Primary ciliary dyskinesia: evaluation using cilia beat frequency assessment via spectral analysis of digital microscopy images

Olm MA, Kogler JE Jr, Macchione M, Shoemark A, Saldiva PH, Rodrigues JC. Primary ciliary dyskinesia: evaluation using cilia beat frequency assessment via spectral analysis of digital microscopy images. J Appl Physiol 111: 295-302, 2011. First published May 5, 2011; doi:10.1152/japplphysiol.00629.2010.-Ciliary beat frequency (CBF) measurements provide valuable information for diagnosing of primary ciliary dyskinesia (PCD). We developed a system for measuring CBF, used it in association with electron microscopy to diagnose PCD, and then analyzed characteristics of PCD patients. 1 The CBF measurement system was based on power spectra measured through digital imaging. Twenty-four patients suspected of having PCD (age 1-19 yr) were selected from a group of 75 children and adolescents with pneumopathies of unknown causes. Ten healthy, nonsmoking volunteers (age >= 17 yr) served as a control group. Nasal brush samples were collected, and CBF and electron microscopy were performed. PCD was diagnosed in 12 patients: 5 had radial spoke defects, 3 showed absent central microtubule pairs with transposition, 2 had outer dynein arm defects, 1 had a shortened outer dynein arm, and 1 had a normal ultrastructure. Previous studies have reported that the most common cilia defects are in the dynein arm. As expected, the mean CBF was higher in the control group (P < 0.001) and patients with normal ultrastructure (P < 0.002), than in those diagnosed with cilia ultrastructural defects (i.e., PCD patients). An obstructive ventilatory pattern was observed in 70% of the PCD patients who underwent pulmonary function tests. All PCD patients presented bronchial wall thickening on chest computed tomography scans. The protocol and diagnostic techniques employed allowed us to diagnose PCD in 16% of patients in this study.

Changes in Management Strategies After Spontaneous Migration of a Retained Intraorbital Metallic Foreign Body

Purpose: Because of the controversial biologic tolerance and management, retained intraorbital metallic foreign body (RIMFb) poses a formidable challenge to surgeons. Besides location of the foreign body, indications for surgical management include neurologic injury, mechanical restriction of the eye movement, and development of local infection or draining fistula. The authors describe an unusual case of spontaneous migration of a RIMFb. Methods: A 26-year-old man had a gunshot injury on the left orbit. The patient was initially managed conservatively because of the posterior position of the bullet fragment. Thereafter, because of the clinical impairments and anterior migration of projectile, surgical treatment was considered. Results: Spontaneous anterior migration has led to mechanical disturbances and inflammatory complications that comprise explicit surgical indications for removal. The patient underwent surgery with complete relief of symptoms. We suppose that extrinsic ocular muscles might play a role in shifting large RIMFb over time, leading to change in the management strategies. Conclusions: Spontaneous migration of RIMFb is a rare clinical situation that can lead to pain, local deformity, as well as changes in the management strategies of the affected patients even in the late phase of follow-up.

Reliability assessment of Cobb angle measurements using manual and digital methods

BACKGROUND CONTEXT: The vertebral spine angle in the frontal plane is an important parameter in the assessment of scoliosis and may be obtained from panoramic X-ray images. Technological advances have allowed for an increased use of digital X-ray images in clinical practice. PURPOSE: In this context, the objective of this study is to assess the reliability of computer-assisted Cobb angle measurements taken from digital X-ray images. STUDY DESIGN/SETTING: Clinical investigation quantifying scoliotic deformity with Cobb method to evaluate the intra- and interobserver variability using manual and digital techniques. PATIENT SAMPLE: Forty-nine patients diagnosed with idiopathic scoliosis were chosen based on convenience, without predilection for gender, age, type, location, or magnitude of the curvature. OUTCOME MEASURES: Images were examined to evaluate Cobb angle variability, end plate selection, as well as intra- and interobserver errors. METHODS: Specific software was developed to digitally reproduce the Cobb method and calculate semiautomatically the degree of scoliotic deformity. During the study, three observers estimated the Cobb angle using both the digital and the traditional manual methods. RESULTS: The results showed that Cobb angle measurements may be reproduced in the computer as reliably as with the traditional manual method, in similar conditions to those found in clinical practice. CONCLUSIONS: The computer-assisted method (digital method) is clinically advantageous and appropriate to assess the scoliotic curvature in the frontal plane using Cobb method. (C) 2010 Elsevier Inc. All rights reserved.

Validity and reliability of a computer method to estimate vertebral axial rotation from digital radiographs

Axial vertebral rotation, an important parameter in the assessment of scoliosis may be identified on X-ray images. In line with the advances in the field of digital radiography, hospitals have been increasingly using this technique. The objective of the present study was to evaluate the reliability of computer-processed rotation measurements obtained from digital radiographs. A software program was therefore developed, which is able to digitally reproduce the methods of Perdriolle and Raimondi and to calculate semi-automatically the rotation degree of vertebra on digital radiographs. Three independent observers estimated vertebral rotation employing both the digital and the traditional manual methods. Compared to the traditional method, the digital assessment showed a 43% smaller error and a stronger correlation. In conclusion, the digital method seems to be reliable and enhance the accuracy and precision of vertebral rotation measurements.

Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFN gamma production

IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN gamma production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFN gamma but was enhanced by prostaglandin E(2) (PGE(2)). IL-23-induced IL-17 production was increased by PGE(2) and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFN gamma-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFN gamma but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNF alpha, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFN gamma production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs.

Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2(-/-) mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naive WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein-coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2(-/-) mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.

Targeting endothelin ET(A) and ET(B) receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ET(A) and ET(B) receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ET(B) receptor antagonism, but rather by endothelin ET(A) receptor antagonism. Furthermore, endothelin ET(A), but not ET(B), receptor antagonism inhibited antigen-induced PGE(2) production, whereas either selective or combined blockade of endothelin ET(A) and/or ET(B) receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: Role of ATP-sensitive potassium channels

In this study, we have addressed the role of H2S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H S synthesis inhibitors, DL-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H2S donors, NaHS or Lawesson`s reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB4. Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K-ATP(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K-ATP(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H`S augments neutrophil adhesion and locomotion, by a mechanism dependent on K-ATP(+) channels.

Aldosterone and angiotensin II synergistically stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways

Objective - Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). Methods and Results - VSMCs from WKY rats were studied. At low concentrations (10(-10) mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone ( Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 ( inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. Conclusions - Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

Phlebotomine salivas inhibit immune inflammation-induced neutrophil migration via an autocrine DC-derived PGE(2)/IL-10 sequential pathway

In the present study, we investigated whether saliva from Phlebotomus papatasi and Phlebotomus duboscqi inhibited antigen-induced neutrophil migration and the mechanisms involved in these effects. The pretreatment of immunized mice with salivary gland extracts (SGE) of both phlebotomines inhibited OVA challenge-induced neutrophil migration and release of the neutrophil chemotactic mediators, MIP-1 alpha, TNF-alpha, and leukotriene B-4 (LTB4). Furthermore, SGE treatment enhanced the production of anti-inflammatory mediators, IL-10 and PGE(2). SGE treatments failed to inhibit neutrophil migration and MIP-1 alpha and LTB4 production in IL-10(-/-) mice, also failing in mice treated with nonselective (indomethacin) or selective (rofecoxibe) cyclooxygenase (COX) inhibitors. COX inhibition resulted in diminished SGE-induced IL-10 production, and PGE(2) release triggered by SGE remained increased in IL-10(-/-) mice, suggesting that prostanoids are acting through an IL-10-dependent mechanism. SGE treatments in vivo reduced the OVA-induced lymphoproliferation of spleen-derived cells. Further, the in vitro incubation of bone marrow-derived dendritic cells (DC) with SGE inhibited the proliferation of CD4(+) T cells from OVA-immunized mice, which was reversed by indomethacin and anti-IL-10 antibody treatments. Supporting these results, SGE induced the production of PGE(2) and IL-10 by DC, which were blocked by COX inhibition. These effects were associated with the reduction of DC-membrane expression of MHC-II and CD86 by SGE treatment. Altogether, the results showed that Phlebotomine saliva inhibits immune inflammation-induced neutrophil migration by an autocrine DC sequential production of PGE(2)/IL-10, suggesting that the saliva constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Neutrophil migration induced by IL-1 beta depends upon LTB4 released by macrophages and upon TNF-alpha and IL-1 beta released by mast cells

In the present study, we investigate whether mast cells and macrophages are involved in the control of IL-1 beta-induced neutrophil migration, as well as the participation of chemotactic mediators. IL-1 beta induced a dose-dependent neutrophil migration to the peritoneal cavity of rats which depends on LTB4, PAF and cytokines, since the animal treatment with inhibitors of these mediators (MK 886, PCA 4248 and dexamethasone respectively) inhibited IL-1 beta-induced neutrophil migration. The neutrophil migration induced by IL-1 beta is dependent on mast cells and macrophages, since depletion of mast cells reduced the process whereas the increase of macrophage population enhanced the migration. Moreover, mast cells or macrophages stimulated with IL-1 beta released a neutrophil chemotactic factor, which mimicked the neutrophil migration induced by IL-1 beta. The chemotactic activity of the supernatant of IL-1 beta-stimulated macrophages is due to the presence of LTB4, since MK 886 inhibited its release. Moreover, the chemotactic activity of IL-1 beta-stimulated mast cells supernatant is due to the presence of IL-1 beta and TNF-alpha, since antibodies against these cytokines inhibited its activity. Furthermore, significant amounts of these cytokines were detected in the supernatant. In conclusion, our results suggest that neutrophil migration induced by IL-1 beta depends upon LTB4 released by macrophages and upon IL-1 beta and TNF alpha released by mast cells.