Congenital neutropenia: Studies of pathogenesis

Congenital neutropenia (CN) was diagnosed in a five-month-old boy. A variety of studies was performed to define the pathogenesis of his disease. Opsonic antineutrophil antibodies were present in his serum. Transfused normal granulocytes circulated poorly. Incubation of the patient's serum with normal granulocytes failed to alter their metabolic or functional activity. The patient's marrow demonstrated increased numbers of colony-forming units (CFUs) in vitro compared with control marrow. The patient's parents had low marrow CFU activity. The patient's serum and peripheral lymphocytes failed to inhibit normal marrow CFU activity. The patient's marrow did inhibit CFU growth of an HLA-identical-sibling's marrow in coculture. Histocompatibility studies demonstrated the HLA-B12 antigen in this patient, a histocompatibility marker previously associated with CN. These studies suggest some cases of CN are associated with a genetically transmitted marrow factor capable of suppressing myelopoiesis in normal marrow.     

Diabetes-induced bradycardia is an intrinsic metabolic defect reversed by carnitine

Rats with streptozotocin-induced diabetes (STZ-D) have reduced serum carnitine levels and bradycardia. Heart rates (HRs) of 24nondiabetic rats (NRs) and 24 STZ-D rats were compared. L-carnitine (C) was added to the drinking water of rats (12 STZ-D+C) to raise their serum carnitine level. The intrinsic HR for each animal was determined after parasympathetic and sympathetic blockade. The HRs of STZ-D rats (278+/-15 beats per minute) were less than those of NRs (348+/-8 beats per minute) (P<.01). STZ-D rats had low serum carnitine compared with control and STZ-D+C rats. The difference in HR of STZ-D rats and NRs continued after blockade, indicating that the bradycardia ofdiabetes is intrinsic to the heart. The metabolic milieu reflected in the rats' urinary organic acid profiles differed between the control and STZ-D rats. The HR of STZ-D+C rats (326+/-5 beats per minute) did not differ from those of NRs. Increasing either the insulin dose or the serum free carnitine reduced urinary organic acids, but normal HRs were associated only with elevated serum carnitine levels. When glucose is compromised as a myocardial energy source (diabetes mellitus), we propose that elevated levels of serum carnitine may increase myocardial fatty acid metabolism sufficiently to correct the bradycardia of STZ-D rats.     

Human lymphocyte colony formation in agar culture: cell phenotype studies on individual colonies indicate a polyclonal origin of such colonies

Monoclonal origin of human lymphocyte colonies grown in agar culture under mitogenic stimulation is still disputed. To solve this question we used different markers: we failed with the G6PD technic and with the successive staining for X and Y chromosomes on individual colonies. Therefore, individual colonies were investigated for the presence of different cell types using membrane receptor identification and cytochemistry. At different stages of the colony formation, presence of a macrophage surrounded by lymphocytes, of a mixture of T cells and B cells, plasma cells and c.Ig negative cells, in the same colony was demonstrated. The mixture of cells from different lineages in individual colonies indicated a polyclonal origin of such colonies, the capacity for the cells to migrate in a short distance, and the involvement of cell-cell contact throughout the colony formation. Human lymphocyte colony formation appears as a new technic for the study of cellular cooperation.     

Antagonic-stress: A therapeutic composition for deceleration of aging. II. Brain lipofuscinolytic activity demonstrated by electron microscopy

Electron microscopic morphology and distribution of brain lipofuscin and ceroid (LP) were studied after 8 weeks of daily treatment with Antagonic-Stress (AS), a new combination of various components with cerebroprotective and anti-aging properties. LP characteristics were investigated in selected brain areas of control adult (CA), control old (CO) and treated old (TO) groups of 30 male Wistar rats. Cellular and regional distributions of LP in the TO group were very different from those of the CO group and resembled that of the CA group. In addition, electron microscopic signs of dissolution of LP were constantly observed in the TO group. Neurons and glia cells of TO group displayed an intense replacement of damaged organelles, especially of mitochondria, with normal organelles, many of them in hyperfunctional stages. Neuronal, glial and capillary LP lysis occurred simultaneously with neuro-glial LP transfer and capillary LP elimination. Numerous microglia cells overloaded with processed LP were identified in the vicinity of the capillary areas. AS treatment of 8 weeks induces lysis of LP which may be useful for therapeutic purposes in various brain pathologies and in deceleration of brain aging in healthy elderly.     

Congenital dyserythropoietic anaemia type II (HEMPAS): characterization of aberrant intracellular organelles by immunogold electron microscopy

Erythrocytes are reticulocytes obtained from peripheral blood of a congenital dyserythropoietic anaemia type II patient were examined by immunoelectron microscopy using anti-band 3 and anti-glycophorin A antibodies. Vacuoles which have membranous structures inside were stained heavily by these antibodies, indicating the presence of band 3 and glycophorin A in these vacuoles. Empty vacuoles which open to the cell surface and those present in cytoplasm were also stained by these antibodies at the inside of the wall. These observations suggest that the vacuoles are functioning for trapping and discarding the defective plasma membranes. On the other hand, small vesicles and ferritin-loaded vacuoles were not stained by these antibodies. In these experiments, peripheral cisternae and most of the intracellular membranous structures at blebs and clefts were not stained by the antibodies. Therefore, they are probably part of the endoplasmic reticulum or are derived from intracellular organelles but not from the plasma membranes.     

Cytoplasmic inclusions in Giardia: an electron microscopy study

Ultrastructural observations of Giardia trophozoites showed two different types of cytoplasmic inclusions. In Giardia muris from hamsters, the inclusions scattered in the cytoplasm were round, about 0.2 micron in diameter, limited by a double membrane, and with some electron-dense contents. In G. duodenalis from domestic rats, the inclusions were polyhedral, about 0.2 micron in diameter, limited by a double membrane, and with less electron dense contents. These inclusions have an unknown nature, and evidences show interaction between the inclusions and the Giardia trophozoites.     

Gap junctions on hippocampal mossy fiber axons demonstrated by thin-section electron microscopy and freeze fracture replica immunogold labeling

Gap junctions have been postulated to exist between the axons of excitatory cortical neurons based on electrophysiological, modeling, and dye-coupling data. Here, we provide ultrastructural evidence for axoaxonic gap junctions in dentate granule cells. Using combined confocal laser scanning microscopy, thin-section transmission electron microscopy, and grid-mapped freeze-fracture replica immunogold labeling, 10 close appositions revealing axoaxonic gap junctions ( approximately 30-70 nm in diameter) were found between pairs of mossy fiber axons ( approximately 100-200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction ( approximately 100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus. Immunogold labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data support computer modeling and in vitro electrophysiological data suggesting that axoaxonic gap junctions play an important role in the generation of very fast (>70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy.     

Scanning electron microscopy of the liver cell cytoskeleton

Rat livers were perfused with 0.5% Triton X-100 for 30 to 60 min and studied by scanning electron microscope. Three-dimensional filamentous networks were visualized in the cytoplasm of hepatocytes in situ. Branching and end-to-side contacts of intermediate filaments, and intermediate filaments which were connected with microtubules and microfilaments were noted. Numerous filaments were observed in the perinuclear region and at the cell border. Filaments were attached to polyribosomes and nuclei. No differences were observed in the cytoplasmic cytoskeleton after treatment of the sample at 4 degrees C. These results support the concept that intermediate filaments mechanically integrate the cytoplasmic space.     

Distribution and differentiation of murine mast cell progenitors determined with soft agar culture

We have examined the distribution and differentiation of mast cell progenitors (mast-CFC) using a sensitive semisolid agar culture stimulated with STIL-3 (leukemic T cell line)-conditioned medium as a source of interleukin 3. The number of mast-CFC in the bone marrow of normal (+/+) mice was much higher than in previously reported data, although the number was almost the same in the spleen and peripheral blood as in previously reported data. Although mast-CFC were detectable in genetically anemic W/Wv mice as well, the concentration in the bone marrow was significantly lower than that of the +/+ mice. In the bone marrow there were more immature mast-CFC forming large-sized colonies (greater than 500 cells), whereas these were very few in the spleen and peripheral blood. Most mast-CFC were in the resting state. We conclude that mast-CFC differentiate to some extent in the bone marrow and then migrate in the peripheral blood to some organs where they proliferate and differentiate.     

Norwalk virus in Norway: an outbreak of gastroenteritis studied by electron microscopy and radioimmunoassay

An outbreak of acute gastroenteritis affected approximately half of 40 children staying at a holiday centre in Southern Norway. By direct electron microscopy Norwalk-like viruses were demonstrated in 4/8 available stool specimens. No other pathogens were detected. Antibody against these viruses was demonstrated by immune electron microscopy in all of 7 convalescent phase sera but in none of 11 acute phase sera collected. Radioimmunoassay examination showed a rise in titre of Norwalk virus antibody in 6 available paired sera. This outbreak of Norwalk virus gastroenteritis in Norway was thus documented by a combined use of direct and immune electron microscopy and radioimmunoassay.     

Acinar cell carcinoma of the pancreas showing finger-print-like zymogen granules by electron microscopy: immunohistochemical study

A rare case of finger‐print‐like zymogen granules shown by electron microscopy is reported. The patient was a 75‐year‐old man who was histologically and ultrastructurally confirmed to have acinar cell carcinoma of the pancreas. Frozen section and postmortem examination revealed that the tumor was made up of solid nests of cells resembling the appearance of normal pancreatic acini, showing polygonal cells which had round or oval nuclei, and rare mitotic figures. Zymogen‐like granules, shown by eosinophilic granular staining, were abundant in the cytoplasm. Electron microscopy showed that the tumor cells were closely packed, occasionally forming small intercellular spaces resembling pancreatic acini (microtubules). The cytoplasm contained characteristic zymogen granules with dark‐to‐medium electron density, measuring 660 nm ± 213 SD in diameter. The granules of medium density were large, and showed finger‐print‐like patterns. Investigation of more cases is necessary to identify whether these finger‐print‐like patterns are an important factor in the genesis of acinar cell carcinoma.     

Architecture of myocardial cells in human cardiac ventricles with concentric and eccentric hypertrophy as demonstrated by quantitative scanning electron microscopy

Summary Scanning electron microscopy (SEM) was used to compare the shapes, size, and connections of cardiocytes in the midwall myofibers in the left ventricles of 5 normal hearts (266±16 g), 5 hearts with concentric hypertrophy (564±99 g) and 5 with eccentric hypertrophy (651±114 g), obtained at autopsy and fixed in formalin. In the myofibers from normal and hypertrophied hearts, intercalated discs demarcated cardiocytes which consisted of a cylindrical trunk and one or more series and/or lateral branches; cell connections at the intercalated discs had 6 common basic patterns. The length (L), width (W) and L/W ratio of the cells and the size and number of the series and lateral branches per cell were measured in 50 cells from each heart and averaged for comparison studies. The cells in the concentrically hypertrophied ventricles were much thicker than normal (33.0 ± 2.8 vs 18.2±1.4µm,P<0.01) but not significantly longer (81.2±9.5 vs 71.2±9.6µm, NS), so that the L/W ratio was greatly decreased (2.6±0.3 vs 4.1±0.7,P<0.01). The cells in the eccentrically hypertrophied ventricles were markedly thickened (25.9±2.4µm,P<0.01) and elongated (102.3±10.5µm,P<0.01), so that the L/W ratio (4.2±0.4, NS) remained the same as in normal hearts. In both types of hypertrophy, series and lateral branches were significantly thicker and longer than in normal hearts; the number of series branches per cell was also significantly increased. The number of lateral branches per cell did not differ between the normal and concentrically hypertrophied hearts (2.2±0.7 vs 2.3±0.6, NS), but it was decreased by approximately one-half in the eccentrically hypertrophied hearts (1.2±0.3,P<0.05). The potential significance of these differences in SEM findings of cardiocytes is discussed with special reference to the differences in the cause, anatomy, and pathophysiology of concentric and eccentric hypertrophy in adult human hearts.This work was supported in part by a Research Grant for Cardiovascular Diseases (62 C-4) from the Ministry of Health and Welfare and a Research Grant for the Pathogenesis and Prevention of Myocardial Cell Injury (# O1624006) from the Ministry of Education, Culture and Science, Japan.     

Tomography of the cell nucleus using confocal microscopy and medium voltage electron microscopy

Changes in nuclear structures are widely used by pathologists as diagnostic and prognostic indicators in cancer cells. Recent studies have demonstrated that the cell nucleus is probably the most complex organelle in the cell. It contains the genome and is the site of all related activities such as DNA repair, DNA duplication, RNA synthesis, RNA processing and RNA transport. These activities take place within dynamic three-dimensional compartments. The detailed study of these compartments requires an approach termed "cell tomography" based on 3D imaging using confocal microscopy and electron tomography. In this paper, we will first summarize the most recent findings concerning the organization of the cell nucleus. We will then describe markers used to identify molecules specific for various nuclear compartments and their use in tomography of the cell nucleus by confocal microscopy and electron tomography.     

Stimulation of persisting colonies in agar cultures by sera from patients with CML and AML

Cord plasma contains colony-stimulating activity (CSA) which stimulates the in vitro clonal growth of neutrophils, eosinophils, macrophages, erythrocytes, and persisting mast cells in semisolid cultures. Analysis of day 35 colonies in agar cultures was found to be a suitable means of demonstrating this activity and discriminating between it and granulocyte-macrophage colony-stimulating factor (GM-CSF). Serum (10%) from patients with acute and chronic myeloid leukemia (AML and CML) was added to normal human bone marrow cultures to search for similar activity in these patient's serum. Although the number of colonies on day 12 (predominantly neutrophils and macrophages) was not significantly different from the number of colonies in cultures containing normal serum, the number of colonies increased 500% in cultures containing CML serum on day 35. Serum from patients with AML during regeneration also stimulated an increased number of colonies on day 35. Although both eosinophil and mast cell colonies were still present on day 35, only mast cell colonies persisted for 150 days. On day 35, cultures containing 10% CML serum contained predominantly eosinophil colonies (84%), whereas cultures containing AML serum contained predominantly mast cell colonies (76%). Although serum contains various CSFs, the specific factor which stimulates persisting mast cell colonies may be the human equivalent of murine persisting (P) cell-stimulating factor (Multi-CSF).