Application of in-vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action.

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liver-directed AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

Management and surgical outcomes of pediatric retinal detachment associated with familial exudative vitreoretinopathy - Our experience at a tertiary care ophthalmic center in North India.

Purpose: To report the clinical profile, management, and long-term anatomical and visual acuity (VA) outcomes of pediatric macula-off rhegmatogenous retinal detachment (RRD) secondary to familial exudative vitreoretinopathy (FEVR). Methods: This was a prospective, interventional study of 14 eyes of 13 children aged ≤18 years with macula-off FEVR-RRD. The primary outcomes were anatomical reattachment and VA changes. Results: The mean (±SD) age of the study population was 12.14 (±3.23) years (range 6-18 years) with a male preponderance (M:F - 10:3). Of the 14 eyes, 10 underwent vitrectomy with silicone oil injection, while four underwent scleral buckling surgery. Significant improvement in VA was noted at a mean (±SD) follow-up duration of 3.32 (±1.34) years, with the mean (±SD) LogMAR VA improving from 1.42 (±0.48) (Snellen equivalent 2/60; range from 6/36 to counting finger close to face [CFCF]) to 0.6 (±0.31) (Snellen equivalent 6/24; range 6/9-6/36) (P < 0.00001) at the final visit. Successful anatomical reattachment was achieved in 13/14 eyes (92.85%). Screening of the other eye and family members was performed for FEVR and treated with laser photocoagulation when deemed necessary (7/10 contralateral eye; 12/20 siblings; 0/24 parents). Conclusion: To conclude, RRD may arise in eyes with FEVR at a young age and with a male predilection in Indian population. Timely surgical intervention by scleral buckling procedure or vitrectomy, based on the patient profile, can achieve excellent anatomical and VA outcomes. Careful clinical and angiographic screening of the other eye and family members is vital.

A rare case of total anomalous pulmonary venous connection with intact interatrial septum.

The presence of interatrial communication is considered obligatory in total anomalous pulmonary venous connection (TAPVC). Even a restriction in this communication leads to obstructive TAPVC. We report a rare case of obstructed supracardiac TAPVC with the absence of interatrial communication and with multiple ventricle septal defects (VSDs) in a 3-month-old child.

Truncus Arteriosus With Double Aortic Arch.

Truncus arteriosus (TA) or common arterial trunk is a congenital cardiac anomaly having high association with arch anomalies such as right aortic arch or aortic arch interruption. However, TA with double aortic arch (DAA) is a rare occurrence. We report a case of TA with DAA where the diagnosis of DAA was missed initially.

Type II Aortopulmonary Window with Isolated Left Subclavian Artery from Left Pulmonary Artery.

Type II Aortopulmonary window (APW) accounts for only 10% of total cases of APW, which by itself is a rare congenital anomaly. Various cardiac malformations have been reported to be associated with this rare anomaly. We report one such association of origin of left subclavian artery (LSCA) from left pulmonary artery (LPA) via ductus arteriosus that was surgically repaired.

Type ii aortopulmonary window with isolated left subclavian artery from left pulmonary artery

Abstract Type II Aortopulmonary window (APW) accounts for only 10% of total cases of APW, which by itself is a rare congenital anomaly. Various cardiac malformations have been reported to be associated with this rare anomaly. We report one such association of origin of left subclavian artery (LSCA) from left pulmonary artery (LPA) via ductus arteriosus that was surgically repaired.

Does addition of bi-directional cavo-pulmonary shunt to tricuspid repair in advanced cases of Ebstein anomaly result in better outcomes?

OBJECTIVES: Ebstein anomaly can be managed by single ventricular, bi-ventricular and one and a half ventricular repairs. We present midterm results of Comprehensive Tricuspid Valve repair (CTVR) with bi-directional cavo-pulmonary shunt (BCPS). METHODS: In this prospective observational study (Jan2012-July2018), 69 patients underwent surgery for Ebstein anomaly. In Group I (n = 48; 69.6%), all patients got CTVR and a BCPS (one and a half ventricle repair). Group II (n = 15; 21.8%) consisted of a similar repair without BCPS (bi-ventricle repair). All patients were echocardiographed at six monthly intervals. RESULTS: Median age of the cohort was 17 years (range 1-68). 12 (17.4%) patients were Carpentier type B, 51 (73.9%) were type C and 6 (8.7%) were type D. There were two early mortalities (2.89%). At a mean follow up of 3.2 ± 1.2 years, there were no late deaths and one delayed repair-failure in each group. Group I had significantly lower mean TR grade (1.2 ± 0.4vs1.6 ± 0.5, p = 0.03) as compared to Group II without a significant difference in the mean gradients (1.5 ± 0.5vs1.6 ± 0.6, p = 0.4). Mean indexed TAPSE (15.0 ± 6.7vs.16.6 ± 5.6 mm/m2, p = 0.21), NYHA class (1.2 ± 0.4vs1.3 ± 0.4) and six-minute walk distance (506 vs 507 m, p = 0.7) was similar in both groups. CONCLUSION: One and a half ventricle repair of Ebstein anomaly gives a more functionally competent, non-stenotic and durable tricuspid valve as compared to a two-ventricle repair. BCPS doesn't result in facial swelling or AV malformations. Preload reduction by BCPS may allow the myopathic ventricle to remodel.

Anomalous origin of a pulmonary artery in a teenager with tetralogy of Fallot with associated double aortic arch and coronary arteriovenous fistula.

We report the case of a 14-year-old female who had tetralogy of Fallot along with anomalous origin of the left pulmonary artery from the ascending aorta with co-dominant double aortic arch forming a complete vascular ring compressing the oesophagus along with a left main coronary artery to right ventricular outflow tract fistula. She underwent surgical correction without conduit placement.

Intramural Aortic Course Should Always Be Considered for Anomalous Origin of the Left Coronary Artery From the Right Pulmonary Artery.

Anomalous origin of the left coronary artery (LCA) from the right pulmonary artery (ALCARPA) is an extremely rare subset of an already rare entity, anomalous origin of the LCA from the pulmonary artery. Whenever it is diagnosed preoperatively, one should be extremely vigilant about the potential intramural course of the descending part of the LCA in the aorta. Preoperative imaging frequently fails to delineate this intramural course. We report our experience with one such case where we had accidentally injured the LCA during dissection from the right pulmonary artery. Although it was successfully managed, it reinforces our aforementioned point concerning the importance of vigilance in seeking to identify intramurality as a component of this anomaly of coronary artery origin.

Ebstein's Anomaly: "The One and a Half Ventricle Heart".

OBJECTIVE: Ebstein's anomaly remains a relatively ignored disease. Lying in the 'No Man's land' between congenital and valve surgeons, it largely remains inadequately studied. We report our short-term results of treating it as a 'one and a half ventricle heart' and propose that the true tricuspid annulus (TTA) 'Z' score be used as an objective criterion for estimation of 'functional' right ventricle (RV). METHODS: 22 consecutive patients undergoing surgery for Ebstein's anomaly were studied. Echocardiography was performed to assess the type and severity of the disease, tricuspid annular dimension and its 'Z' score. Patients were operated by a modification of the cone repair, with addition of annuloplasty, bidirectional cavopulmonary shunt (BCPS) and right reduction atrioplasty to provide a comprehensive repair. TTA 'Z' score was correlated later with postplication indexed residual RV volume. RESULTS: There was one (4.5%) early and no late postoperative death. There was a significant reduction in tricuspid regurgitation grading (3.40±0.65 to 1.22±0.42, P<0.001). Residual RV volume reduced to 71.96±3.8% of the expected volume and there was a significant negative correlation (rho -0.83) between TTA 'Z' score and indexed residual RV volume. During the follow-up of 20.54±7.62 months, the functional class improved from 2.59±0.7 to 1.34±0.52 (P<0.001). CONCLUSION: In Ebstein's anomaly, a higher TTA 'Z' score correlates with a lower postplication indexed residual RV volume. Hence, a complete trileaflet repair with offloading of RV by BCPS (when the TTA 'Z' score is >2) is recommended. The short-term outcomes of our technique are promising.

Ebstein's anomaly: "The one and a half ventricle heart"

Abstract Objective: Ebstein's anomaly remains a relatively ignored disease. Lying in the 'No Man's land' between congenital and valve surgeons, it largely remains inadequately studied. We report our short-term results of treating it as a 'one and a half ventricle heart' and propose that the true tricuspid annulus (TTA) 'Z' score be used as an objective criterion for estimation of 'functional' right ventricle (RV). Methods: 22 consecutive patients undergoing surgery for Ebstein's anomaly were studied. Echocardiography was performed to assess the type and severity of the disease, tricuspid annular dimension and its 'Z' score. Patients were operated by a modification of the cone repair, with addition of annuloplasty, bidirectional cavopulmonary shunt (BCPS) and right reduction atrioplasty to provide a comprehensive repair. TTA 'Z' score was correlated later with postplication indexed residual RV volume. Results: There was one (4.5%) early and no late postoperative death. There was a significant reduction in tricuspid regurgitation grading (3.40±0.65 to 1.22±0.42, P<0.001). Residual RV volume reduced to 71.96±3.8% of the expected volume and there was a significant negative correlation (rho −0.83) between TTA 'Z' score and indexed residual RV volume. During the follow-up of 20.54±7.62 months, the functional class improved from 2.59±0.7 to 1.34±0.52 (P<0.001). Conclusion: In Ebstein's anomaly, a higher TTA 'Z' score correlates with a lower postplication indexed residual RV volume. Hence, a complete trileaflet repair with offloading of RV by BCPS (when the TTA 'Z' score is >2) is recommended. The short-term outcomes of our technique are promising.

Mesenchymal Stem Cell Therapy in Ischemic Stroke: A Meta-analysis of Preclinical Studies.

Numerous preclinical studies have been carried out using mesenchymal stem cells (MSCs) therapy for ischemic stroke. The purpose of the present meta-analysis is to review the quality of preclinical studies. In all, 4,361 articles were identified, out of which 64 studies were included (excluding in vitro studies). The results were obtained across species, route, and time of administration, immunogenicity, and doses. The median quality score 4.90/10, confidence interval 95%, and large effect size were observed, which strongly supports the translation potential of MSC therapy for ischemic stroke.

Btn2a2, a T cell immunomodulatory molecule coregulated with MHC class II genes.

Evidence has recently emerged that butyrophilins, which are members of the extended B7 family of co-stimulatory molecules, have diverse functions in the immune system. We found that the human and mouse genes encoding butyrophilin-2A2 (BTN2A2) are regulated by the class II trans-activator and regulatory factor X, two transcription factors dedicated to major histocompatibility complex class II expression, suggesting a role in T cell immunity. To address this, we generated Btn2a2-deficient mice. Btn2a2(-/-) mice exhibited enhanced effector CD4(+) and CD8(+) T cell responses, impaired CD4(+) regulatory T cell induction, potentiated antitumor responses, and exacerbated experimental autoimmune encephalomyelitis. Altered immune responses were attributed to Btn2a2 deficiency in antigen-presenting cells rather than T cells or nonhematopoietic cells. These results provide the first genetic evidence that BTN2A2 is a co-inhibitory molecule that modulates T cell-mediated immunity.

γ-Secretase Activity Is Required for Regulated Intramembrane Proteolysis of Tumor Necrosis Factor (TNF) Receptor 1 and TNF-mediated Pro-apoptotic Signaling.

The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.

Embolization of the Device to the Left Pulmonary Artery after the Interventional Closure of Ruptured Sinus of Valsalva Aneurysm.

Formation of an aneurysm in the sinus of Valsalva of the aortic root is usually due to an area of congenital weakness in its wall. This aneurysm may progressively dilate and rupture into any of the cardiac chambers or into the pericardial cavity. Though this is conventionally treated by surgery, interventional therapy using various closure devices is becoming more common. Embolization of these closure devices may occur. We report a case of embolization of such a device into the left pulmonary artery which during surgical retrieval, unmasked the hidden ventricular septal defect (VSD). Therefore one has to be cautious while making a diagnosis of rupture of the sinus of Valsalva of right coronary sinus without VSD.

Potential role of increased oxygenation in altering perinatal adrenal steroidogenesis.

BACKGROUND: At birth, the large fetal adrenal involutes rapidly, and the patterns of steroidogenesis change dramatically; the event(s) triggering these changes remain largely unexplored. Fetal abdominal viscera receive hypoxic blood having a partial pressure of oxygen of only ~2 kPa (20-23 mm Hg); perinatal circulatory changes change this to adult values (~20 kPa). We hypothesized that transition from fetal hypoxia to postnatal normoxia participates in altering perinatal steroidogenesis. METHODS: We grew midgestation human fetal adrenal cells and human NCI-H295A adrenocortical carcinoma cells in 2% O2, then transitioned them to 20% O2 and quantitated steroidogenic mRNAs by quantitative PCR and microarrays. RESULTS: Transitioning fetal adrenal cells from hypoxia to normoxia increased mRNAs for 17α-hydroxylase/17,20 lyase (P450c17), 3ß-hydroxysteroid dehydrogenase (3ßHSD2), and steroidogenic acute regulatory protein (StAR). We repeated the protocol with NCI-H295A cells acclimated to hypoxia for 15 d, quantitating 31,255 transcripts by microarray. Using an arbitrary 1.5-fold difference, 1 d of normoxia increased 4 transcripts and decreased 56, whereas 2 d of normoxia increased 62 transcripts and decreased 105. P450c17, 3ßHSD2, and StAR were ranked among the top eight increased transcripts. CONCLUSION: These data suggest that the hypoxic/normoxic transition at birth contributes to perinatal changes in adrenal steroidogenesis.

Extraadrenal 21-hydroxylation by CYP2C19 and CYP3A4: effect on 21-hydroxylase deficiency.

CONTEXT: 21-Hydroxylase deficiency (21OHD) is caused by CYP21A2 gene mutations disrupting the adrenal 21-hydroxylase, P450c21. CYP21A2 mutations generally correlate well with the 21OHD phenotype, but some children with severe CYP21A2 mutations have residual 21-hydroxylase activity. Some hepatic P450 enzymes can 21-hydroxylate progesterone, but their physiological relevance in modifying 21OHD is not known. OBJECTIVE: We determined the ability of CYP2C19 and CYP3A4 to 21-hydroxylate progesterone and 17-hydroxyprogesterone (17OHP), determined the impact of the common P450 oxidoreductase (POR) variant A503V on these activities, and examined correlations between CYP2C19 variants and phenotype in patients with 21OHD. METHODS: Bacterially expressed, N-terminally modified, C-His-tagged human P450c21, CYP2C19, and CYP3A4 were combined with bacterially expressed wild-type and A503V POR. The 21-hydroxylation of radiolabeled progesterone and 17OHP was assessed, and the Michaelis constant (Km) and maximum velocity (Vmax) of the reactions were measured. CYP2C19 was genotyped in 21OHD patients with genotypes predicting severe congenital adrenal hyperplasia. RESULTS: Compared to P450c21, the Vmax/Km for 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 were 17 and 10%, respectively. With both forms of POR, the Km for P450c21 was approximately 2.6 microm, the Km for CYP2C19 was approximately 11 microm, and the Km for CYP3A4 was approximately 110 microm. Neither CYP2C19 nor CYP3A4 could 21-hydroxylate 17OHP. The CYP2C19 ultrametabolizer allele CYP2C19 17 was homozygous in one of five patients with a 21OHD phenotype that was milder than predicted by the CYP21A2 genotype. CONCLUSIONS: CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not 17OHP, possibly ameliorating mineralocorticoid deficiency, but not glucocorticoid deficiency. Multiple enzymes probably contribute to extraadrenal 21-hydroxylation.

P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia.

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

Seat belt-induced chylothorax: a cause of idiopathic chylothorax?

Chylothoraces are associated with multiple etiologies including non-Hodgkin lymphoma and surgical trauma, representing 50% and 25% of all chylothoraces, respectively. Intrathoracic operations such as repair of coarctation of the aorta and esophagectomy are commonly associated with surgical trauma. Idiopathic chylothoraces may account for up to 15% of all chylothoraces. When a thorough evaluation finding is negative, further history to identify possible blunt, nonpenetrating trauma to the chest is warranted.