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Characteristics of eosinophilic inclusions within Schistosoma japonicum eggs.

Although eosinophilic bar- or droplet-like inclusions are frequently detectable inside eggs deposited in the livers of Schistosoma japonicum-infected animals, little is known of their exact nature. In the livers of mice implanted with freshly laid eggs, inclusion-positive eggs were found in 28.7 and 46.2% of deposited eggs at 2 and 4 weeks, respectively, after implantation, but in 4.3% at 5 weeks when most of the eggs had already degenerated. When the extent of granuloma formation was investigated, granulomas around inclusion-positive eggs were smaller than those around negative eggs. Host factors associated with the formation of inclusion were sought using in vivo and in vitro studies. Following the administration of anti-egg antigen serum into egg-implanted mice, no increase in occurrence of inclusion-positive eggs was seen. In a co-culture of mature eggs with infected rabbit or mouse serum, inclusions were rarely found. In contrast, they were found in 17.9% of eggs in the presence of splenic cells. The present study is the first to show that there is decreased granuloma formation in the presence of eosinophilic inclusions inside eggs and our in vitro study suggests that host cell-egg interaction is responsible for the formation of inclusions.

Animals↗

Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli. 1. Composition.

We have determined the macromolecular composition of inclusion bodies formed by overexpressing beta-lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition. Specifically, contaminating polypeptides ranged from less than 5% to over 50% of the total protein content. Phospholipids composed 0.5-13% of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature beta-lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown at basic pH. Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.

Bacterial Proteins↗

Ultrastructural study of intranuclear inclusion bodies of pulmonary adenocarcinoma.

Intranuclear inclusion bodies are sometimes observed in pulmonary adenocarcinoma by light microscopy. Electron microscopic characteristics of lung cancer cells with intranuclear inclusion bodies were studied. In addition, polymerase chain reaction (PCR) was performed using primers coding for human papillomavirus (HPV) types 16, 18, and 33. Eosinophilic intranuclear inclusion bodies were observed in 22 out of 285 cases by light microscopy. Immunohistochemically, cancer cell nuclei stained with PE-10. Three types of intranuclear inclusion bodies were classified electron microscopically. Type A showed aggregation of electron dense particles (30-40 nm) with an electron-dense core and was most frequently observed. Type B consisted of a mass of branching and whirling tubular structures. Type B intranuclear inclusions had a relationship with inner nuclear membrane. In type C, several spherical inclusions were observed in one nucleus. HPV DNA was detected using PCR and type-specific probes in a case with type A inclusion bodies. This study suggests that intranuclear inclusion bodies in pulmonary adenocarcinoma are formed by several different mechanisms.

Adenocarcinoma, Papillary↗

An electron microscope study of intranuclear inclusions in mouse liver and hepatoma.

An electron microscope study of intranuclear inclusions which occur in giant cells in a transplantable mouse hepatoma and in enlarged liver cells in mice fed a diet containing bentonite demonstrates that these inclusions are formed by invaginations of the nuclear envelope, and corroborates a previous histochemical study which revealed that the contents of the inclusions are of cytoplasmic origin. In the hepatoma cells the intranuclear inclusions are abundant, small, and situated close to the border of the nucleus, and there are wide openings from the cytoplasm into the invaginations whose contents include lipid droplets, ergastoplasm, and structurally normal mitochondria. In the enlarged liver cells the inclusions are fewer in number, generally much larger than those in the hepatoma, hence they extend deeper into the nucleus, and the interior is continuous with the cytoplasm through only a small opening. Some normal ergastoplasm is present within the inclusions but all other constituents are abnormal. Both normal and degenerating mitochondria occur in the cytoplasm but only degenerating ones are found within the inclusions. Both types of inclusions arise in greatly enlarged cells in which an attempt is made to maintain the normal nuclear surface/nuclear volume ratio by the development of the invaginations of the nuclear envelope.

Animals↗

Induction of inclusion formation and disruption of lamin A/C structure by premutation CGG-repeat RNA in human cultured neural cells.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects some adult carriers of pre-mutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. FXTAS is thought to be caused by a toxic 'gain-of-function' of the expanded CGG-repeat FMR1 mRNA, which is found in the neuronal and astrocytic intranuclear inclusions associated with the disorder. Using a reporter construct with a FMR1 5' untranslated region harboring an expanded (premutation) CGG repeat, we have demonstrated that intranuclear inclusions can be formed in both primary neural progenitor cells and established neural cell lines. As with the inclusions found in post-mortem tissue, the inclusions induced by the expanded CGG repeat are alphaB-crystallin-positive; however, inclusions in culture are not associated with ubiquitin, indicating that incorporation of ubiquitinated proteins is a later event in the disease process. The absence of ubiquitinated proteins also argues against a model in which inclusion formation is due to a failure of the proteasomal degradative machinery. The presence of the expanded CGG repeat, as RNA, results in reduced cell viability as well as the disruption of the normal architecture of lamin A/C within the nucleus. This last observation, and the findings that lamin A/C is present in both the inclusions of FXTAS patients and the inclusions in cell culture, suggests that lamin A/C dysregulation may be a component of the pathogenesis of FXTAS; in particular, the Charcot-Marie-Tooth-type neuropathy associated with FXTAS may represent a functional laminopathy.

5' Untranslated Regions↗

Secondary structure characterization of beta-lactamase inclusion bodies.

The secondary structure of proteins in E. coli inclusion bodies was investigated via Raman spectroscopy. Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C. All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples. The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E. coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content. Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures. These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content. Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.

Escherichia coli↗

Parkinson's disease and dementia with neuronal inclusions in the cerebral cortex: Lewy bodies or Pick bodies.

Cortical Lewy bodies may be difficult to differentiate from Pick bodies by the usual staining methods. This problem is illustrated in a case of progressive dementia with parkinsonian features. Lewy bodies were found, not only in the pigmented nuclei in the brainstem and in the cerebral cortex, but also in the dentate fascia, a predilection site for Pick bodies. The inclusion bodies were compared with the inclusion bodies in a case of Pick's disease. Intense argyrophilia of the cortical inclusion bodies argued in favor of Pick bodies; antibodies to phosphorylated neurofilaments reacted with the cortical inclusions and with classical Pick bodies, but antibodies to paired helical filaments reacted only with the Pick bodies. The most convincing evidence, that the inclusions were Lewy bodies, was obtained by electron microscopy. The filaments in the inclusions showed fuzzy deposits of electron dense material, characteristic for filaments of Lewy bodies. This contrasted with the smooth filaments of the Pick bodies. It is concluded that cortical inclusions in brains from patients with Lewy bodies in the pigmented nuclei of the brainstem most likely represent Lewy bodies. This can be confirmed by examining the ultrastructure of the inclusions.

Aged↗

Characterization of ubiquitinated intraneuronal inclusions in a novel Belgian frontotemporal lobar degeneration family.

The most common histologic feature in patients with frontotemporal lobar degeneration (FTLD) is intracellular brain inclusions of yet uncharacterized proteins that react with antiubiquitin (Ub) antibodies, but not with tau or synuclein (FTLD-U). We identified a four-generation Belgian FTLD family in which 8 patients had dominantly inherited FTLD. In one patient, we showed frontotemporal atrophy with filamentous Ub-positive intracellular inclusions in absence of tau pathology or any alterations in the levels of soluble tau. We characterized the cellular and subcellular localization and morphology of the inclusions. Ub-positive inclusions predominantly occurred within neurons (>97%), but were also observed within oligodendroglia (approximately 2%) and microglia (<1%), but not within astroglia. Regarding the subcellular localization, the intranuclear inclusions (INI) were up to approximately four-fold more frequent than the cytoplasmic inclusions, although the latter were more specific to neurons. The INIs frequently appeared spindle-shaped and 3-dimensional confocal reconstructions identified flattened, leaf-like structures. Ultrastructurally, straight 10- to 18-nm-diameter filaments constituted the spindle-shaped inclusions that occurred in close proximity to the nuclear membrane. Staining for HSP40, p62, and valosin/p97 was observed in only a minority of the inclusions. Whereas the precise nature of the protein remains elusive, characterization of such familial FTLD-U patients would be helpful in identifying a common denominator in the pathogenesis of familial and the more prevalent sporadic FTLD-U.

Aged↗

Viral DNA synthesis in vitro with the inclusions isolated from adenovirus 12-infected cells.

A fraction defined as the inclusions was isolated by banding in CsC1 gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18S, 22S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.

Adenoviruses, Human↗

Ultrastructural cytochemistry of eosinophilic inclusions in the cells of hyperplastic nodules and hepatomas in mouse liver.

Intracisternal inclusions in the cells of 48 hyperplastic nodules and hepatomas in mouse liver were examined by electron microscopy to determine the precise compositions of the inclusions in relation to their ultrastructure, and some preliminary attempts at isolation and chemical analysis of the inclusions were performed. We classified the inclusions into two types. One was mainly of larger size, consisting of a single electron-lucent core and a granular cortical zone of high electron density. The other type was smaller, with a number of tiny, electron-lucent areas crowded into the central area instead of a single core. The cortical material of the inclusions was digested by pepsin treatment of thin sections, whereas the core and the electron-lucent areas within the cortical zone were not extracted. On the other hand, in materials treated with ethanol before post-osmication, only the core and electron-lucent areas within the cortical zone were partially extracted. The ultrastructure of the isolated inclusions was very similar to that of inclusions in situ. The chemical composition of the isolated fractions was estimated to be 60% protein and 35% lipid. Electrophoretically, the protein of this fraction showed a single band. We conclude that the cortical substance is proteinaceous in nature, probably consisting of a single protein or a group of proteins with identical electrophoretic mobility, whereas the core is composed of lipid. The possibility that the inclusions are due to an impairment in the mechanism of intracellular lipoprotein transport is discussed.

Alpha-Globulins↗

Regulation of the packaging of Bacillus thuringiensis delta-endotoxins into inclusions.

During sporulation, many Bacillus thuringiensis subspecies synthesize several related delta-endotoxins which are packaged into bipyramidal intracellular inclusions. These inclusions are solubilized in the alkaline, reducing conditions of the midguts of susceptible insect larvae and are converted by proteolysis to active toxins. The toxins insert into the membranes of cells lining the midgut and form cation-selective channels, which results in lethality. There are three delta-endotoxins, Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions produced by a B. thuringiensis subsp. aizawai cell. While the ratio of the steady-state mRNAs for these three protoxins has been shown to differ (cry1Ab3/cry1Ca1/cry1Da1 mRNA ratio, 4:2:1), the half-lives of the cry1Da1 and cry1Ab3 mRNAs were found to be similar, indicating that there were differences in the transcription rates. The relative contents of these delta-endotoxins in purified inclusions from B. thuringiensis subsp. aizawai have been measured previously, and an even greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1) was found. In order to account for this deficiency, other steps which could be involved in inclusion formation, such as translation and packaging, were examined. The three cry genes have the same dual overlapping promoters, but the ribosome binding sequence for the cry1Da1 gene was not the consensus sequence. Translation was enhanced about fourfold by changing to the consensus sequence. In addition, the relative amount of Cry1Da1 protoxin in inclusions was twofold lower when cells were sporulated in Luria-Bertani (LB) medium than when cells were sporulated in a glucose-yeast extract medium. This difference was attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the delta-endotoxin composition of an inclusion.

Amino Acid Sequence↗

Characterization of intracytoplasmic hydrocarbon inclusions from the hydrocarbon-oxidizing Acinetobacter species HO1-N.

The ultrastructure of Acinetobacter sp. strain HO1-N grown on hydrocarbon and nonhydrocarbon substrates was compared using thin sections and freeze-etching. Hydrocarbon-grown cells were characterized by the presence of intracytoplasmic membrane-bound hexadecane inclusions. This membrane did not exhibit a typical unit membrane structure but appeared as a monolayer. The freeze-etch technique revealed the internal structure of the hexadecane inclusions and provided evidence for the presence of a smooth-surfaced limiting membrane. Freeze-etching also revealed intracytoplasmic membranes in the hexadecane-grown cells. These ultrastructural modifications were not present in nonhydrocarbon-grown cells. The hexadecane inclusions were isolated from Acinetobacter. Negative-staining of the inclusions revealed electron-transparent vesicles approximating the size of the inclusions seen in whole cells. Freeze-etching of the purified inclusions revealed membrane-bound vesicles. The purified inclusions exhibited a relatively high value of lipid phosphorus to protein. The lipid composition and the electrophoretic banding pattern of the inclusions on sodium dodecyl sulfate-polyacrylamide gels were determined and compared with other membrane fractions (outer membrane and cytoplasmic membrane) previously isolated from this organism.

Acinetobacter↗

Attenuated nuclear shrinkage in neurones with nuclear inclusions of SCA1 brains.

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is one of the autosomal dominant neurodegenerative disorders commonly linked to pathological expansion of the CAG repeat of the relevant gene. Nuclear inclusions and neurodegeneration are both triggered by this pathological expansion of the CAG/polyglutamine repeat on ataxin-1, but it remains to be determined whether or not nuclear inclusion formation is associated with accelerated neurodegeneration. OBJECTIVE: To examine the influence of nuclear inclusions on nuclear size and deformity in human brains from patients suffering from SCA1. MATERIAL: Pontine sections of brains obtained at necropsy from seven patients with SCA1 and five controls. METHODS: The size and deformity of each neuronal nucleus was quantified. Nuclei with and without inclusions were examined separately to assess the possible influence of nuclear inclusions on neurodegeneration. RESULTS: Nuclear shrinkage and deformity were more marked in SCA1 brains than in controls. This shrinkage was attenuated in neurones containing nuclear inclusions. CONCLUSIONS: The existence of nuclear inclusions in SCA1 is presumably linked to a mechanism that attenuates rather than accelerates nuclear shrinkage. This in vivo finding may provide a clue to constructing a rational therapeutic strategy for combating neurodegeneration associated with nuclear inclusions.

Adult↗

Viral behavior of paracrystalline inclusions in osteoclasts of Paget's disease of bone.

Fresh tissues from six patients with Paget's disease of bone were examined ultrastructurally to investigate whether the characteristic paracrystalline inclusions in pagetic osteoclasts revealed viral behavior. These inclusions appeared as microfilamentous aggregates in both nuclei and cytoplasm of the osteoclasts in all six cases. The filamentous elements of the inclusions with a diameter of 11-15 nm showed tubular structures with a central electron-lucent zone measuring 5-7 nm in diameter. Viral budding-like structures containing these inclusions were found at the peripheral cytoplasm or cell processes in the ruffled border of some pagetic osteoclasts in two cases. The inclusions in the budding-like structures were often arrayed in a parallel fashion on the cytoplasmic side of the cell membranes of extruded cytoplasm or cell processes. Virion-like particles were also found in the extracellular spaces of the ruffled border. Marked nuclear degeneration was often seen in pagetic osteoclasts of three cases, although other nuclei in the same osteoclasts appeared normal. The degenerated nuclei showed nuclear ring formation where destroyed nuclear membranes were seen and disappearance of nuclear matrices was noted. Since the modifications were always associated with the accumulation of abundant inclusions, they were probably caused by the inclusions. These findings suggested that the inclusions showed viral behavior in pagetic osteoclasts, and that the nuclear modifications were caused by virus infection.

Crystallization↗

Ultrastructural analysis of granulocyte inclusions in genetically confirmed MYH9-related disorders.

BACKGROUND AND OBJECTIVES: MYH9-related disorders are autosomal dominant hereditary macrothrombocytopenias caused by mutations in the MYH9 gene. This gene encodes the non-muscular myosin heavy chain type II A (MHCIIA). Among these disorders, May-Hegglin anomaly (MHA), Sebastian syndrome (SS), and Fechtner syndrome (FS) are associated with different types of ribosome inclusions in granulocytes. FS also exhibits Alport-like manifestations: nephropathy, neurosensory deafness, and cataracts. The aim of our study was to assess the granulocyte inclusion ultrastructure in genetically confirmed MYH9-related disorders. DESIGN AND METHODS: Ten individuals were studied. All fulfilled the clinical and laboratory findings to be diagnosed as having an MYH9-related disorder. The ultrastructure of 50 granulocyte sections for each patient was examined, and the percentages of the different types of inclusion were established. Mutations of the MYH9 gene were also analyzed. RESULTS: The patients were classified as having MHA if the inclusions contained parallel longitudinal filaments. If not, they were classified as having SS or FS. FS patients also showed Alport-like manifestations. In all syndromes we observed a wide variability of the inclusion ultrastructure. Moreover, a small number of inclusions typical of other syndromes was observed. A new cross-striated inclusion variant was identified in SS. A significant number of pure ribosome aggregates were identified in all syndromes. INTERPRETATION AND CONCLUSIONS: Like other MYH9-related traits, the variation and partial overlap in the inclusion ultrastructure could be attributed to specific changes in the polymerization, assembly, or stability of the MHCIIA. These changes might be associated with MYH9 gene mutations as well as with its heterogeneous expression.

Adult↗

Leiomyoma of the gastrointestinal tract with intracytoplasmic inclusion bodies. Report of three cases.

We report three cases of leiomyoma of the gastrointestinal tract with intracytoplasmic inclusion bodies similar to those characteristic of inclusion body fibromatosis (IBF). The first two cases represent leiomyoma of the stomach: one in a 70-year-old female and the other in a 72-year-old female. In both instances inclusion bodies were present in a large amount. In the third case the leiomyoma was located in the esophagus of a 63-year-old male and inclusion bodies in this case were rare. In all three cases an immunohistochemical analysis showed positivity of the tumor cells for muscle specific actin HHF35 (MSA), alpha-smooth muscle actin (SMA), h-caldesmon and desmin. The first case showed some inclusion bodies with positivity for cytokeratin CAM 5.2 and focal weak positivity for cytokeratin 18. In the second case the inclusion bodies were positive at the periphery with antibodies directed against MSA and SMA. In the third case the inclusion bodies were immunohistochemically entirely negative. Ultrastructurally, the inclusion bodies in the first case were composed of aggregated filaments, some with entrapped cytoplasmic organels and others with finely granular dense cores.

Actins↗

Intranuclear inclusions in Schwann cells of aged fowl ciliary ganglia.

Schwann cells in ciliary ganglia of fowls aged five to seven years were found to contain numerous intranuclear inclusions and pseudo-inclusions. Similar inclusions were usually absent from both neurons and non-neuronal cells, including connective tissue cells, and were rare in Schwann cells of chickens aged less than five years. Inclusions were of two different types: filamentous bundles and granulofibrillar bodies. Individual nuclei contained one to three inclusions. Pseudo-inclusions, i.e. cytoplasmic pockets invaginated into the nuclei, were found more rarely and accompanied one or both types of 'true' inclusions. The possible significance of these findings in relation to ageing phenomena is discussed. It is concluded that intranuclear inclusions appear to be a consequence of nuclear/cellular activation and may be regarded as aggregates of previously dispersed intranuclear proteins.

Aging↗

Cytoplasmic inclusions in rabbit extraocular muscle.

Cytoplasmic inclusion bodies, similar to those previously described in abnormal and normal human extraocular muscle, were observed in the orbital surface layer of the superior rectus muscle in rabbit. These inclusion bodies are composed of a flocculent material of low density studded with granular foci of increased density. In sequential samples of serially reconstructed muscle fibers visualized by electron microscopy, cytoplasmic inclusion bodies were seen in 4.5% of 1187 samples through multiply innervated fibers that vary systematically in diameter along their length; inclusion bodies were also seen in 0.8% of 354 samples through multiple innervated fibers of constant diameter. Cytoplasmic inclusion bodies were not seen in 1838 samples through singly innervated fibers. These data suggest that such inclusion bodies may occur preferentially in multiply innervated fibers. The present findings are not compatible with previous suggestions that such cytoplasmic inclusion bodies may be indicative of a pathologic or aging process. These findings are consistent with previous suggestions that such inclusion bodies are to be considered as normal structures in extraocular muscle.

Animals↗