DNA-based fluorescent probes of NOS2 activity in live brains.

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.

Second International Guidelines for the Diagnosis and Management of Hereditary Hemorrhagic Telangiectasia.

DESCRIPTION: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease with an estimated prevalence of 1 in 5000 that is characterized by the presence of vascular malformations (VMs). These result in chronic bleeding, acute hemorrhage, and complications from shunting through VMs. The goal of the Second International HHT Guidelines process was to develop evidence-based consensus guidelines for the management and prevention of HHT-related symptoms and complications. METHODS: The guidelines were developed using the AGREE II (Appraisal of Guidelines for Research and Evaluation II) framework and GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodology. The guidelines expert panel included expert physicians (clinical and genetic) in HHT from 15 countries, guidelines methodologists, health care workers, health care administrators, patient advocacy representatives, and persons with HHT. During the preconference process, the expert panel generated clinically relevant questions in 6 priority topic areas. A systematic literature search was done in June 2019, and articles meeting a priori criteria were included to generate evidence tables, which were used as the basis for recommendation development. The expert panel subsequently convened during a guidelines conference to conduct a structured consensus process, during which recommendations reaching at least 80% consensus were discussed and approved. RECOMMENDATIONS: The expert panel generated and approved 6 new recommendations for each of the following 6 priority topic areas: epistaxis, gastrointestinal bleeding, anemia and iron deficiency, liver VMs, pediatric care, and pregnancy and delivery (36 total). The recommendations highlight new evidence in existing topics from the first International HHT Guidelines and provide guidance in 3 new areas: anemia, pediatrics, and pregnancy and delivery. These recommendations should facilitate implementation of key components of HHT care into clinical practice.

Applicability of the Curaçao Criteria for the Diagnosis of Hereditary Hemorrhagic Telangiectasia in the Pediatric Population.

OBJECTIVE: To evaluate the accuracy of the clinical Curaçao criteria in the diagnosis of hereditary hemorrhagic telangiectasia (HHT) in children and adolescents. STUDY DESIGN: This was a retrospective, multicenter chart review of 673 patients evaluated between 2002 and 2016; 290 were eligible for the study. Genetic testing for a pathogenic mutation was considered the gold standard against which the clinical Curaçao criteria were compared. Patients were divided into 4 age categories: 0-5, 6-10, 11-15, and 16-21-years. Sensitivity and specificity were calculated for each age group, and for the overall population. RESULTS: Overall the Curaçao criteria had a sensitivity of 68% (95% CI 60%-76%) and a specificity of 98% (95% CI 91%-100%). Sensitivity was lowest in the 0- to 5-year group, and increased with advancing age. The Curaçao criteria had the highest sensitivity in the 16- to 21-year-olds. Specificity was 100% in all age groups except for the 11- to 15-year-olds. CONCLUSIONS: This study evaluated the use of the Curaçao criteria for the diagnosis of HHT in the pediatric population with a family history of HHT. In those between the age of 0 and 21 years who meet 1 criterion (unlikely HHT) or 2 criteria (possible HHT), genetic testing is preferred for diagnosis. The Curaçao criteria appear to reliably diagnose HHT in children and adolescents who meet 3 or 4 criteria (definite HHT).

A novel association between high red blood cell alloimmunization rates and hereditary hemorrhagic telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder associated with multiple arteriovenous malformations. HHT patients may require red blood cell (RBC) transfusion due to spontaneous hemorrhage or surgical bleeding. Because HHT-associated hemorrhage often occurs in submucosa we hypothesized that RBC alloimmunization rates in HHT patients may be higher than those observed in other transfused patients and investigated this in a retrospective study. STUDY DESIGN AND METHODS: Eighty-five patients with HHT who were transfused at our tertiary care facility were identified. A group of randomly selected, chronically transfused patients without HHT were used as controls (n = 207). RBC transfusion and alloantibody data were extracted from medical records. RESULTS: Alloimmunization rates among patients with HHT were significantly higher than those of controls (15.29% vs. 2.42%; p < 0.001), while HHT patients received fewer RBC transfusions at our institution compared to controls (4.27 units vs. 8.84 units; p < 0.0001). Anti-E, -K, and -c were the three most common alloantibodies identified in HHT patients. There was a trend for HHT patients to make more antibodies per alloimmunized patient than controls (2.38 alloantibodies vs. 1.60 alloantibodies), although this difference was not significant (p = 0.37). CONCLUSION: To our knowledge, this is the first study to evaluate RBC alloimmunization rates in patients with HHT. It is unclear whether the high alloimmunization rates observed are due to lifetime transfusion burden, underlying disease pathophysiology, or other variables. Additional studies are needed to evaluate the generalizability of our findings to other HHT populations and to consider the utility of prophylactic antigen matching for C/E/K.

Tissue-specific mosaicism in hereditary hemorrhagic telangiectasia: Implications for genetic testing in families.

Mosaicism in hemorrhagic telangiectasia (HHT) has been previously identified when testing blood samples of HHT patients. We report the first detection of mosaicism not involving blood of a family proband, and discuss implications for genetic testing algorithms in HHT families. Sanger sequencing and large deletion/duplication analysis in a patient with HHT identified no pathogenic variant in ENG, ACVRL1, or SMAD4. Exome sequencing was then performed on this proband, as well as her affected adult child. A pathogenic ENG variant was detected in the proband's affected child, but not in DNA extracted from peripheral blood of the affected parent/proband. Additional tissue samples (saliva and hair bulbs) were obtained from the proband. The variant was not detected in saliva, but was detected in the hair bulb sample (at 33%). This is the first report of an HHT patient with mosaicism in whom the disease-causing mutation was not detected in blood. The molecular findings in this family suggest that the possibility of mosaicism not present or detectable in blood should be considered if a proband with HHT tests "negative" for a mutation in known genes. This occurrence is particularly suspect for families in which the proband does not have a clearly affected parent. This mechanism may explain some patients with classic HHT in whom a pathogenic variant has not been identified in one of the known HHT genes.

Long-term seizure suppression and optogenetic analyses of synaptic connectivity in epileptic mice with hippocampal grafts of GABAergic interneurons.

Studies in rodent epilepsy models suggest that GABAergic interneuron progenitor grafts can reduce hyperexcitability and seizures in temporal lobe epilepsy (TLE). Although integration of the transplanted cells has been proposed as the underlying mechanism for these disease-modifying effects, prior studies have not explicitly examined cell types and synaptic mechanisms for long-term seizure suppression. To address this gap, we transplanted medial ganglionic eminence (MGE) cells from embryonic day 13.5 VGAT-Venus or VGAT-ChR2-EYFP transgenic embryos into the dentate gyrus (DG) of adult mice 2 weeks after induction of TLE with pilocarpine. Beginning 3-4 weeks after status epilepticus, we conducted continuous video-electroencephalographic recording until 90-100 d. TLE mice with bilateral MGE cell grafts in the DG had significantly fewer and milder electrographic seizures, compared with TLE controls. Immunohistochemical studies showed that the transplants contained multiple neuropeptide or calcium-binding protein-expressing interneuron types and these cells established dense terminal arborizations onto the somas, apical dendrites, and axon initial segments of dentate granule cells (GCs). A majority of the synaptic terminals formed by the transplanted cells were apposed to large postsynaptic clusters of gephyrin, indicative of mature inhibitory synaptic complexes. Functionality of these new inhibitory synapses was demonstrated by optogenetically activating VGAT-ChR2-EYFP-expressing transplanted neurons, which generated robust hyperpolarizations in GCs. These findings suggest that fetal GABAergic interneuron grafts may suppress pharmacoresistant seizures by enhancing synaptic inhibition in DG neural circuits.

Brain arteriovenous malformations associated with hereditary hemorrhagic telangiectasia: gene-phenotype correlations.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disease with a wide spectrum of vascular malformations (VMs) involving multiple organs. Nine to 16% of patients with HHT harbor brain arteriovenous malformations (AVMs), which can cause intracranial hemorrhage (ICH). Our objective was to study clinical manifestations of brain AVMs in patients with HHT and correlate these with the specific gene mutated. We reviewed records of 171 patients with HHT and brain AVMs. A history of ICH was found in 27% (41/152) patients, with a mean age of 26 ± 18 range, (0-68) years. All of patients with ICH were neurologically asymptomatic prior to ICH. Multiple brain AVMs were found in 23% (170/39) of patients on initial examination. Genetic test results were available in 109 (64%) patients. Mutations in ENG, ACVRL1, and SMAD4 were present in 75 (69%), 18 (17%), and 2 (2%), respectively. A history of ICH was reported in 24% of patients with an ENG mutation and 27% of patients with an ACVRL1 mutation, with a mean age of 26 ± 16 (range, 2-50) and 18 ± 21 (0-48) years, respectively. No statistically significant differences in age at first brain AVM diagnosis, prevalence of ICH history, age at ICH, or other manifestations of brain AVMs were observed among gene groups. In conclusion, no evidence for differences in brain AVM characteristics was observed among HHT gene groups, although we cannot exclude clinically important differences. Larger studies are needed to further guide brain AVM screening decisions in patients with HHT.

Incidence of Spontaneous Pulmonary AVM Rupture in HHT Patients.

The spontaneous rupture of pulmonary AVMs, resulting in pulmonary hemorrhage and hydrothorax, is a life-threatening complication. While this phenomenon has been previously reported, the true incidence is not yet known. This study retrospectively reviewed records of 801 HHT patients with pulmonary AVMs to identify a single lifetime episode of hemothorax or pulmonary hemorrhage secondary to pulmonary AVM rupture. The lifetime prevalence and incidence of pulmonary AVM rupture in HHT patients was 2.7% and 0.16% respectively. In these patients, AVM rupture represented the initial presentation of HHT in nine (40.9%) cases and was life-threatening in nine (40.9%) cases. All cases occurred in virgin lesions, and subsequent embolization was curative. While a feared complication, pulmonary AVM rupture is rare and is likely effectively prevented by existing embolization techniques and indications.

Overlapping spectra of SMAD4 mutations in juvenile polyposis (JP) and JP-HHT syndrome.

Juvenile polyposis (JP) and hereditary hemorrhagic telangiectasia (HHT) are clinically distinct diseases caused by mutations in SMAD4 and BMPR1A (for JP) and endoglin and ALK1 (for HHT). Recently, a combined syndrome of JP-HHT was described that is also caused by mutations in SMAD4. Although both JP and JP-HHT are caused by SMAD4 mutations, a possible genotype:phenotype correlation was noted as all of the SMAD4 mutations in the JP-HHT patients were clustered in the COOH-terminal MH2 domain of the protein. If valid, this correlation would provide a molecular explanation for the phenotypic differences, as well as a pre-symptomatic diagnostic test to distinguish patients at risk for the overlapping but different clinical features of the disorders. In this study, we collected 19 new JP-HHT patients from which we identified 15 additional SMAD4 mutations. We also reviewed the literature for other reports of JP patients with HHT symptoms with confirmed SMAD4 mutations. Our combined results show that although the SMAD4 mutations in JP-HHT patients do show a tendency to cluster in the MH2 domain, mutations in other parts of the gene also cause the combined syndrome. Thus, any mutation in SMAD4 can cause JP-HHT. Any JP patient with a SMAD4 mutation is, therefore, at risk for the visceral manifestations of HHT and any HHT patient with SMAD4 mutation is at risk for early onset gastrointestinal cancer. In conclusion, a patient who tests positive for any SMAD4 mutation must be considered at risk for the combined syndrome of JP-HHT and monitored accordingly.

Hindbrain and Spinal Cord Contributions to the Cutaneous Sensory Innervation of the Larval Zebrafish Pectoral Fin.

Vertebrate forelimbs contain arrays of sensory neuron fibers that transmit signals from the skin to the nervous system. We used the genetic toolkit and optical clarity of the larval zebrafish to conduct a live imaging study of the sensory neurons innervating the pectoral fin skin. Sensory neurons in both the hindbrain and the spinal cord innervate the fin, with most cells located in the hindbrain. The hindbrain somas are located in rhombomere seven/eight, laterally and dorsally displaced from the pectoral fin motor pool. The spinal cord somas are located in the most anterior part of the cord, aligned with myomere four. Single cell reconstructions were used to map afferent processes and compare the distributions of processes to soma locations. Reconstructions indicate that this sensory system breaks from the canonical somatotopic organization of sensory systems by lacking a clear organization with reference to fin region. Arborizations from a single cell branch widely over the skin, innervating the axial skin, lateral fin surface, and medial fin surface. The extensive branching over the fin and the surrounding axial surface suggests that these fin sensory neurons report on general conditions of the fin area rather than providing fine location specificity, as has been demonstrated in other vertebrate limbs. With neuron reconstructions that span the full primary afferent arborization from the soma to the peripheral cutaneous innervation, this neuroanatomical study describes a system of primary sensory neurons and lays the groundwork for future functional studies.

Subaortic Membranes in Patients With Hereditary Hemorrhagic Telangiectasia and Liver Vascular Malformations.

Background Patients with hereditary hemorrhagic telangiectasia have liver vascular malformations that can cause high-output cardiac failure (HOCF). Known sequelae include pulmonary hypertension, tricuspid regurgitation, and atrial fibrillation. Methods and Results The objectives of this study were to describe the clinical, echocardiographic, and hemodynamic characteristics and prognosis of hereditary hemorrhagic telangiectasia patients with HOCF who were found to have a subaortic membrane (SAoM). A retrospective observational analysis comparing patients with and without SAoM was performed. Among a cohort of patients with HOCF, 9 were found to have a SAoM in the left ventricular outflow tract by echocardiography (all female, mean age 64.8±4.0 years). The SAoM was discrete and located in the left ventricular outflow tract 1.1±0.1 cm below the aortic annular plane. It caused turbulent flow, mild obstruction (peak velocity 2.8±0.2 m/s, peak gradient 32±4 mm Hg), and no more than mild aortic insufficiency. Patients with SAoM (n=9) had higher cardiac output (12.1±1.3 versus 9.3±0.7 L/min, P=0.04) and mean pulmonary artery pressures (36±3 versus 28±2 mm Hg, P=0.03) compared with those without SAoM (n=19) during right heart catheterization. Genetic analysis revealed activin receptor-like kinase 1 mutations in each of the 8 patients with SAoM who had available test results. The presence of a SAoM was associated with a trend towards higher 5-year mortality during follow-up. Conclusions SAoM with mild obstruction occurs in patients with hereditary hemorrhagic telangiectasia and HOCF. SAoM was associated with features of more advanced HOCF and poor outcomes.

Genotype-Phenotype Correlations in Children with HHT.

Hereditary hemorrhagic telangiectasia (HHT), a rare autosomal dominant disease mostly caused by mutations in three known genes (ENG, ACVRL1, and SMAD4), is characterized by the development of vascular malformations (VMs). Patients with HHT may present with mucocutaneous telangiectasia, as well as organ arteriovenous malformations (AVMs) of the central nervous system, lungs, and liver. Genotype-phenotype correlations have been well described in adults with HHT. We aimed to investigate genotype-phenotype correlations among pediatric HHT patients. Demographic, clinical, and genetic data were collected and analyzed in 205 children enrolled in the multicenter Brain Vascular Malformation Consortium HHT Project. A chi-square test was used to determine the association between phenotypic presentations and genotype. Among 205 patients (age range: 0-18 years; mean: 11 years), ENG mutation was associated with the presence of pulmonary AVMs (p < 0.001) and brain VM (p < 0.001). The presence of a combined phenotype-defined as both pulmonary AVMs and brain VMs-was also associated with ENG mutation. Gastrointestinal bleeding was rare (4.4%), but was associated with SMAD4 genotype (p < 0.001). We conclude that genotype-phenotype correlations among pediatric HHT patients are similar to those described among adults. Specifically, pediatric patients with ENG mutation have a greater prevalence of pulmonary AVMs, brain VMs, and a combined phenotype.

Results of exercise stress testing in patients with diffuse pulmonary arteriovenous malformations.

Patients with diffuse pulmonary arteriovenous malformations (PAVMs) are subject to frequent complications and need to be followed closely. As part of this follow-up, we have employed exercise stress testing (EST) as an aid to assess their status. Twenty patients from a cohort of 35 with diffuse PAVMs have undergone EST using a standard cycle ergometer test. All patients had previously undergone pulmonary angiography, noncontrast chest computed tomography (CT), and repair of large focal PAVMs, prior to EST. Mean room air oxygen saturation at baseline and at maximum exercise (85% of maximum heart rate) were tabulated. Serial studies in six children and young adults were plotted by year and compared using the patient as their own control. Fourteen females and six males ranging in age from 4 to 50 years (mean 22 years) were studied. Baseline mean oxygen saturation was 84% and fell to 73% at maximum exercise. There was no significant difference between those with unilateral and bilateral involvement (P = 0.09). In four of the six patients with serial EST, the baseline and exercise oxygen saturations were quite stable. In the two patients who became symptomatic, with age, growth, and more activity, complete embolization of one or more segments of the lung improved their EST and functionality. Based on our previous work in patients with diffuse PAVMs, EST appears to offer a relatively safe and noninvasive method for assessing these patients. Our limited experience with serial EST suggests a good correlation with decreased functionality in these patients.

New definition and natural history of patients with diffuse pulmonary arteriovenous malformations: twenty-seven-year experience.

BACKGROUND: Patients with diffuse pulmonary arteriovenous malformations (PAVM), a small but important subset of the PAVM population, have significant morbidity and mortality rates. METHODS: Thirty-six patients (21 female and 15 male) with diffuse PAVM from a cohort of 821 consecutive patients with PAVM were evaluated. Diffuse PAVM were categorized angiographically: involvement of one or more segmental pulmonary arteries in one or both lungs. Hereditary hemorrhagic telangiectasia (HHT) status, gender, presence or absence of large (> or = 3-mm diameter artery) focal PAVM, oxygen saturations, complications including hemoptysis, years of follow-up, and survival were tabulated. RESULTS: HHT was present in 29 of 36 patients (81%), and diffuse PAVM were more commonly bilateral (26 of 36 patients, 72%) than unilateral (10 of 36 patients, 28%) [p = 0.02]. Female gender was associated with bilateral diffuse PAVM (19 of 26 patients, 73%) [p = 0.01]. Focal PAVM were present in both groups but more commonly in patients with bilateral involvement (16 of 26 patients, 62%) [p = 0.02]. Initial oxygen saturations (pulse oximetry, standing) of patients with unilateral and bilateral diffuse PAVM were 87 +/- 7% and 79 +/- 8% (mean +/- SD), respectively (p = 0.02). The last or current values for patients with unilateral and bilateral involvement are 95 +/- 3% and 85 +/- 7%, respectively (p < 0.0001). Nine deaths occurred, and all were in patients with bilateral involvement. Deaths were due to hemoptysis of bronchial artery origin (n = 2), hemorrhage from duodenal ulcer (n = 1), spontaneous liver necrosis (n = 3), brain hemorrhage (n = 1), brain abscess (n = 1), and operative death during attempted lung transplant (n = 1). CONCLUSIONS: Patients with diffuse PAVM are a high-risk group, and yearly follow-up is recommended.

Long-term complications of septal dermoplasty in patients with hereditary hemorrhagic telangiectasia.

OBJECTIVE: Septal dermoplasty has been recommended as the treatment of choice for life-threatening epistaxis in patients with hereditary hemorrhagic telangiectasia. This study evaluates the complications of septal dermoplasty in the management of transfusion-dependent epistaxis. STUDY DESIGN: Consecutive retrospective study. SUBJECTS AND METHODS: Between 1994 and 2006, septal dermoplasty was performed on 106 consecutive patients with transfusion dependent epistaxis. Of 103 potential patients, 37 either died or were lost to follow-up, which left 66 patients for study. Data on complications and quality of life were collected on 50 (76%) of 66 patients (mean follow-up, 3.75 years) via phone interview. RESULTS: Seventy-eight percent experienced nasal odor; 72% had nasal crusting; 58% had decreased sense of smell; 30% noted worsened sinus infection; 88% could breathe through their nose; 86% stated improved quality of life. CONCLUSION: Septal dermoplasty remains an effective way of treating transfusion dependent epistaxis in patients with hereditary hemorrhagic telangiectasia and subjectively improves their quality of life.

Outcome of septal dermoplasty in patients with hereditary hemorrhagic telangiectasia.

OBJECTIVES/HYPOTHESIS: Septal dermoplasty has been recommended as the treatment of choice for life-threatening epistaxis in patients with hereditary hemorrhagic telangiectasia. The purpose of the study was to evaluate the effectiveness and outcomes of septal dermoplasty for management of transfusion-dependent epistaxis. STUDY DESIGN: Retrospective study. METHODS: Between 1994 and 2004, septal dermoplasty was performed on 67 consecutive patients with severe epistaxis attributable to hereditary hemorrhagic telangiectasia. The numbers of units of blood received 1 year before and 1 year after septal dermoplasty were obtained. A subjective appraisal of the results of the surgery as well as second procedures after septal dermoplasty was determined. Patients were screened for pulmonary and cerebral arteriovenous malformations, gastrointestinal tract bleeding, and symptomatic liver disease. RESULTS: Data were obtained in 66 of 67 (98%) patients with a mean age of 61.5 years (mean follow-up, 3.9 y). Accurate transfusion requirements 1 year before and 1 year after septal dermoplasty were available in 32 of 66 (48%) patients. In these 32 patients, the mean units of blood received decreased from 21 units (range, 2-100 units) 1 year before septal dermoplasty to 1 unit (range, 0-10 units) in the year after septal dermoplasty (P < .001). Improved quality of life was claimed in 57 patients. Second therapies, ranging from cautery to repeat partial septal dermoplasty, were required in 15 patients during follow-up. Among the 67 patients, 31 (46%) had pulmonary arteriovenous malformation, 14 (21%) had gastrointestinal tract bleeding, 7 (10%) had symptomatic liver disease, and 5 (7%) had cerebral arteriovenous malformation. During the follow-up, 14 patients died of other complications of hereditary hemorrhagic telangiectasia (11 patients) and unrelated causes (3 patients). CONCLUSION: Septal dermoplasty remains an effective way of reducing transfusion requirements in patients with hereditary hemorrhagic telangiectasia and subjectively improves their quality of life. The otolaryngologist caring for patients with hereditary hemorrhagic telangiectasia should be familiar with other organ involvement by hereditary hemorrhagic telangiectasia to prevent complications during surgery.

The Young's procedure for severe epistaxis from hereditary hemorrhagic telangiectasia.

BACKGROUND: Surgical treatment of epistaxis in hereditary hemorrhagic telangiectasia (HHT) has historically been managed with the laser procedure or the septodermoplasty procedure. For transfusion-dependent patients with severe epistaxis we have been performing the Young's procedure or surgical closure of the nostrils. The objective of this study was to report treatment of severe epistaxis related to HHT with the Young's procedure and assess patient outcome. METHODS: Patients with severe iron or blood transfusion-dependent epistaxis who underwent a Young's procedure in three otolaryngology HHT centers were reviewed. Patients were evaluated for postoperative epistaxis and subjective outcome. RESULTS: Forty-three patients underwent a Young's procedure for severe epistaxis and were observed for a mean of 34 months. The procedure was well tolerated by all patients and 30 of 36 patients (83%) experienced complete cessation of bleeding after the Young's procedure. Patients had a mean increase in hemoglobin of 4.68 g/dL after the procedure. The average Glasgow Benefit Inventory score after surgery was 43.56. No patients requested a reversal of the procedure. CONCLUSION: The Young's procedure is a safe and efficacious procedure with complete cessation of epistaxis in most patients with severe epistaxis and HHT.