Shin Nippon Biomedical Laboratories (Japan)

Villitis of unknown etiology (VUE) is a destructive inflammatory lesion of unknown cause in the human placenta. It may be caused by semiallogeneic rejection of fetal tissue by the mother. CD8+ tissue-resident memory T (TRM) cells reside in the peripheral tissues and have an important role in the local defense against reinfection. These cells also contribute to immunopathogenesis under some circumstances and cause local tissue damage and autoimmune disease. In this study, we examine the contribution of CD103+ CD8+ TRM-like cells to the development of VUE. The study included 23 cases of human placenta diagnosed as VUE (high-grade). Double-labeling immunohistochemistry was performed in formalin-fixed paraffin-embedded tissues. CD103+ CD8+ cell accumulation in actively inflamed areas was observed in all cases. The double positivity rate for CD103 and CD8 among the total CD103+ and CD8+ cells was 95% (71-100%) and 58% (18-90%) [median (range)], respectively. This indicated that the vast majority of CD103+ cells in VUE are CD103+ CD8+ TRM-like cells. CD103+ cells exhibited a significantly high proliferative activity based on a Ki-67 labeling index in CD103+ cells of 52% (13-90%). CD103+ cells were aligned beneath E-cadherin+ syncytiotrophoblast cells (subtrophoblast alignment) in the peripheral areas of the VUE lesions. Syncytiotrophoblast cells in VUE were also characterized by the induction of human leukocyte antigens A, B, and C expression. CD103+ cells also expressed granzyme B. Our results suggest that proliferating CD103+ CD8+ TRM-like cells work as cytotoxic effector cells and play an important role in VUE pathogenesis.

BackgroundThe blood-brain barrier (BBB) expresses transporters, receptors, and tight junction proteins that regulate the exchange of substances between the blood and brain. The differences in the expression of these proteins in the BBB among different species and cultured BBB model cells should be clarified to interpret the BBB function in model animals and cells. This study aimed to elucidate species differences among humans, monkeys, and mice and in vitro–in vivo differences in the BBB proteome using deep proteomic analysis.MethodsBrain microvessels (BMVs) were isolated from frozen cerebral cortices of human and monkey and frozen mouse cerebrums. BMVs and cultured brain microvascular endothelial cells (BMECs), such as hCMEC/D3, HBMEC/ciβ, and primary BMECs, were analyzed via data-independent acquisition using liquid chromatography-mass spectrometry.ResultsProteomics identified 7,149–8,274 proteins in the BMV fractions and 6,657–7,534 proteins in the brain lysates. Comparative analysis revealed distinct proteomic profiles among the three species, with the human profile being more similar to that of monkeys than that of mice. The expression profile of the solute carrier organic anion transporter family was found to vary among mouse, monkey, and human BMVs. The expression levels of SLC22A6/Slc22a6 and SLC22A8/Slc22a8 were higher in mice than in monkeys and humans, whereas SLC43A3/Slc43a3 expression levels were lower in mice. The expression of amino acid transporters, such as SLC7A5 and SLC3A2/Slc3a2, was higher in BMVs, whereas that of SLC1A5/Slc1a5 and SLC38A9/Slc38a9 was higher in cultured BMECs. MFSD2A/Mfsd2a and SLC27A1/Slc27a1 were highly expressed in BMVs. The expression of tight junction proteins, particularly the claudin family, varied between BMVs and cultured BMECs and among cell lines. Specifically, the expression of claudin-5 was higher in BMVs, and claudin-11 expression was higher in cultured BMECs.ConclusionsDeep proteomic analysis revealed species-specific differences in transport-related proteins in the BBB. Furthermore, in vitro and in vivo differences were observed in the transporter and claudin protein expression. This study provides a BBB proteome profile dataset and offers insights for a comprehensive understanding of BBB protein expression across species and between in vivo and in vitro conditions.

The epidermis is an essential organ for life by retaining water and as a protective barrier. The epidermis is maintained through metabolism, in which basal cells produced from epidermal stem cells differentiate into spinous cells, granular cells and corneocytes, and are finally shed from the epidermal surface. This is epidermal turnover, and with aging, there is a decline in epidermis function. Other factors that may affect epidermal turnover include ultraviolet damage and genetic factors. These genetic factors are of particular interest as little is known. Although recent skin-focused genome-wide association studies (GWAS) have been conducted, the genetic regions associated with epidermal turnover are almost uninvestigated. Therefore, we conducted a GWAS on epidermal turnover in the Japanese population, using the corneocyte area, which correlates to the rate of epidermal turnover, as an indicator. As a result, rs2278431 (p = 1.29 × 10-7 ) in 19q13.2 was associated with corneocyte size. Furthermore, eQTL analysis suggested that rs2278431 was related to the SPINT2 gene. In addition, SPINT2 knockdown studies using epidermal keratinocytes revealed that SPINT2 is involved in keratinocyte proliferation and in corneocyte size regulation in reconstructed epidermis. These results suggest that rs2278431 is involved in the expression of SPINT2 and affects epidermal turnover.

In new drug development, cells or animals are treated with the selected candidate compound to confirm its efficacy and safety in nonclinical studies. Clinical laboratory tests are carried out using samples from experimental animals in these studies. The clinical laboratory test method validation in nonclinical fields should be conducted keeping in mind that the circumstances differ from those in clinical settings. However, the validation procedures have not been systematically integrated into any standard. The considerations in this paper set out systematically practical guidance for the validation of quantitative analytical methods for fluid samples collected from animal studies, for the purpose of ensuring that laboratory test method validation is conducted in nonclinical fields at an enough level.

The development of embryonic external genitalia (eExG) into characteristic male structures, such as urethra and penile erectile tissues, depends on 5α-dihydrotestosterone (DHT). Although the corpus cavernosum (CC) is well known as essential for erectile function in adults, its developmental process and its dependency on DHT have been unknown. To reveal the dimorphic formation of the murine CC from the embryonic stage, we first analyzed the production of the protein vascular endothelial growth factor receptor-2 (FLK1) via its expression (hereinafter referred as "expression of FLK1") and the expression of alpha-smooth muscle actin (ACTA2) and collagen type 1 (COL1A1) in developing external genitalia. The 5-α reductase type 2 encoded by the SRD5A2 gene has been suggested to be a crucial enzyme for male sexual differentiation, as it converts testosterone (T) into DHT in the local urogenital organs. In fact, SRD5A2 mutation results in decreased synthesis of DHT, which leads to various degrees of masculinized human external genitalia (ExG). We further investigated the expression profile of SRD5A2 during the formation of the murine CC. We observed that SRD5A2 was expressed in smooth muscle of the CC. To determine the role of SRD5A2 in CC formation, we analyzed the formation of erectile tissue in the male Srd5a2 KO mice and measured the levels of androgens in the ExG by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Intriguingly, there were no obvious defects in the CCs of male Srd5a2 KO mice, possibly due to increased T levels. The current study suggests possible redundant functions of androgens in CC development.

The cynomolgus macaque is a non-human primate species widely used in drug metabolism studies. Despite the importance of genetic polymorphisms in cytosolic aldehyde oxidase (AOX) 1 in humans, genetic variants have not been investigated in cynomolgus or rhesus macaques. Genetic variants in AOX1 were identified and allele frequencies were assessed using the genomes of 24 cynomolgus and 8 rhesus macaques. The analysis identified 38 non-synonymous variants, some of which were unique to cynomolgus macaques (bred in Cambodia, Indochina, or Indonesia) or rhesus macaques, whereas many variants were shared by the two lineages. Among the variants observed at relatively high frequencies, eight were selected for functional analysis. Recombinant P605L and V1338I AOX1 variants showed substantially lower phthalazine and carbazeran oxidation activities than the wild-type AOX1 protein. In liver cytosolic fractions from cynomolgus and rhesus macaques genotyped for P605L and V1338I AOX1, groups of cytosolic fractions with P605L and/or V1338I AOX1 variants showed significantly lower phthalazine and carbazeran oxidation activities than the wild type. These results indicate that AOX1 is polymorphic in cynomolgus and rhesus macaques, just as it is in humans. Further investigation is needed to reveal the functional significance of these AOX1 variants in drug metabolism.

Plasma concentrations of acetaminophen, its glucuronide and sulfate conjugates, and cysteinyl acetaminophen were experimentally determined after oral administrations of 10 mg/kg in humanised-liver mice, control mice, rats, common marmosets, cynomolgus monkeys, and minipigs; the results were compared with reported human pharmacokinetic data. Among the animals tested, only rats predominantly converted acetaminophen to sulfate conjugates, rather than glucuronide conjugates. In contrast, the values of area under the plasma concentration curves of acetaminophen, its glucuronide and sulfate conjugates, and cysteinyl acetaminophen after oral administration of acetaminophen in marmosets and minipigs were consistent with those reported in humans under the present conditions. Physiologically based pharmacokinetic (PBPK) models (consisting of the gut, liver, and central compartments) for acetaminophen and its primary metabolite could reproduce and estimate, respectively, the plasma and hepatic concentrations of acetaminophen in experimental animals and humans after single virtual oral doses. The values of area under the curves of hepatic concentrations of acetaminophen estimated using PBPK models were correlated with the measured levels of cysteinyl acetaminophen (a deactivated metabolite) in plasma fractions in these species. Consequently, using simple PBPK models and plasma data to predict hepatic chemical concentrations after oral doses could be helpful as an indicator of in vivo possible hepatotoxicity of chemicals such as acetaminophen.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments.