Exacerbations and Pseudomonas aeruginosa colonization are associated with altered lung structure and function in primary ciliary dyskinesia.

BACKGROUND: Recurrent bacterial infections of the respiratory tract are one of the major clinical features of the primary ciliary dyskinesia (PCD), a rare genetic disease due to malfunctioning of motile cilia. Chronic infections and persistent inflammation of the respiratory system result in progressive lung disease. Aim of the study was to highlight the main factors associated with clinical, functional and anatomical deterioration in PCD patients. METHODS: We retrospectively analyzed data from 58 patients with PCD, 37 adults and 21 children. The demographic and clinical data, forced expiratory volume at 1 s (FEV1) and forced vital capacity (FVC), sputum microbiology and imaging results (chest CT scores-modified Bhalla) were recorded. Patients were stratified according to the number of exacerbations (< 2/year vs ≥ 2/year) and chronic Pseudomonas aeruginosa (PA) colonization. The possible correlations between lung function and chest CT scores were assessed; we also evaluated the correlation between these parameters and the severity scores for bronchiectasis (BSI, FACED and e-FACED). RESULTS: Chest CT scores showed a significant correlation with FEV1 (p = 0.0002), age (p <  0.0001), BMI (p = 0.0002) and number of lung lobes involved (p <  0.0001). PA colonization had an overall prevalence of 32.6%: no significant difference in FEV1 between PA colonized and non-colonized patients was found (p = 0.70), while chest CT score was significantly worse in chronic PA colonized patients (p = 0.009). Patients with a high number of exacerbation (≥ 2/year) were older (p = 0.01), had lower FEV1 (p = 0.03), greater number of lobes involved (p < 0.001) and worse CT score than patients with low number of exacerbations (p = 0.001); they also had higher prevalence of PA chronic bronchial infection (33.3% versus 13.6%, p = 0.10). Multivariable linear regression analyses adjusted for gender, age and BMI showed positive associations between PA colonization and number of exacerbations with severity of disease (number of lobes involved, CT score, BSI, FACED, and e-FACED). CONCLUSIONS: In our PCD population the number of exacerbations (≥ 2/year) and PA colonization were the two most relevant factors associated with severity of disease.

Emerging ciliopathies: are respiratory cilia compromised in Usher syndrome?

PURPOSE: Usher syndrome is a ciliopathy involving photoreceptors and cochlear hair cells (sensory cilia): since sensory and motor ciliopathies can overlap, we analysed the respiratory cilia (motile) in 17 patients affected by Usher syndrome and 18 healthy control subject. PATIENTS AND METHODS: We studied the mucociliary transport time with the saccharine test, ciliary motility and ultrastructure of respiratory cilia obtained by nasal brushing; we also recorded the classical respiratory function values by spirometry. RESULTS: All enrolled subjects showed normal respiratory function values. The mean mucociliary transport time with saccharine was 22.33 ± 17.96 min, which is in the range of normal values. The mean ciliary beat frequency of all subjects was 8.81 ± 2.18 Hz, which is a value approaching the lower physiological limit. None of the classical ciliary alterations characterizing the "ciliary primary dyskinesia" was detected, although two patients showed alterations in number and arrangement of peripheral microtubules and one patient had abnormal ciliary roots. CONCLUSIONS: Respiratory cilia in Usher patients don't seem to have evident ultrastructural alterations, as expected, but the fact that the ciliary motility appeared slightly reduced could emphasize that a rigid distinction between sensory and motor ciliopathies may not reflect what really occurs.

Cetuximab ± chemotherapy enhances dendritic cell-mediated phagocytosis of colon cancer cells and ignites a highly efficient colon cancer antigen-specific cytotoxic T-cell response in vitro.

Cetuximab is a human/mouse chimeric IgG1 monoclonal antibody (mAb) to epidermal growth factor receptor, approved for colorectal carcinoma treatment in combination with chemotherapy. The immune-mediated effects elicited by its human fraction of crystallization moiety might critically contribute to the overall anti-tumor effectiveness of the antibody. We therefore investigated cetuximab ability to promote colon cancer cell opsonization and phagocytosis by human dendritic cells (DCs) that are subsequently engaged in antigen-cross presentation to cytotoxic T-lymphocyte (CTL) precursors. Human colon cancer cell lines were evaluated for susceptibility to DC-mediated phagocytosis before and after treatment with chemotherapy ± cetuximab in vitro. Human DCs loaded with control or drug-treated cetuximab-coated colon cancer cells were used to in vitro generate cytotoxic T cell clones from peripheral blood mononuclear cells of human leucocyte antigen-A(*)02.01(+) donors. T-cell cultures were characterized for immune-phenotype and tumor-antigen specific CTL activity. The results confirmed that treatment of tumor cells with irinotecan + L-folinate + 5-flurouracil (ILF) or with gemcitabine + ILF increased tumor antigen expression. Moreover, malignant cells exposed to chemotherapy and cetuximab were highly susceptible to phagocytosis by human DCs and were able to promote their activation. The consequent DC-mediated cross-priming of antigens derived from mAb-covered/drug-treated cancer cells elicited a robust CTL anti-tumor response. On the basis of our data, we suggest a possible involvement of CTL-dependent immunity in cetuximab anti-cancer effects.

Mutations in DNAH5 account for only 15% of a non-preselected cohort of patients with primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is characterised by recurrent infections of the upper respiratory airways (nose, bronchi, and frontal sinuses) and randomisation of left-right body asymmetry. To date, PCD is mainly described with autosomal recessive inheritance and mutations have been found in five genes: the dynein arm protein subunits DNAI1, DNAH5 and DNAH11, the kinase TXNDC3, and the X-linked retinitis pigmentosa GTPase regulator RPGR. METHODS: We screened 89 unrelated individuals with PCD for mutations in the coding and splice site regions of the gene DNAH5 by denaturing high performance liquid chromatography (DHPLC) and sequencing. Patients were mainly of European origin and were recruited without any phenotypic preselection. RESULTS: We identified 18 novel (nonsense, splicing, small deletion and missense) and six previously described mutations. Interestingly, these DNAH5 mutations were mainly associated with outer + inner dyneins arm ultrastructural defects (50%). CONCLUSION: Overall, mutations on both alleles of DNAH5 were identified in 15% of our clinically heterogeneous cohort of patients. Although genetic alterations remain to be identified in most patients, DNAH5 is to date the main PCD gene.

Increased lung surfactant phosphatidylcholine in patients affected by lysosomal storage diseases.

Sandhoff disease, Gaucher disease type I and sialidosis type I are lysosomal storage disorders caused, respectively, by deficiency of activity of beta-hexosaminidase (storage of GM(2) and GA(2) ganglioside), glucosylceramidase (storage of glucosylceramide) and alpha-neuraminidase (storage of glucopeptides and/or oligosaccharides). Progressive clinical systemic and neurological dysfunctions are observed. In these pathologies, respiratory infections often lead to death. Elevation of the lung surfactant phosphatidylcholine (PC) has previously been reported in the Hexb mouse, a model of Sandhoff disease. We evaluated phospholipids in the lung surfactant of patients affected by the described lysosomal diseases, observing a statistically significant increase of total lipid phosphate in the patients as compared with controls. Moreover, higher levels of PC in patients affected by sialidosis (3.6-fold) and Gaucher (4-fold) disease, and of PC (4.15-fold) and phosphatidylethanolamine (2.3-fold) in a patient affected by Sandhoff disease were noted. The latter confirms the previous results in the Hexb mouse. We suggest that changes in phospholipid metabolism can be common in different lysosomal storage disorders and can increase the susceptibility to respiratory infections, usually present in these disorders.

Apoptotic index: discriminant feature for the differentiation of cutaneous diffuse malignant follicular center cell lymphomas from lymphoid hyperplasia.

Diffuse subtypes of cutaneous lymphoid hyperplasia (CLH; n = 18) and primary malignant follicular center cell lymphoma of the skin (FCCL, n = 11) were diagnosed by conventional histology, immunophenotyping on paraffin sections, and gene rearrangement analysis. We then counted on semithin, Azur A-stained sections of resin-re-embedded biopsy specimens the relative numbers of apoptotic bodies among all lymphoid cells (apoptotic index [AI]). The diagnostic value of AI was compared to that of mitotic indices (MI) and percentages of various cell types in the cutaneous infiltrate. Features of cellular infiltrates distinguishing to two groups of lesions, in the order of decreasing significance, were percent large lymphoid cells, percent medium-sized lymphoid cells (both higher in FCCL); percent small lymphoid cells, percent epithelioid/giant cells, and percent histiocytes/macrophages (all three higher in CLH). However, of all parameters tested, AI had the greatest discriminant value (median in FCCL 1.11%, in CLH 0.14%; p = 8 x 10(-6)). Two cases, diagnosed as CLH with all morphologic and immunologic methods used, showed B-cell monoclonality at the DNA level. Linear discriminant analysis determined the following order of distinctive power of variables: 1) AI; 2) MI; 3) percent small lymphoid cells; 4) percent medium-sized lymphoid cells; 5) percent large lymphoid cells; 6) percent epithelioid/giant cells; and 7) percent histiocytes/macrophages. The present study thus establishes AI as an important parameter in the differentiation of diffuse CLH from diffuse cutaneous FCCL.

Ultrastructural features in active chronic hepatitis with changes resembling Wilson's disease.

A case of chronic hepatitis with ultrastructural changes resembling alterations usually occurring in Wilson's disease is presented in an elderly man. At the time of the diagnosis, the patient did not show clinical and laboratory data consistent with the diagnosis of Wilson's disease. Subsequently, the patient developed neurologic symptoms similar to that resulting from hepatolenticular degeneration. The possibility that such lesions are caused by an abnormal copper metabolism in consequence of acquired liver disease is considered.

Intimal-type primary sarcoma of the aorta. Report of a case with evidence of rhabdomyosarcomatous differentiation.

We report an intimal sarcoma presenting as an aortic aneurysm. A 68-year-old man suffered from chest pain and speech disturbance. Computed tomography showed a sacciform aneurysm of the aorta, which was resected, revealing a polypoid tumour measuring 1.5x2x2.5 cm projecting into the lumen. This proved to be a poorly differentiated high-grade sarcoma having morphological, immunophenotypic and ultrastructural features consistent with rhabdomyosarcomatous differentiation. Primary sarcomas of the aorta are extremely rare. Many cases have been diagnosed as "intimal" on the basis of their site of origin, and they are not easy to classify from their histological pattern. Electron microscopy and the use of a more comprehensive panel of immunohistochemical markers should be applied in the histological classification of"intimal" sarcoma.

Toxic injury to rat gut musculature following intraperitoneal administration of 2-t-butyl-4-methoxyphenol.

The 100-fold increase in toxicity of intraperitoneal (i.p.) rather than orally administered 2-t-butyl-4-methoxyphenol (BHA) is adduced to the depressive effect which this compound exerts on the contractility of the gut musculature. A structure/activity relation study shows the t-butyl group on the benzene ring as being the major determinant of i.p. BHA toxicity. Contractile activity, elicited by field electrical stimulation, acetylcholine or Ba2+, of the ileum longitudinal muscle preparation from BHA-treated rats was greatly reduced 30 min after i.p. injection, and almost absent during the subsequent 48 h. Electron-microscope examination of ileum longitudinal muscle also showed partial destruction of cell membranes 4 h after BHA administration with subsequent mitochondrial swelling and destruction of cristae, myofibrillar fragmentation and cell necrosis. Comparable suppression of contractile activity and morphological damage were observed in BHA or t-butylbenzene incubated ileum segments where longitudinal smooth muscle contractility was irreversibly depressed in a time- and dose-dependent manner. These convergent findings point to the toxic effect of i.p. BHA on gut musculature with consequent impairment of intestinal transit.

Severe teratospermia in an infertile man with bronchiectasis.

Morphologic examination of sperm samples from an infertile man with bronchiectasis revealed a severe teratospermia characterized by rare abnormalities that were present in most of his spermatozoa. These abnormalities were represented by round-headed spermatozoa, changes in chromatin condensation, acrosomal alterations, multiple tails, and complete subcellular derangement. The similarity between the above-reported abnormalities and those observed in various species of laboratory animals strongly suggests that the observed defects might be the result of a defective function of the manchette during spermiogenesis. The absence of specific defects of the axonemal morphologic features in sperm tails and in respiratory cilia, together with some laboratory data, suggests that association between infertility and bronchiectasis cannot be related, in our patient, to a generalized syndrome.

The saccharin method for testing mucociliary function in patients suspected of having primary ciliary dyskinesia.

In order to evaluate the clinical value of the saccharin test as a practical and simple measure of mucociliary clearance, nasal mucociliary clearance (NMCC) and ciliary ultrastructure were studied in 22 patients suspected of having primary ciliary dyskinesia (PCD) based on the saccharin test. Ten patients fulfilling the diagnostic criteria of PCD had a pathological response to the saccharin test (transport time greater than 60 minutes), and this was consistently associated with ultrastructural defects, specific for PCD. These results validate the suitability of the clinical use of the saccharin test as a screening procedure for NMCC. The false-negative results obtained in three cases of PCD, all with borderline values, cannot be ascribed to ineffectiveness of the test, but rather to the persistence of some motility by certain defective cilia, detectable by microphoto-oscillographic investigation of specimens obtained by nasal biopsy or brushing.

Ultrastructural ciliary defects in children with recurrent infections of the lower respiratory tract.

One hundred fifty-four children with recurrent or chronic infections of the lower respiratory tract compatible with the diagnosis of primary ciliary dyskinesia (PCD) were evaluated for the presence of ultrastructural ciliary abnormalities. Studies were performed on multiple samples of respiratory mucosa obtained by nasal and bronchial brushing. Twenty-eight children showed ultrastructural ciliary defects compatible with the diagnosis of PCD: Twenty-four presented dynein arm deficiency (either as isolated defect or in association with microtubular abnormalities), two had ciliary aplasia, and two showed microtubular abnormalities. Eleven patients with PCD had situs viscerum inversus, bronchiectasis, and chronic sinusitis (Kartagener's syndrome); one child with Kartagener's syndrome had normal ciliary structure. The appearance of respiratory symptoms within the first month of life, the colonization by Haemophilus influenzae, and a history of recurrent rhinitis and otitis were characteristically present in children with PCD. The clinical status of those patients who reached adolescence was, in our experience, remarkably good. An early diagnosis with adequate prevention and therapy of respiratory infections may have an important role in minimizing irreversible lung damage.

Nasal ciliary function and ultrastructure in Down syndrome.

OBJECTIVES/HYPOTHESIS: To investigate the in vivo nasal ciliary beat and the ciliary ultrastructure in Down syndrome because, although in this condition an increased susceptibility to respiratory tract infections has been reported by several authors, the nature of this phenomenon is not fully understood. STUDY DESIGN: Experimental study of 18 subjects with Down syndrome and 18 healthy control subjects. METHODS: Ciliary beat frequency (CBF) was measured on samples of ciliated epithelium obtained from the inferior nasal turbinate; a further brushing for ultrastructural analysis was performed only in subjects showing a CBF reduction or a ciliary movement alteration. RESULTS: The mean CBF in the group with Down syndrome was 7 +/- 2.82 Hz, and in the control subjects it was 10.94 +/- 0.65 Hz. In the same 66.6% of subjects with Down syndrome, we observed a fibrillatory movement of cilia and no metachronicity was present. Moreover, in 14 subjects with Down syndrome as hyperproduction of mucus was present. Ultrastructural evaluation at transmission election microscopy instead revealed a normal architecture of cilia. CONCLUSION: We attribute the nature of the mucociliary defect in Down syndrome to recurrent respiratory tract infections causing changes in mucus properties as in rheological parameters and not to a primitive defect of cilia.

Telomerase activity, Ki-67, cyclin D1 and A expression, and apoptosis in solitary fibrous tumors: additional features of a predictable course?

Solitary fibrous tumors (SFTs) are infrequent soft tissue neoplasms which are usually benign and surgically curable. However, their behavior is not always predictable, although several clinical and pathological criteria of malignancy have been established. In many cancers, including some soft tissue tumors, telomerase activity (TA) has been shown to be a new reliable pathological marker of malignancy. Overexpression of some cyclins is associated with higher degrees of malignancy and predictive of the clinical course. In this study, we evaluated TA, mitotic and apoptotic indices (MI, AI), and the expression of Ki-67, cyclins D1 and A in five typical and two clinicopathologically atypical SFTs, the latter two of which had also recurred. High TA was demonstrated in the two atypical cases, which also showed a higher labeling index to Ki-67, as well as higher cyclin D1 and A expression, and either none or very few apoptoses. We suggest that TA, Ki-67, cyclin expression, and AI be evaluated in SFTs as possible adjunctive pathological criteria of behavior.

Quantitatively evaluated ultrastructural findings can add to the differential diagnosis between keratoacanthoma and well differentiated squamous cell carcinoma.

The differential diagnosis between keratoacanthoma (KA) and well differentiated squamous cell carcinoma (WDSCC) is not always easy to perform. Seven cases of KA and seven cases of WDSCC have been here analyzed by morphometry on ultrastructural sections and compared with normal epidermis. Parameters expressing the cohesivity among epithelial cells (numerical and surface density of desmosomes; volume density of intercellular space) were significantly different in KA and WDSCC, so that they may be useful in differential diagnosis. The Authors also questioned the nature of KA, suggesting a continuum between this lesion and WDSCC.

Histological and ultra-structural effects of rapid muscle expansion through intramuscular administration of carbon dioxide: an intra-operative study in an animal model.

INTRODUCTION: Carbon dioxide (CO2) therapy refers to trans-cutaneous or sub-cutaneous administration of CO2 for therapeutic purposes, and recent studies have pointed out that it produces a vasodilation effect after it is locally injected, which helps amplify the reconstructive potentiality of an expanded-muscle flap. MATERIALS AND METHODS: Thirty male Wistar rats, weighting between 350 and 400 g, were randomly divided into three groups of 10. In the first group, single intra-operative rapid expansion was carried out under the right latissimus dorsi muscle. In the second group, for five days prior to surgery, a pre-treatment with intramuscular injections of CO2 was performed. The third group served as controls. For each group, the latissimus dorsi muscle was fixed as soon possible after mice died, and ultrathin sections of it examined with transmission electron microscope. RESULTS: In the treated group, the majority of expanded muscles showed a normal striation pattern, whereas a few fibers showed mild disorganization of the myo-filaments in the sarcomeres, which appeared overstretched (average 2.37 µm). CONCLUSIONS: This evidence could demonstrate a greater capacity of muscle recovery after treatment by CO2 expansion.