FUS immunoreactivity of neuronal and glial intranuclear inclusions in intranuclear inclusion body disease.

AIMS: Recent studies have shown that fused-in-sarcoma (FUS) protein is a component of 'neuronal' intranuclear inclusion bodies (INIBs) in the brains of patients with intranuclear inclusion body disease (INIBD). However, the extent and frequency of FUS-immunoreactive structures in INIBD are uncertain. METHODS: We immunohistochemically examined the brain, spinal cord and peripheral ganglia from five patients with INIBD and five control subjects, using anti-FUS antibodies. RESULTS: In controls, the nuclei of both neurones and glial cells were intensely immunolabelled with anti-FUS and neuronal cytoplasm was weakly positive for FUS. In INIBD, neuronal and glial INIBs in the brain and spinal cord were positive for FUS. FUS-positive INIBs were also found in the peripheral ganglia. The proportion of FUS-positive neuronal INIBs relative to the total number of inclusion-bearing neurones ranged from 55.6% to 83.3% (average 73.2%) and that of FUS-positive glial INIBs ranged from 45.9% to 85.7% (average 62.7%). The nucleus and cytoplasm of inclusion-bearing neurones and glial cells showed no FUS immunoreactivity. CONCLUSIONS: These findings suggest that FUS is incorporated into INIBs in both neurones and glial cells and that loss of normal FUS immunoreactivity may result from reduced protein expression and/or sequestration within inclusions.

Historical changes in epidemiology of diffuse panbronchiolitis.

BACKGROUND AND OBJECTIVE: Japanese pulmonologists, experienced in treating patients with diffuse panbronchiolitis (DPB) prior to the 1980s, have uniformly observed that new incidences of DPB are now a rare event in Japan. However, there is no epidemiological data to support this observation. We examined epidemiological trends of the number of patients with DPB in a large company. DESIGN: The computerized health records of JR East Company employees were used to identify patients with DPB and then these were followed up using the assessments of these patients in JR Tokyo General Hospital and two other JR hospitals. The whole study period was 27 years (1976-2003), although detailed analyses were carried out for three specific periods; the first was 1976-1980, the second was 1989-1993, and the third was 1999-2003. RESULTS: In the first period, 11 DPB cases (four incidence, and seven prevalence) were detected among a total of 355,572 workers. In the second period, three DPB cases (one incidence, and two prevalence) were identified from a total of 180,359 workers. In the third period, no case was found in a total of 144,485 workers. CONCLUSION: This epidemiological trend suggests that both the incidence and prevalence of DPB may have decreased.

Clinical impact of hyperglycemia on days 0-7 after allogeneic stem cell transplantation.

In order to clarify the association between hyperglycemia during the early period after allogeneic stem cell transplantation (allo-SCT) and adverse outcomes, we retrospectively analyzed 563 consecutive patients who underwent allo-SCT at our institute between 2008 and 2015. Patients were categorized into three groups according to mean fasting blood glucose levels on days 0-7 (normoglycemia group<110 mg/dL, n=347; mild hyperglycemia group 110-149 mg/dL, n=192 and moderate/severe hyperglycemia group≥150 mg/dL, n=24). The median follow-up was 2.7 years. Patients in the moderate/severe hyperglycemia group had significantly worse characteristics. The cumulative incidences of 2-year non-relapse mortality (NRM) and the probabilities of 2-year overall survival (OS) in the normoglycemia, mild hyperglycemia and moderate/severe hyperglycemia groups were 7.5%, 19% and 29%, respectively (P<0.01), and 69%, 53% and 33%, respectively (P<0.01). In multivariate analyses, hyperglycemia was an independent predictor of high NRM (vs normoglycemia; mild hyperglycemia, hazard ratio (HR) 2.56, 95% confidence interval (CI) 1.56-4.18; moderate/severe hyperglycemia, HR 4.46, 95% CI 1.92-10.3) and poor OS (vs normoglycemia; mild hyperglycemia, HR 1.54, 95% CI 1.14-2.07; moderate/severe hyperglycemia, HR 1.61, 95% CI 0.89-2.91). In conclusion, hyperglycemia on days 0-7 after allo-SCT was associated with inferior outcomes.

Ultrastructural and biochemical observations on antineutrophil antibody- and complement-induced immuno-injury to equine neutrophils.

Antibody-induced damage to neutrophils was studied to elucidate processes associated with destruction of neutrophils in immune-mediated neutropenias. Cytomorphological changes and release of certain cellular constituents were determined for neutrophils treated with an antineutrophil serum in the presence or absence of rabbit complement. Neutrophils exposed to the antineutrophil serum alone showed endocytotic vacuoles and degranulation. In contrast, neutrophils exposed to the antineutrophil serum and complement showed marked morphologic changes. The plasma membrane developed numerous vesicles, villous processes and minute areas of bilayer discontinuity. Highly damaged cells exhibited cellular and nuclear swellings, disruption of cytoplasmic integrity and disordered distribution of lysosomal granules. Cytoplasmic constituents (K+ and lactate dehydrogenase) were released extracellularly from neutrophils exposed to the antineutrophil serum with or without complement. Cytological changes induced by the antineutrophil serum and complement were analogous to those reported for leucocytes exposed to the activated complement components C5b-9 (the membrane attack complex) and bacterial toxins. It was concluded that the cytological abnormalities observed were most probably associated with immune-mediated damage to the cell membrane, leading to leakage of cytoplasmic constituents like K+, colloidal osmotic swelling, and disruption of the cytoskeletal system.

Fusiform erythrocytes resembling sickle cells in angora goats: light and electron microscopic observations.

Fresh blood from nine mature non-pregnant angora goats was found to contain a varying proportion of spindle-shaped, fusiform, triangular, pear-shaped, and other bizarre forms of erythrocytes in addition to normal discoid biconcave erythrocytes. The number of spindled and fusiform erythrocytes varied from 2 to 66 per cent, with two goats having such cells in excess of 50 per cent. Other aberrant forms of erythrocytes ranged from 3 to 54 per cent. Although the aberrant erythrocytes seemed to be present intravascularly, all goats studied were clinically normal and exhibited no other haematological abnormality. Light and electron microscopic observations indicated that polymerisation of haemoglobin in the form of longitudinal tubular fibres was responsible for conferring the fusiform and spindle shapes to erythrocytes, a phenomenon akin to that seen in sickle-shaped human and deer erythrocytes.

Fusiform erythrocytes resembling sickle cells in angora goats: observations on osmotic and mechanical fragilities and reversal of cell shape during anaemia.

Osmotic and mechanical fragilities of erythrocytes were determined for seven goats having 3.4-71 per cent fusiform erythrocytes. The osmotic fragility was related to the erythrocyte shape in that the osmotic resistance was considerably higher for bloods containing more than 26 per cent fusiform erythrocytes. A decrease in the proportion of fusiform erythrocytes in the same goats was related to an increase in the osmotic fragility. Anaemia was induced in two goats by removal of 200-400 ml of blood at three or four day intervals for eight weeks. Red cell values decreased by 28-43 per cent within three weeks, but further bleeding produced either no or less (0-21 per cent) reductions in these values. Slight reticulocytosis was seen during the anaemic phase and there was a concomitant increase in the mean corpuscular volume and mean corpuscular haemoglobin values. Reticulocytosis diminished before the start of recovery from anaemia and disappeared during the recovery phase. The most significant finding was the change in the erythrocyte morphology during production of and recovery from anaemia. The development of anaemia was associated with a gradual reduction in the proportion of fusiform erythrocytes or discoid cells and simultaneous increase in the proportion of erythrocytes exhibiting distinct poikilocytosis. Recovery from the anaemia was rapid (within five weeks), but reversal of the erythrocyte shape took several months. Severe blood loss anaemia in the goat is known to induce synthesis of haemoglobin C, and in these anaemic goats formation of a new haemoglobin, most likely haemoglobin C, was demonstrated by electrophoretic and column chromatographic analyses. It was concluded that the formation of haemoglobin C was responsible for the morphological changes in the erythrocytes.

Scanning electron microscopic studies of megakaryocytes and platelet formation in the dog and rat.

Megakaryocyte morphology and platelet formation in canine and murine bone marrows were studied by scanning electron microscopy. In situ-fixed bone marrow preparations and cell suspensions of bone marrow provided complementary information for the 2 species (dogs and rats). Cylindrical processes (proplatelets) of variable length and thickness, originating from the megakaryocyte surface, were in the larger marrow sinusoids and the central vein. Regional constrictions along the length of proplatelets, particularly near their apical region, and the presence of fragments of such processes supported the concept of platelet formation through segmentation of proplatelets. Megakaryocytes presented varied morphology. Surface features resembling platelets were observed on megakaryocytes, indicating that platelets may have been released through surface budding. In conclusion, megakaryocytes formed long proplatelet processes that actively migrated to venous sinusoids to release platelets by fragmentation. Scanning electron microscopy analysis revealed a complex and variable megakaryocyte surface topography. The platelet-like structures on megakaryocyte surfaces may represent platelet release by a budding mechanism. The similarity between murine platelet release and canine platelet release demonstrates that data from rodent models may be applicable to nonrodents.

Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin.

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P less than 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.

In vitro platelet release by rat megakaryocytes: effect of metabolic inhibitors and cytoskeletal disrupting agents.

Development of an in vitro visual assay facilitated the study of large numbers of megakaryocytes undergoing proplatelet formation in short-term cultures. Approximately 9% of megakaryocytes formed platelets during a 24-hour period. In the presence of an inhibitor of anaerobic glycolysis (NaF), proplatelet formation was inhibited, whereas inhibitors of respiration (NaCN) did not significantly (P greater than 0.05) decrease proplatelet formation. Presence of the microtubule-disrupting agents colchicine and vincristine sulfate in culture medium inhibited proplatelet formation, whereas the microfilament-disrupting agent cytochalasin B had a less pronounced inhibition.

In vitro platelet release by rat megakaryocytes: effect of heterologous antiplatelet serum.

A visual assay to study megakaryocyte platelet release via proplatelet formation in vitro was established. Samples of megakaryocyte-enriched rat bone marrow were incubated (37 C) in RPMI-1640 medium with 15% autologous serum in specially prepared chambers. In the culture system, approximately 6% of megakaryocytes formed proplatelet processes within 24 hours. Inclusion of a heterologous antiplatelet antibody in the culture system inhibited proplatelet formation, compared with that in controls.

Scanning electron microscope study of platelet release by canine megakaryocytes in vitro.

Megakaryocytes were isolated from bone marrow from healthy dogs, using a combination of density-gradient centrifugation and polysucrose-velocity sedimentation techniques. The 2-step separation technique resulted in a preparation comprising 30% to 35% megakaryocytes of total nucleated cells. Accessibility to large numbers of viable canine megakaryocytes allowed investigation of platelet release by these cells in short-term cultures. Megakaryocytes were observed to form long cytoplasmic processes that gradually developed segmental constrictions and subsequently fragmented into platelet-sized pieces. Some platelet-sized cytoplasmic pieces of megakaryocytes presumably underwent discoid transformation.

Circulating proplatelets: isolation and quantitation in healthy rats and in rats with induced acute blood loss.

A technique to isolate megakaryocyte proplatelet processes from blood of rats' hearts, using colloidal silica coated with polyvinylpyrrolidone density gradient, was developed. The proplatelet concentration in blood from right ventricles was significantly higher (P less than 0.001) than that in blood from left ventricles in healthy rats, as well as in rats with induced acute blood loss. The proplatelet concentration of blood from the heart, 24 hours after acute blood loss was induced was significantly (P less than 0.001) increased, indicating that platelet production was accelerated. The demonstration of proplatelets entering the pulmonary circulation indicates platelet release via proplatelet formation. Seemingly, proplatelets are fragmented in the lungs at predesignated locations along the proplatelet process.

Intramedullary hemorrhage caused by arteriovenous malformation: a case of mixed lateral and medial medullary syndrome.

A 48-year-old man with no known risk factor for cerebrovascular disease, other than cigarette smoking, experienced the sudden onset of a mixed lateral and medial medullary syndrome. Computed tomography scan failed to show any definite abnormality. Magnetic resonance imaging scans revealed hemorrhage restricted to the left dorsolateral medulla. Angiography showed abnormal arteries originating from the left vertebral artery with small niduses located on the surface of the medulla and contralateral cerebellum. Small brain-stem hemorrhages are a contraindication to thrombolytic or anticoagulant therapy, and therefore must be recognized in the acute stage.

The platelet factor-3 test for detection of canine antiplatelet antibody.

Autoimmune thrombocytopenia can be diagnosed by demonstrating antiplatelet antibody in patient's serum using a platelet factor-3 test. A procedure was developed to conduct the platelet factor-3 test using a Fibrometer to record coagulation time. The procedure provides a more practical and consistent way of reading results than making visual observations. It is also simple enough for use as a laboratory method to evaluate thrombocytopenic patients. With this procedure, positive results were obtained for about one-third of the thrombocytopenic dogs examined so far.