Considerations for the Identification and Conveyance of Clinical Pathology Findings in Preclinical Toxicity Studies: Results From the 9th ESTP International Expert Workshop.

The European Society of Toxicologic Pathology (ESTP) organized a panel of 24 international experts from many fields of toxicologic clinical pathology (e.g., industry, academia, and regulatory) that came together in 2021 to align the use of terminology to convey the importance of clinical pathology findings in preclinical toxicity studies. An additional goal consisted of how to identify important findings in standard and nonstandard clinical pathology associated endpoints. This manuscript summarizes the information and opinions discussed and shared at the ninth ESTP International Expert Workshop, April 5 to 6, 2022. In addition to terminology usage, the workshop considered topics related to the identification and conveyance of the importance of test item-related findings. These topics included sources of variability, comparators, statistics, reporting, correlations to other study data, nonstandard biomarkers, indirect/secondary findings, and an overall weight-of-evidence approach.

Parents make the difference: a randomized-controlled trial of a parenting intervention in Liberia.

BACKGROUND: The objective of this study was to evaluate the impact of a brief parenting intervention, 'Parents Make the Difference'(PMD), on parenting behaviors, quality of parent-child interactions, children's cognitive, emotional, and behavioral wellbeing, and malaria prevention behaviors in rural, post-conflict Liberia. METHODS: A sample of 270 caregivers of children ages 3-7 were randomized into an immediate treatment group that received a 10-session parent training intervention or a wait-list control condition (1:1 allocation). Interviewers administered baseline and 1-month post-intervention surveys and conducted child-caregiver observations. Intent-to-treat estimates of the average treatment effects were calculated using ordinary least squares regression. This study was pre-registered at ClinicalTrials.gov (NCT01829815). RESULTS: The program led to a 55.5% reduction in caregiver-reported use of harsh punishment practices (p < 0.001). The program also increased the use of positive behavior management strategies and improved caregiver-child interactions. The average caregiver in the treatment group reported a 4.4% increase in positive interactions (p < 0.05), while the average child of a caregiver assigned to the treatment group reported a 17.5% increase (p < 0.01). The program did not have a measurable impact on child wellbeing, cognitive skills, or household adoption of malaria prevention behaviors. CONCLUSIONS: PMD is a promising approach for preventing child abuse and promoting positive parent-child relationships in low-resource settings.

In vitro development of resistance to human immunodeficiency virus protease inhibitor GW640385.

Development of in vitro resistance to GW640385, a new human immunodeficiency virus type 1 protease inhibitor, was studied. Variants characterized included one with <4-fold resistance and amino acid substitutions Q58E/A71V (protease) and P452K (Gag) and one with >50-fold resistance and amino acid substitutions L10F/G16E/E21K/A28S/M46I/F53L/A71V (protease) and L449F/P453T (Gag). The A28S substitution substantially reduced replication capacity.

Selection and interpretation of clinical pathology indicators of hepatic injury in preclinical studies.

This position paper delineates the expert recommendations of the Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathology for the use of preclinical, clinical pathology endpoints in assessment of the potential for drug-induced hepatic injury in animals and humans. Development of these guidelines has been based on current recommendations in the relevant preclinical and human clinical trial literature; they are intended to provide a method for consistent and rigorous interpretation of liver-specific data for the identification of hepatic injury in preclinical studies and potential liability for hepatic injury in human patients.

2-Amino-6-arylsulfonylbenzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1.

A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-activity relationship (SAR) studies suggested that a meta substituent, particularly a meta methyl substituent, invariably increased antiviral activities. However, optimal antiviral activities were manifested by compounds where both meta groups in the arylsulfonyl moiety were substituted and one of the substituents was a methyl group. Such a disubstitution led to compounds 3v, 3w, 3x, and 3y having IC50 values against HIV-1 in the low nanomolar range. When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u-z and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against the V106A and P236L strains and submicromolar to low nanomolar activity against strains E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v (739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure allowed for the rationalization of SAR data and potencies of the compounds against the mutants.

Proximal trisomy of 1q mosaicism in a girl with hypertrophic cardiomyopathy associated with Wolff-Parkinson-White syndrome and multiple congenital anomalies.

We report an African American female who is mosaic for partial trisomy of 1q due to a direct duplication of 1q12 to 1q25. The child has hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. The physical features include micrognathia, cleft palate, low set ears, posteriorly placed thumbs, and syndactyly of the second and third toes of both feet. Other abnormalities include intestinal malrotation, scoliosis, mental retardation, cerebral palsy, and hydrocephalus. There was also a selective deficiency of antibody responses to polysaccharide antigens. Proximal duplication of chromosome 1q is rare and has not been previously associated with hypertrophic cardiomyopathy. Most known gene disorders related to hypertrophic cardiomyopathy are autosomal dominant missense mutations in sarcomeric protein genes; however, none of the sarcomeric genes previously linked to hypertrophic cardiomyopathy are in this region. This finding thus highlights the possibility of additional genetic mechanisms for hypertrophic cardiomyopathy.

Physiological responses to mixing in large scale bioreactors.

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

At the maize/Agrobacterium interface: natural factors limiting host transformation.

BACKGROUND: Agrobacterium tumefaciens has been successfully harnessed as the only natural vector for the incorporation of foreign genes into higher plants, but its use in the grain crops is often limited. Low transformation efficiency has been partly attributed to a failure in the initial events in the transformation process, specifically in the capacity of the VirA/VirG two-component system to induce expression of the virulence genes. RESULTS: Here we show that the root exudate of Zea mays seedlings specifically inhibits virulence gene expression, determine that 2-hydroxy-4,7-dimethoxybenzoxazin-3-one (MDIBOA), which constitutes > 98% of the organic exudate of the roots of these seedlings, is the most potent and specific inhibitor of signal perception in A. tumefaciens-mediated gene transfer yet discovered, and develop a model that is able to predict the MDIBOA concentration at any distance from the root surface. Finally, variants of A. tumefaciens resistant to MDIBOA-mediated inhibition of vir gene expression have been selected and partially characterized. CONCLUSIONS: These results suggest a strategy in which a plant may resist pathogen invasion by specifically blocking virulence gene activation and yet ensure that the 'resistance factor' does not accumulate to levels sufficient to impose toxicity and selection pressure on the pathogen. The data further establish that naturally occurring inhibitors directed against signal perception by the VirA/VirG two-component regulatory system can play an important role in host defense. Finally, selected variants resistant to specific MDIBOA inhibition may now be used to extend the transformation efficiency of maize and possibly other cereals.

Deletions in the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase are observed in HIV-1 isolated from subjects during long-term antiretroviral therapy.

OBJECTIVES: To examine the effect of in-frame deletions in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on plasma viremia and phenotypic resistance to antiretroviral drugs. STUDY DESIGN/METHODS: Plasma HIV-1 RNA was isolated from 168 antiretroviral therapy-experienced subjects for quantification of plasma viremia, RT sequence analysis, and phenotypic resistance assays. RESULTS: Four patients were found to harbor HIV-1 strains possessing in-frame, 3-nucleotide deletions at RT codons 67, 69, and 70. In these subjects, phenotypic resistance and high plasma viremia were observed only in a background of multiple resistance mutations. A recombinant virus engineered with an in-frame deletion of RT codon 67 did not have increased resistance to nucleoside reverse transcriptase inhibitors (NRTIs). CONCLUSIONS: Selection for deletions within the beta3-beta4 hairpin loop of the HIV-1 RT is an uncommon event most likely to occur in subjects with long-term antiretroviral experience. The codon 67 deletion does not appear to cause increased phenotypic resistance or increased viremia in the absence of concomitant RT mutations.

Newborn infant with inherited ring and de novo interstitial deletion on homologous chromosome 22s.

A 2-day-old infant was evaluated and suspected of having 22q11.2 deletion based on microcephaly, short and narrow palpebral fissures, a prominent nose with hypoplastic alae nasi, thin fingers, and a right aortic arch. He also had an imperforate anus, which is not in the del 22q11.2 syndrome. Karyotype analysis identified a ring 22, while fluorescence in situ hybridization (FISH) for the DiGeorge syndrome critical region identified a 22q deletion on the other homologue. The karyotype designation was 46,XY,r(22)(p13q13.3).ish del(22)(q11.2q11.2) (D22S75-). Both parents function in the mildly mentally retarded range. The father's karyotype was normal whereas the mother had the ring 22 that was inherited by her son. This is the first case reported for abnormalities on both 22 homologues.

Characterization of (+) strand initiation and termination sequences located at the center of the equine infectious anemia virus genome.

Permeabilized preparations of equine infectious anemia virus (EIAV) are shown here to support efficient and accurate synthesis of full-length double-stranded proviral DNA. When (-) and (+) strand products were analyzed by Southern blotting, a discontinuity, mapping approximately to the center of the EIAV genome, could be demonstrated for the (+) strand, predicting a second site for initiation of DNA synthesis and a specific mechanism of (+) strand termination. Precise localization of this (+) strand origin within the integrase (IN) coding region was achieved through its in vitro selection and extension into, and excision from, nascent DNA by purified recombinant p66/p51 EIAV reverse transcriptase (RT), suggesting that the EIAV genome harbors a central polypurine tract (cPPT). In addition, a model system was developed for evaluating whether sequences immediately downstream of the cPPT would terminate (+) strand synthesis in the context of strand displacement. Such a sequence was indeed discovered which functions in a manner analogous to that of the central termination sequence (CTS) of HIV, where A-tract-induced minor groove compression has been suggested to induce localized distortion of the nucleic acid duplex and termination of (+) strand synthesis. This interpretation is reinforced by experiments indicating that read-through of the CTS can be efficiently promoted by substituting 2,6-diaminopurine for adenine, thereby relieving minor groove compression. The nucleotide substitution can also shift the site of termination in strand displacement (+) strand synthesis. Collectively, our data support proposals that lentiviruses may have evolved specialized mechanisms for initiating and terminating (+) strand DNA synthesis at the center of their genomes.

Use of human recombinant erythropoietin and prednisone for treatment of myelodysplastic syndrome with erythroid predominance in a dog.

A 7-year-old German Shepherd dog was referred for evaluation of severe nonregenerative anemia (PCV, 10%; reticulocyte fraction, 0.2%). Cytologic examination of a bone marrow aspirate indicated erythroid predominance and dyserythropoiesis, and a diagnosis of myelodysplastic syndrome (MDS) with erythroid predominance was made. The dog was given a single blood transfusion and was treated with prednisone and recombinant human erythropoietin (EPO). Eight weeks later, anemia had resolved. The dog remained clinically normal 30 months after treatment, with a PCV of 45%. Results suggest that EPO may be useful in the treatment of dogs with MDS with erythroid predominance or erythroleukemia. Additional studies are required to confirm the benefit of EPO to manage MDS-associated anemia in dogs.

On becoming a parasite: evaluating the role of wall oxidases in parasitic plant development.

BACKGROUND: The temporal and spatial control of the transition from vegetative to parasitic growth is critical to any parasite, but is essential to the sessile parasitic plants. It has been proposed that this transition in Striga spp. is controlled simply by an exuded oxidase that converts host cell-surface phenols into benzoquinones which act as developmental signals that mediate the transition. An understanding of this mechanism may identify the critical molecular events that made possible the evolution of parasitism in plants. RESULTS: PoxA and PoxB are identified as the only apoplastic phenol oxidases in Striga asiatica seedlings, and the genes encoding them have been cloned and sequenced. These peroxidase enzymes are capable of oxidizing the 60 known inducing phenols into a small set of benzoquinones, and it is these quinones that induce parasitic development. Analysis of the reaction requirements and comparisons to host enzymes, however, lead us to argue that PoxA and PoxB are not necessary for host recognition. CONCLUSIONS: A new model is proposed where constitutive production of an activated oxygen species (in the case of Striga, H2O2) mediates host recognition. This strategy would allow a parasite to exploit abundant host enzymes to produce the diffusible recognition signals by converting a standard host defense into a parasitic offense.

Recombinant human CXC-chemokine receptor-4 in melanophores are linked to Gi protein: seven transmembrane coreceptors for human immunodeficiency virus entry into cells.

This article describes the transient expression of the CXC chemokine receptor-4 in Xenopus laevis melanophores and the resulting functional assay for the endogenous ligand for this receptor stromal cell-derived factor (SDF)-1alpha. Specifically, it will be shown that SDF-1alpha produces increased light transmittance in transfected cells that is consistent with the activation of Gi protein. This stimulus pathway is further implicated by the abolition of this response after pretreatment of the cells with pertussis toxin, a known method for the inactivation of Gi protein. The fact that SDF-1alpha does not produce responses in nontransfected cells and that treatment of the cells with 12G5, an antibody specific for the CXC chemokine receptor-4, eliminates this response indicates that this ligand produces responses by activation of this receptor in these cells. The possible relevance to human immunodeficiency virus (HIV) entry into cells was explored by observing the effects of SDF-1alpha on HIV-mediated cell fusion. It was found that SDF-1alpha blocked cell-to-cell fusion (as has been previously reported) at concentrations 1200-fold greater than those required to produce Gi protein mediated responses. The implications of the functional assay to screening for new drugs to block HIV-mediated fusion is discussed.

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

A molecular anatomical analysis of mosaic trisomy 16.

A one-month-old child presenting with an aortic coarctation was found to have a left single transverse palmar crease and proportionate growth delay on physical examination, prompting a peripheral blood chromosome analysis. This showed a mosaic trisomy of chromosome 16, subsequently observed to decrease with the passage of time. As her phenotype was relatively benign, further analysis was performed to define more precisely the extent of her mosaicism given the supposedly lethal nature of the aneuploid cell line. Fluorescence in situ hybridisation and CA repeat polymorphism studies demonstrated the aneuploidy in multiple tissues, including the structurally affected aorta. Molecular analysis showed both maternal chromosomes 16 to be present in the trisomic cells, but maternal heterodisomy was not present in the diploid cells. Given the increasing number of individuals described with aneuploid mosaicism, we suggest that the study of multiple tissues is a necessary approach, the eventual goal being the appreciation of the relationship between the characteristics of a somatic mosaicism and the phenotype it imparts.

DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs.

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-beta-D enantiomers of some pairs were preferred as substrates. In contrast, the 1-beta-L enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-beta-D enantiomer required for the same inhibitory effect. This preference for the 1-beta-L enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol.

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

"Balanced" karyotypes in six abnormal offspring of balanced reciprocal translocation normal carrier parents.

Among 6800 consecutive blood samples studied for clinical cytogenetic diagnosis, we identified 30 families in which one parent of the proband had a balanced reciprocal autosomal translocation (excluding Robertsonian rearrangements). Twenty-eight of the 30 families had a malformed and/or mentally retarded proband: 19 with an unbalanced derived chromosome, 3 with abnormalities involving chromosomes other than those in the translocation, 5 with a "balanced" reciprocal translocation, and 1 with a normal karyotype. We hypothesize that the latter 6 affected probands with "balanced" karyotypes could be abnormal due to submicroscopic deletions and duplications as was originally suggested by Jacobs [1984]. Particularly in these 6 families, 83% of translocation breakpoints were associated with fragile sites, more than expected by chance (P < 0.025). This supports the report of an association between fragile sites and constitutional chromosome breakpoints by Hecht and Hecht [1984]. To explain these findings, we propose that autosomal fragile sites are unstable areas which predispose to breaks and unequal crossing over near the fragile site breakpoints creating minute duplications and deletions. Consequently, newborn infants inheriting a seemingly "balanced" karyotype from a normal parent with a balanced reciprocal translocation may still be at an increased risk of being malformed and/or developmentally delayed because of submicroscopic chromosomal imbalances.