Estimation of in vivo reticuloendothelial system phagocytic activity in rats by direct blood clearance techniques and nuclear scintigraphy.

There has been considerable interest in the examination of reticuloendothelial system phagocytic blockade. In this study, the kinetics of phospholipid liposome-mediated and intraperitoneal silica-mediated phagocytic blockade were examined using five methods of analysis of in vivo 99mTc-labeled albumin clearance and reticuloendothelial cell uptake. Two direct blood sampling techniques revealed significant impairment in 99mTc-labeled albumin clearance after treatment with silica (P less than 0.05), while liposome treatment was not associated with such impairment. A method utilizing nuclear scintigraphy for the determination of blood clearance was incapable of detecting silica-mediated blockade but demonstrated significant impairment by liposomes at 2 hr (P less than 0.001), 6 hr (P less than 0.05), and 24 hr (P less than 0.001). Gamma camera imaging methods for determination of hepatic uptake demonstrated significant (P less than 0.05) and reversible impairment of 99mTc-labeled albumin uptake by liposomes. The most promising of these techniques utilizes deconvolutional analysis of liver region of interest time-activity curves to correct for continuously changing blood concentrations of tracer and for intracellular tracer processing and catabolism. Measurements of reticuloendothelial system phagocytic activity should include methods that take into account the observed discrepancies between blood clearance determinations and reticuloendothelial cell uptake.

Metabolism of liposome-encapsulated heparin.

In order to elucidate the metabolism of liposome encapsulated heparin (LIPO-HEP), LIPO-HEP containing 3H-heparin (3H-HEP) and/or 14C-phosphatidylcholine (14C-PC) was intravenously administered into rats and the radioactivity as well as the biological activity in plasma and certain organs was investigated. The amount of 3H-radioactivity in plasma was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin. The amount of 14C-radioactivity in plasma of rats receiving LIPO-HEP, however, was not proportional to the amount of 3H-radioactivity in the same rats, indicating the dissociation of liposome and heparin in plasma. Incorporation of 3H-radioactivity into various organs examined, i.e., liver, spleen, lung, was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin, e.g. 4.7 and 11.8 times higher in the liver and the spleen, respectively at 150 min after the injection. Thereby, in contrast to the untreated heparin, LIPO-HEP was selectively incorporated into the reticuloendothelial system (RES) and it may be suggested that prolonged biological activity in LIPO-HEP is due to a gradual release of heparin from the liposomes entrapped in RES, and that it is not due to prolonged circulation in blood.

Immunohistochemical assessment of ferritin in bone marrow trephine biopsies: correlation with marrow hemosiderin.

In 340 bone marrow biopsies we compared ferritin, stained with an immunoperoxidase method, with hemosiderin, stained with Perls' reaction. Ferritin and hemosiderin showed the same distribution in reticuloendothelial cells. All the Perls-positive cases (n = 177) were ferritin-positive too. None of the ferritin-negative cases (n = 13) were Perls-positive. Of 163 cases with negative Perls' reaction in bone marrow, 13 (12.5%) were also ferritin-negative: these patients were mainly affected by polycythemia vera or by untreated iron deficiency anemia. Thus, immunohistochemical assessment of bone marrow ferritin can be a more sensitive tool for the evaluation of body iron stores in iron deficiency than Perls' reaction.

Hemosiderosis in rodents and the effect of acetohydroxamic acid on urinary iron excretion.

Previously reported animal models of hemosiderosis fall short of simulating the human disease state of transfusion-induced hemosiderosis. An explanation for this was found by reexamining heat-treated red blood cell (rbc) loading in the mouse. After 18 intraperitoneal transfusions, each equal to two-thirds of mouse rbc volume, liver Fe reaches a level of 0.3% Fe (dry weight), which is far below the 2%-8% found in heavily transfused patients. Using the easily synthesized chelate compound ferric acetohydroxamate, Fe(AHA)3, liver Fe levels in the rat of up to 2% were achieved after 38 intraperitoneal injections. Fe was distributed in both the reticuloendothelial (RE) system and parenchymal cells, as ascertained by light microscopy. No definite histological or biochemical abnormalities could be demonstrated in loaded rats. Cardiac Fe was approximately doubled. Thus, chelate loading, while producing Fe liver levels similar to those of humans with hemosiderosis, may still be of limited usefulness in studying long-term sequelae. On the other hand, this model can be used in determining responses to chelating agents. To explore this, Fe stores were first labeled by giving 59Fe as 59Fe(AHA)3 prior to loading. In animals loaded to 0.7% liver Fe (calculated), desferroxamine, at a dose of 400 mg/kg, induced a 20-fold rise in urinary Fe. This was duplicated by AHA at a dose of 800-1600 mg/kg. It is concluded that Fe(AHA)3-loaded rats are a potentially useful model of hemosiderosis and that further studies are needed to determine whether AHA can be effective in the treatment of transfusion-induced hemosiderosis.

Post-embedding direct immunogold detection of a protein antigen in insect tissue.

An immunocytochemical study was performed to localize the site of hemoglobin synthesis in larvae and embryos of the insect Chironomus thummi. Heterologous antisera specific for C. thummi hemoglobins were prepared using a highly purified hemoglobin extract. Tissue samples were prepared by glutaraldehyde fixation of whole dissected larvae or whole embryos without osmium tetroxide postfixation. Epoxy resin-embedded thin sections were labeled with a direct immunogold conjugate. Immune label was localized in rough endoplasmic reticulum of fat body cells of larvae. Immune label was also present in embryos. The technique, which did not require chemical etching of the sections, proved very useful for demonstration of this intracellular protein antigen.

A neuroendocrine marker in tissues of the immune system.

Antibodies to chromogranin, a secretory protein marker for the diffuse neuroendocrine system, were used to analyze rat lymphoreticular tissues by means of immunochemistry and immunohistochemistry. Chromogranin-positive cells were present in spleen, lymph node, thymus, and fetal liver. When these organs were gently dispersed and separated on a Ficoll gradient, the chromogranin-immunoreactive cells became enriched in the dense red-cell pellets. The unexpected distribution of these neuroendocrine cells in all immunologically relevant structures suggests that they may link the nervous and immunological systems.

[Stage-classification of reticuloendothelial function in hepatic injuries in the rat].

The serial changes of RE function were classified into 3 stages according to changes in opsonic activity and phagocytic index in hepatectomized, CCl4-treated cirrhotic rats; enhanced stage, compensating stage and critical stage. The phagocytic index was measured by initial disappearance rate after 4 mg/kg i.v. injection of 51Cr-labelled LPS. An opsonic activity was determined by the bioassay method using cultured RE cells prepared from the normal rat liver. The enhanced stage was characterized by an enhancement in both phagocytic index and opsonic activity--3 to 21 day-70% hepatectomized, 2 to 5 day-40% hepatectomized, 3 week-cirrhotic rats. The compensating stage by increased opsonic activity and normal phagocytic index was 2 day-70% hepatectomized and 9 week-cirrhotic rats. The critical stage by decreased phagocytic index was 5 hour to 1 day-70% hepatectomized, 13 week-cirrhotic rats.

Immunohistochemical and immunoelectron microscopic localization of S-100 protein in the interdigitating reticulum cells of the human lymph node.

Lymph nodes of 5 cases with non-specific lymphadenitis and of 4 cases with dermatopathic lymphadenitis were examined by the peroxidase anti-peroxidase (PAP) method and by immunoelectron microscopy using monospecific antibody against nervous system specific S-100 protein. S-100 protein was detected in dendritic shaped cells of the thymus-dependent area of non-specific lymphadenitis. The paracortical area of dermatopathic lymphadenitis showed a marked accumulation of S-100 protein-containing dendritic shaped cells. Immunoelectron micrographs revealed that S-100 protein-containing dendritic shaped cells of both non-specific and dermatopathic lymphadenitis corresponded to interdigitating reticulum cells (IRC). The detection of S-100 protein in the nucleus and on the cytoplasmic and nuclear membrane suggested that IRCs themselves synthesized S-100 protein. S-100 protein is a very useful cell marker for the identification of IRC in the lymphoid tissue.

Functional studies on histiocytic and interdigitating reticulum cells from human lymphoid tissue.

Histiocytic (HRC) and interdigitating (IDC) reticulum cells were obtained from human lymphoid tissue (lymph nodes, tonsils), suspended in cell culture medium and cultured. The suspended cells, which were identified by morphological and enzyme histochemical criteria, were studied for surface binding properties for Fc-receptors using erythrocytes coated with IgG. HRC show strong surface binding, IDC, somewhat weaker, but definitely positive surface binding. Both types of reticulum cells possess Fc-receptors; on IDC, however, they are fewer in number and less dense. In connection with recently discovered Fc-receptors on Langerhans cells of the epidermis, functional relationships between blood monocytes, Langerhans cells in skin and IDC in lymphoid tissue are discussed.

Comparative study of reticuloendothelial system pools of the normal and immunologically activated rabbit synovium with radiocolloids.

A quantitative study of the uptake of radiocolloids by the joint synovium relative to other reticuloendothelial system organs was performed in the rabbit model. The radiocolloids of 198Au and 99mTc-sulfide colloid were administered intravenously and distribution in synovial pad, liver, spleen, bone marrow, blood, lung, and other tissues was determined in a gamma counter. The uptake of radiocolloids by both normal and immunologically activated synovial pad was very small in comparison with the reticuloendothelial cells in liver, spleen, and bone marrow.

[A new type of sialidosis with kidney disease: nephrosialidosis. II. Anatomic study]. / Un nouveau type de sialidose avec atteinte rénale: la nephrosialidose. II.--Etude anatomique.

The anatomical, macroscopical, histological, histochemical and ultrastructural findings in nephrosialidosis have certain similarities to those found in mucolipidoses. (Excess of complex lipid in neural tissue and an excess of light coloured material in the reticulo-endothelial system that is not easily characterised). Several distinct features help to distinguish nephrosialidosis from closely related conditions (renal lesions, storage of material in sympathetic ganglia).

Histamine biosynthesis in shock.

The histidine decarboxylase activity of the lung and spleen was determined in rats made resistant to trauma either by prior sublethal exposure or by injection of extracts prepared from the spleens and plasma of trauma-resistant rats. The data describe the posttraumatic period in the normal animal as being associated with an increased histidine decarboxylase activity. In trauma-resistant animals, changes in the enzyme activity were prevented in the lung and were less pronounced in the spleen. The administration of extracts from trauma-resistant rats was similarly effective in impeding the changes in enzyme induction following trauma. It is suggested that an active humoral factor previously shown to be elaborated during conditioning and associated with the RES may act by inhibiting the activation of histidine decarboxylase.

Investigations into human thorotrastosis. Tissue concentrations of 232-Th and late effects in 13 autopsy cases.

The subjects of investigation were 13 dead thorotrast patients, 10 male, 3 female, with ages ranging from 45 to 79 years. Two thousand organ and tissue specimens were investigated by means of autopsy and by both microscopic-autoradiographic and neutron activation analysis in order to detect late effects and to determine on approximate mean concentration of 232Th (mg per g of tissue). A comparison between late effects and concentrations of the dye medium led to the following conclusions: 1. 232Th is, after intravascular injection, deposited in all organs and tissues of the human body. 2. The highest mean concentrations are shown in the spleen, liver, bone marrow, and lymph nodes. 3. The distribution of 232Th is inhomogeneous in all organs and tissues. The variations of maximum and minimum concentration lie around factor 2.2 X 10(0)-2.4 X 10(5). 4. Late effects occur only in spleen, liver, lymph nodes, and bone marrow, but not in organs and tissues that show a mean concentration of 232Th under 10(-1) mg per g tissue. 5. It is highly probable that tumors of thorotrast patients in organs other than spleen, liver lymph nodes, and bone marrow are not caused by deposition of 232Th or ThO2.

Interdigitating reticulum cells in the popliteal lymph node of the rat. An ultrastructural and cytochemical study.

Electronmicroscopic and cytochemical studies were performed to localize interdigitating reticulum cells (IDC) in the popliteal lymph node of the rat. The morphological features of the IDC of the rat correspond to those described for other species, but also show similarities to normal macrophages in the rat. This is considered to be an argument in favour of the common origin of IDC's and macrophages. Ultrahistochemical studies with horseradish peroxidase (HRP) reveal no phagocytotic capacity of IDC's. After perfusion fixation containing ruthenium red (RR) the surface coat stains heavily: RR is also found deep in the membrane invaginations of the IDC, indicating the presence of polyanionic sialoglycoproteins. The post-capillary-venules (PVC) are very permeable to both HRP and RR. The phosphotungstic acid-chromic acid stain (PTA-CrA) also reveals glycoproteins in the surface coat; these glycoproteins are susceptible to alpha-neuraminidase, whereas glycoproteins in the Golgi complexes, lysosomes and in the vesicular complexes of IDC are not. The glycoproteins of the latter are susceptible to 0.1 N NaOH. These findings indicate that IDC produce different kinds of glycoprotein, one of which may be secreted and act as a factor for stimulating peripheral T-lymphocytes. Intimate contact between IDC's and PCV's could be observed. It is therefore conceivable that IDC's play an important role in the homing of T-lymphocytes.

Quantitative measurement of splenic and hepatic red-cell destruction.

A method has been developed by means of which independent measurement can be made of the amount of red-cell destruction occurring in the spleen and the liver. The technique involves a standard red-cell survival study and surface-counting measurements together with quantitative scanning of the spleen and liver with 113mIn colloid in order to calibrate the surface counter. The rate of destruction in each organ is obtained by fitting the measured uptake curve for the organ to a theoretical uptake curve by computer. In addition, if whole body counting is also performed, the amount of red-cell destruction occurring in the rest of the reticuloendothelial system may be deduced. Results are given for measurements on a series of 11 patients.