Exploring NICU nurses' views of a novel genetic point-of-care test identifying neonates at risk of antibiotic-induced ototoxicity: A qualitative study.

AIM: To explore the views of neonatal intensive care nursing staff on the deliverability of a novel genetic point-of-care test detecting a genetic variant associated with antibiotic-induced ototoxicity. DESIGN: An interpretive, descriptive, qualitative interview study. METHODS: Data were collected using semi-structured interviews undertaken between January and November 2020. Participants were neonatal intensive care nursing staff taking part in the Pharmacogenetics to Avoid Loss of Hearing trial. RESULTS: Thematic analysis resulted in four themes: perceived clinical utility; the golden hour; point-of-care device; training and support. Recommendations were made to streamline the protocol and ongoing training and support were considered key to incorporating the test into routine care. CONCLUSION: Exploring the views of nurses involved in the delivery of the point-of-care test was essential in its implementation. By the study endpoint, all participants could see the value of routine clinical introduction of the point-of care test. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE: Nurses are in a key position to support the delivery of point-of-care genetic testing into mainstream settings. This study has implications for the successful integration of other genetic point-of-care tests in acute healthcare settings. IMPACT: The study will help to tailor the training and support required for routine deployment of the genetic point-of-care test. The study has relevance for nurses involved in the development and delivery of genetic point-of-care tests in other acute hospital settings. REPORTING METHOD: This qualitative study adheres to the Standards for Reporting Qualitative Research EQUATOR guidelines and utilizes COREQ and SRQR checklists. PATIENT OR PUBLIC CONTRIBUTION: All staff working on the participating neonatal intensive care units were trained to use the genetic point-of-care test. All inpatients on the participating units were eligible to have testing via the point-of-care test. The Pharmacogenetics to Avoid Loss of Hearing Patient and Public Involvement and Engagement group provided valuable feedback. TRIAL AND PROTOCOL REGISTRATION: Registered within the University of Manchester. Ethics approval reference numbers: IRAS: 253102 REC reference: 19/NW/0400. Also registered with the ISRCTN ref: ISRCTN13704894.

Rapid Point-of-Care Genotyping to Avoid Aminoglycoside-Induced Ototoxicity in Neonatal Intensive Care.

Importance: Aminoglycosides are commonly prescribed antibiotics used for the treatment of neonatal sepsis. The MT-RNR1 m.1555A>G variant predisposes to profound aminoglycoside-induced ototoxicity (AIO). Current genotyping approaches take several days, which is unfeasible in acute settings. Objective: To develop a rapid point-of-care test (POCT) for the m.1555A>G variant before implementation of this technology in the acute neonatal setting to guide antibiotic prescribing and avoid AIO. Design, Setting, and Participants: This pragmatic prospective implementation trial recruited neonates admitted to 2 large neonatal intensive care units between January 6, 2020, and November 30, 2020, in the UK. Interventions: Neonates were tested for the m.1555A>G variant via the rapid POCT on admission to the neonatal intensive care unit. Main Outcomes and Measures: The primary outcome assessed the proportion of neonates successfully tested for the variant of all infants prescribed antibiotics. Secondary outcomes measured whether implementation was negatively associated with routine clinical practice and the performance of the system. The study was statistically powered to detect a significant difference between time to antibiotic administration before and after implementation of the MT-RNR1 POCT. Results: A total of 751 neonates were recruited and had a median (range) age of 2.5 (0-198) days. The MT-RNR1 POCT was able to genotype the m.1555A>G variant in 26 minutes. Preclinical validation demonstrated a 100% sensitivity (95% CI, 93.9%-100.0%) and specificity (95% CI, 98.5%-100.0%). Three participants with the m.1555A>G variant were identified, all of whom avoided aminoglycoside antibiotics. Overall, 424 infants (80.6%) receiving antibiotics were successfully tested for the variant, and the mean time to antibiotics was equivalent to previous practice. Conclusions and Relevance: The MT-RNR1 POCT was integrated without disrupting normal clinical practice, and genotype was used to guide antibiotic prescription and avoid AIO. This approach identified the m.1555A>G variant in a practice-changing time frame, and wide adoption could significantly reduce the burden of AIO.

Pharmacogenetics to Avoid Loss of Hearing (PALOH) trial: a protocol for a prospective observational implementation trial.

INTRODUCTION: In conjunction with a beta-lactam, aminoglycosides are the first-choice antibiotic for empirical treatment of sepsis in the neonatal period. The m.1555A>G variant predisposes to ototoxicity after aminoglycoside administration and has a prevalence of 1 in 500. Current genetic testing can take over 24 hours, an unacceptable delay in the acute setting. This prospective-observational trial will implement a rapid point of care test (POCT), facilitating tailored antibiotic prescribing to avoid hearing loss. METHODS AND ANALYSIS: The genedrive POCT can detect the m.1555A>G variant in 26 min from buccal swab. This system will be integrated into the clinical pathways at two large UK neonatal centres over a minimum 6-month period. The primary outcome is the number of neonates successfully tested for the variant out of all babies prescribed antibiotics. As a secondary outcome, clinical timings will be compared with data collected prior to implementation, measuring the impact on routine practice. ETHICS AND DISSEMINATION: Approval for the trial was granted by the Research Ethics Committee (REC) and Human Research Authority in August 2019. Results will be published in full on completion of the study. TRIAL REGISTRATION NUMBER: ISRCTN13704894. PROTOCOL VERSION: V 1.3.

Transcriptome analysis of IPF fibroblastic foci identifies key pathways involved in fibrogenesis.

INTRODUCTION: Fibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection. METHODS: The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression. RESULTS: WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor ß1 (TGF-ß1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-ß1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts. CONCLUSION: These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.

Diagnosing and Preventing Hearing Loss in the Genomic Age.

Over the past two decades, significant technological advances have facilitated the identification of hundreds of genes associated with hearing loss. Variants in many of these genes result in severe congenital hearing loss with profound implications for the affected individual and their family. This review collates these advances, summarizing the current state of genomic knowledge in childhood hearing loss. We consider how current and emerging genetic technologies have the potential to alter our approach to the management and diagnosis of hearing loss. We review approaches being taken to ensure that these discoveries are used in clinical practice to detect genetic hearing loss as soon as possible to reduce unnecessary investigations, provide information about reproductive risks, and facilitate regular follow-up and early treatment. We also highlight how rapid sequencing technology has the potential to identify children susceptible to antibiotic-induced hearing loss and how this adverse reaction can be avoided.

Development and clinical validation of the Genedrive point-of-care test for qualitative detection of hepatitis C virus.

OBJECTIVE: Recently approved direct acting antivirals provide transformative therapies for chronic hepatitis C virus (HCV) infection. The major clinical challenge remains to identify the undiagnosed patients worldwide, many of whom live in low-income and middle-income countries, where access to nucleic acid testing remains limited. The aim of this study was to develop and validate a point-of-care (PoC) assay for the qualitative detection of HCV RNA. DESIGN: We developed a PoC assay for the qualitative detection of HCV RNA on the PCR Genedrive instrument. We validated the Genedrive HCV assay through a case-control study comparing results with those obtained with the Abbott RealTime HCV test. RESULTS: The PoC assay identified all major HCV genotypes, with a limit of detection of 2362 IU/mL (95% CI 1966 to 2788). Using 422 patients chronically infected with HCV and 503 controls negative for anti-HCV and HCV RNA, the Genedrive HCV assay showed 98.6% sensitivity (95% CI 96.9% to 99.5%) and 100% specificity (95% CI 99.3% to 100%) to detect HCV. In addition, melting peak ratiometric analysis demonstrated proof-of-principle for semiquantification of HCV. The test was further validated in a real clinical setting in a resource-limited country. CONCLUSION: We report a rapid, simple, portable and accurate PoC molecular test for HCV, with sensitivity and specificity that fulfils the recent FIND/WHO Target Product Profile for HCV decentralised testing in low-income and middle-income countries. This Genedrive HCV assay may positively impact the continuum of HCV care from screening to cure by supporting real-time treatment decisions. TRIAL REGISTRATION NUMBER: NCT02992184 .

An in vitro diagnostic certified point of care single nucleotide test for IL28B polymorphisms.

Numerous genetic polymorphisms have been identified as associated with disease or treatment outcome, but the routine implementation of genotyping into actionable medical care remains limited. Point-of-care (PoC) technologies enable rapid and real-time treatment decisions, with great potential for extending molecular diagnostic approaches to settings with limited medical infrastructure (e.g., CLIA certified diagnostic laboratories). With respect to resource-limited settings, there is a need for simple devices to implement biomarker guided treatment strategies. One relevant example is chronic hepatitis C infection, for which several treatment options are now approved. Single nucleotide polymorphisms (SNPs) in the IL-28B / IFNL3 locus have been well described to predict both spontaneous clearance and response to interferon based therapies. We utilized the Genedrive® platform to develop an assay for the SNP rs12979860 variants (CC, CT and TT). The assay utilizes a hybrid thermal engine, permitting rapid heating and cooling, enabling an amplification based assay with genetic variants reported using endpoint differential melting cure analysis in less than 60 minutes. We validated this assay using non-invasive buccal swab sampling in a prospective study of 246 chronic HCV patients, achieving 100% sensitivity and 100% specificity (95% exact CI: 98.8-100%)) in 50 minutes as compared to conventional lab based PCR testing. Our results provide proof of concept that precision medicine is feasible in resource-limited settings, offering the first CE-IVD (in vitro diagnostics) validated PoC SNP test. We propose that IL-28B genotyping may be useful for directing patients towards lower cost therapies, and rationing use of costly direct antivirals for use in those individuals showing genetic risk.

Cystatin F is a biomarker of prion pathogenesis in mice.

Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer's disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.

Loss of epithelial Gq and G11 signaling inhibits TGFß production but promotes IL-33-mediated macrophage polarization and emphysema.

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling links hundreds of G protein-coupled receptors with four G protein signaling pathways. Two of these, one mediated by Gq and G11 (Gq/11) and the other by G12 and G13 (G12/13), are implicated in the force-dependent activation of transforming growth factor-ß (TGFß) in lung epithelial cells. Reduced TGFß activation in alveolar cells leads to emphysema, whereas enhanced TGFß activation promotes acute lung injury and idiopathic pulmonary fibrosis. Therefore, precise control of alveolar TGFß activation is essential for alveolar homeostasis. We investigated the involvement of the Gq/11 and G12/13 pathways in epithelial cells in generating active TGFß and regulating alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation that was primarily caused by alternatively activated (M2-polarized) macrophages, enhanced matrix metalloproteinase 12 (MMP12) production, and age-related alveolar airspace enlargement consistent with emphysema. Mice with impaired Gq/11 signaling had reduced stretch-mediated generation of TGFß by epithelial cells and enhanced macrophage MMP12 synthesis but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the cytokine interleukin-33 (IL-33) was increased in these alveolar epithelial cells, resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFß. Our results suggest that alveolar Gq/11 signaling maintains alveolar homeostasis and likely independently increases TGFß activation in response to the mechanical stress of the epithelium and decreases epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/11 signaling promotes inflammatory emphysema but protects against mechanically induced lung injury.

Low sputum MMP-9/TIMP ratio is associated with airway narrowing in smokers with asthma.

Asthmatic smokers have poor symptom control and accelerated decline in lung function. A reduced ratio of matrix metalloproteinase (MMP)-9/tissue inhibitors of metalloproteinases (TIMPs) in nonsmokers with asthma has been implicated in airway remodelling. We tested the hypothesis that sputum MMP-9 activity/TIMPs ratios are reduced in smokers compared with never-smokers with asthma and are associated with reduced lung function and altered computed tomography (CT) measures of airway wall dimensions. Lung function, airway dimensions by CT, and induced sputum concentrations (and activity) of MMP-9 and TIMP-1 and -2 were measured in 81 asthmatics and 43 healthy subjects (smokers and never-smokers). Respiratory epithelial MMP9 and TIMP mRNA was quantified in 31 severe asthmatics and 32 healthy controls. Sputum MMP-9 activity/TIMP-1 and TIMP-2 ratios, and nasal epithelial MMP9/TIMP1 and MMP9/TIMP2 expression ratios were reduced in smokers with asthma compared with never-smokers with asthma. Low sputum ratios in asthmatic smokers were associated with reduced post-bronchodilator forced expiratory volume in 1 s (FEV1), FEV1/forced vital capacity ratio and segmental airway lumen area. The association of a low sputum MMP-9 activity/TIMP-1 ratio with persistent airflow obstruction and reduced CT airway lumen area in smokers with asthma may indicate that an imbalance of MMP-9 and TIMPs contributes to structural changes to the airways in this group.

Comparative gene expression profiling identifies common molecular signatures of NF-κB activation in canine and human diffuse large B cell lymphoma (DLBCL).

We present the first comparison of global transcriptional changes in canine and human diffuse large B-cell lymphoma (DLBCL), with particular reference to the nuclear factor-kappa B (NF-κB) pathway. Microarray data generated from canine DLBCL and normal lymph nodes were used for differential expression, co-expression and pathway analyses, and compared with analysis of microarray data from human healthy and DLBCL lymph nodes. The comparisons at gene level were performed by mapping the probesets in canine microarrays to orthologous genes in humans and vice versa. A considerable number of differentially expressed genes between canine lymphoma and healthy lymph node samples were also found differentially expressed between human DLBCL and healthy lymph node samples. Principal component analysis using a literature-derived NF-κB target gene set mapped to orthologous canine array probesets and human array probesets clearly separated the healthy and cancer samples in both datasets. The analysis demonstrated that for both human and canine DLBCL there is activation of the NF-κB/p65 canonical pathway, indicating that canine lymphoma could be used as a model to study NF-κB-targeted therapeutics for human lymphoma. To validate this, tissue arrays were generated for canine and human NHL and immunohistochemistry was employed to assess NF-κB activation status. In addition, human and canine B-cell lymphoma lines were assessed for NF-κB activity and the effects of NF-κB inhibition.

Sputum matrix metalloproteinase-9 is associated with the degree of emphysema on computed tomography in COPD.

BACKGROUND: Matrix-metalloproteinase (MMP)-9 has been implicated in the pathogenesis of COPD, although its link to disease severity is unclear. The purpose of the study was to examine the relationship between disease severity assessed by lung function and computed tomography (CT) and sputum MMP-9 expression, concentration and activity in patients with COPD. FINDINGS: In 53 COPD subjects, smokers and ex-smokers; 46 healthy controls, smokers and never smokers, we measured sputum MMP-9 concentrations (ELISA) and enzyme activity (FRET), sputum MMP-9 mRNA expression, spirometry, diffusing capacity for carbon monoxide (DLco) and CT assessment of emphysema (% low attenuation areas below-950 Hounsfield units). Sputum MMP-9 concentrations and mRNA expression in COPD subjects were significantly greater than in healthy never-smokers (p = 0.007 and p = 0.001 respectively) and similar to those in healthy smokers. Disease severity when assessed by the extent of emphysema measured by CT, but not by spirometry or DLco values, was directly associated with sputum MMP-9 concentrations [r = 0.442 (0.171, 0.634), p = 0.020], and MMP-9 activity [r = 0.447 (0.219, 0.643), p = 0.010]. In moderate to severe COPD, increased MMP-9 mRNA expression levels were associated with reduced post-bronchodilator FEV1 [r = -0.530 (-0.686, -0.327), p < 0.001], FEV1/FVC ratio [r = -0.551 (-0.701, -0.354), p < 0.001] and reduced DLco [r = -0.399 (-539, -0.102), p = 0.048]. CONCLUSIONS: Sputum MMP-9 concentrations in COPD are directly associated with the extent of emphysema measured by CT and MMP-9 expression levels are inversely associated with DLco. These findings support a role for MMP-9 in the pathogenesis of COPD.

Simultaneous non-negative matrix factorization for multiple large scale gene expression datasets in toxicology.

Non-negative matrix factorization is a useful tool for reducing the dimension of large datasets. This work considers simultaneous non-negative matrix factorization of multiple sources of data. In particular, we perform the first study that involves more than two datasets. We discuss the algorithmic issues required to convert the approach into a practical computational tool and apply the technique to new gene expression data quantifying the molecular changes in four tissue types due to different dosages of an experimental panPPAR agonist in mouse. This study is of interest in toxicology because, whilst PPARs form potential therapeutic targets for diabetes, it is known that they can induce serious side-effects. Our results show that the practical simultaneous non-negative matrix factorization developed here can add value to the data analysis. In particular, we find that factorizing the data as a single object allows us to distinguish between the four tissue types, but does not correctly reproduce the known dosage level groups. Applying our new approach, which treats the four tissue types as providing distinct, but related, datasets, we find that the dosage level groups are respected. The new algorithm then provides separate gene list orderings that can be studied for each tissue type, and compared with the ordering arising from the single factorization. We find that many of our conclusions can be corroborated with known biological behaviour, and others offer new insights into the toxicological effects. Overall, the algorithm shows promise for early detection of toxicity in the drug discovery process.

Sputum matrix metalloproteinase-12 in patients with chronic obstructive pulmonary disease and asthma: relationship to disease severity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 has been implicated in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and asthma. The influence of disease severity on sputum MMP-12 concentrations and activity is not known. OBJECTIVES: We sought to examine the relationship between disease severity assessed by means of lung function and computed tomography (CT) and induced sputum MMP-12 concentrations and activity in patients with asthma and COPD. METHODS: In 208 subjects (109 asthmatic patients, smokers and never smokers, mild, moderate, and severe; 53 patients with COPD, smokers and exsmokers, mild, moderate, and severe; and 46 healthy control subjects, smokers and never smokers), we measured induced sputum MMP-12 concentrations (ELISA) and enzyme activity (fluorescence resonance energy transfer), sputum cell MMP12 mRNA expression (quantitative PCR [qPCR]), diffusing capacity for carbon monoxide (Dlco), and CT assessment of emphysema (percentage of low-attenuation areas at less -950 Hounsfield units). RESULTS: Sputum MMP-12 concentrations are greater in patients with COPD and smokers with asthma than in healthy nonsmokers (P = .003 and P = .035, respectively) but similar to those seen in healthy smokers. In patients with COPD, disease severity, when measured by means of CT-assessed emphysema, but not by means of spirometry or Dlco values, is directly associated with sputum MMP-12 concentrations and activity. In the asthma groups there is no significant association between disease severity and sputum MMP-12 concentrations or activity. CONCLUSIONS: Sputum MMP-12 concentrations and activity in patients with COPD are directly associated with the extent of emphysema measured by means of CT. This finding supports a role for MMP-12 in the pathogenesis of COPD and might suggest that blocking MMP-12 activity in patients with COPD could prevent the further development of emphysema.

Global array-based transcriptomics from minimal input RNA utilising an optimal RNA isolation process combined with SPIA cDNA probes.

BACKGROUND: Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficulty in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.

Engulfment of cerebral apoptotic bodies controls the course of prion disease in a mouse strain-dependent manner.

Progressive accumulation of PrP(Sc), a hallmark of prion diseases, occurs when conversion of PrP(C) into PrP(Sc) is faster than PrP(Sc) clearance. Engulfment of apoptotic bodies by phagocytes is mediated by Mfge8 (milk fat globule epidermal growth factor 8). In this study, we show that brain Mfge8 is primarily produced by astrocytes. Mfge8 ablation induced accelerated prion disease and reduced clearance of cerebellar apoptotic bodies in vivo, as well as excessive PrP(Sc) accumulation and increased prion titers in prion-infected C57BL/6 × 129Sv mice and organotypic cerebellar slices derived therefrom. These phenotypes correlated with the presence of 129Sv genomic markers in hybrid mice and were not observed in inbred C57BL/6 Mfge8(-/-) mice, suggesting the existence of additional strain-specific genetic modifiers. Because Mfge8 receptors are expressed by microglia and depletion of microglia increases PrP(Sc) accumulation in organotypic cerebellar slices, we conclude that engulfment of apoptotic bodies by microglia may be an important pathway of prion clearance controlled by astrocyte-borne Mfge8.

Liposome-siRNA-peptide complexes cross the blood-brain barrier and significantly decrease PrP on neuronal cells and PrP in infected cell cultures.

BACKGROUND: Recent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrP(C) expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrP(C) expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs), acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation. CONCLUSIONS/SIGNIFICANCE: Liposome-siRNA-peptide complexes (LSPCs) delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrP(C) expression and eliminated PrP(RES) formation in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrP(C)-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.

Peroxisome proliferator-activated receptor alpha and alpha/gamma agonists do not cause impairment in renal function in the rat.

BACKGROUND/AIMS: Patients treated with peroxisome proliferator-activated receptor analogs (PPAR) alpha or alpha/gamma may develop a transient and reversible increase in serum creatinine, the mechanism of which remains obscure. This study evaluates whether treatment with either PPAR-alpha or -alpha/gamma analogs, fenofibrate or tesaglitazar, may cause deterioration in renal hemodynamics or exert direct tubular or glomerular nephrotoxic effects in rats. METHODS: Male Sprague-Dawley rats (300-320 g) were treated per os with fenofibrate (300 mg/kg/day), tesaglitazar (1.2 mg/kg/day) or vehicle, for 14 days. Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured by inulin clearance and ultrasonic flowmetry, and cumulative excretion of sodium and creatinine were assessed. Biomarkers of glomerular and tubular injury were measured, including urinary albumin excretion and renal mRNA levels of kidney injury molecule 1 (Kim-1), lipocalin 2 (Lcn2), and osteopontin (Spp1). RESULTS: Fenofibrate and tesaglitazar improved the lipid profile, but caused no detectable decrease in GFR or RBF compared with vehicle-treated rats. Furthermore, the cumulative excretions of sodium and creatinine were not altered by the drugs. Finally, there was no significant difference between drug- and vehicle-treated groups in urinary albumin excretion or in the expression of renal injury biomarkers. CONCLUSIONS: In the rat, no direct nephrotoxic effect or deterioration in renal hemodynamics and function were observed following treatment with fenofibrate or tesaglitazar.

Urinary alpha1-antichymotrypsin: a biomarker of prion infection.

The occurrence of blood-borne prion transmission incidents calls for identification of potential prion carriers. However, current methods for intravital diagnosis of prion disease rely on invasive tissue biopsies and are unsuitable for large-scale screening. Sensitive biomarkers may help meeting this need. Here we scanned the genome for transcripts elevated upon prion infection and encoding secreted proteins. We found that alpha(1)-antichymotrypsin (alpha(1)-ACT) was highly upregulated in brains of scrapie-infected mice. Furthermore, alpha(1)-ACT levels were dramatically increased in urine of patients suffering from sporadic Creutzfeldt-Jakob disease, and increased progressively throughout the disease. Increased alpha(1)-ACT excretion was also found in cases of natural prion disease of animals. Therefore measurement of urinary alpha(1)-ACT levels may be useful for monitoring the efficacy of therapeutic regimens for prion disease, and possibly also for deferring blood and organ donors that may be at risk of transmitting prion infections.

alpha-Hemoglobin stabilizing protein is not a suitable marker for a screening test for variant Creutzfeldt-Jakob disease.

BACKGROUND: A test is needed to identify blood donors who are in the preclinical phase of variant Creutzfeldt-Jakob disease (CJD). alpha-Hemoglobin stabilizing protein (AHSP; syn. ERAF, EDRF) transcript levels are reduced in the blood of mice incubating transmissible spongiform encephalopathy. STUDY DESIGN AND METHODS: Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to measure AHSP transcript and protein levels in normal blood donors, patients with CJD, and patients with other neuronal and hematologic diseases. Temporal AHSP expression was measured in sheep incubating bovine spongiform encephalopathy (BSE). RESULTS: Quantitation of AHSP in peripheral blood from normal blood donors revealed that protein levels, but not transcript levels, are influenced by sex with higher levels found in males, suggesting posttranslational regulation involving the product of an X-linked gene. When AHSP mRNA and protein levels were quantitated in peripheral blood from patients with variant and sporadic CJD, no consistent differences from normal were found. Serial quantitation of AHSP in individual BSE-infected sheep did not reveal any disease-related changes. CONCLUSION: We conclude that quantitation of AHSP is not likely to be useful for detection of preclinical prion disease in man.