Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy.

BACKGROUND: We reported that PARP-1 regulates genes whose products are crucial for asthma, in part, by controlling STAT6 integrity speculatively through a calpain-dependent mechanism. We wished to decipher the PARP-1/STAT6 relationship in the context of intracellular trafficking and promoter occupancy of the transcription factor on target genes, its integrity in the presence of calpains, and its connection to autophagy. METHODS: This study was conducted using primary splenocytes or fibroblasts derived from wild-type or PARP-1-/- mice and Jurkat T cells to mimic Th2 inflammation. RESULTS: We show that the role for PARP-1 in expression of IL-4-induced genes (e.g. gata-3) in splenocytes did not involve effects on STAT6 phosphorylation or its subcellular trafficking, rather, it influenced its occupancy of gata-3 proximal and distal promoters in the early stages of IL-4 stimulation. At later stages, PARP-1 was crucial for STAT6 integrity as its inhibition, pharmacologically or by gene knockout, compromised the fate of the transcription factor. Calpain-1 appeared to preferentially degrade JAK-phosphorylated-STAT6, which was blocked by calpastatin-mediated inhibition or by genetic knockout in mouse fibroblasts. The STAT6/PARP-1 relationship entailed physical interaction and modification by poly(ADP-ribosyl)ation independently of double-strand-DNA breaks. Poly(ADP-ribosyl)ation protected phosphorylated-STAT6 against calpain-1-mediated degradation. Additionally, our results show that STAT6 is a bonafide substrate for chaperone-mediated autophagy in a selective and calpain-dependent manner in the human Jurkat cell-line. The effects were partially blocked by IL-4 treatment and PARP-1 inhibition. CONCLUSIONS: The results demonstrate that poly(ADP-ribosyl)ation plays a critical role in protecting activated STAT6 during Th2 inflammation, which may be synthetically targeted for degradation by inhibiting PARP-1.

Comparison between the endoscopic scores of bolgerization versus partial resection of the middle turbinate for management of the postoperative lateralization in the early follow-up period after endoscopic frontal sinusotomy: A randomized controlled study.

BACKGROUND: Frontal sinusotomy is a challenging procedure that needs meticulous handling due to its unique anatomical position. Postoperative middle turbinate lateralization is critical comorbidity for the success rate, and many techniques are adopted to prevent it. The study aimed to compare the effect of middle turbinate bolgerization and partial resection on the postoperative endoscopic scores and assess their impact on the middle meatus and the frontal recess outcome. PATIENT AND METHODS: This prospective study was conducted on forty-one patients undergoing bilateral frontal sinusotomy for chronic frontal sinusitis. Nasal cavities were randomized so that partial middle turbinate resection technique was done alternately with bolgerization approach in every patient. Each participant acted as their control. Both sides were compared using Lund Kennedy Endoscopic Score (LKES) and Perioperative Sinus Endoscopy Score (POSE) at the baseline, 1st, 3rd, and 12th-month intervals postoperatively. Also, middle turbinate status was assessed at the end of the 12th-month interval using POSE score. RESULTS: The total frontal sinus patency rate was 82.9% (63/76 operated sinus). Baseline scores, LKES (3.79 ± 0.777 vs 4.05 ± 0.769, p = 0.142, for the side of resection and the side for bolgerization respectively) and POSE (1.79 ± 0.413 vs 1.82 ± 0.393, p = 0.777, for the side of resection and the side for bolgerization respectively). Regarding LKES, the differences between both operated sides were fluctuating with p values: 0.001*, 0.171, and 0.044* for the 1st, 3rd, and 12th months follow-up intervals, respectively. Regarding the POSE score of the frontal sinus, the difference between both groups was steadily increasing with p values: 0.318, 0.119, and 0.017* for the 1st, 3rd, and 12th months follow-up intervals. The middle turbinate's POSE score at the 12th month was significantly higher in the side allocated for bolgerization (p-value = 0.008*). CONCLUSION: Partial middle turbinate resection showed favorable endoscopic outcomes than bolgerization at the 12th month follow up period in patients undergoing primary ESS for chronic frontal sinusitis.

The impact of bolgerization versus partial resection of the middle turbinate on frontal sinusotomy patency outcome: A randomised controlled study.

OBJECTIVES: This study aimed to compare the effects of middle turbinate resection vs bolgerization on the incidence of middle meatus synechia and their prognostic value on the patency outcomes after frontal sinusotomy. DESIGN: A randomised controlled study. SETTING: Tertiary centre hospital. MAIN OUTCOME MEASURES: Thirty-eight patients undergoing bilateral frontal sinusotomy for chronic frontal sinusitis were included. Partial middle turbinate resection was alternated with bolgerization in both nasal cavities of every patient. The Lund-Kennedy endoscopic scores (LKESs) for both sides were compared at the first, third and sixth months postoperatively. Middle meatus synechia was assessed using the visual analogue score (VAS). Sinus patency was assessed at the end of the sixth month using a 70° nasal endoscope. RESULTS: The sinus patency outcome was significantly higher in the resected group (34\38) than the bolgerized group (26\38), (P = .047*). The VAS scores suggested that the middle turbinate bolgerization group showed a significantly higher incidence of middle meatal synechia than the partial middle turbinate resection group (4.47 ± 2.617 vs 3.29 ± 2.301; P = .040*). CONCLUSION: Middle turbinate resection showed more favourable results than bolgerization concerning the sinus patency outcome after frontal sinusotomy. It also showed a lower incidence of middle meatus synechia postoperatively.

The prognostic variables affecting the frontal sinusotomy patency outcome and how to manage: A prospective study.

OBJECTIVES: The study aimed to assess the factors affecting the frontal sinus patency after endoscopic frontal sinusotomy. DESIGN: A prospective cohort study. SETTING: Tertiary centre hospital. MAIN OUTCOME MEASURES: Fifty patients with refractory chronic frontal sinusitis (83 operated frontal sinuses) had frontal sinusotomy and followed up for six months. Multiple operative factors were included the type of the procedure, intraoperative sinus findings, degree of mucosal preservation and middle turbinate stability. Other factors were also assessed, including smoking, the presence of allergic rhinitis, asthma, gastroesophageal reflux and other associated medical comorbidities. RESULTS: The sinus patency success rate was 75.9%. There was a significant difference regarding the intraoperative anteroposterior sinus ostium diameter (5.36 ± 1.45 mm vs 8.88 ± 2.38 mm, P-value: .001* in the failed group and the success group, respectively). There was a significant association between the patency outcome and the presence of associated medical comorbidities (P-value: .001*), the presence of allergic rhinitis (P-value: .001*), the degree of sinus mucosal preservation (P-value: .012*) and the degree of middle turbinate stability (P-value: .001*). The multivariate analysis showed that the intraoperative anteroposterior diameter of the sinus ostium, middle turbinate stability and presence of allergic rhinitis were significant predictors (P-value: .012*, .042* and .013*, respectively). CONCLUSION: Sinuses with anteroposterior ostium diameters less than 5.36 mm are more susceptible to restenosis. The flail middle turbinate increases the risk of postoperative middle meatus synechia and frontal sinus patency failure. The presence of allergic rhinitis has a negative impact on the patency outcome.

The preoperative radiological findings associated with failure of frontal sinusotomy: A prospective study.

OBJECTIVES: The study aimed to assess the association between the preoperative CT findings and the patency outcome of the frontal sinus after endoscopic frontal sinusotomy in the early follow-up period. DESIGN: A prospective cohort study. SETTING: Tertiary hospital centre. MAIN OUTCOME MEASURES: The study measures the association between the frontal sinusotomy outcome and the standard preoperative radiological scores, including Harvard, Kennedy and Lund-Mackay. It also measures the impact of the degree of sinus mucosal thickness on the outcome. Furthermore, it measures the effect of the anteroposterior lengths of both the frontal sinus ostium and the frontal recess on postoperative frontal sinus patency. RESULTS: Harvard, Kennedy and modified Lund-Mackay scores showed no evidence of association with the frontal sinusotomy patency outcome (P-values .397, .487 and .501), respectively. Still, the Lund-Mackay score showed a negative correlation with symptom improvement. Sinuses with a high-grade mucosal thickness on CT scan were associated with high failure rates (P-value: .009*). The anteroposterior length of the frontal sinus ostium significantly affects the outcome (P-value: .001*). In contrast, there was no association between the anteroposterior length of the frontal recess and the outcome (P-value: .965). CONCLUSION: The Harvard, Kennedy and Lund-Mackay scores could not predict the frontal sinusotomy patency outcome. Failed cases were associated with advanced degrees of mucosal pathology in the preoperative CT scan. Sinuses ostia with anteroposterior diameters less than 5.36 mm showed more susceptibility for sinus restenosis postoperatively. The variability of the anteroposterior length of the frontal recess did not affect the surgical outcome.

Microparticles in systemic sclerosis: Potential pro-inflammatory mediators and pulmonary hypertension biomarkers.

BACKGROUND AND OBJECTIVE: Endothelial microparticles (EMP) are submicron vesicles released from endothelial cells. We aimed to determine the utility of EMP as biomarkers of pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) patients and the pathogenic role of microparticles (MP) in vascular inflammation. METHODS: Levels of EMP (CD144+, CD31+, CD62E+ and CD143+) were compared between three groups (10 SSc patients with PAH, 10 SSc patients without pulmonary hypertension (no-PH) and 10 healthy age- and sex-matched controls). Human pulmonary artery endothelial cells (HPAEC) were exposed in vitro to MP obtained from SSc patients or healthy controls, and levels of cytokines and inflammatory adhesion molecules were compared. RESULTS: CD144+ EMP were significantly higher in the SSc-PAH group compared to either the SSc-no PH or healthy controls (diagnostic accuracy 80%, P = 0.02). Compared to controls, SSc patients had higher CD31+/CD62E+ ratios, indicating larger contributions of apoptosis to EMP release (P = 0.04). Patients with limited SSc had significantly higher levels of CD143+ EMP compared to those with diffuse subtype (P = 0.008). When HPAEC were exposed to MP from SSc patients, there was a significant increase in inflammatory cytokines and adhesion molecules. Interestingly, exposure to healthy control MP caused a reduction in inflammatory markers. CONCLUSION: EMP (particularly CD144+) are promising biomarkers of PAH in SSc but require further study. MP isolated from SSc patients induced an increase in endothelial cell inflammation and may be an important pathogenic factor in SSc.

PARP-1 Is Critical for Recruitment of Dendritic Cells to the Lung in a Mouse Model of Asthma but Dispensable for Their Differentiation and Function.

Dendritic cells (DCs) are critical in asthma and many other immune diseases. We previously demonstrated a role for PARP-1 in asthma. Evidence on PARP-1 playing a role in Th2-associated DC function is not clear. In this study, we examined whether PARP-1 is critical for DC differentiation and function using bone marrow progenitors and their migration to the lung in an ovalbumin-based mouse model of asthma. Results show that changes in PARP-1 levels during GM-CSF-induced DC differentiation from bone marrow progenitors were cyclic and appear to be part of an array of changes that included STAT3/STAT5/STAT6/GRAIL/RAD51. Interestingly, PARP-1 gene deletion affected primarily STAT6 and γH2AX. PARP-1 inhibition significantly reduced the migration of DCs to the lungs of ovalbumin-challenged mice, which was associated with a concomitant reduction in lung levels of the adhesion molecule VCAM-1. The requirement of PARP-1 for VCAM-1 expression was confirmed using endothelial and lung smooth muscle cells. PARP-1 expression and activity were also required for VCAM-1 in differentiated DCs. An assessment of CD11b+/CD11c+/MHCIIhigh DCs in spleens and lymph nodes of OVA-sensitized mice revealed that PARP-1 inhibition genetically or by olaparib exerted little to no effect on DC differentiation, percentage of CD80+/CD86+/CD40+-expressing cells, or their capacity to promote proliferation of ovalbumin-primed (OTII) CD4+ T cells. These findings were corroborated using GM-CSF-induced differentiation of DCs from the bone marrow. Surprisingly, the PARP-1-/- DCs exhibited a higher intrinsic capacity to induce OTII CD4+ T cell proliferation in the absence of ovalbumin. Overall, our results show that PARP-1 plays little to no role in DC differentiation and function and that the protective effect of PARP-1 inhibition against asthma is associated with a prevention of DC migration to the lung through a reduction in VCAM-1 expression. Given the current use of PARP inhibitors (e.g., olaparib) in the clinic, the present results may be of interest for the relevant therapies.

Sulfated non-anticoagulant heparin blocks Th2-induced asthma by modulating the IL-4/signal transducer and activator of transcription 6/Janus kinase 1 pathway.

BACKGROUND: The efficacy of heparins and low-MW-heparins (LMWH) against human asthma has been known for decades. However, the clinical utility of these compounds has been hampered by their anticoagulant properties. Much effort has been put into harnessing the anti-inflammatory properties of LMWH but none have been used as therapy for asthma. Sulfated-non-anticoagulant heparin (S-NACH) is an ultra-LMWH with no systemic anticoagulant effects. OBJECTIVE: The present study explored the potential of S-NACH in blocking allergic asthma and examined the potential mechanism by which it exerts its effects. METHODS: Acute and chronic ovalbumin-based mouse models of asthma, splenocytes, and a lung epithelial cell line were used. Mice were challenged with aerosolized ovalbumin and administered S-NACH or saline 30 min after each ovalbumin challenge. RESULTS: Sulfated-non-anticoagulant heparin administration in mice promoted a robust reduction in airway eosinophilia, mucus production, and airway hyperresponsiveness even after chronic repeated challenges with ovalbumin. Such effects were linked to suppression of Th2 cytokines IL-4/IL-5/IL-13/GM-CSF and ovalbumin-specific IgE without any effect on IFN-γ. S-NACH also reduced lung fibrosis in mice that were chronically-exposed to ovalbumin. These protective effects of S-NACH may be attributed to modulation of the IL-4/JAK1 signal transduction pathway through an inhibition of STAT6 phosphorylation and a subsequent inhibition of GATA-3 and inducible NO synthase expression. The effect of the drug on STAT6 phosphorylation coincided with a reduction in JAK1 phosphorylation upon IL-4 treatment. The protective effects of S-NACH treatment was associated with reduction of the basal expression of the two isoforms of arginase ARG1 and ARG2 in lung epithelial cells. CONCLUSIONS: Our study demonstrates that S-NACH constitutes an opportunity to benefit from the well-known anti-asthma properties of heparins/LMWH while bypassing the risk of bleeding. Our results show, for the first time, that such anti-asthma effects may be associated with reduction of the IL-4/JAK1/STAT6 pathway.

Treatment with intranasal iloprost reduces disease manifestations in a murine model of previously established COPD.

Pulmonary endothelial prostacyclin appears to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The effect of treatment with a prostacyclin analog in animal models of previously established COPD is unknown. We evaluated the short- and long-term effect of iloprost on inflammation and airway hyperresponsiveness (AHR) in a murine model of COPD. Nineteen mice were exposed to LPS/elastase, followed by either three doses of intranasal iloprost or saline. In the long-term treatment experiment, 18 mice were exposed to LPS/elastase and then received 6 wk of iloprost or were left untreated as controls. In the short-term experiment, iloprost did not change AHR but significantly reduced serum IL-5 and IFN-γ. Long-term treatment with iloprost for both 2 and 6 wk significantly improved AHR. After 6 wk of iloprost, there was a reduction in bronchoalveolar lavage (BALF) neutrophils, serum IL-1ß (30.0 ± 9.2 vs. 64.8 ± 7.4 pg/ml, P = 0.045), IL-2 (36.5 ± 10.6 vs. 83.8 ± 0.4 pg/ml, P = 0.01), IL-10 (75.7 ± 9.3 vs. 96.5 ± 3.5 pg/ml, P = 0.02), and nitrite (15.1 ± 5.4 vs. 30.5 ± 10.7 µmol, P = 0.01). Smooth muscle actin (SMA) in the lung homogenate was also significantly reduced after iloprost treatment (P = 0.02), and SMA thickness was reduced in the small and medium blood vessels after iloprost (P < 0.001). In summary, short- and long-term treatment with intranasal iloprost significantly reduced systemic inflammation in an LPS/elastase COPD model. Long-term iloprost treatment also reduced AHR, serum nitrite, SMA, and BALF neutrophilia. These data encourage future investigations of prostanoid therapy as a novel treatment for COPD patients.

Potential of Inducible Nitric Oxide Synthase as a Therapeutic Target for Allergen-Induced Airway Hyperresponsiveness: A Critical Connection to Nitric Oxide Levels and PARP Activity.

Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AHR by olaparib-mediated PARP inhibition may be associated with the partial but not the complete blockade of iNOS expression. Indeed, L-NIL administration prevented olaparib-mediated protection against AHR in chronically HDM-exposed mice. Our study suggests that the amount of iNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOS as a therapeutic target for asthma.

DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4⁺ T-cell function without causing severe combined immunodeficiency.

BACKGROUND: We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases. OBJECTIVE: We sought to investigate the role of DNA-PK in asthma. METHODS: Cell culture and ovalbumin (OVA)- or house dust mite-based murine asthma models were used in this study. RESULTS: DNA-PK was essential for monocyte adhesion to TNF-α-treated endothelial cells. Administration of the DNA-PK inhibitor NU7441 reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness, and OVA-specific IgE production in mice prechallenged with OVA. Such effects correlated with a marked reduction in lung vascular cell adhesion molecule 1 expression and production of several cytokines, including IL-4, IL-5, IL-13, eotaxin, IL-2, and IL-12 and the chemokines monocyte chemoattractant protein 1 and keratinocyte-derived chemokine, with a negligible effect on IL-10/IFN-γ production. DNA-PK inhibition by gene heterozygosity of the 450-kDa catalytic subunit of the kinase (DNA-PKcs(+/-)) also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma by using house dust mite, a human allergen. Remarkably, such protection occurred without causing severe combined immunodeficiency. Adoptive transfer of TH2-skewed OT-II wild-type CD4(+) T cells reversed IgE and TH2 cytokine production but not airway hyperresponsiveness in OVA-challenged DNA-PKcs(+/-) mice. DNA-PK inhibition reduced IL-4, IL-5, IL-13, eotaxin, IL-8, and monocyte chemoattractant protein 1 production without affecting IL-2, IL-12, IFN-γ, and interferon-inducible protein 10 production in CD3/CD28-stimulated human CD4(+) T cells, potentially by blocking expression of Gata3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4(+) T cells in vitro DNA-PK inhibition severely blocked CD3/CD28-induced Gata3 and T-bet expression in CD4(+) T cells and prevented differentiation of TH1 and TH2 cells under respective TH1- and TH2-skewing conditions. CONCLUSION: Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.

PARP inhibition by olaparib or gene knockout blocks asthma-like manifestation in mice by modulating CD4(+) T cell function.

BACKGROUND: An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. METHODS: We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. RESULTS: Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4(+) T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1(-/-) mice. Adoptive transfer of Th2-skewed OT-II-WT CD4(+) T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1(-/-)mice suggesting a role for PARP-1 in CD4(+) T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4(+) T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4(+) T cells. CONCLUSIONS: Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.

PARP is activated in human asthma and its inhibition by olaparib blocks house dust mite-induced disease in mice.

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.

Computed Tomography Staging of Middle Ear Cholesteatoma.

BACKGROUND: To establish computed tomography (CT) staging of middle ear cholesteatoma and assess its impact on the selection of the surgical procedure. MATERIAL/METHODS: Prospective study was conducted on 61 consecutive patients (mean age 26.8 years) with middle ear cholesteatoma. CT scan of the temporal bone and surgery were performed in all patients. CT staging classified cholesteatoma according to its location in the tympanic cavity (T); extension into the mastoid (M); and associated complications (C). Cholesteatoma was staged as stage I (T1, T2), stage II (T3, M1, M2, C1), and stage III (C2). RESULTS: The overall sensitivity of CT staging of cholesteatoma compared to surgery was 88% with excellent agreement and correlation between CT findings and intra-operative findings (K=0.863, r=0.86, P=0.001). There was excellent agreement and correlation of CT staging with surgical findings for T location (K=0.811, r=0.89, P=0.001), good for M extension (K=0.734, r=0.88, P=0.001), and excellent for associated C complications (K=1.00, r=1.0, P=0.001). Atticotympanotomy was carried out in stage I (n=14), intact canal wall surgery was performed in stage II (n=38), and canal wall down surgery was done in stage III (n=5) and stage II (n=4). CONCLUSIONS: We established CT staging of middle ear cholesteatoma that helps surgeons to select an appropriate surgery.

Apoptotic DNA fragmentation may be a cooperative activity between caspase-activated deoxyribonuclease and the poly(ADP-ribose) polymerase-regulated DNAS1L3, an endoplasmic reticulum-localized endonuclease that translocates to the nucleus during apoptosis.

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca(2+) chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca(2+) independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca(2+) chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca(2+)-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.

Endoscopic ossiculoplasty in traumatic conductive hearing loss with intact tympanic membrane: a five-year experience.

BACKGROUND: Exploratory tympanotomy in cases of traumatic ossicular disruption with intact tympanic membrane is crucial for both diagnostic and therapeutic purposes. Performing this procedure using the endoscope is gaining popularity. Hence, this study aimed to demonstrate varieties of ossicular pathology and their management in our institution. METHODS: A retrospective evaluation was conducted of 136 ears in patients with traumatic ossicular disruption with an intact tympanic membrane, who underwent endoscopic exploratory tympanotomy. A proposed algorithm was followed, to incorporate different traumatic ossicular possibilities. Assessment of hearing outcomes and surgical complications was performed six months post-operatively. RESULTS: Incudostapedial dislocation was the most commonly encountered type of traumatic ossicular disruption (35.3 per cent). Air conduction threshold improved significantly following endoscopic ossiculoplasty, from 50.9 ± 6.35 dB pre-operatively to 22.35 ± 3.27 dB post-operatively, with successful air-bone gap closure. CONCLUSION: Endoscopic ear surgery is effective in the diagnosis and management of challenging cases of post-traumatic ossicular disruption with an intact tympanic membrane.

Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex.

BACKGROUND: The DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID. METHODS: We investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/-, or Ku70+/- mice used in this study. RESULTS: A ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α - induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system. CONCLUSIONS: This study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions.

Endoscopic-Assisted Cochlear Implantation in Far Advanced Otosclerosis.

OBJECTIVES: To evaluate the effectiveness of cochlear implantation (CI) in case of far advanced otosclerosis and to evaluate the value of using intraoperative otoendoscopy to facilitate the identification of the round window membrane and the scala tympani without the need to remove the posterior canal wall or to perform a subtotal petrosectomy. STUDY DESIGN: Retrospective case-series study. SETTING: Tertiary academic CI center. PATIENTS AND METHODS: This study was conducted on patients with far advanced otosclerosis who underwent endoscopic-assisted CI between January 2010 and June 2020 at the same CI center. The minimum follow-up period was 2 years after surgery. RESULTS: Fourteen patients were included in the study. Ten patients had undergone a previous stapedotomy. Electrode insertion in the scala tympani was successfully accomplished in all cases included in the study. There was a statistically significant improvement in pure-tone average and speech discrimination scores in all cases of the study group (p < 0.0001). There were no statistically significant differences in postoperative pure-tone average or speech discrimination scores between cases with and without cochlear ossification or between cases with and without a previous stapedotomy (p > 0.05). CONCLUSION: Endoscopic-assisted CI is an effective option for hearing restoration in patients with far advanced otosclerosis. Otoendoscopy can facilitate visualization and access to the scala tympani without the need to remove the posterior canal wall or to perform a subtotal petrosectomy.

Discriminant Analysis of the Prognostic Factors for Hearing Outcomes in Patients with Idiopathic Sudden Sensorineural Hearing Loss.

BACKGROUND: This study aims to determine and assess prognostic variables that might affect the hearing result in patients with idiopathic sudden sensorineural hearing loss following intratympanic steroid injection. METHODS: In total, 190 patients with idiopathic sudden sensorineural hearing loss received intratympanic steroid injection. Two hearing indices (recovery and nonrecovery) will be analyzed as dependent variables; patient's age, time period between the onset of hearing loss and treatment, initial level of hearing (hearing loss pre), type of audiogram curve (upsloping, downsloping, and flat), presence of vertigo, presence of tinnitus, and diabetes) will be analyzed as prognostic factor variables. RESULTS: Recovery was seen in 72% of the patients. Different preinjection audiogram curves and hearing grades had a significant effect on recovery, absence of vestibular symptoms and no diabetic history were noted to have a good prognosis. Delay in treatment by more than 30 days from the onset of hearing loss was associated with a worse prognosis. CONCLUSION: Idiopathic sudden sensorineural hearing loss associated with late treatment plan more than 1 month, presence of vertigo, diabetes, and profound prehearing loss were negative prognostic factors. Whereas age, gender, and presence of tinnitus did not affect prognosis. More stable response was obtained when intratympanic steroids were added within 1 month after diagnosis, and the patient presented with mild or moderate hearing loss grade, flat or downsloping pure tone audiometery curve, and absence of vertigo and nondiabetic with significantly good results.