The genetic framework of primary ciliary dyskinesia assessed by soft computing analysis.

BACKGROUND: International guidelines disagree on how best to diagnose primary ciliary dyskinesia (PCD), not least because many tests rely on pattern recognition. We hypothesized that quantitative distribution of ciliary ultrastructural and motion abnormalities would detect most frequent PCD-causing groups of genes by soft computing analysis. METHODS: Archived data on transmission electron microscopy and high-speed video analysis from 212 PCD patients were re-examined to quantitate distribution of ultrastructural (10 parameters) and functional ciliary features (4 beat pattern and 2 frequency parameters). The correlation between ultrastructural and motion features was evaluated by blinded clustering analysis of the first two principal components, obtained from ultrastructural variables for each patient. Soft computing was applied to ultrastructure to predict ciliary beat frequency (CBF) and motion patterns by a regression model. Another model classified the patients into the five most frequent PCD-causing gene groups, from their ultrastructure, CBF and beat patterns. RESULTS: The patients were subdivided into six clusters with similar values to homologous ultrastructural phenotype, motion patterns, and CBF, except for clusters 1 and 4, attributable to normal ultrastructure. The regression model confirmed the ability to predict functional ciliary features from ultrastructural parameters. The genetic classification model identified most of the different groups of genes, starting from all quantitative parameters. CONCLUSIONS: Applying soft computing methodologies to PCD diagnostic tests optimizes their value by moving from pattern recognition to quantification. The approach may also be useful to evaluate atypical PCD, and novel genetic abnormalities of unclear disease-producing potential in the future.

Longitudinal Nitric Oxide Levels and Infections by Ultrastructure and Genotype in Primary Ciliary Dyskinesia.

BACKGROUND: We hypothesized that differences in nasal nitric oxide (nNO) and fractional exhaled nitric oxide (Feno) relate to prognosis in primary ciliary dyskinesia (PCD). RESEARCH QUESTION: What is the relationship between baseline values and longitudinal evolution of nNO and Feno and ultrastructure, genotype, and respiratory infections in PCD? STUDY DESIGN AND METHODS: Prospective, longitudinal, single-center study in adults and children evaluated biannually for up to 10 years. We compared cross-sectional and longitudinal values of nNO and Feno in ultrastructural (inner dynein arm [IDA] and microtubular disorganization [MTD]) and genetic (CCDC39 and CCDC40) groups known to have worse pulmonary function with patients within the ultrastructural and genetic groups with a better prognosis. Linear mixed-effects models were used to evaluate longitudinal associations. RESULTS: One hundred forty-one patients with PCD underwent 1,014 visits. At enrollment, no differences were found in children in nNO or Feno between the IDA and MTD group and the other ultrastructural groups. In adults, nNO (P = .038) and Feno (P = .032) were significantly lower in the IDA and MTD group than in all other combined ultrastructural groups. Feno values were significantly lower in the CCDC39 and CCDC40 group than in the DNAH5 and DNAH11 combined genotype group (P = .033) and in all other genotypes (P = .032). The IDA and MTD group showed a significant decline in nNO with age (P < .01) compared with other ultrastructural groups who showed stable levels. The CCDC39 and CCDC40 group showed the steepest decline in nNO over time (P < .01) compared with all other genotypes. A higher nNO was associated with lower likelihood of any positive bacterial isolate from the lower respiratory tract (P = .008). Changes in Feno over time did not differ between structural groups or genotypes. INTERPRETATION: Lower nNO in patients with PCD with genetic and ultrastructural changes associated with greater lung function decline may be related to worse prognosis, but whether a low nNO is causal needs further study. If lower nNO directly results in a poorer prognosis, strategies augmenting upper airway nitric oxide production may be worth evaluating.

Longitudinal Lung Volume Changes by Ultrastructure and Genotype in Primary Ciliary Dyskinesia.

Rationale: Genotype-phenotype relationships are emerging in primary ciliary dyskinesia (PCD), but little is known about lung volume changes over time. Objectives: To investigate the evolution of static lung volumes with ultrastructural defects, gene mutations, body mass index, and specific infections in PCD. Methods: Prospective, longitudinal, single-center study in children and adults evaluated twice yearly for up to 10 years. Linear mixed-effects models were used to assess associations between ciliary morphology, genetic mutations, and clinical features. Results: A total of 122 patients had 1,096 visits. At enrollment, almost all spirometric and, especially in adults, plethysmographic parameters were significantly worse in absent inner dynein arms (IDAs), central apparatus (CA) defects, and microtubular disorganization (MTD) (IDA/CA/MTD) compared with patients with normal electron microscopy (EM) results. The mean trend increase with time for residual volume (RV) was significantly higher in IDA/CA/MTD group compared with groups with outer dynein arm defect and normal EM results. The mean trend of RV/total lung capacity in the IDA/CA/MTD group was significantly worse than in all other groups. The steepest rise in lung volumes was in CCDC39 and CCDC40, whereas hyperinflation increased less in DNAH5 and DNAH11 groups. RV/total lung capacity showed a significantly steeper rise in patients with Pseudomonas aeruginosa compared with patients with other infections or patients without infection. Conclusions: Patients with IDA/CA/MTD defects or CCDC39 and CCDC40 mutations had the greatest increase in hyperinflation, whereas those with outer dynein arm defect and normal EM results or DNAH11 and DNAH5 mutations had less severe changes. We have robustly confirmed the worse prognosis for some genetic and ultrastructural defects, which association hitherto rested solely on spirometry.

Olfactory dysfunction is worse in primary ciliary dyskinesia compared with other causes of chronic sinusitis in children.

Cilia have multiple functions including olfaction. We hypothesised that olfactory function could be impaired in primary ciliary dyskinesia (PCD). Olfaction, nasal nitric oxide (nNO) and sinus CT were assessed in patients with PCD and non-PCD sinus disease, and healthy controls (no CT scan). PCD and non-PCD patients had similar severity of sinus disease. Despite this, defective olfaction was more common in patients with PCD (P<0.0001) and more severe in patients with PCD with major Transmission Electron Microscopy (TEM) abnormalities. Only in classical PCD did olfaction inversely correlate with sinusitis and nNO. We speculate that defective olfaction in PCD is primary in nature.

Polarized Ends of Human Macula Densa Cells: Ultrastructural Investigation and Morphofunctional Correlations.

The morphology of the kidney macula densa (MD) has extensively been investigated in animals, whereas human studies are scanty. We studied the fine structure of human MD cells focusing on their apical and basal ends and correlating structure and function. The MD region was examined by transmission electron microscopy in six renal biopsies from patients with kidney disease. Ultrastructural analysis of MD cells was performed on serial sections. MD cells show two polarized ends. The apical portion is characterized by a single, immotile cilium associated with microvilli; apically, cells are joined by adhering junctions. In the basal portion, the cytoplasm contains small, dense granules and numerous, irregular cytoplasmic projections extending to the adjacent extraglomerular mesangium. The projections often contain small, dense granules. A reticulated basement membrane around MD cells separates them from the extraglomerular mesangium. Although the fact that tissue specimens came from patients with kidney disease mandates extreme caution, ultrastructural examination confirmed that MD cells have sensory features due to the presence of the primary cilium, that they are connected by apical adhering junctions forming a barrier that separates the tubular flow from the interstitium, and that they present numerous basal interdigitations surrounded by a reticulated basement membrane. Conceivably, the latter two features are related to the functional activity of the MD. The small, dense granules in the basal cytoplasm and in cytoplasmic projections are likely related to the paracrine function of MD cells. Anat Rec, 301:922-931, 2018. © 2017 Wiley Periodicals, Inc.

Up to date on primary ciliary dyskinesia in children.

Primary ciliary dyskinesia (PCD) is a congenital, clinically and ultrastructurally heterogeneous disease due to abnormal structure and/or function of cilia, with impaired mucociliary transport leading to several respiratory disorders. PCD can be diagnosed by the combination of thorough clinical examination with functional and ultrastructural analysis of the cilia. This paper shows progresses in PCD diagnosis obtained by ciliogenesis in culture evaluation of ciliated respiratory cells and by genetic analysis of mutations in candidate genes. Moreover, since to date no specific treatments are available to correct the ciliary dysfunction, the paper shows the proper therapeutical approach by the use of respiratory physiotherapy and regular exercise to favour airways clearance, by antibiotics administration to control acute airway infections. Macrolides administration as antiinflammatory option is suggested.

Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis.

Diagnosis of primary ciliary dyskinesia (PCD) sometimes requires repeated nasal brushing to exclude secondary ciliary alterations. Our aim was to evaluate whether the use of a new method of nasal epithelial cell culture can speed PCD diagnosis in doubtful cases and to identify which are the most informative parameters by means of a multilayer artificial neural network (ANN). A cross-sectional study was performed in patients with suspected PCD. All patients underwent nasal brushing for ciliary motion analysis, ultrastructural assessment and evaluation of ciliary function after ciliogenesis in culture by ANN. 151 subjects were studied. A diagnostic suspension cell culture was obtained in 117 nasal brushings. A diagnosis of PCD was made in 36 subjects (29 of whom were children). In nine out of the 36 patients the diagnosis was made only after a second brushing, because of equivocal results of both tests at first examination. In each of these subjects diagnosis of PCD was confirmed by cell culture results. Cell culture in suspension evaluated by means of ANN allows the separation of PCD from secondary ciliary dyskinesia patients after only 5 days of culture and allows diagnosis to be reached in doubtful cases, thus avoiding the necessity of a second sample.

Giant mesenteric cyst of mesothelial origin in a haemodialysis patient with previous peritoneal dialysis therapy.

A 55-year-old female haemodialysis patient presented progressive abdominal liquid formation after having been excluded from peritoneal dialysis therapy because of recurrent peritonitis. Ultrasound was suspicious for ascites secondary to sclerosing peritonitis. Computed tomography revealed a thin-walled mesenteric cyst extending from the epigastric to the pelvic region. The cyst was excised incompletely as extensive adhesions were present. Histology was consistent with a mesothelial cyst of inflammatory origin. Three months after surgery, ultrasound detected a local recurrence at the descending colon. This case emphasizes the relation between mesenteric cyst, persistent inflammatory status and preceding peritoneal dialysis complicated by peritonitis.

Ribosome-lamella complexes in injured podocytes: description of a case and review of the literature.

Ribosome-lamella complexes (RLCs) are mainly observed in a variety of hematological disorders and occasionally in solid neoplasms and in nonneoplastic diseases. These intracytoplasmic organelles are held to arise from rough endoplasmic reticulum, but, in agreement with more recent literature data, their function is still unclear. Ultrastructural analysis of glomeruli from a patient with focal segmental glomerulosclerosis secondary to metabolic syndrome disclosed significant foot process loss and abundant cytoskeletal proteins in major podocyte processes; two of the latter also displayed RLCs. This is the second report of RLCs in human renal glomerulus. Their close association with cytoskeletal proteins and lysosomes suggests a relationship with abnormal protein biosynthesis.

Intermitochondrial cement (nuage) in a spermatocytic seminoma: comparison with classical seminoma and normal testis.

The nuage, an ultrastructural marker of normal human germ cells (spermatogonia type A and primary spermatocytes), may be found associated with mitochondria (intermitochondrial cement) and/or free in the cytoplasm. Eight specimens from germ cell-related tumours were reviewed to assess whether the nuage could have diagnostic significance in testicular neoplasms. The nuage of neoplastic cells from seven classical seminomas and one spermatocytic seminoma was compared with that from two normal testes. The ultrastructural study demonstrated that only spermatocytic seminoma cells contained both types of nuage and that significantly fewer spermatocytic seminoma cells (28%) contained intermitochondrial cement compared with control spermatogonia type A (81.1%) and primary spermatocytes (47.6%). The data indicate that (1) the detection of the nuage confirms that the phenotype of spermatocytic seminoma is more differentiated than that of classical seminoma; (2) the intermitochondrial cement is an additional example of how a distinctive organelle of a normal cell is preserved in its neoplastic counterpart and (3) if the intermitochondrial cement were found in other cases of spermatocytic seminoma, this organelle of the normal germ cell lineage could be considered as a new ultrastructural marker of the neoplasm.

Torquetenovirus infection and ciliary dysmotility in children with recurrent pneumonia.

BACKGROUND: The aim of the study was to assess Torquetenovirus (TTV) loads within respiratory ciliated cells and to verify the existence of a correlation between TTV loads and functional or structural ciliary abnormalities, in a group of children with recurrent or persistent pneumonia. METHODS: Nasal brushing samples of 55 children (28 male) were evaluated for ciliary motion and ultrastructural assessment, as well as for detection and quantification of TTV. Moreover, presence and load of TTV within ciliated cells, obtained from 5 patients by laser capture microdissection, were determined. RESULTS: The nasal samples of 47 (85%) children with persistent or recurrent pneumonia resulted positive for TTV (loads = 2.1-7.3 log10 copies/microg total DNA). TTV were demonstrated also within microdissected ciliated cells. No significant difference between primary (11 subjects) and secondary ciliary dyskinesia (44 subjects) for TTV prevalence and mean loads were found. A significant correlation was observed between nasal TTV loads and ciliary beat frequency score (r = 0.305; P < 0.05), but not between TTV loads and presence of abnormal motion patterns, in patients with secondary ciliary abnormalities. As expected no correlations were found between nasal TTV loads and ciliary motion analysis in primary ciliary dyskinesia. CONCLUSIONS: The presence of TTV in nasal samples demonstrates TTV ability to infect respiratory ciliated cells and suggests that these cells are potentially able to support virus replication. Moreover, TTV may behave in respiratory cells in a similar way to other viruses, that is, they disrupt the mucociliary escalator.

Brush cells in the human duodenojejunal junction: an ultrastructural study.

Brush cells have been identified in the respiratory and gastrointestinal tract mucosa of many mammalian species. In humans they are found in the respiratory tract and the gastrointestinal apparatus, in both the stomach and the gallbladder. The function of brush cells is unknown, and most morphological data have been obtained in rodents. To extend our knowledge of human brush cells, we performed an ultrastructural investigation of human small intestine brush cells. Six brush cells identified in five out of more than 300 small intestine biopsies performed for gastrointestinal tract disorders were examined by transmission electron microscopy. Five brush cells were located on the surface epithelium and one in a crypt. The five surface brush cells were characterized by a narrow apical pole from which emerged microvilli that were longer and thicker than those of enterocytes. The filamentous core extended far into the cell body without forming the terminal web. Caveolae were abundant. Filaments were in the form of microfilaments and intermediate filaments. Cytoplasmic projections containing filaments were found on the basolateral surface of brush cells. In a single cell, axons containing vesicles and dense core granules were in close contact both with the basal and the lateral surface of the cell. The crypt brush cell appeared less mature. We concluded that human small intestine brush cells share a similar ultrastructural biology with those of other mammals. They are polarized and well-differentiated cells endowed with a distinctive cytoskeleton. The observation of nerve fibres closely associated with brush cells, never previously described in humans, lends support to the hypothesis of a receptor role for these cells.

Nasal nitric oxide in atypical primary ciliary dyskinesia.

BACKGROUND: Atypical cases of primary ciliary dyskinesia (PCD) may present with minimal transmission electron microscopy (TEM) defects. The diagnostic role of nasal nitric oxide (nNO) levels was evaluated in those patients. METHODS: Sixty-four children with recurrent pneumonia were studied with ciliary motion analysis, TEM, and nNO. RESULTS: Investigations indicated PCD in 12 patients, secondary ciliary dyskinesia (SCD) in 50 patients, and normal results in 2 patients. In 4 of 50 children with SCD, atypical PCD was considered possible. The mean (+/- SD) nNO was 130 +/- 46.95 parts per billion in children affected by PCD, 127.79 +/- 68.58 parts per billion in atypical patients, and 760 +/- 221 parts per billion in children with SCD. Three to 5 months later, the nNO level was 132.75 +/- 55.76 parts per billion in children with atypical disease and 778.00 +/- 197 parts per billion in children with SCD. CONCLUSION: Low levels of nNO may help to identify patients with atypical PCD.

Unusual ultrastructural features in microvillous inclusion disease: A report of two cases.

Microvillous Inclusion Disease (MID) is an inherited disorder characterized by intractable diarrhea in infancy. Ultrastructural detection of pathognomonic microvillous inclusions in the enterocytes is essential for diagnosis. The aim of this research is to contribute to the knowledge of MID studying enterocytes and goblet cells (gc). Samples of duodenal mucosa from two young infants with MID (aged 75 days and 3 months, respectively) were studied by light and electron microscopy. Detection in the intestinal villi of immature gc (with microvilli) in one of the cases led us to seek them in control samples. The total number of gc with microvilli (immature) and without microvilli (mature) were counted. In both MID specimens, light microscopy showed atrophy of villi and PAS-positive material in the enterocyte cytoplasm. The ultrastructure of villous enterocytes was characterized by brush-border abnormalities, microvillous inclusions, dense apical granules, and lysosomes. Intermediate structures between microvillous inclusions and lysosomes were also detected within a cell, as were rare microvilli on the lateral membrane of the enterocytes. In one MID specimen, immature gc were also identified in the absorptive compartment. Only mature gc were observed in the controls. The significance of the latter finding requires further studies.