Ultrastructural and biochemical characterization of glycosaminoglycans in HNK-1-positive large granular lymphocytes

Natural killer (NK) cells are large granular lymphocytes (LGLs) that contain distinct lysosomal granules. The present study was undertaken to determine if these lysosomes contain glycosaminoglycans (GAGs) similar to those previously described in myeloid cells. Mononuclear cells from human blood were stained with HNK-1 fluoresceinated monoclonal antibody, and the NK cell population reactive with this antibody were isolated with a fluorescence-activated cell sorter (FACS). Specific staining of sulfated macromolecules with the cationic reagent, high iron diamine, was observed in the lysosomal granules of 90% of the HNK-1 positive cells. Staining in the same location was also observed in the unsorted LGLs, presumed to be NK cells, and intense staining of the cell surface was also a prominent feature of these cells. Surface staining was not evident in the majority of the FACS- separated NK cells. Digestion with chondroitinase ABC or treatment with nitrous acid reduced the staining in both locations; after sequential treatment with both chondroitinase and nitrous acid, little or no staining was seen. The presence of chondroitin sulfate (and/or dermatan sulfate) and heparan sulfate was also shown by the finding that incubation of the isolated NK cells with 35S-sulfate yielded cell- associated radiolabeled macromolecules with the characteristics of these two groups of GAGs. Of the labeled GAG pool, 60% was degraded by chondroitinase and 40% was susceptible to nitrous acid treatment. LGLs of a patient with Chediak-Higashi syndrome was also stained, and intracellular sulfate staining was clearly localized to the enlarged granules, supporting the conclusion that the lysosomes are the major site of intracellular accumulation of GAGs in normal NK cells.     

Human marrow erythropoiesis in culture. I. Characterization of methylcellulose colony assay

We examined the morphological and functional characteristics of human marrow erythrocytes cultured with a recently developed methylcellulose colony assay technique. Erythrocytic cells in various stages of development were observed, and a significant degree of maturational synchrony within individual colonies was noted. By light microscopy, colonies consisting of late normoblasts appeared compact, had an orange hue attributable to their hemoglobin, and demonstrated pseudoperoxidase activity, whereas colonies composed of early erythroblasts grew less compact or in clusters of smaller cell aggregates and showed no reddish tinge. Colonies possessing intermediate features were also observed. Maturational synchrony of individual colonies was confirmed using ransmission and scanning electron microscopy. The ultrastructure and cytochemistry of most immature cells were normal. The mature erythrocytes, however, were severely microcytic and hypochromic and contained one to several Heinz bodies. These defects in the cytoplasmic maturation of erythrocytes corresponded with impaired granulocytic maturation in culture, which we observed previously, and suggest environmental or nutritional defects in culture. Linearity of the method was confirmed using five normal bone marrows. Erythropoietin dose-responses observed in ten normal marrows were comparable to the previously reported results and revealed significant variation in individual plating efficiencies.     

Human islet mass,morphology, and survival after cryopreservation using the Edmonton protocol

The aim of this study was to assess recovery, cell death, and cell composition of post-thaw cultured human islets. Cryopreserved islets were provided by the Clinical Islet Transplant Program, Edmonton, Canada. Islets were processed using media prepared in accordance with Pre-Edmonton and Edmonton protocols. Cryopreserved islets were rapidly thawed and cultured for 24 h, 3 d, 5 d, and 7 d, following which they were processed for histology. Islet quantification, integrity, morphology and tissue turnover were studied via hematoxylin and eosin stained sections. Ultrastructure was studied by electron microscopy and endocrine cell composition by immunohistochemistry. Using the Pre-Edmonton protocol, islet recovery was 50.1% and islet survival was 50% at 24 h while for the Edmonton protocol, the islet recovery was 69.4% (p < 0.001) and islet survival, 50% at ≈2.5 d. With an increasing culture duration although the physical integrity was retained there was an increasing loss of cohesivity both at light microscopic and at ultrastructure level regardless of the protocols used. Percentage islet survival and tissue turnover correlated negatively with culture duration in both protocols. The Edmonton protocol appears to preserve the islets better. However, culture duration adversely affects islet survival and quality, indicating the need for more optimal cryopreservation and culture techniques.