Head and neck venous thrombosis secondary to pediatric otolaryngologic infection.

OBJECTIVES: Head and neck venous thrombosis is a rare but potentially devastating complication of childhood otolaryngologic infections. This study examines the presentation and management of this condition. METHODS: A retrospective chart review was performed on all pediatric patients with otolaryngologic infections complicated by cranial and cervical venous thrombosis at a tertiary children's hospital from 2007 to 2018. Patient demographics, presentation, site of infection, thrombosis location, implicated pathogen, length of hospital stay, need for surgery, and anticoagulant regimen were assessed. RESULTS: This study included 33 patients (mean age, 7.5 years; age range, 0.8-17 years; 19 [58%] male). The most common infection source was otologic (n = 20), followed by ophthalmic and sinonasal pathology (n = 9), and neck infections (n = 4). The most common site of thrombosis secondary to ear pathology was the sigmoid sinus. The ophthalmic veins were the most common site of thrombosis for ophthalmic/sinonasal infections. Nine CN VI palsies, one CN VII palsy, and one CN III palsy were observed. Twenty-six subjects (79%) required surgical intervention. All those who experienced a nerve palsy required surgery. Length of hospitalization significantly differed with the stay for a neck infection complicated by thrombosis longer compared to otologic and sinonasal infections (F[2,30] = 7.08, p = 0.003). Length of hospital stay was significantly correlated with admission temperature (r = 0.506, p = 0.003) and CRP (r = 0.400, p = 0.03) but not WBC (r = 0.181, p = 0.31). Culture growth predominantly isolated a single causative organism rather than polymicrobial involvement. Forty-eight species were identified, most (n = 41/48, 85%) being Gram-positive bacteria. Alpha-hemolytic Streptococcus was the most common isolate from children with vessel thrombosis secondary to ear infections, with Streptococcus pyogenes predominant in sinonasal infections and Staphylococcus aureus the most common in neck abscesses. There was significant variability in anticoagulation management within the patient population, but no bleeding complications were documented. Most patients had no evidence of underlying thrombophilia (n = 15); for those with positive hypercoagulability screens, the most common positive marker was the presence of lupus inhibitor (n = 6). CONCLUSION: Venous thrombosis resulting from adjacent otolaryngologic infection is a serious complication requiring proper recognition and management. The involved vasculature and cranial nerve findings are dependent on the anatomic location of the underlying infection. Cranial neuropathies in the presence of these infections should prompt evaluation for possible thrombosis.

Endoscopic Versus Microscopic Stapedotomy: A Single-Blinded Randomized Control Trial.

OBJECTIVE: To demonstrate non-inferiority of endoscopic stapedotomy to microscopic stapedotomy for the treatment of otosclerosis. STUDY DESIGN: Single-blinded randomized control trial. SETTING: Tertiary, academic otology-neurotology practice. PATIENTS: Adult subjects with a diagnosis of otosclerosis and a preoperative air-bone gap (ABG) more than or equal to 20 dB undergoing primary stapedotomy. INTERVENTION: Endoscopic or microscopic stapedotomy. MAIN OUTCOME MEASURES: Primary audiometric outcome was postoperative ABG. Secondary audiometric outcomes included speech reception threshold (SRT), word recognition score (WRS), bone- and air-conduction pure tone averages (PTA), change in ABG, and ABG closure rates to less than or equal to 10 dB and less than or equal to 20 dB. RESULTS: Twenty-two patients were recruited. Eleven patients underwent endoscopic stapedotomy and 11 underwent microscopic stapedotomy. The endoscopic group was non-inferior to the microscopic group in terms of postoperative audiometric outcomes (endoscope versus microscope, p-value): ABG (8.1 dB versus 8.1 dB, <0.001), SRT (27.7 dB versus 25.9 dB, <0.001), WRS (92% at 65 dB versus 98% at 62 dB, <0.001), air-conduction PTA (33.5 dB versus 30.8 dB, <0.01), and change in ABG (23.0 dB versus 20.7 dB, <0.0001). ABG closure rates to less than or equal to 10 dB (72.7% versus 81.2%, p = 1.0) and less than or equal to 20 dB (90.9% versus 100%, p = 1.0) were not significantly different. There was no significant difference in operative time, necessity of scutum curettage, or postoperative dysgeusia. No patients required chorda tympani sacrifice. Preoperative tinnitus resolved in three patients in each group postoperatively. CONCLUSIONS: This study is the first randomized control trial to demonstrate non-inferiority of endoscopic to microscopic stapedotomy.

Identification of a novel class of RIP1/RIP3 dual inhibitors that impede cell death and inflammation in mouse abdominal aortic aneurysm models.

Receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) are essential mediators of cell death processes and participate in inflammatory responses. Our group recently demonstrated that gene deletion of Rip3 or pharmacological inhibition of RIP1 attenuated pathogenesis of abdominal aortic aneurysm (AAA), a life-threatening degenerative vascular disease characterized by depletion of smooth muscle cells (SMCs), inflammation, negative extracellular matrix remodeling, and progressive expansion of aorta. The goal of this study was to develop drug candidates for AAA and other disease conditions involving cell death and inflammation. We screened 1141 kinase inhibitors for their ability to block necroptosis using the RIP1 inhibitor Necrostatin-1s (Nec-1s) as a selection baseline. Positive compounds were further screened for cytotoxicity and virtual binding to RIP3. A cluster of top hits, represented by GSK2593074A (GSK'074), displayed structural similarity to the established RIP3 inhibitor GSK'843. In multiple cell types including mouse SMCs, fibroblasts (L929), bone marrow derived macrophages (BMDM), and human colon epithelial cells (HT29), GSK'074 inhibited necroptosis with an IC50 of ~3 nM. Furthermore, GSK'074, but not Nec-1s, blocked cytokine production by SMCs. Biochemical analyses identified both RIP1 and RIP3 as the biological targets of GSK'074. Unlike GSK'843 which causes profound apoptosis at high doses (>3 µM), GSK'074 showed no detectable cytotoxicity even at 20 µM. Daily intraperitoneal injection of GSK'074 at 0.93 mg/kg significantly attenuated aortic expansion in two mouse models of AAA (calcium phosphate: DMSO 66.06 ± 9.17% vs GSK'074 27.36 ± 8.25%, P < 0.05; Angiotensin II: DMSO 85.39 ± 15.76% vs GSK'074 36.28 ± 5.76%, P < 0.05). Histologically, GSK'074 treatment diminished cell death and macrophage infiltration in aneurysm-prone aortae. Together, our data suggest that GSK'074 represents a new class of necroptosis inhibitors with dual targeting ability to both RIP1 and RIP3. The high potency and minimum cytotoxicity make GSK'074 a desirable drug candidate of pharmacological therapies to attenuate AAA progression and other necroptosis related diseases.

Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury.

RATIONALE: Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKCδ (protein kinase C-delta) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-endothelial cells communication. OBJECTIVE: To test whether PKCδ-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury. METHODS AND RESULTS: Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared with the wild-type controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKCδ-infected SMCs increased endothelial wound closure without affecting their proliferation. A polymerase chain reaction-based array analysis identified Cxcl1 and Cxcl7 among others as PKCδ-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKCδ regulates Cxcl7 expression through STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization. CONCLUSIONS: SMCs promote reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of endothelial cells from uninjured endothelium.

Necroptosis in cardiovascular disease - a new therapeutic target.

Contrary to the apoptosis-necrosis binary view of cell death, recent experimental evidence demonstrates that several forms of necrosis, represented by necroptosis, are regulated or programmed in nature. Multiple death stimuli known to be associated with cardiovascular disease are capable of causing either apoptosis or necroptosis. Whether a cell dies from apoptosis or necroptosis has distinct consequences on inflammation. It is known that apoptosis, a non-lytic form of death mediated by the caspase family of proteases, does not generally evoke an immune response. Necroptosis, on the other hand, is a lytic form of cell death. Due to the rapid loss of plasma membrane integrity, cells dying from necroptosis release proinflammatory intracellular contents and subsequently cause inflammation. Our review delineates various genetic and biochemical evidence that demonstrates a compelling role of necroptosis in the pathogenesis and/or progression of cardiovascular disease including myocardial infarction, atherosclerosis, and aortic aneurysm. Through recent studies of necroptosis in cardiovascular diseases, we attempt to discuss the role of necroptosis in vascular inflammation as well as the potential of necroptosis inhibitors in future clinical management of cardiovascular events. Inhibiting necroptosis in the vasculature has an overall protective role and necroptosis may represent a new therapeutic target to prevent the development and progression of cardiovascular diseases.

Deficits in Col5a2 Expression Result in Novel Skin and Adipose Abnormalities and Predisposition to Aortic Aneurysms and Dissections.

Classic Ehlers-Danlos syndrome (cEDS) is characterized by fragile, hyperextensible skin and hypermobile joints. cEDS can be caused by heterozygosity for missense mutations in genes COL5A2 and COL5A1, which encode the α2(V) and α1(V) chains, respectively, of collagen V, and is most often caused by COL5A1 null alleles. However, COL5A2 null alleles have yet to be associated with cEDS or other human pathologies. We previously showed that mice homozygous null for the α2(V) gene Col5a2 are early embryonic lethal, whereas haploinsufficiency caused aberrancies of adult skin, but not a frank cEDS-like phenotype, as skin hyperextensibility at low strain and dermal cauliflower-contoured collagen fibril aggregates, two cEDS hallmarks, were absent. Herein, we show that ubiquitous postnatal Col5a2 knockdown results in pathognomonic dermal cauliflower-contoured collagen fibril aggregates, but absence of skin hyperextensibility, demonstrating these cEDS hallmarks to arise separately from loss of collagen V roles in control of collagen fibril growth and nucleation events, respectively. Col5a2 knockdown also led to loss of dermal white adipose tissue (WAT) and markedly decreased abdominal WAT that was characterized by miniadipocytes and increased collagen deposition, suggesting α2(V) to be important to WAT development/maintenance. More important, Col5a2 haploinsufficiency markedly increased the incidence and severity of abdominal aortic aneurysms, and caused aortic arch ruptures and dissections, indicating that α2(V) chain deficits may play roles in these pathologies in humans.

Inhibition of Receptor-Interacting Protein Kinase 1 with Necrostatin-1s ameliorates disease progression in elastase-induced mouse abdominal aortic aneurysm model.

Abdominal aortic aneurysm (AAA) is a common aortic disease with a progressive nature. There is no approved pharmacological treatment to effectively slow aneurysm growth or prevent rupture. Necroptosis is a form of programmed necrosis that is regulated by receptor-interacting protein kinases (RIPs). We have recently demonstrated that the lack of RIP3 in mice prevented aneurysm formation. The goal of the current study is to test whether perturbing necroptosis affects progression of existing aneurysm using the RIP1 inhibitors Necrostatin-1 (Nec-1) and an optimized form of Nec-1, 7-Cl-O-Nec-1 (Nec-1s). Seven days after aneurysm induction by elastase perfusion, mice were randomly administered DMSO, Nec-1 (3.2 mg/kg/day) and Nec-1s (1.6 mg/kg/day) via intraperitoneal injection. Upon sacrifice on day 14 postaneurysm induction, the aortic expansion in the Nec-1s group (64.12 ± 4.80%) was significantly smaller than that of the DMSO group (172.80 ± 13.68%) (P < 0.05). The mean aortic diameter of Nec-1 treated mice appeared to be smaller (121.60 ± 10.40%) than the DMSO group, though the difference was not statistically significant (P = 0.1). Histologically, the aortic structure of Nec-1s-treated mice appeared normal, with continuous and organized elastin laminae and abundant αActin-expressing SMCs. Moreover, Nect-1s treatment diminished macrophage infiltration and MMP9 accumulation and increased aortic levels of tropoelastin and lysyl oxidase. Together, our data suggest that pharmacological inhibition of necroptosis with Nec-1s stabilizes pre-existing aneurysms by diminishing inflammation and promoting connective tissue repair.

Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes.

In this work, we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this, we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α values) that quantify interactions of glycerol, tetraEG, and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O, and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence, tetraEG and PEG300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD), while glycerol and di- and monoethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α values reported here, chemical contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA) and separated from excluded volume effects.