Aberrant Multiciliogenesis in Idiopathic Pulmonary Fibrosis.

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.

Use of electron microscopy when screening liver biopsies from neonates and infants: experience from a single tertiary children's hospital (1991-2017).

Background: Although the role of electron microscopy is diminishing in several areas of adult pathology, it remains an essential tool for the study of pediatric liver biopsies.Methods: Clinical charts, histologic slides and EM materials of native liver biopsies from patients <1 year old (1991-2017) were reviewed.Results: 677 biopsies were performed on 353 males and 324 females. This study presents the concrete numbers for both the indications and the diseases, and describes the role of EM. EM was performed on 24.7% of liver biopsies and demonstrated key pathologic findings in 10 cases (6%), which led to the appropriate biochemical and/or genetic testing to confirm the diagnoses. The cases included five cases of glycogen storage disease with characteristic findings with cytoplasmic glycogen accumulation, two cases of mitochondrial disorder with pleomorphic mitochondria with crystalloid inclusions and one case each of Niemann-Pick Disease with abundant myelinosomes, Alpha-1 antitrypsin deficiency with deposits in the endoplasmic reticulum and infantile Refsum disease with trilamellar inclusions and lack of peroxisomes. In this study, we describe the detailed histologic and EM findings of each case .Conclusion: EM played an important screening and diagnostic role in the challenging cases and was also used to rule out detectable pathologic conditions.

Histologic and ultrastructural features in early and advanced phases of Zellweger spectrum disorder (infantile Refsum disease).

Zellweger spectrum disorders (ZSD) are rare autosomal recessive inherited metabolic disorders and include severe (Zellweger syndrome) and milder phenotypes [neonatal adrenoleukodystrophy and infantile Refsum disease (IRD)]. ZSD are characterized by impaired peroxisomal functions and lack of peroxisomes detected by electron microscopy (EM). ZSD are caused by mutations in any of the 14 PEX genes. Patients with ZSD commonly demonstrate nonspecific hepatic symptoms within the first year, often without clinical suspicion of ZSD. Thus, recognition of pathologic findings in the liver is critical for the early diagnosis. We herein demonstrate the histologic and ultrastructural features in liver biopsies in the early and advanced phases from a 16-year-old male with IRD. The initial biopsy at 5 months of age showed a lack of peroxisomes by EM, and this finding played a critical role in the early diagnosis. In contrast, the second biopsy at 14 years of age, after long-term diet therapy, demonstrated significant disease progression with near-cirrhotic liver. In addition to lack of peroxisomes, EM revealed abundant trilamellar inclusions within large angulated lysosomes in many of the hepatocytes and Kupffer cells. Mitochondrial abnormalities were identified only in the second biopsy and were mainly identified in damaged cells; thus they were likely nonspecific secondary changes. This is the first report demonstrating histological and ultrastructural features of liver biopsies in the early and advanced phases from a child with ZSD. Trilamellar inclusions are considered to be an ultrastructural hallmark of ZSD, but they may not be apparent in the early phases.

Ultrastructural features of NUT midline carcinoma.

Midline carcinomas associated with the nuclear protein in testis (NUT) gene rearrangement are rare, aggressive tumors that have been diagnosed most commonly in the head, neck, mediastinum, and upper aerodigestive tract. The ultrastructural features associated with this tumor have thus far received only brief comment and have never been illustrated. The authors provide a more extensive description and illustrate the electron microscopic findings in a typical case of NUT midline carcinoma, confirmed by cytogenetic and fluorescence in situ hybridization studies. This tumor was composed of cells displaying large, irregularly shaped nuclei with prominent compact nucleoli and abundant cytoplasm containing prominent bundles of tonofilaments, occasional clusters of pleomorphic granules, small numbers of lipid inclusions, and rare glycogen deposits. The cells exhibited stubby microvillous projections, were intermittently enveloped by basal lamina, and were interjoined by numerous well-formed desmosomal-type junctions and occasional junctional complexes. The authors propose that this constellation of ultrastructural features can prove helpful in discriminating NUT midline carcinoma from similar appearing entities.

Papillary tumor of the pineal region: ultrastructural study of a case.

Papillary tumor of the pineal region (PTPR) is a recently classified neuroepithelial tumor for which there has been little comprehensive ultrastructural study. Here, we describe the radiographic, intraoperative, histologic, immunohistochemical, and in-depth ultrastructural findings in a case of PTPR. This study corroborates that PTPR has concomitant ependymal, neuroendocrine, and secretory features, and details novel ultrastructural as well as immunohistochemical features that further this argument. Discrepancies with prior descriptions of PTPR are described, as these differences may reflect phenotypic variability in this rare tumor, and the ultrastructural features that relate to the putative ependymal origin of the entity are emphasized.

Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Plexiform fibrohistiocytic tumor: ultrastructural studies may aid in discrimination from cellular neurothekeoma.

Plexiform fibrohistiocytic tumor is a low-grade soft tissue malignancy that can at times be difficult to differentiate from the less biologically aggressive cellular neurothekeoma. The two entities, which may display identical clinical and histological features, cannot be distinguished by immunohistochemical or molecular diagnostic means. Electron microscopy may enable the accurate identification of problematic examples and thus aid in resolving these occasionally occurring diagnostic dilemmas. To illustrate typical variations in the ultrastructural appearance of plexiform fibrohistiocytic tumor, the authors present two diagnostically noncontroversial examples, and to demonstrate the potential diagnostic utility of electron microscopy in this setting, they present an example of plexiform fibrohistiocytic tumor that could not otherwise have been distinguished from cellular neurothekeoma.

Renal medullary carcinoma: ultrastructural studies may benefit diagnosis.

Renal medullary carcinoma is a recently described highly aggressive malignancy that in most instances exhibits a constellation of clinical and light microscopic features sufficiently distinctive to enable a quick and confident diagnosis. Presented are three examples where, because of unusual elements in the clinical presentation, electron microscopic examination proved beneficial in establishing the diagnosis.

Marijuana medusa: The many pulmonary faces of marijuana inhalation in adolescent males.

Marijuana use has risen dramatically over the past decade. Over this same time period, pediatric hospitals have seen an increase in presentation of adolescents with acute respiratory symptoms after recent marijuana inhalation. We report a case series of three adolescent males with significant findings of bilateral pulmonary nodules and ground glass opacities on chest imaging associated with recent marijuana inhalation. Lung biopsies in two of the three patients confirmed silica-induced pneumoconiosis. The third patient was diagnosed with acute hypersensitivity pneumonitis without lung biopsy. Improvement in clinical symptoms and lung function testing were noted in two of three patients after marijuana inhalation cessation. This case series highlights the variety of severe pulmonary presentations in adolescents following recent marijuana inhalation. Future studies are required to assess whether these presenting pulmonary complications are from direct marijuana exposure or indirect associations with marijuana inhalation injuries.

Filamin C Truncation Mutations Are Associated With Arrhythmogenic Dilated Cardiomyopathy and Changes in the Cell-Cell Adhesion Structures.

OBJECTIVES: The purpose of this study was to assess the phenotype of Filamin C (FLNC) truncating variants in dilated cardiomyopathy (DCM) and understand the mechanism leading to an arrhythmogenic phenotype. BACKGROUND: Mutations in FLNC are known to lead to skeletal myopathies, which may have an associated cardiac component. Recently, the clinical spectrum of FLNC mutations has been recognized to include a cardiac-restricted presentation in the absence of skeletal muscle involvement. METHODS: A population of 319 U.S. and European DCM cardiomyopathy families was evaluated using whole-exome and targeted next-generation sequencing. FLNC truncation probands were identified and evaluated by clinical examination, histology, transmission electron microscopy, and immunohistochemistry. RESULTS: A total of 13 individuals in 7 families (2.2%) were found to harbor 6 different FLNC truncation variants (2 stopgain, 1 frameshift, and 3 splicing). Of the 13 FLNC truncation carriers, 11 (85%) had either ventricular arrhythmias or sudden cardiac death, and 5 (38%) presented with evidence of right ventricular dilation. Pathology analysis of 2 explanted hearts from affected FLNC truncation carriers showed interstitial fibrosis in the right ventricle and epicardial fibrofatty infiltration in the left ventricle. Ultrastructural findings included occasional disarray of Z-discs within the sarcomere. Immunohistochemistry showed normal plakoglobin signal at cell-cell junctions, but decreased signals for desmoplakin and synapse-associated protein 97 in the myocardium and buccal mucosa. CONCLUSIONS: We found FLNC truncating variants, present in 2.2% of DCM families, to be associated with a cardiac-restricted arrhythmogenic DCM phenotype characterized by a high risk of life-threatening ventricular arrhythmias and a pathological cellular phenotype partially overlapping with arrhythmogenic right ventricular cardiomyopathy.

Atypical presentation of neuronal ceroid lipofuscinosis type 8 in a sibling pair and review of the eye findings and neurological features.

PURPOSE: To report atypical presentation of neuronal ceroid lipofuscinoses type 8 (CLN8) to the eye clinic and review clinical features of CLN8. OBSERVATIONS: Detailed eye exam by slit lamp exam, indirect ophthalmoscopy, fundus photography, optical coherence tomography, visual fields and electroretinogram (ERG). Molecular genetic testing using Next Generation Sequencing panel (NGS) and array Comparative Genomic Hybridization (aCGH).The siblings in this study presented to the eye clinic with retinitis pigmentosa and cystoid macular edema, and a history of seizures but no severe neurocognitive deficits or regression. Genetic testing identified a c.200C > T (p.A67V) variant in the CLN8 gene and a deletion encompassing the entire gene. Electron microscopy of lymphocytes revealed fingerprint inclusions in both siblings. CONCLUSIONS: and Importance: Pathogenic variants in CLN8 account for the retinitis pigmentosa and seizures in our patients however, currently, they do not have regression or neurocognitive decline. The presentation of NCL can be very diverse and it is important for ophthalmologists to consider this in the differential diagnosis of retinal disorders with seizures or other neurological features. Molecular genetic testing of multiple genes causing isolated and syndromic eye disorders using NGS panels and aCGH along with additional complementary testing may often be required to arrive at a definitive diagnosis.

Ultrastructural features of eosinophilic oesophagitis: impact of treatment on desmosomes.

AIMS: A growing body of evidence suggests a role for altered epithelial barrier function in the pathophysiology of eosinophilic oesophagitis (EoE), but few have described the epithelial structure during inflammation. The purpose of this study was to define ultrastructural features of active, inactive EoE and control subject's oesophageal epithelia. METHODS: We prospectively enrolled patients undergoing diagnostic upper endoscopy for evaluation of EoE. Mucosal pinch biopsies were obtained from the distal oesophagus and processed for routine histology and electron microscopic assessment. Clinical features of enrolled subjects were analysed and subjects were divided into four groups: normal, gastroesophageal reflux disease (GERD), inactive EoE and active EoE. Representative photomicrographs of the basal and superficial epithelia were reviewed for abnormalities. Desmosomes were quantified on the surface of epithelia three to four prickle-cell layers above the basal layer. RESULTS: Twenty-nine paediatric cases (ages 2-18 years) were enrolled in the study. We observed a significant decrease in the number of desmosomes per cell (DPC) of subjects with active EoE compared with inactive EoE, GERD and normal epithelia. With respect to DPC, no significant differences were found between inactive EoE compared with GERD or normal subjects. Additional ultrastructural features observed included epithelial microplicae and evidence of eosinophil transmigration, degranulation, and sombrero formation. CONCLUSIONS: Consistent with clinical and molecular findings, our ultrastructural data provide support for an altered oesophageal barrier in paediatric cases with active EoE, which may improve following treatment.

Primary intracranial Ewing's sarcoma with unusual features.

Pediatric primary "small round blue cell" tumors in the CNS represent several entities, some more common than others. Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is rare and must be distinguished from other tumors such as medulloblastoma [1, 2], atypical rhabdoid/teratoid tumor, ependymomal tumors, metastatic sarcomas, hematologic malignancies, and other mimics. Although therapy for ES/pPNET is effective, it brings severe side effects, including cardiac toxicity, making correct recognition important [3]. As small blue cell tumors look similar, diagnosis often depends on special stains, immunohistochemistry, and molecular techniques. While the combination of membranous immunohistochemical reactivity for CD99 with cytoplasmic glycogen provides effective screening, demonstration of characteristic translocations of EWSR1 (chromosome 22) or FUS (chromosome 16) by fluorescent in situ hybridization (FISH) can confirm the diagnosis. We are reporting three primary ES/pPNET of the CNS, two of which occurred in children. While the adult case demonstrates the classic histopathology, the two pediatric cases have histopathology that significantly deviates from the usual. One is suggestive of a primary sarcoma, and the other mimics an ependymoma, but all three cases are confirmed with FISH. These observations suggest that primary ES in the CNS may have histology different from the classic morphology and a high index of suspicion should be maintained in order to make the correct diagnosis. A search of the literature suggests that these tumors are most frequently seen in children and young adults. Imaging often shows a supratentorial enhancing mass that touches the leptomeninges. Survival over three years is good but long term prognosis is unknown [3, 4].

Mitochondrial energy failure in HSD10 disease is due to defective mtDNA transcript processing.

Muscle, heart and liver were analyzed in a male subject who succumbed to HSD10 disease. Respiratory chain enzyme analysis and BN-PAGE showed reduced activities and assembly of complexes I, III, IV, and V. The mRNAs of all RNase P subunits were preserved in heart and overexpressed in muscle, but MRPP2 protein was severely decreased. RNase P upregulation correlated with increased expression of mitochondrial biogenesis factors and preserved mitochondrial enzymes in muscle, but not in heart where this compensatory mechanism was incomplete. We demonstrate elevated amounts of unprocessed pre-tRNAs and mRNA transcripts encoding mitochondrial subunits indicating deficient RNase P activity. This study provides evidence of abnormal mitochondrial RNA processing causing mitochondrial energy failure in HSD10 disease.

Ciliary inclusion disease: report of a new primary ciliary dyskinesia variant.

Biopsies from 6 children with clinical presentations suggestive of primary ciliary dyskinesia (PCD) displayed respiratory epithelial cells with disorganized accumulations of basal bodies within the cytoplasm and large intracytoplasmic vesicles into which projected numerous microvilli and cilia. Microvilli, but few cilia, were present at the cell surface. Ultrastructural study revealed a variety of nonspecific abnormalities but demonstrated the cilia generally to be morphologically normal, suggesting that the cause of cilia malfunction was not any recognized primary cause or secondary effect. Repeat studies from 2 patients produced similar findings. It is proposed that this entity, termed ciliary inclusion disease, represents a variant form of PCD manifesting as a consequence of improper ciliogenesis caused by inhibited cytoskeleton-regulated migration of basal bodies to the luminal surface of the airway respiratory epithelial cells.

Features of intraventricular tanycytic ependymoma: report of a case and review of literature.

Tanycytic ependymoma is the rarest variant of ependymoma and occurs primarily in the spinal cord. Intracranial cases are even rarer. Only 9 ventricular and 5 subcortical tanycytic ependymoma have been reported in the literature. Amongst the 9 ventricular cases, only one tumor arose from the third ventricle. We report here another case of tanycytic ependymoma arising from the third ventricle completed with immunohistochemical, ultrastructural, and molecular pathology study. The patient was a 44 year-old male who presented with headache, nausea and visual disturbances of a few months duration. Neuroradiological findings showed a well-defined mass arising from the posterolateral wall of third ventricle. Histologically the tumor was composed of monotonous spindle cells arranged in fascicles without definitive perivascular rosettes. The tumor cells were diffusely positive for glial fibrillary acidic protein and epithelial membrane antigen, showed faint immunoreactivity for synaptophysin but were negative for neurofilament proteins and Ki-67 was less than 1%. Molecular studies showed absence of isocitrate dehydrogenase gene 1 and 2 mutation. A diagnosis of tanycytic ependymoma (TE) was made. From literature review with our current case included, intraventricular tanycytic ependymomas ranged from 1.8 to 4.0 cm. The age of patients ranged from 3.5 to 75 years with a mean age of 37.5 and a male predominance. The tumors occurred as well-defined, solitary ventricular mass without significant peritumoral edema with or without cystic changes. Histopathology and immunohistochemical profile are rather similar among different tumors. The immediate to short term outcome is excellent but long term follow up data is lacking.

Primary Ciliary Dyskinesia in Children: A Review for Pediatricians, Allergists, and Pediatric Pulmonologists.

Primary ciliary dyskinesia (PCD) is a rare genetic condition that causes impaired mucociliary clearance due to poorly functioning cilia. PCD is one disease manifestation of the many recently recognized associations with ciliary malfunction, referred to as "ciliopathies." Manifestations of PCD commonly begin in the neonatal period with cough, pneumonia, and chronic ear infections or effusions. Approximately half of the affected individuals have situs inversus totalis. The diagnosis is often made in later childhood or early adulthood, because symptoms mimic more common childhood illnesses and because the definitive diagnosis of PCD can be challenging. Treatment recommendations are largely based on therapies used for other conditions with impaired mucociliary clearance in the absence of evidence-based research specific for PCD. Early recognition and initiation of both otolaryngologic and pulmonary management might reduce potential long-term morbidities. The purpose of this article is to update primary care providers, allergists, and pediatric pulmonologists on recent advances in this interesting condition.