratio of oxidized glutathione to total glutathione increased. The excretion rate of glutathione from the hepatocytes in iron-loaded mice also increased. These observations suggest that chronic iron-loading of mice stimulates lipid peroxidation and oxidation of glutathione and that peroxidized molecules may be catabolized using reduced glutathione.

[The effect of cepharanthine on the hemopoietic suppression by X-ray irradiation--hematological and histological studies].

The effects of cepharanthine on the suppression of hemopoiesis by X-ray irradiation were studied. A whole body X-irradiation (3 Gy) induced decrease of leucocyte count, nucleated cell count of bone marrow, myeloid stem cell count (CFU-C), and spleen weight. Oral administration of cepharanthine (25 mg/kg BW or 50 mg/kg BW) tended to decrease these damage on hemopoiesis, and increased spleen weight on 5th day after irradiation. Histological examinations revealed that the administration of cepharanthine accelerated the hemopoietic recovery in the red pulp of spleen.

Initiation and recovery processes of endotoxin induced disseminated intravascular coagulation (DIC): scanning and transmission electron microscopic observations of rat renal tissues.

To clarify the initiation, development and recovery processes of disseminated intravascular coagulation (DIC), rat glomerular capillaries and fibrin thrombi were examined under transmission and scanning electron microscopes. DIC was induced in rats by a single intraperitoneal injection of endotoxin (Et., 7.5 mg/kg lipopolysaccharide:B, E. coli 026:B6). At 2 h after Et. injection, the endothelial surface of the glomerular capillary became irregular with projections like a sea anemone. At 4 h after Et. injection, agglomerated fibrin thrombi composed of fibrin fiber bundles with fine cross-striated fibriform structures were observed in the capillary lumen. The fibrin thrombi gradually changed into fine reticular systems suggesting a degradation process by 6 h after Et. injection, and formed a coarse granular agglomerate by 8 h after Et. injection. These fibrin thrombi disappeared within 12 h of Et. injection, but the endothelial surface remained edematous. At 24 h after Et. injection, the microstructure of the glomerular capillaries returned normal. Based on these observations, we concluded that DIC was primarily initiated by injury to the capillary endothelium, and that changes on the endothelial surface contributed to the development of DIC.

Pancreatic islets after repeated injection of Fe3+-NTA. An ultrastructural study of diabetic rats.

Pancreatic islet cells were examined ultrastructurally in rats after repeated intraperitoneal injections of ferric nitrilotriacetate (Fe3+-NTA) to produce a model of bronze diabetes. Despite diabetic signs such as glycosuria and ketouria, no ultrastructural alterations were found in islet cells up to 90 days after the beginning of the Fe3+-NTA injections. After 120 days, however, degenerative changes appeared, with most B cells of the islets of Langerhans showing clumped nuclear chromatin, a dilated nuclear envelope, vacuolated and dilated endoplasmic reticulum, and a loss of cell polarization toward the capillary lumen. The cells contained a number of light secretory granules with an electron-lucent core and a narrow halo. Numerous electron-dense ferritin-like particles were also found in the cytoplasmic matrix, and A and D cells were almost intact. Repeated venesection therapy of rats injected with Fe3+-NTA for 120 days resulted in an increase of morphologically normal B cells with a smaller number of necrotizing cells. This process was accompanied by recovery from diabetic symptoms. The toxic effect of injected iron on B cells was thus clarified.

Importance of immunoenzyme histochemical reaction in diagnosis of disseminated intravascular coagulation in human and animal material.

Renal tissues from 208 human necropsies were observed histologically for disseminated intravascular coagulation (DIC). The tissues were stained with hematoxylin-eosin, Mallory's phosphotungstic acid hematoxylin (PTAH) and cationic ferric hydroxide colloid stabilized with cacodylate (Fe-Cac), and tested by immunoenzyme histochemical (IEH) reaction for fibrin-related materials (FRMs). The use of the IEH method increased FRM recognition, and FRMs were detected in a total of 80 cases (38.5%). In 26 cases diagnosed clinically as DIC, FRMs were shown in 23 of the cases (88.5%). Thus, 57 patients with FRMs were clinically asymptomatic. In rats with DIC induced by endotoxin injection, glomerulus FRM was effluxed into the tubulus through the Bowman's capsule and was excreted into urine. The electric charge was reduced on the endothelial surface of the glomerular capillaries in both human and rat DIC. Under the scanning electron microscopy, the endothelial surface appeared coarse in the glomerular capillary and fibrin degradation was present. Our conclusions are: (a) PTAH is non-specific for FRMs, (b) IEH aids the pathohistological diagnosis of DIC, especially in asymptomatic forms including the compensated DIC state, (c) FRMs in tubuli suggest DIC, and (d) DIC is possibly initiated by a reduction in the capillary electric surface charge.

Capillary growth from reversed rat aortic segments cultured in collagen gel.

The process of angiogenesis from aortic segments turned inside out and embedded in collagen gel was studied. Two to three days after inoculation, fibroblastic cells migrated from both ends of the segments. Later, capillary sprouts also appeared from both ends of the segments but not from the outer surface, even though there was a covering of endothelial cells. If the outer surface was injured, capillaries sometimes appeared at the damaged site. This may suggest that endothelial cells have more affinity for basement membrane than collagen gel and that they migrate only from an injured site. Immunohistochemical staining demonstrated factor VIII-related antigen in the capillary structures but not in the fibroblastic cells. Electron microscopically, capillary lumina were lined with several endothelial cells, and fibroblastic cells had the characteristics of smooth muscle cells. Since these fibroblastic cells have been known to appear under angiogenetic conditions in vivo, they may play an important role in angiogenesis, and the present culture technique may be a useful model for studying this process.