Safety Testing of an Antisense Oligonucleotide Intended for Pediatric Indications in the Juvenile Göttingen Minipig, Including an Evaluation of the Ontogeny of Key Nucleases

The adult Göttingen Minipig is an acknowledged model for safety assessment of antisense oligonucleotide (ASO) drugs developed for adult indications. To assess whether the juvenile Göttingen Minipig is also a suitable nonclinical model for pediatric safety assessment of ASOs, we performed an 8-week repeat-dose toxicity study in different age groups of minipigs ranging from 1 to 50 days of age. The animals received a weekly dose of a phosphorothioated locked-nucleic-acid-based ASO that was assessed previously for toxicity in adult minipigs. The endpoints included toxicokinetic parameters, in-life monitoring, clinical pathology, and histopathology. Additionally, the ontogeny of key nucleases involved in ASO metabolism and pharmacologic activity was investigated using quantitative polymerase chain reaction and nuclease activity assays. Similar clinical chemistry and toxicity findings were observed; however, differences in plasma and tissue exposures as well as pharmacologic activity were seen in the juvenile minipigs when compared with the adult data. The ontogeny study revealed a differential nuclease expression and activity, which could affect the metabolic pathway and pharmacologic effect of ASOs in different tissues and age groups. These data indicate that the juvenile Göttingen Minipig is a promising nonclinical model for safety assessment of ASOs intended to treat disease in the human pediatric population.

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Nonclinical Safety Assessment of a Synthetic Peptide Thrombopoietin Agonist

International Journal of Toxicology ◽

10.1177/1091581811404708 ◽

2011 ◽

Vol 30 (4) ◽

pp. 385-404 ◽

Cited By ~ 5

Author(s):

Elaine Knight ◽

Gary Eichenbaum ◽

Verna Hillsamer ◽

Tony Greway ◽

Alfred Tonelli ◽

...

Keyword(s):

Antibody Formation ◽

Safety Assessment ◽

Chemical Nature ◽

Osseous Metaplasia ◽

Repeat Dose ◽

Stopping Criterion ◽

Platelet Volume ◽

Repeat Dose Toxicity ◽

Pharmacologic Effect ◽

Pharmacologic Activity

RWJ-800088 is a novel, potent polyethylene glycol (PEG)-conjugated thrombopoietin (TPO) mimetic that increases platelet levels and protects against thrombocytopenia. A nonclinical safety program was customized for this peptide that takes into account its protein-like structure, synthetic chemical nature, agonist pharmacologic activity, and mode of administration. In repeat-dose toxicity studies, the salient findings were dose-related increases in circulating platelet counts, mean platelet volume, and megakaryocytes in the bone marrow with no antibody formation. Reversible myelofibrosis and hyperostosis were observed in rats, but not dogs, when the circulating platelet levels exceeded 3× those of vehicle controls. The bone effects were due to the exaggerated pharmacologic effect and excessive stimulation and elevation of megakaryocytes by TPO, which results in intramedullary proliferation of fibroblasts and mesenchymal cells followed by osseous metaplasia. These findings support the use of platelet elevations of >3× as a stopping criterion to prevent potential adverse bone-related effects in humans.

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Distribution of Human Cytomegalovirus Glycoprotein H Genotypes in Different Samples from Chinese Children

Journal of Pediatric Infectious Diseases ◽

10.1055/s-0041-1733868 ◽

2021 ◽

Author(s):

Wei Li ◽

Lu-Yan Chen ◽

Ran Tao ◽

Shi-Qiang Shang

Keyword(s):

Breast Milk ◽

Human Cytomegalovirus ◽

Throat Swab ◽

Quantitative Polymerase Chain Reaction ◽

Age Groups ◽

Urine Samples ◽

Serum Samples ◽

Predominant Genotype ◽

Milk Samples ◽

Glycoprotein H

Abstract Objective This study aimed to investigate characteristics of human cytomegalovirus (HCMV) glycoprotein H (gH) genotypes in urine, throat swab, and serum from children and breast milk from children's mothers. Methods Fresh urine samples, throat swabs, or serum samples from children and breast milk samples from children's mothers were collected for HCMV DNA detection. The positive samples of HCMV DNA were further detected by fluorescent quantitative polymerase chain reaction (PCR) with gH typing. Results Of 1,703 HCMV DNA-positive samples, the highest proportion (83.3%, 85/102) of children aged between 21 days and 3 months was detected positive in breast milk samples (p = 0.002), and the highest proportion (70.5%, 110/156) of children aged above 3 months was detected positive in throat swab samples (p = 0.002). HCMV in throat swab specimens is mainly high copy (p < 0.0001), and low-copy HCMV is prevalent in breast specimens (p < 0.0001). Among them, 1,059 samples were identified as gH1 genotype, 530 samples were gH2, and 114 samples were coinfection (gH1/2). There had the highest gH2 rates (32.3%) and lowest gH1 (61.0%) rates in urine samples (p = 0.041), whereas the highest gH1 rates (71.6%) and lowest gH2 rates (19.6%) were found in breast milk samples (p = 0.032). Concerning age groups, patients aged between 21 days and 3 months had the highest gH1 proportion (p = 0.017), while patients aged above 3 months had the highest gH1 and gH2 HCMV coinfection proportion (p = 0.002). Among 43 pairs of maternal and child samples corresponding to positive samples, gH genotype of 35 pairs of samples was consistent with a rate of 81.4%. Conclusion gH1 is the predominant genotype of HCMV in each kind of sample in China. However, the distribution of the HCMV gH genotype is different among different samples.

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Etiology of Diarrhea Requiring Hospitalization in Bangladesh by Quantitative Polymerase Chain Reaction, 2014–2018

Clinical Infectious Diseases ◽

10.1093/cid/ciaa840 ◽

2020 ◽

Author(s):

Mami Taniuchi ◽

Kamrul Islam ◽

Md Abu Sayeed ◽

James A Platts-Mills ◽

Md Taufiqul Islam ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Vibrio Cholerae ◽

Coefficient Of Variation ◽

Quantitative Polymerase Chain Reaction ◽

Age Groups ◽

Public Health Problem ◽

Major Public Health Problem ◽

Surveillance Systems ◽

Chain Reaction ◽

Polymerase Chain

Abstract Background Diarrhea remains a major public health problem and characterization of its etiology is needed to prioritize interventions. However, most data are from single-site studies of children. We tested samples from participants of any age from 11 geographically diverse hospitals in Bangladesh to describe pathogen-specific burdens of diarrhea. Methods We utilized 2 existing diarrhea surveillance systems: a Nationwide network at 10 sentinel hospitals and at the icddr,b hospital. We tested stools from enrolled participants and nondiarrheal controls for enteropathogens using quantitative polymerase chain reaction and calculated pathogen-specific attributable fractions (AFs) of diarrhea. Results We analyzed 5516 patients with diarrhea and 735 controls. Overall, rotavirus had the highest attributable burden of diarrhea (Nationwide AF, 17.7%; 95% confidence interval [CI], 14.3–20.9%; icddr,b AF, 39.9%; 38.0–41.8%), followed by adenovirus 40/41 (Nationwide AF, 17.9%; 95% CI: 13.9–21.9%; icddr,b AF, 16.6%; 95% CI, 14.4–19.4%) and Vibrio cholerae (Nationwide AF, 10.2%; 95% CI, 9.1–11.3%; icddr,b AF, 13.3%; 95% CI: 11.9–15.1%). Rotavirus was the leading pathogen in children <5 years and was consistent across the sites (coefficient of variation = 56.3%). Adenovirus 40/41 was the second leading pathogen in both children and adults. Vibrio cholerae was the leading pathogen in individuals >5 years old, but was more geographically variable (coefficient of variation = 71.5%). Other attributable pathogens included astrovirus, norovirus, Shigella, Salmonella, ETEC, sapovirus, and typical EPEC. Conclusions Rotavirus, adenovirus 40/41, and V. cholerae were the leading etiologies of infectious diarrhea requiring hospitalization in Bangladesh. Other pathogens were important in certain age groups or sites.

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Association of Laboratory Methods, Colonization Density, and Age With Detection of Streptococcus pneumoniae in the Nasopharynx

American Journal of Epidemiology ◽

10.1093/aje/kwz191 ◽

2019 ◽

Vol 188 (12) ◽

pp. 2110-2119 ◽

Cited By ~ 4

Author(s):

Catherine G Sutcliffe ◽

Lindsay R Grant ◽

Emily Cloessner ◽

Keith P Klugman ◽

Jorge E Vidal ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Enrichment Culture ◽

Quantitative Polymerase Chain Reaction ◽

Age Groups ◽

The United States ◽

Healthy Individuals ◽

Navajo Nation ◽

White Mountain Apache ◽

Colonization Density ◽

Culture Positive

Abstract Culture-based methods for detecting Streptococcus pneumoniae in the nasopharynx lack sensitivity. In this study, we aimed to compare the performance of culture and molecular methods in detecting pneumococcus in the nasopharynx of healthy individuals and to evaluate the associations of age and colonization density with detection. Between 2010 and 2012, nasopharyngeal specimens were collected from healthy individuals living on Navajo Nation and White Mountain Apache Tribal lands in the United States. Pneumococci were detected by means of broth-enrichment culture and autolysin-encoding gene (lytA) quantitative polymerase chain reaction (qPCR). Among 982 persons evaluated (median age, 18.7 years; 47% male), 35% were culture-positive and an additional 27% were qPCR-positive. Agreement between culture and qPCR was 70.9% but was higher among children (age <18 years) (75.9%–84.4%) than among adults (age ≥18 years) (61.0%–74.6%). The mean density of colonization was lower for culture-negative samples (3.14 log10 copies/mL) than for culture-positive samples (5.02 log10 copies/mL), overall and for all age groups. The percent culture-positive increased with increasing density, exceeding 80% at densities of ≥10,000 copies/mL. Mean colonization density decreased with age. Use of qPCR improved detection of pneumococcus in the nasopharynx of healthy individuals. This finding was most notable among adults, probably because of improved detection of low-density colonization.

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A Functionally Improved Locked Nucleic Acid Antisense Oligonucleotide Inhibits Bcl-2 and Bcl-xL Expression and Facilitates Tumor Cell Apoptosis

Oligonucleotides ◽

10.1089/1545457042258297 ◽

2004 ◽

Vol 14 (3) ◽

pp. 199-209 ◽

Cited By ~ 18

Author(s):

A. Paula Simões-Wüst ◽

Sally Hopkins-Donaldson ◽

Brigitte Sigrist ◽

Larisa Belyanskaya ◽

Rolf A. Stahel ◽

...

Keyword(s):

Nucleic Acid ◽

Tumor Cell ◽

Cell Apoptosis ◽

Antisense Oligonucleotide ◽

Locked Nucleic Acid ◽

Tumor Cell Apoptosis

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High cases of submicroscopic Plasmodium falciparum infections in a suburban population of Lagos, Nigeria

Malaria Journal ◽

10.1186/s12936-019-3073-7 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 1

Author(s):

Florence A. Umunnakwe ◽

Emmanuel T. Idowu ◽

Olusola Ajibaye ◽

Blessed Etoketim ◽

Samuel Akindele ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Large Scale ◽

Quantitative Polymerase Chain Reaction ◽

Age Groups ◽

Malaria Diagnosis ◽

School Age Children ◽

Asymptomatic Malaria ◽

High Prevalence ◽

Study Population ◽

Low Sensitivity

Abstract Background Asymptomatic malaria parasites are significant sources of infections for onward malaria transmission. Conventional tools for malaria diagnosis such as microscopy and rapid diagnostic test kits (RDT) have relatively low sensitivity, hence the need for alternative tools for active screening of such low-density infections. Methods This study tested var acidic terminal sequence-based (varATS) quantitative polymerase chain reaction (qPCR) for screening asymptomatic Plasmodium falciparum infections among dwellers of a sub-urban community in Lagos, Nigeria. Clinically healthy participants were screened for malaria using microscopy, RDT and varATS qPCR techniques. Participants were stratified into three age groups: 1–5, 6–14 and > 14 years old. Results Of the 316 participants screened for asymptomatic malaria infection, 78 (24.68%) were positive by microscopy, 99 (31.33%) were positive by RDT and 112 (35.44%) by varATS qPCR. Participants aged 6–14 years had the highest prevalence of asymptomatic malaria, with geometric means of ~ 116 parasites/µL and ~ 6689 parasites/µL as detected by microscopy and varATS, respectively. Conclusion This study has revealed high prevalence of asymptomatic malaria in the study population, with varATS detecting additional sub-microscopic infections. The highest concentration of asymptomatic malaria was observed among school-age children between 6 and 14 years old. A large-scale screening to identify other potential hotspots of asymptomatic parasites in the country is recommended.

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Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa

Genes ◽

10.3390/genes10050363 ◽

2019 ◽

Vol 10 (5) ◽

pp. 363 ◽

Cited By ~ 3

Author(s):

Sarah Naessens ◽

Laurien Ruysschaert ◽

Steve Lefever ◽

Frauke Coppieters ◽

Elfride De Baere

Keyword(s):

Retinitis Pigmentosa ◽

Autosomal Dominant ◽

Quantitative Polymerase Chain Reaction ◽

Dominant Negative ◽

Locked Nucleic Acid ◽

Dominant Negative Effect ◽

Acid Modification ◽

Autosomal Dominant Retinitis Pigmentosa ◽

Knock Down ◽

Negative Effect

The recurrent missense variant in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), c.166G>A, p.(Gly56Arg) or G56R, underlies 1%–2% of cases with autosomal dominant retinitis pigmentosa (adRP), a frequent, genetically heterogeneous inherited retinal disease (IRD). The mutant NR2E3 protein has a presumed dominant negative effect (DNE) by competition for dimer formation with Cone-Rod Homeobox (CRX) but with abolishment of DNA binding, acting as a repressor in trans. Both the frequency and DNE of G56R make it an interesting target for allele-specific knock-down of the mutant allele using antisense oligonucleotides (AONs), an emerging therapeutic strategy for IRD. Here, we designed gapmer AONs with or without a locked nucleic acid modification at the site of the mutation, which were analyzed for potential off-target effects. Next, we overexpressed wild type (WT) or mutant NR2E3 in RPE-1 cells, followed by AON treatment. Transcript and protein levels of WT and mutant NR2E3 were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot respectively. All AONs showed a general knock-down of mutant and WT NR2E3 on RNA and protein level, showing the accessibility of the region for AON-induced knockdown. Further modifications are needed however to increase allele-specificity. In conclusion, we propose the first proof-of-concept for AON-mediated silencing of a single nucleotide variation with a dominant negative effect as a therapeutic approach for NR2E3-associated adRP.

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A 13-Week Dermal Repeat-Dose Neurotoxicity Study of Hydrodesulfurized Kerosene in Rats

International Journal of Toxicology ◽

10.1177/1091581813514282 ◽

2013 ◽

Vol 33 (1_suppl) ◽

pp. 68S-77S ◽

Cited By ~ 5

Author(s):

Rudolph Breglia ◽

Quang Bui ◽

Donald Burnett ◽

Francis Koschier ◽

Elizabeth Lapadula ◽

...

Keyword(s):

Clinical Chemistry ◽

Skin Irritation ◽

Exposure Period ◽

Test Material ◽

High Dose ◽

Test Substance ◽

Repeat Dose ◽

Sprague Dawley ◽

Microscopic Evaluation ◽

Aviation Turbine Fuel

A 13-week dermal repeat-dose toxicity study was conducted with hydrodesulfurized (HDS) kerosene, a test material that also met the commercial specifications for aviation turbine fuel (jet A). The objectives were to assess the potential for target organ toxicity and neurotoxicity. The HDS kerosene was applied to the shaved backs of Sprague-Dawley CD rats, 12/sex/group, 6 h/d, 5 d/wk in doses of 0 (vehicle control), 165 mg/kg (20% HDS kerosene), 330 mg/kg (40% HDS kerosene), or 495 mg/kg (60% HDS kerosene). Additional rats (12/sex) from the control and the high-dose groups were held without treatment for 4 weeks to assess recovery. Standard parameters of toxicity were investigated during the in-life phase. At necropsy, organs were weighed and selected tissues were processed for microscopic evaluation. Neurobehavioral evaluations included tests of motor activity and functional observations that were conducted pretest, at intervals during the exposure period and after recovery. No test substance-related effects on mortality, clinical observations (except dermal irritation), body weight, or clinical chemistry values were observed. A dose-related increase in skin irritation, confirmed histologically as minimal, was evident at the dosing site. The only statistically significant change considered potentially treatment related was an increase in the neutrophil count in females at 13 weeks. No test article-related effects were observed in the neurobehavioral assessments or gross or microscopic findings in the peripheral or central nervous system tissues in any of the dose groups. Excluding skin irritation, the no observed adverse effect level value for all effects was considered 495 mg/kg/d.

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Establishment of Reference Intervals for Alkaline Phosphatase in Pakistani Children Using a Data Mining Approach

Laboratory Medicine ◽

10.1093/labmed/lmz096 ◽

2019 ◽

Vol 51 (5) ◽

pp. 484-490 ◽

Cited By ~ 1

Author(s):

Sibtain Ahmed ◽

Jakob Zierk ◽

Aysha Habib Khan

Keyword(s):

Data Mining ◽

Alkaline Phosphatase ◽

Clinical Decision Making ◽

Clinical Chemistry ◽

Age Groups ◽

Clinical Decision ◽

German Society ◽

Reference Intervals ◽

Photometric Method ◽

Data Mining Approach

Abstract Objective To establish reference intervals (RIs) for alkaline phosphatase (ALP) levels in Pakistani children using an indirect data mining approach. Methods ALP levels analyzed on a Siemens Advia 1800 analyzer using the International Federation of Clinical Chemistry’s photometric method for both inpatients and outpatients aged 1 to 17 years between January 2013 and December 2017, including patients from intensive care units and specialty units, were retrieved. RIs were calculated using a previously validated indirect algorithm developed by the German Society of Clinical Chemistry and Laboratory Medicine’s Working Group on Guide Limits. Results From a total of 108,845 results, after the exclusion of patients with multiple specimens, RIs were calculated for 24,628 males and 18,083 females with stratification into fine-grained age groups. These RIs demonstrate the complex age- and sex-related ALP dynamics occurring during physiological development. Conclusion The population-specific RIs serve to allow an accurate understanding of the fluctuations in analyte activity with increasing age and to support clinical decision making.

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miR-378 reduces mesangial hypertrophy and kidney tubular fibrosis via MAPK signalling

Clinical Science ◽

10.1042/cs20160571 ◽

2017 ◽

Vol 131 (5) ◽

pp. 411-423 ◽

Cited By ~ 14

Author(s):

Bo Wang ◽

Kevin Yao ◽

Andrea F. Wise ◽

Ricky Lau ◽

Hsin-Hui Shen ◽

...

Keyword(s):

Type Iv Collagen ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Mesangial Cell ◽

Quantitative Polymerase Chain Reaction ◽

Locked Nucleic Acid ◽

Type I ◽

Protective Function ◽

Type Iv ◽

Tgf Β1

The regulatory role of a novel miRNA, miR-378, was determined in the development of fibrosis through repression of the MAPK1 pathway, miR-378 and fibrotic gene expression was examined in streptozotocin (STZ)-induced diabetic mice at 18 weeks or in unilateral ureteral obstruction (UUO) mice at 7 days. miR-378 transfection of proximal tubular epithelial cells, NRK52E and mesangial cells was assessed with/without endogenous miR-378 knockdown using the locked nucleic acid (LNA) inhibitor. NRK52E cells were co-transfected with the mothers against decapentaplegic homolog 3 (SMAD3) CAGA reporter and miR-378 in the presence of transforming growth factor-β (TGF-β1) was assessed. Quantitative polymerase chain reaction (qPCR) showed a significant reduction in miR-378 (P<0.05) corresponding with up-regulated type I collagen, type IV collagen and α-smooth muscle actin (SMA) in kidneys of STZ or UUO mice, compared with controls. TGF-β1 significantly increased mRNA expression of type I collagen (P<0.05), type IV collagen (P<0.05) and α-SMA (P<0.05) in NRK52E cells, which was significantly reduced (P<0.05) following miR-378 transfection and reversed following addition of the LNA inhibitor of endogenous miR-378. Overexpression of miR-378 inhibited mesangial cell expansion and proliferation in response to TGF-β1, with LNA–miR-378 transfection reversing this protective effect, associated with cell morphological alterations. The protective function of MAPK1 on miR-378 was shown in kidney cells treated with the MAPK1 inhibitor, selumetinib, which inhibited mesangial cell hypertrophy in response to TGF-β1. Taken together, these results suggest that miR-378 acts via regulation of the MAPK1 pathway. These studies demonstrate the protective function of MAPK1, regulated by miR-378, in the induction of kidney cell fibrosis and mesangial hypertrophy.

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