Richard T. Parmley

Ultrastructural cytochemistry and radioautography of hemoglobin—iron absorption☆

The subcellular route of hemoglobin-iron absorption by canine intestinal epithelial cells was investigated with cytochemical and radioautographic methods and compared to that observed for inorganic iron absorption. A solution of rabbit [59Fe]hemoglobin or an inorganic-iron solution was injected into closed duodenal loops of beagle dogs and mucosal biopsies were obtained 15, 60, and 120 min thereafter. Mucosal retention of radioactivity in dogs given hemoglobin iron was 6.9–10.2% after 120 min, and portal venous blood radioactivity was confined to the inorganic iron fraction. In the same animals diaminobenzidine (DAB)-reactive heme was visualized in microendocytic caveolae at the bases of microvilli and in membrane-bound tubulovesicular structures and granules of the apical cytoplasm. DAB-reactive heme was not identified in the lateral intercellular space or the basal extracellular space. In the same specimens, acid ferrocyanide stained ferric iron in a few apical cytoplasmic vesicles as well as along the outer lateral and basal plasmalemma but failed to identify inorganic iron in microvilli. Both the microvilli and the lateral plasmalemma appeared stained in specimens from animals given inorganic iron. In radioautographic specimens of animals given [59Fe]hemoglobin, silver grains were most frequently associated with the apical cytoplasm and the lateral plasmalemma. We conclude that at least one mechanism of hemoglobin-iron absorption involves endocytosis and degradation of hemoglobin in membrane-bound organelles with subsequent conversion of its iron to an inorganic form, which is then transported to the lateral intercellular space where subsequent processing is similar to that observed for absorbed inorganic iron.

Ultrastructural Cytochemistry and Radioautography of Complex Carbohydrates in Heterophil Granulocytes from Rabbit Bone Marrow

The subcellular route of incorporation of complex carbohydrates into rabbit heterophil primary granules and their subsequent intragranular distribution during granule maturation were studied with ultrastructural, cytochemical, and radioautographic methods. High iron diamine (HID) staining of sulfated glycoconjugates in primary granules was partially diminished after treatment with chondroitinase ABC or after removal of N-sulfate groups with nitrous acid, but was not altered by exposure to hyaluronidase, trypsin, or HCl. Subsequent thiocarbohydrazide-silver proteinate (TCH-SP) straining of thin sections increased the density of the HID reaction product. Golgi-derived spherules and very immature morular granules stained weakly with HID-TCH-SP and labeled intensely after a 10 min incubation with 35SO4. After a 60 min 35SO4 pulse and a 60 min chase, an increase in radiolabeling was observed in granules with HID stained, fused morular material, and some labeling was present in more mature rim stained granules. Fully mature granules lacked HID or HID-TCH-SP staining, but contained most of the 35SO4 labels after a 60 min pulse and 18 hr chase in vitro. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing complex carbohydrates in Golgi-associated small vesicles. These vesicles lacked HID-TCH-SP staining and apparently contained neutral glycoprotein. They frequently bordered, in a rosette arrangement, the immature morular granules, but not the more mature primary granules. The PA-TCH-SP method localized complex carbohydrates in the rim of granules precursors and enclosed a spherule or morula, but failed to stain the sulfate-containing material in the morulas or spherules. PA-TCH-SP reactivity was diffusely distributed in moderately mature granules and was decreased in fully mature granules. These results indicate that heterophil primary granule contain several complex carbohydrates including O-sulfated and N-sulfated glycosaminoglycans, as well as vicinal glycol-containing glycoproteins. These complex carbohydrates are transported to immature primary granules by different Golgi-derived organelles. The complex carbohydrates are subsequently distributed differently within primary granules and become masked to staining as the granule matures.

Ultrastructural localization of the transferrin receptor and transferrin on marrow cell surfaces

Summary. The monoclonal antibody OKT9 (a known transferrin receptor antibody) and a monoclonal antibody to transferrin (ATfn) were used to localize the transferrin receptor and transferrin on marrow cells. After incubation of cell suspensions with the antibody, the cells were reacted with an affinity purified Fab fragment of goat anti‐mouse IgG conjugated to horseradish peroxidase (GAM‐HRP), which was in turn visualized by reaction with 3,3′‐diaminobenzidine (DAB). Erythroblast cell surfaces stained intensely with OKT9‐GAM‐HRP‐DAB, weaker staining was observed on reticulocyte surfaces, whereas erythrocyte surfaces lacked staining. Staining was present on surface caveolae, which often contained endogenous ferritin particles. Moderate to strong OKT9 staining was observed on less than 10% of presumed lymphoid cells. Monocytes, macrophages, promyelocytes, granulocytes, megakaryocytes and platelets consistently lacked OKT9 staining. ATm‐GAM‐HRP‐DAB staining of erythroid cells was similar to that observed with OKT9 staining; however, in contrast to the latter staining, ATfn stained the surfaces of megakaryocytes, platelets, monocytes and most lymphocytes. Promyelocytes stained weakly, whereas late granulocytes lacked staining. These results indicate that the T9 transferrin receptor (1) is largely confined to erythroid cells in marrow, (2) is diffusely distributed on the surface of early erythroid cells, (3) decreases with cell maturation, and (4) is lost when haemoglobin synthesis is completed. Transferrin appears in a similar distribution on erythroid cell surfaces but also appears to bind to some cell surfaces lacking the T9 receptor.

Ultrastructural cytochemistry and immunocytochemistry of sulfated glycosaminoglycans in epiphyseal cartilage.

Cartilage proteoglycan monomers contain sulfated glycosaminoglycans (GAGs), which consist of chondroitin 4and 6-sulfate, and keratan sulfate. We have examined newborn rat epiphyseal cartilage to demonstrate the nature of reactive sites in the matrix granule with the high iron diamine (HID) and HID-thiocarbohydrazide-silver proteinate (HID-TCH-SP) methods for sulfated glycoconjugates and an immunoferritin method specific for chondroitin 4-sulfate. HID strongly stained matrix granules in osmicated specimens but weakly stained these granules in unosmicated specimens. HI D-TCH-SP staining produced two types of stain deposits, averaging 1 1 and 7 nm in diameter, in the matrix granule and was not influenced by postosmication. Testicular hyaluronidase digestion of specimens removed most of the 1 1 nm stain deposits in the matrix granule. The size of the HID-TCH-SP reaction products appeared to be related to molecular weight and! or sulfate content of the GAG chains, as evidenced by the

Ultrastructural cytochemistry of complex carbohydrates in osteoblasts, osteoid, and bone matrix

SummaryGlycosaminoglycans (GAGs) and glycoproteins are essential components for osteogenesis. We have examined rat osteoblasts, osteoid, transitional zone, and fully calcified bone matrix, utilizing Spicers high-iron diaminethiocarbohydrazide-silver protein (HID-TCH-SP) method for sulfated glycoconjugates and Thiérys periodate-TCH-SP (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained cytoplasmic granules of osteoblasts. Stain deposits in the extracellular matrix were observed in decreasing amounts in osteoid, the transitional zone, and fully calcified bone matrix. Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits. PA-TCH-SP staining was observed with increasing intensity in rough endoplasmic reticulum, Golgi saccules, and cytoplasmic granules. Collagen fibrils in osteoid were weakly stained with PA-TCH-SP, and their staining appeared even weaker in fully calcified bone matrix. In contrast, collagen fibrils in calcified cartilage stained intensely with the PA-TCH-SP method. Focal circular profiles (0.1–0.5µm in diameter), which lacked collagen fibrils but reacted moderately with PA-TCH-SP, were frequently seen in the transitional zone and fully calcified bone matrix, but were only occasionally present in osteoid. The presence of testicular hyaluronidase-resistant GAG and acid phosphatase in these focal areas suggests that they represent sites of GAG degradation. The eventual loss of HID-TCH-SP staining in the bone matrix suggests that removal of sulfated glycoconjugates may be a requisite for expansion of initial calcification sites and/or complete calcification.

Endocytic activity and ultrastructural cytochemistry of lysosome-related organelles in epiphyseal chondrocytes.

The relationship of lysosomes, multivesicular bodies (MVBs), and tubulovesicular structures (TVS) of the chondrocyte from rat epiphyseal cartilage was studied with ultrastructural cytochemical methods for the localization of acid phosphatase, endocytosed horseradish peroxidase (HRP), and sulfated and vicinal glycol-containing complex carbohydrates. Lysosomes contained intense uniformly distributed acid phosphatase activity. MVBs with an abundant fibrogranular matrix (dark) demonstrated strong acid phosphatase staining, whereas MVBs with a sparse matrix (light) stained less intensely or lacked staining. TVS associated with Golgi elements were acid phosphatase positive, whereas other TVS lacked staining. Incubation of viable chondrocytes with HRP resulted in macroendocytic (phagocytic) and microendocytic (pinocytic) engulfment with significant incorporatin into MVBs and some TVS as demonstrated by diaminobenzidine staining. Both acid phosphatase-positive and HRP-containing TVS appeared to contact and fuse with MVBs. High iron diamine (HID) failed to stain sulfated glycoconjugates in lysosomes and TVS in contrast to heavily stained secretory granules. MVBs stained weakly or not at all with the HID method. Periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) strongly stained vicinal glycol-containing glycoconjugates in lysosomes. MVBs, TVS, and microendocytic caveolae. The PA-TCH-SP reactivity of the limiting membranes of MVBs, lysosomes, and TVS was much stronger that the reactivity associated with the plasmalemma and secretory granule membranes. These studies indicate chondrocyte lysosomes, MVBs and TVS represent part of a complex system engaged in processing phagocytosed material. This endocytic activity as measured by HRP incorporation was greatest in chondrocytes of the hypertrophic zone and may be related to matrix modification necessary for calcification and/or matrix reutilization.

Ultrastructural Cytochemistry of Complex Carbohydrates in Leukocyte Granules

proteinate staining. The distributions of reactive vicinal glycols and stained sulfate groups differed in rabbit heterophils and suggested that the high iron diamine-stained sulfated glycoconjugate was a glycosaminoglycan rather than a sulfated glycoprotein. The overlapping distribution of glycosaminoglycan and acid phosphatase staining in these granules, the increased cytochemical reactivity in immature granules and apparent masking of staining in mature granules, and biochemical data indicating an inhibitory effect of glycosaminoglycan on acid hydrolases, suggest that glycosaminoglycans may function in storage of some acid hydrolases and in prevention of premature granule destruction.

Isolated marrow lymphoma: an entity of possible T-cell derivation.

Seven adults had a distinct clinicopathologic type of lymphoproliferative disorder of the bone marrow. All patients presented with weakness and pancytopenia; no evidence of gross extramedullary involvement was found. In 5 cases severe and prolonged bone marrow hypoplasia was associated with combination chemotherapy; 1 patient died of infection during initial therapy. In 6 of the 7 cases, clinical improvement occurred following therapy. As a terminal event, 2 patients developed a leukemic phase. Tumor cells from 4 patients were studied immunologically, and in 2 patients surface marker characteristics suggestive of T‐cell tumor origin were found. In 2 cases, ultrastructural studies of lymphoid cells were compatible with a T‐cell neoplasm. The above data suggest that these cases represent a distinct type of chemotherapy‐sensitive lymphoma in which conservative initial treatment may induce a response without prolonged bone marrow hypoplasia.

Phagocytosis of neutrophils by marrow macrophages in childhood chronic benign neutropenia

Chronic benign neutropenia of childhood is a heterogeneous disorder. This study describes two severely neutropenic children with a benign clinical course and the unique bone marrow finding of macrophage engulfment of band and segmented neutrophils. Phagocytic vacuoles in the majority of macrophages contained neutrophils in various stages of digestion at both the light and electron microscope level (with as many as four neutrophils observed in single macrophages). Ultrastructural studies demonstrated that the neutrophils were morphologically normal, and apparently viable at the time of phagocytosis. This type of neutrophil phagocytosis was not observed in five other consecutively studied children with CBN. All of these children with CBN had ultrastructurally normal neutrophils, lacked demonstrable antineutrophil antibodies or serum inhibitors to in vitro myelopoiesis, and had normal colony-forming cells and colony-stimulating activity in vitro. Thus despite many similar clinical and laboratory features in children with CBN, only a unique subgroup of these patients demonstrates abnormal neutrophil-macrophage interaction.

Ultrastructural cytochemistry of basophils in chronic myelocytic leukemia

Abstract Cytochemical methods were used to determine the ultrastructural distribution of antimonate precipitable cations, anionic complex carbohydrate, acid phosphatase, and peroxidase in blood basophils from patients with chronic myelocytic leukemia and markedly elevated basophil counts. The morphology of these basophils varied but was generally similar to that reported for normal basophils. Antimonate precipitable cations were sparse in specific granules but more abundant in extra-granular cytoplasm and in vesicular structures of many basophils and, although varying with the fixation method, were distributed atypically in nuclei in a pattern similar to that seen in mast cells. The dialyzed iron technique diffusely stained anionic complex carbohydrate in the specific granules of basophils, and high iron diamine staining disclosed a sulfated mucosubstance in these granules. Acid phosphatase and peroxidase were distributed in a pattern similar to that of the acid mucosubstance in the basophil granules, and their presence suggested a lysosomal function for these granules