Typical Inclusion Bodies Research Articles

“Salt and Pepper Endothelium” Recurring After Descemet Membrane Endothelial Keratoplasty

To describe the presence of "salt and pepper endothelium", that is, typical cellular inclusion bodies in a patient with Fuchs endothelial corneal dystrophy (FECD), that recurred in the donor corneal endothelial cells after Descemet membrane endothelial keratoplasty (DMEK). A 76-year-old man underwent DMEK for FECD in his left eye. Routine specular microscopy imaging, best-corrected visual acuity measurements, and pachymetry measurements were performed before and after surgery. Besides large guttae indicating FECD, preoperative specular microscopy images showed variable-sized dark cellular inclusion bodies in the endothelial cells. One month after DMEK, donor endothelial cells appeared normal; however, at 3 months, the typical inclusion bodies reappeared and progressed slowly within a 4-year follow-up period. Both best-corrected visual acuity and pachymetry were stable throughout the study period. "Salt and pepper endothelium" recurred after the host tissue was exchanged by donor Descemet membrane, that is, a DMEK graft. These changes may indicate that either the donor corneal endothelial cell morphology is modified by adjacent tissue structures or that it is completely replaced by recipient endothelium within the first months after surgery.

Retinal Dystrophy and Optic Nerve Pathology in the Mouse Model of Mucolipidosis IV

Mucolipidosis IV is a debilitating developmental lysosomal storage disorder characterized by severe neuromotor retardation and progressive loss of vision, leading to blindness by the second decade of life. Mucolipidosis IV is caused by loss-of-function mutations in the MCOLN1 gene, which encodes the transient receptor potential channel protein mucolipin-1. Ophthalmic pathology in patients includes corneal haze and progressive retinal and optic nerve atrophy. Herein, we report ocular pathology in Mcoln1(-/-) mouse, a good phenotypic model of the disease. Early, but non-progressive, thinning of the photoreceptor layer, reduced levels of rhodopsin, disrupted rod outer segments, and widespread accumulation of the typical storage inclusion bodies were the major histological findings in the Mcoln1(-/-) retina. Electroretinograms showed significantly decreased functional response (scotopic a- and b-wave amplitudes) in the Mcoln1(-/-) mice. At the ultrastructural level, we observed formation of axonal spheroids and decreased density of axons in the optic nerve of the aged (6-month-old) Mcoln1(-/-) mice, which indicates progressive axonal degeneration. Our data suggest that mucolipin-1 plays a role in postnatal development of photoreceptors and provides a set of outcome measures that can be used for ocular therapy development for mucolipidosis IV.

Inclusion body myositis with granuloma formation in muscle tissue

Inclusion body myositis is a form of inflammatory myopathy. We identified 4 cases of inclusion body myositis showing granuloma formation in muscle tissue and aimed to assess the features of this atypical form of inclusion body myositis. We retrospectively reviewed consecutive patients who satisfied European Neuromuscular Centre IBM Research Diagnostic Criteria 2011. Then, we assessed clinical profiles and pathological findings in patients with inclusion body myositis with granuloma and compared these findings with those of typical inclusion body myositis without granuloma. We identified 15 patients with inclusion body myositis. Four patients showed granuloma formation in muscle tissue in addition to typical pathological features of inclusion body myositis. Granulomas comprised a mixture of inflammatory cells, such as macrophages, epithelioid histiocytic cells, and lymphocytes. One patient was found to have mediastinal granulomatous lymphadenopathy; however, the evidence in other patients was insufficient for a diagnosis of systemic sarcoidosis. There were no significant differences between groups with and without granuloma regarding clinical manifestations, laboratory findings, response to immunomodulating therapies, or myopathological profiles. We established a new form of inclusion body myositis showing granuloma formation in muscle tissue. Inclusion body myositis and granuloma formation could have identical pathomechanisms concerning dysregulation of autophagy.

Cytomegalovirus Identification and Quantification from the Saliva of Human Immunodeficiency Virus Seropositive and Seronegative Patients Using Real Time Polymerase Chain Reaction

Cytomegalovirus Identification and Quantification from the Saliva of Human Immunodeficiency Virus Seropositive and Seronegative Patients Using Real Time Polymerase Chain Reaction

Molecular identification of erythrocytic necrosis virus (ENV) from the blood of Pacific herring (Clupea pallasii)

Viral erythrocytic necrosis (VEN) is a condition affecting the red blood cells of more than 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. Among populations of Pacific herring (Clupea pallasii) on the west coast of North America the disease causes anemia and elevated mortality in periodic epizootics. Presently, VEN is diagnosed by observation of typical cytoplasmic inclusion bodies in stained blood smears from infected fish. The causative agent, erythrocytic necrosis virus (ENV), is unculturable and a presumed iridovirus by electron microscopy. In vivo amplification of the virus in pathogen-free laboratory stocks of Pacific herring with subsequent virus concentration, purification, DNA extraction, and high-throughput sequencing were used to obtain genomic ENV sequences. Fragments with the highest sequence identity to the family Iridoviridae were used to design four sets of ENV-specific polymerase chain reaction (PCR) primers. Testing of blood and tissue samples from experimentally and wild infected Pacific herring as well as DNA extracted from other amphibian and piscine iridoviruses verified the assays were specific to ENV with a limit of detection of 0.0003ng. Preliminary phylogenetic analyses of a 1448bp fragment of the putative DNA polymerase gene supported inclusion of ENV in a proposed sixth genus of the family Iridoviridae that contains other erythrocytic viruses from ectothermic hosts. This study provides the first molecular evidence of ENV's inclusion within the Iridoviridae family and offers conventional PCR assays as a means of rapidly surveying the ENV-status of wild and propagated Pacific herring stocks.

TGN exit of the cation-independent mannose 6-phosphate receptor does not require acid hydrolase binding.

The cation-independent mannose 6-phosphate (Man-6-P) receptor (CI-MPR) binds newly synthesized, Man-6-P-containing lysosomal acid hydrolases in the trans-Golgi network (TGN) for clathrin-mediated transport to endosomes. It has remained unresolved, however, whether acid hydrolase binding is required for exit of the CI-MPR from the TGN. To address this question we used a B cell line derived from a Mucolipidosis type II (MLII)/I-cell disease patient. In MLII patients, acid hydrolases do not acquire the Man-6-P recognition marker and as a consequence do not bind to the CI-MPR. This causes secretion of the majority of the acid hydrolases and a decreased lysosomal activity resulting in typical inclusion bodies. In agreement herewith, ultrastructural analysis of the MLII patient derived B cells showed numerous inclusion bodies with undigested material, which we defined as autolysosomes. By quantitative immuno-electron microscopy we then studied the distribution of the CI-MPR in these cells. We found that the level of co-localization of TGN-localized CI-MPR and clathrin was similar in MLII and control B cells. Moreover, the CI-MPR was readily found in endosomes of MLII cells and the TGN-to-early endosome ratio of CI-MPR labeling was unaltered. These data show that there is no block in TGN exit of the CI-MPR in the absence of Man-6-P-modified acid hydrolases. Notably, late endosomes and inclusion bodies in MLII B cells contained increased levels of the CI-MPR, which likely reflects the reduced degradative capacity of these compartments.

Clinico-pathological findings in naturally infected cases of canine parvovirus infection

The present study was conducted to evaluate the haematological parameters in parvo virus infected dogs and the gross and microscopic changes in the tissues of cases dying due to this infection. The haematology revealed low mean values of haemoglobin, total leukocyte count, total erythrocyte count, packed cell volume. The gross changes were found to be more severe in the heart and intestine with changes like round heart appearance, serosal haemorrhage and presence of chocolaty to reddish brown contents with mucosal haemorrhages in the intestine. Microscopically, cardiac muscle fibres evinced presence of basophilic intranuclear inclusion body, a typical feature of myocardial form of canine parvovirus. Similarly, typical intranuclear inclusion bodies were also found in the intestinal epithelial cells.

Virus Factories of Cauliflower Mosaic Virus Are Virion Reservoirs That Engage Actively in Vector Transmission

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.

Cytomegalovirus infection in the patients with inflammatory bowel disease

Cytomegalovirus infection of the gastrointestinal tract is a relatively common occurrence in patients receiving immunosuppressive drugs, those infected with HIV, or those who are transplantant recepients. CMV infection may be diagnosed if typical intranuclear inclusion bodies are seen on standard hematoxylin and eosin (H&E) stain. However, the sensitivity of H&E exam for CMV infection is low. Immunohistochemistry (IHC) exam performed on colon biopsy specimens is more sensitive than routine histologic exam and has excellent specificity. Currently IHC is considered the gold standard for detecting CMV CMV infection in patients with refractory or complicated IBD should be ruled out before aggressive immunosuppressive therapy for treatment-resistant disease to decrease morbidity and mortality.

MYH9-related disease: Five novel mutations expanding the spectrum of causative mutations and confirming genotype/phenotype correlations

MYH9-related disease (MYH9-RD) is a rare autosomal dominant syndromic disorder caused by mutations in MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA (myosin-9). MYH9-RD is characterized by congenital macrothrombocytopenia and typical inclusion bodies in neutrophils associated with a variable risk of developing sensorineural deafness, presenile cataract, and/or progressive nephropathy. The spectrum of mutations responsible for MYH9-RD is limited. We report five families, each with a novel MYH9 mutation. Two mutations, p.Val34Gly and p.Arg702Ser, affect the motor domain of myosin-9, whereas the other three, p.Met847_Glu853dup, p.Lys1048_Glu1054del, and p.Asp1447Tyr, hit the coiled-coil tail domain of the protein. The motor domain mutations were associated with more severe clinical phenotypes than those in the tail domain.

Presentation of an unusual patient with Lafora disease

Lafora disease is a rare, fatal, autosomal recessive progressive myoclonic epilepsy. The condition is characterised by seizures, myoclonus and dementia. In this case report, a patient who presented with generalised tonic-clonic seizures at the age of 30 is discussed. Until the age of 48, the patient did not have myoclonic jerks or ataxia clinically, but had well controlled seizures. He developed dementia and late extrapyramidal signs. Axillary skin biopsy revealed typical Lafora inclusion bodies. Genetic analysis showed a mutation in the EMP2B gene. To our knowledge, this is the first description of a patient suffering from a Lafora disease without disabling myoclonus and ataxia but rather rare seizures, extrapyramidal signs, and dementia.

Detection and molecular chracterization of porcine type 3 orthoreoviruses circulating in South Korea

Orthoreoviruses infect virtually all mammalian species, causing systemic infections including mild gastrointestinal and respiratory illnesses. However, little is known about the prevalence or genetic diversity of porcine orthoreoviruses in South Korea. We examined 237 diarrheic fecal samples collected from 78 pig farms around the country. RT-PCR utilizing primers specific for the L1 gene of mammalian orthoreoviruses showed that 45 (19.0%) samples were positive. The 10 strains isolated from orthoreovirus-positive samples formed typical perinuclear cytoplasmic inclusion bodies and had an atypical hemagglutination pattern; these are characteristics of type 3 orthoreovirus. Phylogenetic analysis of the S1 gene in these 10 Korean and other strains showed that type 3 orthoreoviruses could be divided into four lineages; the 10 Korean strains were included in porcine lineage IV, along with T3/porcine/Sichuan/2006. Sequence analysis showed that strains in lineage IV had nucleotide identities of 97.0–98.1% and deduced amino acid identities of 96.4–98.2%. Sequence analysis of the σ1 protein, a viral attachment protein, revealed that the amino acid sequences associated with neurotropism (amino acids 198–204, 249I, 350D, and 419E) were highly conserved among the Korean strains, confirming that neural tropism was present. In conclusion, our findings suggest that porcine orthoreovirus infections are endemic in pig farms in South Korea and that the 10 novel Korean porcine orthoreoviruses belong to porcine lineage IV of type 3 orthoreovirus. In addition, sequence analysis of S1 genes encoding the σ1 protein showed that the 9 of 10 Korean porcine orthoreoviruses exhibited neural tropism.

Distal myopathy with rimmed vacuoles: clinical and muscle morphological characteristics and spectrum of GNE gene mutations in 53 Chinese patients

Objectives: Distal myopathy with rimmed vacuoles (DMRV) is a typical autosomal recessive hereditary inclusion body myopathy, characterized by slowly progressive distal muscle weakness with relative sparing of the quadriceps. This study aimed to investigate the variability of clinical and morphological presentation and the spectrum of Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) mutations in Chinese DMRV patients.Methods: We retrospectively reviewed the medical records of 37 patients with DMRV in PLA General Hospital from 1986 to 2011, and further conducted a review of 16 reported Chinese DMRV patients from other hospitals. We systematically analyzed the clinical, muscle morphological features and GNE gene mutation status of all DMRV patients.Results: A total of 53 DMRV patients were studied. Fourteen cases had family history and other 39 cases were sporadic. Fifteen cases showed atypical pathological presentation as mononuclear cell invasion into necrotic or non-necrotic muscle fibers. Rare initial symptom, earlier age of onset and more dysmorphic presentations were shown in sporadic patients. Eighteen mutations in GNE gene were identified. c.317T>C (p.I106T) was a novel GNE gene mutation. c.1892C>T (p.A631V), c.527A>T (p.D176V) and c.1523T>C (p.L508S) were the common GNE mutations in Chinese DMRV patients.Discussion: The clinical, pathological and genetic characteristics of DMRV are distinct in Chinese patients.

Detection of feline herpes virus 1 via polymerase chain reaction and immunohistochemistry in cats with ulcerative facial dermatitis, eosinophilic granuloma complex reaction patterns and mosquito bite hypersensitivity

Ulcerative dermatitis caused by feline herpes virus 1 (FHV-1) is an uncommon disease characterized by cutaneous ulcers secondary to epidermal, adnexal and dermal necrosis. Differential diagnoses for FHV-1 lesions include, but are not limited to, mosquito bite hypersensitivity and eosinophilic granuloma complex. Histopathological diagnosis of FHV-1 dermatitis is based on the detection of the intranuclear inclusion bodies. In cases where intranuclear inclusions are missing but clinical and histological findings are compatible with FHV-1 dermatitis, immunohistochemistry (IHC) and PCRs have been used. In this retrospective study, we evaluated the presence of FHV-1 by IHC and PCR in skin biopsies and compared the results of the two tests. Sixty-four skin biopsy specimens from cats with compatible lesions were reviewed and tested via PCR and IHC for evidence of FHV-1. Polymerase chain reaction was positive in 12 of 64 biopsies; PCR and IHC were positive only in two of 64 biopsies, and these cases were considered true positive cases. The higher number of PCR-positive cases was possibly attributed to amplification of viral DNA from a live attenuated vaccination, but a previous FHV-1 infection with subsequent amplification of latently inserted FHV-1 could not be excluded. If clinical signs and histopathology suggest FHV-1 infection in the absence of typical inclusion bodies, IHC is the preferred diagnostic test; PCR may be useful for initial screening, but due to false positives is not sufficient for a definitive diagnosis.

Isolation of Chlamydophila psittaci from pet birds in Iran

Avian chlamydiosis is one of the most important infectious diseases of birds. Despite the rapid growth of exotic bird populations in Iran, there is little or no information on the specific infections that these types of birds carry. In this study, conventional isolation methods were used in cell culture to study occurrence of infection in pet birds. Samples from the conjunctiva, choana, and cloaca and/or droppings were provided from 17 birds of different species. The samples were used to infect McCoy cell culture to isolate Chlamydophila psittaci. The inoculated cells were fixed, stained by Giemsa, mounted on slides using Entellan® and observed by light microscope for the presence of typical chlamydial inclusion bodies. Chlamydophila psittaci was isolated from four birds including a ring-necked parakeet (Psittacula krameri), an Alexandrine parakeet (Psittacula eupatria), an African grey parrot (Psittacus erithacus), and a Timneh grey parrot (Psittacus erithacus timneh). All negative cultures were passaged a further two times. To the best of our knowledge; the report represents the first isolation of chlamydia from birds in Iran.

Monoclonal Antibodies Against Lipopolysaccharide ofChlamydia trachomatiswith Cross Reactivity to Human ApoB

Splenocytes obtained from mice immunized with whole purified elementary bodies of Chlamydia trachomatis were used for hybridoma construction. The resulting clones were screened with ELISA using chlamydial lipopolysaccharide (LPS) and full-length human apolipoprotein B (ApoB). One analyzed clone producing IgG1 (MAb 7B5) showed simultaneous recognition of chlamydial LPS and human ApoB, suggesting the presence of common antigenic epitopes in their structures. MAb 7B5 exhibited agreeable activity in immunoblot analysis conducted using chlamydial extracts or full-length human ApoB as well as in immunofluorescence (IF) detecting typical inclusion bodies of C. trachomatis and C. pneumoniae in the infected eukaryotic host cells. The removal of LPS from chlamydial suspensions with lauroyl sarcosyl led to a complete disappearance of IF associated with the elementary bodies of C. trachomatis. Therefore, immunologic response to chlamydial antigen may be associated with the generation of ApoB-specific antibody. Molecular mimicry and subsequent formation of cross-reactive antibodies might be an essential mechanism explaining the appearance of circulating auto-antibodies against low density lipoproteins (LDL) in patients with atherosclerosis. Moreover, newly generated MAb 7B5 can be a useful tool in the laboratory diagnosis of chlamydial infections.

Hepatitis and hydropericardium syndrome associated with adenovirus infection in goslings

Two outbreaks of severe acute disease characterised by hepatitis and hydropericardium were observed in young goslings on large-scale farms in Hungary. Histological examination revealed multifocal necrotic areas and two types of intranuclear inclusion bodies adjacent to necrotic areas in the liver. The most prominent type of inclusion bodies showed strong basophilic staining and completely filled the enlarged nucleus. The other type was eosinophilic and occupied the centre of the nucleus, which had margination of chromatin. In the heart, haemorrhage was associated with multifocal necrosis in the myocardium. The presence of fowl adenovirus DNA in different organs of the naturally infected goslings was detected by polymerase chain reaction (PCR). The virus was isolated, and identified as a goose adenovirus by genomic analysis. This is the first report on the involvement of a goose adenovirus in severe acute disease associated with hepatitis and hydropericardium.

Antifungal activity of a recombinant defensin CADEF1 produced by Escherichia coli

The defensin CADEF1 has been known to be up-transcripted in the pepper Capsicum annuum L., upon bacterial attack, abiotic elicitors and environmental stresses. However, the native form of CADEF1 has not been purified from the pepper, and its activity against fungi has been uncertain. In this study, the full-length cDNA of CADEF1 was obtained by fragment piecing-together method. Thereafter, its mature peptide coding sequence was inserted into bacterial expression vector pET28a(+), and the recombinant vector was transformed into Escherichia coli BL21 (DE3). Based on SDS–PAGE and Western blotting analysis, the recombinant CADEF1 was produced as a 5.6 kDa protein in the form of inclusion body in E. coli BL21 (DE3) and the expression level accounted for 15% of the bacterial total protein. Further processing through protein refolding and purification, converted the inclusion body type of CADEF1 into active form with the yield about 13 μg/ml of culture. The refolded CADEF1 significantly showed the activity against growth of the fungal pathogen Verticillium dahliae on average by 67.9% in vitro assay. These results verified the novel activity of CADEF1, which might benefit in prevention of the fungus-related disease in agricultural production.

In Vivo Formation of Protein Based Aqueous Microcompartments

In this paper, we report the formation of protein based liquid droplets resulting in the formation of in vivo microcompartments in E. coli or tobacco cells. These microcompartments were generated by expressing elastin-like polypeptides (ELP), which have the ability to undergo a reversible phase transition, resulting in the formation of an aqueous two-phase system (ATPS) in the cytoplasm of the cell. We prove that these microcompartments are liquid by expressing a fusion protein consisting of ELP and GFP and by performing fluorescence recovery after photobleaching (FRAP) experiments at different stages of cell cultivation. In the initial phases of cell growth, the fusion protein concentration is low and is not sufficient to drive the formation of a second aqueous phase. As the intracellular fusion protein concentration increases with longer cultivation time, droplets start forming, and as protein expression continues, the droplets coalesce at the poles of the E. coli cells. FRAP experiments with cells at different growth stages reveals that the protein in these ELP based droplets is comprised of aqueous and not solid aggregates, as seen in typical inclusion bodies. Staining of the ribosomes and coimaging of the ELP-GFP fusion protein showed that these compartments exclude the protein making machinery of the cell, acting as depots for newly formed protein. It is also shown, in vitro, that ELP based droplets result in the exclusion of proteases, protecting proteins from degradation. Additional studies are still required to test this possibility in vivo. To the best of our knowledge, this is the first report characterizing the formation of an engineered extra aqueous phase in a living organism.

A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or alpha1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to 18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.