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Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries., (© 2024. The Author(s).)

Photocatalytic reduction of CO 2 is a promising strategy to mitigate the effects of global warming by converting CO 2 into valuable energy-dense products. Silver bismuth iodide (SBI) is an attractive material owing to its tunable bandgap and favorable band-edge positions for efficient CO 2 photoreduction. In this study, SBI materials, including AgBi 2 I 7 , AgBiI 4 , Ag 2 BiI 5 , and Ag 3 BiI 6 are first synthesized, through gas-solid reaction by controlling the stoichiometric ratio of reactants. The X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) results revealed that the distance between Ag-I is proportional to the degree of Ag ions delocalization, which occupies the vacant sites. That greatly retards the charge recombination at vacant sites. In addition, the surface potential via photo-assisted Kelvin probe force measurements of various SBI catalysts shows that Ag 3 BiI 6 exhibits the highest surface potential change due to the rich delocalized Ag ions. This results in effective charge carrier transport and prevention of charge recombination at vacant sites. Taking the above advantages, the averaged CO and CH 4 production rates for Ag 3 BiI 6 achieved 0.23 and 0.10 µmol g -1  h -1 , respectively. The findings suggest that Ag 3 BiI 6 has a high potential as a novel photocatalyst for CO 2 reduction and sheds light on the possibility of solving environmental contamination and sustainable energy crises., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Abstract: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

Background: The CDC and ACIP recommend COVID-19 vaccination for patients with inborn errors of immunity (IEI). Not much is known about vaccine safety in IEI, and whether vaccination attenuates infection severity in IEI., Objective: To estimate COVID-19 vaccination safety and examine effect on outcomes in patients with IEI., Methods: We built a secure registry database in conjunction with the US Immunodeficiency Network to examine vaccination frequency and indicators of safety and effectiveness in IEI patients. The registry opened on January 1, 2022, and closed on August 19, 2022., Results: Physicians entered data on 1245 patients from 24 countries. The most common diagnoses were antibody deficiencies (63.7%). At least one COVID-19 vaccine was administered to 806 patients (64.7%), and 216 patients received vaccination prior to the development of COVID-19. The most common vaccines administered were mRNA-based (84.0%). Seventeen patients were reported to seek outpatient clinic or emergency room care for a vaccine-related complication, and one patient was hospitalized for symptomatic anemia. Eight hundred twenty-three patients (66.1%) experienced COVID-19 infection. Of these, 156 patients required hospitalization (19.0%), 47 required ICU care (5.7%), and 28 died (3.4%). Rates of hospitalization (9.3% versus 24.4%, p < 0.001), ICU admission (2.8% versus 7.6%, p = 0.013), and death (2.3% versus 4.3%, p = 0.202) in patients who had COVID-19 were lower in patients who received vaccination prior to infection. In adjusted logistic regression analysis, not having at least one COVID-19 vaccine significantly increased the odds of hospitalization and ICU admission., Conclusion: Vaccination for COVID-19 in the IEI population appears safe and attenuates COVID-19 severity., (© 2024. The Author(s).)

The synthesis and characterization of a linear two-coordinate Cr(II) amido complex, Cr{N( t Bu)Dipp} 2 (Dipp=2,6-diisopropylphenyl), from the reaction of 1 molar equivalent (equiv) of CrCl 2 and 2 equiv. of LiN( t Bu)Dipp is reported. Single-crystal X-ray diffractometry (SC-XRD) analysis revealed that it has a short Cr-N bond distance of 1.8878(9) Å, which could be attributed to the relatively less bulky nature of the amido ligand compared with reported systems. Furthermore, the oxidation reaction of the two-coordinate Cr(II) complex was explored. The oxidation reaction of Cr{N( t Bu)Dipp} 2 with the one-electron oxidants AgOTf and [FeCp 2 ][BAr F 4 ] (BAr F 4 - =[B{C 6 H 3 -3,5-(CF 3 ) 2 } 4 ] - ) afforded the trigonal planar three- and bent two-coordinate Cr(III) complexes Cr{N( t Bu)Dipp} 2 (OTf) and [Cr{N( t Bu)Dipp} 2 ][BAr F 4 ], respectively. The reaction of Cr{N( t Bu)Dipp} 2 with 1 equiv. of the organic azides AdN 3 (Ad=1-adamantyl) and PhN 3 afforded the three-coordinate Cr(IV) imido complexes Cr{N( t Bu)Dipp} 2 (NAd) and Cr{N( t Bu)Dipp} 2 (NPh), respectively. The reaction of Cr{N( t Bu)Dipp} 2 and two equiv. of Me 3 NO afforded the Cr(VI) dioxo complex Cr{N( t Bu)Dipp} 2 (O) 2 . The reaction of Cr{N( t Bu)Dipp} 2 with 1 equiv. of CyN=C=NCy resulted in the insertion of the carbodiimide into the Cr-N bond, with the formation of a three-coordinate Cr(II) complex. Finally, density functional theory (DFT) calculations were used to elucidate the electronic structure of these complexes., (© 2023 Wiley-VCH GmbH.)

Mutations of the human interferon alpha and beta receptor subunit 1 (IFNAR1) gene are associated with severe viral infections. Individuals homozygous for the Glu386∗ variant have impaired type I interferon signalling and can suffer severe illness when exposed to certain viruses and live attenuated virus vaccines. Glu386∗ heterozygotes are clinically unaffected, but can pass the variant allele to their descendants. We aimed to develop an assay that can identify IFNAR1 Glu386∗ homozygotes and heterozygotes to support urgent clinical diagnosis, and that can use dried blood spots (DBS) sent at ambient temperature to overcome geographical logistical challenges in the South Pacific region. The tri-allelic genotyping assay interrogates a single nucleotide polymorphism (rs201609461) located in IFNAR1. The reference allele G encodes for wild-type IFNAR1. Minor alleles A (c.1156G>A) and T (c.1156G>T) encode for Glu386Lys and a truncated IFNAR1 protein (p.Glu386∗), respectively. Synthetic oligonucleotides were mixed in equal molar ratio to create six different genotypes which were randomly assigned to 960 genotyping reactions by R software. Three different fluorescence probes were designed to discriminate the three alleles (G, T and A) within a pair of flanking primers in a single genotyping reaction. The assay discriminated all three alleles using DBS from Guthrie cards randomly spiked with synthetic oligonucleotides. We correctly identified all the different genotypes in 960 reactions in these blinded experiments. These findings validate the genotyping assay for rapidly identifying the IFNAR1 Glu386∗ variant from DBS., (Copyright © 2023 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Background: Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC)., Methods: Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein-protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients., Results: We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC., Conclusions: Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract., (© 2023. The Author(s).)

Human-exhaled breath mainly contains water, oxygen, carbon dioxide, and endogenous gases closely related to human metabolism. The linear relationship between breath acetone and blood glucose concentration has been revealed when monitoring diabetes patients. Considerable attention has been directed toward developing a highly sensitive volatile organic compounds (VOCs) sensing material that can detect breath acetone. In this study, we propose a tungsten oxide/tin oxide/silver/poly (methyl methacrylate) (WO 3 /SnO 2 /Ag/PMMA) sensing material fabricated using the electrospinning technique. By monitoring the evolution of sensing materials' extinction spectra, low concentrations of acetone vapor can be detected. Moreover, the interfaces between SnO 2 and WO 3 nanocrystals construct n-n junctions, which generate more electron-hole pairs than those without such structure when the light strikes. This helps to improve the sensitivity of sensing materials when they are subjected to acetone surroundings. The established sensing materials (WO 3 /SnO 2 /Ag/PMMA) exhibit a sensing limit of 20 ppm for acetone vapor and show specificity for acetone even in ambient humidity.

A Z-scheme heterojunction with spatially separated cocatalysts is proposed for overcoming fundamental issues in photocatalytic water splitting, such as inefficient light absorption, charge recombination, and sluggish reaction kinetics. For efficient light absorption and interfacial charge separation, Z-scheme organic/inorganic heterojunction photocatalysts are synthesized by firmly immobilizing ultrathin g-C 3 N 4 on the surface of TiO 2 hollow spheres via electrostatic interactions. Additionally, two cocatalysts, Pt and IrO x , are spatially separated along the Z-scheme charge-transfer pathway to enhance surface charge separation and reaction kinetics. The as-prepared Pt/g-C 3 N 4 /TiO 2 /IrO x (PCTI) hollow sphere photocatalyst exhibits an exceptional H 2 evolution rate of 8.15 mmol h -1 g -1 and a remarkable apparent quantum yield of 24.3% at 330 nm in the presence of 0.5 wt% Pt and 1.2 wt% IrO x cocatalysts on g-C 3 N 4 and TiO 2 , respectively. Photoassisted Kelvin probe force microscopy is used to systematically analyze the Z-scheme charge-transfer mechanism within PCTI. Furthermore, the benefits of spatially separating cocatalysts in the PCTI system are methodically investigated in comparison to randomly depositing them. This work adequately demonstrates that the combination of a Z-scheme heterojunction and spatially separated cocatalysts can be a promising strategy for designing high-performance photocatalytic platforms for solar fuel production., (© 2022 Wiley-VCH GmbH.)

Umbilical cord blood cells have therapeutic potential for neurological disorders, through a paracrine mechanism of action. A greater understanding of the safety and immunological effects of allogeneic donor cord blood cells in the context of a healthy recipient immune system, such as in cerebral palsy, is needed. This study aimed to determine how quickly donor cord blood cells were cleared from the circulation in children with cerebral palsy who received a single intravenous infusion of 12/12 human leucocyte antigen (HLA)-matched sibling cord blood cells. Twelve participants with cerebral palsy aged 2-12 years received cord blood cell infusions as part of a phase I trial of umbilical blood infusion for cerebral palsy. Digital droplet PCR analysis of DNA copy number variants specific to donor and recipient was used to assess donor DNA clearance at five timepoints post-infusion, a surrogate measure of cell clearance. Donor cells were cleared by 3 months post-infusion in 11/12 participants. When detected, donor DNA was at a fraction of 0.01-0.31% of total DNA with no signs of graft-versus-host disease in any participant. The donor DNA clearance times provided by this study have important implications for understanding the safety of allogeneic cord blood cell infusion for cerebral palsy and translational tissue engineering or regenerative medicine research in other disorders., (© 2022 S. Karger AG, Basel.)

High absorption ability and direct bandgap makes lead-based perovskite to acquire high photovoltaic performance. However, lead content in perovskite becomes a double-blade for counterbalancing photovoltaic performance and sustainability. Herein, we develop a methylammonium bismuth iodide (MBI), a perovskite-derivative, to serve as a lead-free light absorber layer. Owing to the short carrier diffusion length of MBI, its film quality is a predominant factor to photovoltaic performance. Several candidates of non-polar solvent are discussed in aspect of their dipole moment and boiling point to reveal the effects of anti-solvent assisted crystallization. Through anti-solvent engineering of toluene, the morphology, crystallinity, and element distribution of MBI films are improved compared with those without toluene treatment. The improved morphology and crystallinity of MBI films promote photovoltaic performance over 3.2 times compared with the one without toluene treatment. The photovoltaic device can achieve 0.26% with minor hysteresis effect, whose hysteresis index reduces from 0.374 to 0.169. This study guides a feasible path for developing MBI photovoltaics.

Cerebral palsy (CP) is a nonprogressive neurological disorder and the most common physical disability of childhood. There is no cure for CP, but stem cells have the potential to improve brain injury and hence function. This phase 1 clinical trial investigated the safety of the intravenous infusion of full-matched sibling cord blood cells for children with CP aged 1 to 16 years. Preliminary efficacy outcomes were also investigated. Twelve participants received 12/12 HLA-matched sibling cord blood cell infusions. One treatable serious adverse reaction to cryoprotectant was observed, and no adverse reactions occurred beyond 24 h after infusion. Gross motor function measure (GMFM-66) scores did not improve compared with baseline beyond what could be expected from developmental levels, and participants had varied changes in the Quality of Upper Extremity Skills Test (QUEST) and Vineland Adaptive Behavior Scales (VABS-II) scores. In conclusion, matched sibling cord blood cell infusion for children with CP is relatively safe when conducted in an appropriate facility. Australian and New Zealand Clinical Trials Registry (ACTRN12616000403437) and Clinicaltrials.gov (NCT03087110)., Competing Interests: Declaration of Competing Interest Cell Care Australia is a private cord blood bank with a representative on the Trial Steering Committee. There is, therefore, a potential conflict of interest which has been declared to HREC and Steering Committee and is well recognized. No one affiliated with Cell Care Australia was involved in data analysis or interpretation., (Copyright © 2022 International Society for Cell & Gene Therapy. All rights reserved.)

Globally, autosomal recessive IFNAR1 deficiency is a rare inborn error of immunity underlying susceptibility to live attenuated vaccine and wild-type viruses. We report seven children from five unrelated kindreds of western Polynesian ancestry who suffered from severe viral diseases. All the patients are homozygous for the same nonsense IFNAR1 variant (p.Glu386*). This allele encodes a truncated protein that is absent from the cell surface and is loss-of-function. The fibroblasts of the patients do not respond to type I IFNs (IFN-α2, IFN-ω, or IFN-β). Remarkably, this IFNAR1 variant has a minor allele frequency >1% in Samoa and is also observed in the Cook, Society, Marquesas, and Austral islands, as well as Fiji, whereas it is extremely rare or absent in the other populations tested, including those of the Pacific region. Inherited IFNAR1 deficiency should be considered in individuals of Polynesian ancestry with severe viral illnesses., (© 2022 Bastard et al.)

Introduction: Probiotic and Peanut Oral Immunotherapy (PPOIT) is effective at inducing sustained unresponsiveness (SU) at end-of-treatment and this effect persists up to four years post-treatment, referred to as persistent SU. We sought to evaluate (i) how PPOIT altered peanut-specific humoral immune indices, and (ii) how such longitudinal indices relate to persistent SU., Methods: Longitudinal serum/plasma levels of whole peanut- and peanut component- (Ara-h1, -h2, -h3, -h8, -h9) specific-IgE (sIgE) and specific-IgG4 (sIgG4) antibodies were measured by ImmunoCAP and salivary peanut-specific-IgA (sIgA) by ELISA in children (n=62) enrolled in the PPOIT-001 randomised trial from baseline (T0) to 4-years post-treatment (T5). Multivariate regression analyses of log-transformed values were used for point-in-time between group comparisons. Generalised estimating equations (GEE) were used for longitudinal comparisons between groups., Results: PPOIT was associated with changes in sIgE and sIgG4 over time. sIgE levels were significantly reduced post-treatment [T5, PPOIT v.s. Placebo ratio of geometric mean (GM): Ara-h1 0.07, p=0.008; Ara-h2 0.08, p=0.007; Ara-h3 0.15, p=0.021]. sIgG4 levels were significantly increased by end-of-treatment (T1, PPOIT v.s. Placebo ratio of GM: Ara-h1 3.77, p=0.011; Ara-h2 17.97, p<0.001; Ara-h3 10.42, p<0.001) but levels in PPOIT group decreased once treatment was stopped and returned to levels comparable with Placebo group by T5. Similarly, salivary peanut sIgA increased during treatment, as early as 4 months of treatment (PPOIT v.s. Placebo, ratio of GM: 2.04, p=0.014), then reduced post-treatment., Conclusion: PPOIT was associated with broad reduction in peanut specific humoral responses which may mediate the clinical effects of SU that persists to 4-years post-treatment., (This article is protected by copyright. All rights reserved.)

Background : The microarray analysis of whole-genome expression indicated that the gene encoding the protein lumican, which is associated with extracellular matrix (ECM) interaction, was highly expressed in osteotropic lung cancer cell lines with an enhanced capacity of bone metastasis. Methods : The expression of lumican in the osteotropic lung cancer cells was downregulated, and the in vitro migration, invasion, and adhesion of cancer cells to ECM components, and the in vivo bone metastasis capacity of these cells were examined. Exogenous lumican was provided to study the autocrine regulation mechanism of lumican in the bone metastasis of lung cancer cells. Results : Transfection with lumican-specific short hairpin RNA (shRNA) in the osteotropic lung cancer cells reduced the establishment of in vivo bone metastasis, but not lung metastasis. Reduction in the expression of lumican also decreased the attachment of lung osteotropic cancer cells to several components of the ECM and suppressed cell migration and invasion in vitro. Exogenous lumican restored these reduced capacities of lumican knockdown cells and promoted the seeding of lung cancer cells in the bone microenvironment. Conclusions : These results suggested that lumican promotes the metastasis of lung cancer cells to the bones via an autocrine regulatory mechanism, and blocking this interaction may provide a new therapeutic approach to reduce bone metastasis in cases of lung cancer.

Background: We previously reported that probiotic and peanut oral immunotherapy (PPOIT) was effective at inducing sustained unresponsiveness compared with placebo in a double-blind, placebo-controlled randomized trial. This study evaluated the impact of PPOIT on health-related quality of life (HRQL)., Method: Fifty-one participants (PPOIT 24; placebo 27) from the PPOIT trial completed Food Allergy Quality of Life Questionnaire (FAQLQ-PF) and Food Allergy Independent Measure (FAIM) at pre-treatment, end-of-treatment and 3 months after end-of-treatment. A total of 42 participants (20 PPOIT; 22 placebo) completed measures at 12 months post-treatment. Changes over time in PPOIT and placebo groups were examined by repeated-measures analysis of variance and paired t tests., Results: Probiotic and peanut oral immunotherapy was associated with significant improvement in FAQLQ-PF (F = 3.63, P = .02), with mean difference 0.8 at 3 months post-treatment (P = .05) and 1.3 at 12 months post-treatment (P = .005), exceeding the 0.5 minimal clinically important difference for FAQLQ-PF. For FAIM, mean difference was 0.5 (P = .03) at 3 months and 0.4 (P = .04) at 12 months post-treatment. In placebo group, post-treatment FAQLQ and FAIM remained unchanged from pretreatment. Improvement in FAQLQ-PF and FAIM scores related specifically to acquisition of sustained unresponsiveness rather than to receiving PPOIT treatment or participation in the trial., Conclusions: Probiotic and peanut oral immunotherapy has a sustained beneficial effect on psychosocial impact of food allergy at 3 and 12 months after end-of-treatment. Treatment was not associated with reduced HRQL relative to baseline in either PPOIT or placebo groups, indicating that PPOIT was well tolerated and psychological well-being was not negatively impacted. Improved HRQL was specifically associated with acquisition of sustained unresponsiveness., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Oral immunotherapy has attracted much interest as a potential treatment for food allergy, yet little is known about its long-term effects. We aimed to assess long-term outcomes in participants who completed a randomised, double-blind, placebo-controlled trial of combined probiotic and peanut oral immunotherapy (PPOIT), which was previously shown to induce desensitisation and 2-week sustained unresponsiveness., Methods: All participants who completed the PPOIT randomised trial were eligible to participate in this follow-up study 4 years after treatment cessation. Peanut intake and adverse reactions to peanut in the 4 years after treatment cessation were systematically documented with a structured questionnaire administered by allergy nurses. Additionally, participants were invited to undergo peanut skin prick tests, measurement of peanut sIgE and sIgG4 concentrations, and double-blind placebo-controlled peanut challenge to assess 8-week sustained unresponsiveness., Findings: 48 (86%) of 56 eligible participants were enrolled in the follow-up study. Mean time since stopping treatment was 4·2 years in both PPOIT (SD 0·6) and placebo (SD 0·7) participants. Participants from the PPOIT group were significantly more likely than those from the placebo group to have continued eating peanut (16 [67%] of 24 vs one [4%] of 24; absolute difference 63% [95% CI 42-83], p=0·001; number needed to treat 1·6 [95% CI 1·2-2·4]). Four PPOIT-treated participants and six placebo participants reported allergic reactions to peanut after intentional or accidental intake since stopping treatment, but none had anaphylaxis. PPOIT-treated participants had smaller wheals in peanut skin prick test (mean 8·1 mm [SD 7·7] vs 13·3 mm [7·6]; absolute difference -5·2 mm [95% CI -10·3 to 0·0]; age-adjusted and sex-adjusted p=0·035) and significantly higher peanut sIgG4:sIgE ratios than placebo participants (geometric mean 67·3 [95% CI 10·3-440·0] vs 5·2 [1·2-21·8]; p=0·031). Seven (58%) of 12 participants from the PPOIT group attained 8-week sustained unresponsiveness, compared with one (7%) of 15 participants from the placebo group (absolute difference 52% [95% CI 21-82), p=0·012; number needed to treat 1·9 [95% CI 1·2-4·8])., Interpretation: PPOIT provides long-lasting clinical benefit and persistent suppression of the allergic immune response to peanut., Funding: Murdoch Childrens Research Institute and Australian Food Allergy Foundation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The benefit of reducing the risk of stroke against increasing the risk of renal progression associated with antiplatelet therapy in patients with advanced chronic kidney disease (CKD) is controversial., Methods: We enrolled 1301 adult patients with advanced CKD treated with erythropoiesis stimulating agents from January 1, 2002 to June 30, 2009 from the 2005 Longitudinal Health Insurance Database in Taiwan. All of the patients were followed until the development of the primary or secondary endpoints, or the end of the study (December 31, 2011). The primary endpoint was the development of ischemic stroke, and the secondary endpoints included hospitalization for bleeding events, cardiovascular mortality, all-cause mortality, and renal failure. The adjusted cumulative probability of events was calculated using multivariate Cox proportional regression analysis., Results: Adjusted survival curves showed that the usage of aspirin was not associated with ischemic stroke, hospitalization for bleeding events, cardiovascular mortality or all-cause mortality, however, it was significantly associated with renal failure. In subgroup analysis, aspirin use was associated with renal failure in the patients with no history of stroke (HR, 1.41; 95% CI, 1.14-1.73), and there was a borderline interaction between previous stroke and the use of aspirin on renal failure (interaction p=0.0565)., Conclusions: There was no significant benefit in preventing ischemic stroke in the patients with advanced CKD who received aspirin therapy. Furthermore, the use of aspirin was associated with the risk of renal failure in the patients with advanced CKD without previous stroke., (Copyright © 2016 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

In situ neutron diffraction was used to characterize the effect of temperature on the crystal structure of LiNiPO 4 . LiNiPO 4 adopts an ordered olivine structure at room temperature, but, with increasing temperature, this work shows that a significant amount of Li and Ni cation exchange occurs, for example, ∼15% at 900 °C. The antisite disorder is detected by residual nuclear densities on the M1 and M2 octahedral sites in the olivine structure using difference Fourier maps and by changes in cation site occupancies, lattice parameters, and mean ⟨M-O⟩ bond distances. The antisite disorder is also responsible for chemical expansion of the crystal lattice in addition to thermal expansion. Antisite defect formation at high temperature and its reversibility on cooling can be understood as an entropically driven feature of the crystal structure of LiNiPO 4 . The lithium ion diffusion pathway, that follows a curved trajectory along the b axis in the olivine structure, is, therefore, susceptible to be blocked if synthesis conditions are not carefully controlled and should also be influenced by the chemically expanded lattice of the disordered structure if this is preserved to ambient temperature by rapid cooling.

Hydrogenated titanium dioxide has attracted intensive research interests in pollutant removal applications due to its high photocatalytic activity. Herein, we demonstrate hydrogenated TiO 2 nanofibers (H:TiO 2 NFs) with a core-shell structure prepared by the hydrothermal synthesis and subsequent heat treatment in hydrogen flow. H:TiO 2 NFs has excellent solar light absorption and photogenerated charge formation behavior as confirmed by optical absorbance, photo-Kelvin force probe microscopy and photoinduced charge carrier dynamics analyses. Photodegradation of various organic dyes such as methyl orange, rhodamine 6G and brilliant green is shown to take place with significantly higher rates on our novel catalyst than on pristine TiO 2 nanofibers and commercial nanoparticle based photocatalytic materials, which is attributed to surface defects (oxygen vacancy and Ti 3+ interstitial defect) on the hydrogen treated surface. We propose three properties/mechanisms responsible for the enhanced photocatalytic activity, which are: (1) improved absorbance allowing for increased exciton generation, (2) highly crystalline anatase TiO 2 that promotes fast charge transport rate, and (3) decreased charge recombination caused by the nanoscopic Schottky junctions at the interface of pristine core and hydrogenated shell thus promoting long-life surface charges. The developed H:TiO 2 NFs can be helpful for future high performance photocatalysts in environmental applications.

Background: We evaluated the ability of matrix metalloproteinase (MMP)-2, MMP-9, myeloperoxidase, osteopontin and stromal cell-derived factor 1 to predict mortality in hemodialysis (HD) patients., Methods: One hundred forty HD patients were enrolled and followed from December 2007 until December 2012. At the end of this 5-year period, data were compared between the patients who were alive and those who had died., Results: The patients who alive were younger (56 vs. 63y), with lower frequency of diabetes mellitus (34.34% vs. 58.53%), higher concentrations of albumin (4.13 vs. 3.91mg/dl) and lower concentrations of MMP-2 (430.76 vs. 521.59ng/ml). Multivariate analysis showed that age (HR=1.03, p=0.02), diabetes mellitus (HR=2.395, p=0.012), albumin (HR=0.475, p=0.047) and MMP-2 (HR=1.003, p=0.005) were independent factors predicting mortality in HD patients. Receiver operating characteristic curve analysis showed that albumin (AUC=0.628, p=0.027) and MMP-2 (AUC=0.643, p=0.004) had a similar ability (p=0.76) to predict survival of HD patients., Conclusions: Compared with albumin, serum MMP-2 is a non-inferior prognostic marker for predicting the survival of HD patients., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Our previous studies suggest that antibodies against ENO1 (anti-ENO1 Ab) have a protective role in patients with non-small cell lung carcinoma. In this study, we evaluated the prognostic value of anti-ENO1 Ab levels in non-small cell lung carcinoma patients undergoing surgery. Circulating levels of anti-ENO1 Ab were assessed in 85 non-small cell lung carcinoma patients before and after surgery, and were correlated with clinical outcome. After surgery, patients with a higher increase of anti-ENO1 Ab had a lower hazard ratio and a better progression-free survival. Using animal models, we demonstrated that tumor cells reduce the circulating levels of anti-ENO1 Ab through physical absorption and neutralization of anti-ENO1 Ab with surface-expressed and secreted ENO1, respectively. Mice transplanted with ENO1-overexpressing tumors generated ENO1-specific regulatory T cells to suppress the production of anti-ENO1 Ab. Our results suggest that the increase of anti-ENO1 Ab may reflect anti-tumor immune responses and serve as a prognostic marker in postoperative lung cancer patients.

Pediatric patients with severe or nonsevere combined immunodeficiency have increased susceptibility to severe, life-threatening infections and, without hematopoietic stem cell transplantation, may fail to thrive. A subset of these patients have the radiosensitive (RS) phenotype, which may necessitate conditioning before hematopoietic stem cell transplantation, and this conditioning includes radiomimetic drugs, which may significantly affect treatment response. To provide statistical criteria for classifying cellular response to ionizing radiation as the measure of functional RS screening, we analyzed the repair capacity and survival of ex vivo irradiated primary skin fibroblasts from five dysmorphic and/or developmentally delayed pediatric patients with severe combined immunodeficiency and combined immunodeficiency. We developed a mathematical framework for the analysis of γ histone 2A isoform X foci kinetics to quantitate DNA-repair capacity, thus establishing crucial criteria for identifying RS. The results, presented in a diagram showing each patient as a point in a 2D RS map, were in agreement with findings from the assessment of cellular RS by clonogenic survival and from the genetic analysis of factors involved in the nonhomologous end-joining repair pathway. We provide recommendations for incorporating into clinical practice the functional assays and genetic analysis used for establishing RS status before conditioning. This knowledge would enable the selection of the most appropriate treatment regimen, reducing the risk for severe therapy-related adverse effects., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Aim: To investigate the comorbid disease could be the predictors for the elective colectomy in colonic diverticulitis., Methods: A retrospective chart review of 246 patients with colonic diverticulitis admitted between 2000 and 2008 was conducted, and 19 patients received emergent operation were identified and analyzed. Data were collected with regard to age, sex, albumin level on admission, left or right inflammation site, the history of recurrent diverticulitis, preoperative comorbidity, smoking habits, medication, treatment policy, morbidity, and mortality. Preoperative comorbid diseases included cardiovascular disease, diabetes, pulmonary disease, peptic ulcer disease, gouty arthritis, and uremia. Medications in use included non-steroidal anti-inflammatory drugs, acetylsalicylic acid (Aspirin), and corticosteroids. Univariate and multivariate logistic regression analyses were performed to identify the relevant risk factors correlating to colectomy., Results: The mean age of the 246 patients was 69.5 years (range, 24-94 years). Most diverticulitis could be managed with conservative treatment (n = 227, 92.3%), and urgent colectomy was performed in 19 patients (7.7%). There were three deaths in the surgical group and four deaths in the nonsurgical group. The overall mortality rate in the study was 1.7% among patients with conservative treatment and 15.7% among patients undergoing urgent colectomy. Multiple logistic regression analysis indicated that comorbidities were risk factors for urgent colectomy for diverticulitis., Conclusion: To avoid high mortality and morbidity related to urgent colectomy, we suggest that patients with colonic diverticulitis and comorbid diseases may require elective colectomy.

In previous research, we found α-enolase to be inversely correlated with progression-free and overall survival in lung cancer patients and detected α-enolase on the surface of lung cancer cells. Based on these findings, we hypothesized that surface α-enolase has a significant role in cancer metastasis and tested this hypothesis in the current study. We found that α-enolase was co-immunoprecipitated with urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen in lung cancer cells and interacted with these proteins in a cell-free dot blotting assay, which can be interrupted by α-enolase-specific antibody. α-Enolase in lung cancer cells co-localized with these proteins and was present at the site of pericellular degradation of extracellular matrix components. Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion. Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells. Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone. This study demonstrated that surface α-enolase promotes extracellular matrix degradation and invasion of cancer cells and that targeting surface α-enolase is a promising approach to suppress tumor metastasis.

Aim: To study potential predictive factors for early radical resection in two-stage resection for left malignant colonic obstruction., Methods: Thirty-eight cases of left-sided obstructive colon cancer undergoing two-stage operations were reviewed between January 1998 and August 2008. Patients were classified into two groups (n = 19 each): early radical resection (interval ≤ 10 d) and late radical resection (interval > 10 d). Baseline demographics, post-diversion outcome, perioperative data, tumor characteristics, outcome and complications were analyzed., Results: The baseline demographics revealed no differences except for less pre-diversion sepsis in the early group (P < 0.001) and more obstruction days in the late group (P = 0.009). The mean intervals of early and late radical resections were 7.9 ± 1.3 d and 17.8 ± 5.5 d, respectively (P < 0.001). After diversion, the presence of bowel sounds, flatus, removal of the nasogastric tube and the resumption of oral feeding occurred earlier in the early group. The operation time and duration of hospital stay were both significant reduced in the early group. Complication rates did not differ between groups., Conclusion: The earlier recovery of bowel function seems to be predictive of early radical resection. In contrast, pre-diversion sepsis and more obstruction days were predictive of delayed radical resection.

Purpose: Current medical treatments for slow transit constipation (STC) are often ineffective, and total colectomy with ileorectal anastomosis has been the procedure of choice for selected patients with refractory STC. Today, minimally invasive approaches are being utilized in a greater number of procedures as surgeons become more familiar with the techniques involved. The aim of this study was to assess the safety and utility of hand-assisted laparoscopic total colectomy for STC., Method: From January 2002 to December 2005, 44 women presented with complaints of intractable constipation and failed to respond to medical treatment. Slow transit constipation was diagnosed after a series of examinations, including a colonic transit test, anal manometry, balloon expulsion test, and barium enema. All eligible patients underwent a hand-assisted laparoscopic total colectomy with ileorectal anastomosis. Main outcome measures included the operative time, conversion to open procedure, blood loss, time to return of flatus, length of postoperative hospital stay, and complications., Result: The mean operative time was 197 min (range, 125-295 min). The mean estimated blood loss was 113 ml (range, 100-300 ml). The mean day of first time to flatus was 2 days, and the mean hospital stay was 7.6 days. There was no conversion to an open procedure and no surgical mortality. In the following period, two patients developed intestinal obstruction, which underwent exploratory laparotomy. However, some 39 patients (88.6%) expressed excellent or good in satisfaction., Conclusion: Hand-assisted laparoscopic total colectomy could be a safe and efficient technique in the treatment of STC.

Background and Purpose: Laparoscopic sacrocolpopexy (LSCP) offers a minimally invasive treatment for vaginal vault prolapse. We describe the surgical technique and offer insight into the learning curve. In addition, we performed a case series review comparing the laparoscopic procedure with its open surgical counterpart with respect to various demographic and perioperative parameters., Patients and Methods: The Institutional Review Board-approved continence database at our institution was queried to identify all patients undergoing sacrocolpopexy between August 1999 and October 2004. The LSCP was performed in 25 patients, and open abdominal sacrocolpopexy (ASCP) was performed in 22 patients. Data were analyzed using Student's t-test and the Fisher exact test., Results: No significant difference was observed in the demographic characteristics of the patients undergoing the two approaches. The mean estimated blood loss (P = 0.0002) and mean length of hospitalization (P < 0.0001) were significantly less for LSCP, whereas the operative time was significantly longer (219.9 minutes v 185.2 minutes; P = 0.045). The success rate for LSCP at 5.9 months was 100%; the ASCP success rate at 11.0 months was 95%., Conclusions: Laparoscopic sacrocolpopexy led to shorter hospitalization, better hemostasis, and less pain than the open procedure. Early follow-up suggests that LSCP is as effective as ASCP for the treatment of vaginal vault prolapse.

Objectives: To describe a rectocele repair reinforced with solvent-dehydrated, gamma-irradiated, human fascia lata and report our early results with a technique we are confident will have a greater, more durable success rate, with a lower incidence of dyspareunia, than the classic repair., Methods: A total of 73 patients, aged 31 to 86 years, with symptomatic (stool trapping and/or vaginal/perineal splinting or postural modifications to facilitate stool evacuation) rectoceles underwent a site-specific repair reinforced with cadaveric fascia. Perioperative questionnaires, retrospective chart review, and telephone interview by a blinded third-party reviewer and physical examination was conducted. Issues thought to be relevant to the rectocele repair were assessed., Results: Of the 73 patients, 62 responded to the postoperative questionnaire and 50 underwent physical examination. The mean follow-up was 13.7 months (range 6 to 23). Of the 62 patients, 52 (93.6%) denied postoperative stool trapping requiring vaginal/perineal splinting. Of the 39 sexually active patients, 4 (10.3%) experienced de novo dyspareunia. Minor complications were seen in 15 patients (24%). One developed a symptomatic enterocele., Conclusions: Interposition of cadaveric fascia lata avoids dependence on weakened native rectovaginal support to facilitate the rectocele repair. Our technique uses fascial interposition, rather than obliteration of the defect, preventing vaginal narrowing, and should thereby decrease the incidence of dyspareunia. Patient symptom improvement and satisfaction rates were competitive with those after traditional rectocele repair. Follow-up is ongoing with the hope that the fascial reinforcement will translate into more durable results.

Stress urinary incontinence (SUI) is defined as leakage of urine with a sudden increase in intra-abdominal pressure, such as that seen with laughing, lifting, or changing position, without a concomitant rise in detrusor (bladder-generated) pressure. The proposed mechanism of SUI is that an increase in intra-abdominal pressure resulting from various activities causes the bladder pressure to rise above the urethral pressure. The pubovaginal sling remains the standard treatment for female SUI in the US. The market has been flooded with innumerable sling materials. This review discusses the currently available sling materials, surgical approaches, and clinical outcomes data. Long-term data on efficacy is lacking, but early results with new materials and delivery techniques indicate that excellent cure rates with minimal morbidity and high patient satisfaction may be achievable.

Background: Dedicated minimally invasive surgery suites are available that contain specialized equipment to facilitate endoscopic surgery. Laparoscopy performed in a general operating room is hampered by the multitude of additional equipment that must be transported into the room. The objective of this study was to compare the preparation times between procedures performed in traditional operating rooms versus dedicated minimally invasive surgery suites to see whether operating room efficiency is improved in the specialized room., Methods: The records of 50 patients who underwent laparoscopic procedures between September 2000 and April 2002 were retrospectively reviewed. Twenty-three patients underwent surgery in a general operating room and 18 patients in an minimally invasive surgery suite. Nine patients were excluded because of cystoscopic procedures undergone prior to laparoscopy. Various time points were recorded from which various time intervals were derived, such as preanesthesia time, anesthesia induction time, and total preparation time. A 2-tailed, unpaired Student t test was used for statistical analysis., Results: The mean preanesthesia time was significantly faster in the minimally invasive surgery suite (12.2 minutes) compared with that in the traditional operating room (17.8 minutes) (P=0.013). Mean anesthesia induction time in the minimally invasive surgery suite (47.5 minutes) was similar to time in the traditional operating room (45.7 minutes) (P=0.734). The average total preparation time for the minimally invasive surgery suite (59.6 minutes) was not significantly faster than that in the general operating room (63.5 minutes) (P=0.481)., Conclusion: The amount of time that elapses between the patient entering the room and anesthesia induction is statically shorter in a dedicated minimally invasive surgery suite. Laparoscopic surgery is performed more efficiently in a dedicated minimally invasive surgery suite versus a traditional operating room.

A 52-year-old man with a significant history of nephrolithiasis was found to have a calcium oxalate dihydrate ureteral calculus that apparently formed on a hair. This nidus may have been introduced during previous instrumentation, perhaps on the tip of the ureteroscope, laser fiber, or even during the placement of a ureteral stent.

Purpose: In an attempt to evaluate our experience with the treatment of pediatric urethral stricture disease we performed a retrospective review of patients undergoing direct vision internal urethrotomy (DVIU)., Materials and Methods: The computerized surgical logs at 2 pediatric hospitals were reviewed to identify patients who underwent DVIU between 1992 and 2001. Hospital and clinical charts were then reviewed. Many variables were analyzed, including patient age, etiology of stricture, technique and clinical outcomes. Minimum followup to be included in clinical outcome analysis was 12 months., Results: A total of 50 patients were identified (mean age 7.7 years, range 6 months to 17 years). The most common etiology for stricture formation was previous hypospadias repair (20 patients [40%]). Forty patients met the 12-month minimum followup requirement for clinical outcome analysis. Of these patients 20 (50%) had no symptoms to suggest recurrent stricture at a median of 2.0 years (mean 2.7 years, range 12 months to 7 years). Seventeen patients (42.5%) had symptoms of recurrent stricture at a median of 8 months (mean 13 months, range 2 months to 5 years). Technical factors did not influence the ultimate success or failure of the procedure., Conclusions: DVIU provides a therapeutic option that successfully treats approximately half of the patients with a reasonably low complication rate. Complications following DVIU should not preclude its use as a therapeutic modality for the treatment urethral strictures in children. If the child fails the initial DVIU, repeat attempts at endoscopic correction of urethral stricture should be abandoned in favor of definitive urethroplasty.

Insulin is thought to elicit its effects by crosslinking the two extracellular alpha-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

BACKGROUND: Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. RESULTS: Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs) which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75) into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFalpha and TNFbeta and induced an unexpected biological response in a TNFR2-specific manner. CONCLUSIONS: To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.

Translocation of colicin across the membrane of sensitive cells has been studied extensively. However, processing of the toxicity domain of colicin during translocation has been the subject of much controversy. To investigate the final translocation product of colicin across the membrane of Escherichia coli, an endogenously expressed His-tagged Im7 protein was constructed to detect any translocation product containing the DNase domain traversed the inner membrane into cytoplasm of the E. coli cells. As a result, a final processed DNase domain of ColE7 was identified in the intracellular space of the cells treated with Col-Im complex. In the presence of periplasmic extracts, in vitro processing of DNase domain of ColE7 was also observed. These results suggest that the processing of ColE7 has occurred for translocation of the DNase-type colicin across the membrane and the process is probably taking place in the periplasmic space of the membrane., (Copyright 2001 Academic Press.)

Monoclonal antibody PAb1620 recognizes a conformational epitope on the transcription factor p53 and, upon binding, allosterically inhibits p53 binding to DNA. A highly diverse (1.5 x 10(10) members) phage-displayed library of peptides containing 40 random amino acids was used to identify the PAb1620 binding site on p53. Panning this library against PAb1620 resulted in three unique peptides which have statistically significant sequence identities with p53 sufficient to identify the binding site as being composed of amino acids 106-113 and 146-156. Based on these results, we propose a mechanism by which PAb1620 can allosterically inhibit p53 binding to DNA through an indirect interaction between the antibody binding site and the L1 loop (amino acids 112-124) of p53, which is a component of the DNA binding region.

Conversion of testosterone to dihydrotestosterone (DHT) has been demonstrated to be catalyzed by two isoforms of steroid 5 alpha-reductase, designated types I and II. Although several classes of steroid-based inhibitors of the type II isoform have been identified, these agents have not demonstrated highly selective pharmacological activity against human type I 5 alpha-reductase. LY191704 is representative of a series of nonsteroidal agents that have potent [apparent inhibitory constant (Ki) = 11.3 nM] inhibitory activity in human scalp skin homogenates (pH 7.5), a source of type I 5 alpha-reductase. [3H]-DHT production in the presence and absence of LY191704 is consistent with a noncompetitive mode of inhibition. In human prostatic homogenates (pH 5.5), a source of type II 5 alpha-reductase, LY191704 is virtually inactive as an inhibitor [concentration of inhibitor producing 50% inhibition of enzymatic activity (IC50) > 1,000 nM] of [3H]-DHT formation. LY191704 does not inhibit the type I or type II isoforms of rat 5 alpha-reductase, nor does the compound compete for binding to the murine androgen receptor expressed in SF9 cells using a baculo virus expression system. The benzoquinolinones, as exemplified by LY191704, possess exquisite pharmacological selectivity and provide a tool to understand the role of human type I 5 alpha-reductase in normal and pathophysiological states. These agents may also find clinical utility in treating androgen-dependent dermatological conditions.

The conversion of testosterone (T) to dihydrotestosterone (DHT) has been demonstrated to be catalysed by at least two isoforms of human steroid 5 alpha-reductase, designated types I and II. Type II 5 alpha-reductase expression predominates in human accessory sex tissues, localized to the fibromuscular stromal compartment. The type I isoform predominates in skin, prostatic epithelia and, to a lesser extent, in prostatic fibromuscular stroma. The significance of the type I isoform to prostatic cellular growth and function remains undefined. In cultured DU145 cells, we evaluated the metabolism of [14C]-T and demonstrated the time-dependent formation of [14C]-DHT. Oxidative metabolism (conversion of [14C]-T to [14C]-androstenedione) and the formation of conjugated androgen metabolites occurred at a relatively low rate in the DU145 cells. Using human type I 5 alpha-reductase cDNA, Northern blot analysis of DU145 cell mRNA revealed high levels of type I isoform expression. Analogous probing of the DU145 cells with a human 5 alpha-reductase II cDNA failed to reveal expression of the type II isoform. The expression of functional type I activity has been confirmed pharmacologically using isoform-selective 5 alpha-reductase inhibitors. Reductive metabolism of [3H]-T in the DU145 cells was inhibited in a concentration-dependent manner by LY306089, a potent non-steroidal type I-selective inhibitor (IC50 = 10.0 nM). SKF105657, a steroidal type II-specific inhibitor was distinctly less active at inhibiting [3H]-DHT formation. LY306089 was a non-competitive inhibitor of type I 5 alpha-reductase in DU145 cellular homogenates with an apparent Ki value of 4.0 nM. These studies have identified and pharmacologically defined type I 5 alpha-reductase activity in an androgen-insensitive prostatic cancer cell line and provide the basis for additional investigations into the significance of type I 5 alpha-reductase to human prostatic pathophysiology.