Effect of sequencing platforms on the sensitivity of chemical mutation detection using Hawk-Seq™.

BACKGROUND: Error-corrected next-generation sequencing (ecNGS) technologies have enabled the direct evaluation of genome-wide mutations after exposure to mutagens. Previously, we reported an ecNGS methodology, Hawk-Seq™, and demonstrated its utility in evaluating mutagenicity. The evaluation of technical transferability is essential to further evaluate the reliability of ecNGS-based assays. However, cutting-edge sequencing platforms are continually evolving, which can affect the sensitivity of ecNGS. Therefore, the effect of differences in sequencing instruments on mutation data quality should be evaluated. RESULTS: We assessed the performance of four sequencing platforms (HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400) with the Hawk-Seq™ protocol for mutagenicity evaluation using DNA samples from mouse bone marrow exposed to benzo[a]pyrene (BP). The overall mutation (OM) frequencies per 106 bp in vehicle-treated samples were 0.22, 0.36, 0.46, and 0.26 for HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400, respectively. The OM frequency of NextSeq2000 was significantly higher than that of HiSeq2500, suggesting the difference to be based on the platform. The relatively higher value in NextSeq2000 was a consequence of the G:C to C:G mutations in NextSeq2000 data (0.67 per 106 G:C bp), which was higher than the mean of the four platforms by a ca. of 0.25 per 106 G:C bp. A clear dose-dependent increase in G:C to T:A mutation frequencies was observed in all four sequencing platforms after BP exposure. The cosine similarity values of the 96-dimensional trinucleotide mutation patterns between HiSeq and the three other platforms were 0.93, 0.95, and 0.92 for NovaSeq, NextSeq, and DNBSeq, respectively. These results suggest that all platforms can provide equivalent data that reflect the characteristics of the mutagens. CONCLUSIONS: All platforms sensitively detected mutagen-induced mutations using the Hawk-Seq™ analysis. The substitution types and frequencies of the background errors differed depending on the platform. The effects of sequencing platforms on mutagenicity evaluation should be assessed before experimentation.

Application of testicular organ culture system for the evaluation of spermatogenesis impairment.

Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo.

Expression analysis of genes including Zfhx4 in mice and zebrafish reveals a temporospatial conserved molecular basis underlying craniofacial development.

BACKGROUND: Embryonic craniofacial development involves several cellular and molecular events that are evolutionarily conserved among vertebrates. Vertebrate models such as mice and zebrafish have been used to investigate the molecular and cellular etiologies underlying human craniofacial disorders, including orofacial clefts. However, the molecular mechanisms underlying embryonic development in these two species are unknown. Therefore, elucidating the shared mechanisms of craniofacial development between disease models is crucial to understanding the underlying mechanisms of phenotypes in individual species. RESULTS: We selected mice and zebrafish as model organisms to compare various events during embryonic craniofacial development. We identified genes (Sox9, Zfhx3 and 4, Cjun, and Six1) exhibiting similar temporal expression patterns between these species through comprehensive and stage-matched gene expression analyses. Expression analysis revealed similar gene expression in hypothetically corresponding tissues, such as the mice palate and zebrafish ethmoid plate. Furthermore, loss-of-function analysis of Zfhx4/zfhx4, a causative gene of human craniofacial anomalies including orofacial cleft, in both species resulted in deformed skeletal elements such as the palatine and ethmoid plate in mice and zebrafish, respectively. CONCLUSIONS: These results demonstrate that these disease models share common molecular mechanisms, highlighting their usefulness in modeling craniofacial defects in humans.

Integrating mathematical approaches (IMAS): Novel methodology for predicting dermal absorption rates of chemicals under finite dose conditions.

Quantitative structure permeation relationship (QSPR) models have gained prominence in recent years owing to their capacity to elucidate the influence of physicochemical properties on the dermal absorption of chemicals. These models facilitate the prediction of permeation coefficient (Kp) values, indicating the skin permeability of a chemical under infinite dose conditions. Conversely, obtaining dermal absorption rates (DAs) under finite dose conditions, which are crucial for skin product safety evaluation, remains a challenge when relying solely on Kp predictions from QSPR models. One proposed resolution involves using Kroes' methodology, categorizing DAs based on Kp values; however, refinement becomes necessary owing to discreteness in the obtained values. We previously developed a mathematical model using Kp values obtained from in vitro dermal absorption tests to predict DAs. The present study introduces a new methodology, Integrating Mathematical Approaches (IMAS), which combines QSPR models and our mathematical model to predict DAs for risk assessments without conducting in vitro dermal absorption tests. Regarding 40 chemicals (76.1 ≤ MW ≤ 220; -1.4 ≤ Log Ko/w ≤ 3.1), IMAS showed that 65.0% (26/40) predictions of DA values were accurate to within twofold of the observed values in finite dose experiments. Compared to Kroes' methodology, IMAS notably mitigated overestimation, particularly for hydrophilic chemicals with water solubility exceeding 57.0 mg/cm3. These findings highlight the value of IMAS as a tool for skin product risk assessments, particularly for hydrophilic compounds.

Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

The inter-laboratory validation study of EpiSensA for predicting skin sensitization potential.

The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories. The purpose of this validation study was to assess transferability of the EpiSensA to new laboratories along with its within- (WLR) and between-laboratory reproducibility (BLR). The validation study was organized into two independent stages. As demonstrated during the first stage, where three sensitizers and one non-sensitizer were correctly predicted by all participating laboratories, the EpiSensA was successfully transferred to all three participating laboratories. For Phase I of the second stage, each participating laboratory performed three experiments with an identical set of 15 coded test chemicals resulting in WLR of 93.3%, 93.3%, and 86.7%, respectively. Furthermore, when the results from the 15 test chemicals were combined with those of the additional 12 chemicals tested in Phase II of the second stage, the BLR for 27 test chemicals was 88.9%. Moreover, the predictive capacity among the three laboratories showed 92.6% sensitivity, 63.0% specificity, 82.7% accuracy, and 77.8% balanced accuracy based on murine local lymph node assay (LLNA) results. Overall, this validation study concluded that EpiSensA is easily transferable and sufficiently robust for assessing the skin sensitization potential of chemicals.

Impact of nCuO containing treated wastewater on soil microbes and dissolved organic matter in paddy field leachate.

Using treated wastewater (TWW) resources in agriculture is a major pathway for disseminating nanoparticles. Copper-oxide nanoparticles (nCuO) offer potential benefits, but their presence in the environment poses risks to agricultural and environmental sustainability. This study examined soil microbial transformations and the composition of leachate dissolved organic matter (DOM) of paddy soils irrigated with nCuO-contaminated TWW at different concentrations (T2: 0.02 mgL-1, T3: 0.2 mgL-1, T4: 2.0 mgL-1) and examined the differences in Cu source (T5: 0.2 mgL-1 CuSO4). Results showed negative impacts on the absolute microbial abundance with up to 46 % reduction relative to the control treatment (T1). Changes in relative abundance of specific microbes at the genus level deviated from the corresponding phyla. Acidobacteria, Actinobacteria, Chloroflexi, and Verrucomicrobia phyla increased in the surface (0-3 cm) and subsurface (3-15 cm) layers responding differently to nCuO. In the 0-3 cm layer, Nitrospirae, Euryarchaeota, and Crenarchaeota increased, but only Dechloromonas genus from Proteobacteria increased with increasing nCuO. No significant variations were observed in the DOM composition, except in T4, which had a significantly low content of dissolved organic carbon (DOC), total dissolved nitrogen, and terrestrial humic-like and protein-like components. Ninety-eight distinct genera were identified, of which 44%, including 15 bacteria and two archaea, varied between the surface and subsurface, among treatments, and significantly correlated with more DOM parameters in the subsurface. T4 had the highest microbial diversity in the 0-3 layer, and Cu treatments slightly increased the diversity index in the subsurface. Moreover, the effects differed by Cu source, with T3 showing 10 % more reduction in the subsurface and 17 % less reduction in the surface than T5. The variable microbial responses to nCuO and their strong correlations with DOM highlight the need to consider the potential consequences of low nCuO concentrations on biogeochemical cycles.

Tolerogenic phenotype of dendritic cells is induced after hapten sensitization followed by attenuated contact hypersensitivity response in atopic dermatitis model NC/Nga mice.

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are common inflammatory diseases. We previously reported attenuated contact hypersensitivity (CHS) responses in AD model mice using 2,4-dinitrofluorobenzene, reflecting clinical experiments. However, previous studies have not addressed the commonality of findings across haptens and mechanisms focused on dendritic cells (DCs). Thus, this study evaluated CHS responses to fluorescein isothiocyanate (FITC) and DC migration and maturation in the sensitization phase of CHS in AD. CHS responses to FITC were compared between NC/Nga mice without and with AD induction (non-AD and AD mice, respectively). T-cell responses and DC migration and maturation after FITC-induced sensitization were examined in the draining lymph nodes of non-AD and AD mice. AD mice demonstrated reduced CHS responses to FITC under decreased T-cell proliferation following sensitization and interferon-γ production by hapten-specific T cells compared with non-AD mice. In addition, the number of FITC+CD11c+MHC class IIhigh migratory DCs 24 h after FITC sensitization was comparable between non-AD and AD mice. However, FITC+CD11c+MHC class IIhigh migratory DCs in AD mice exhibited lower expression levels of CD80 and CD86 and higher expression levels of PD-L1 and mRNA of transforming growth factor beta than non-AD mice. These findings suggest that attenuated CHS responses may be hapten-independent and the induction of the tolerogenic phenotype of hapten-bearing DCs can contribute to reduced T-cell proliferation after sensitization and CHS responses in AD.

Evaluation of MTT reducers and strongly colored substances in the Short Time Exposure test method for assessing eye irritation potential.

The Short Time Exposure (STE) test evaluates eye irritation potential using a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assays may underpredict results for some substances that directly reduce MTT (i.e., MTT reducers) or interfere with absorbance because of their strong color (i.e., strongly colored substances). Based on previous research, we selected 25 substances as MTT reducers. Of these, 13 were expected to be MTT reducers at 5% dilution (5% MTT reducers) of the STE test condition. These 13 substances were then tested to determine whether the results were interfered from direct MTT reduction. Those 5% MTT reducers that were classified as irritants based on in vivo data were identified as irritants by the STE test. In addition, the low cell viability results at 5% dilution suggested that direct MTT reduction had not occurred. Next, the remaining 5% MTT reducers that were classified as non-irritants based on in vivo data were identified as non-irritants by the STE test. We then examined two strongly colored substances. One was classified as an irritant based on in vivo data and was confirmed as an irritant by the STE test. The other was classified as a non-irritant by the STE test. This was further evaluated using a medium that did not contain MTT; the result indicated that it was a non-irritant correctly. In conclusion, the STE test is useful for evaluating eye irritation potential without the drawback of underprediction for MTT reducers and strongly colored substances.

CTLA-4 suppresses hapten-induced contact hypersensitivity in atopic dermatitis model mice.

Atopic dermatitis (AD) patients with skin barrier dysfunction are considered to be at a higher risk of allergic contact dermatitis (ACD), although previous studies showed that attenuated ACD responses to strong sensitizers in AD patients compared to healthy controls. However, the mechanisms of ACD response attenuation in AD patients are unclear. Therefore, using the contact hypersensitivity (CHS) mouse model, this study explored the differences in CHS responses to hapten sensitization between NC/Nga mice with or without AD induction (i.e., non-AD and AD mice, respectively). In this study, ear swelling and hapten-specific T cell proliferation were significantly lower in AD than in non-AD mice. Moreover, we examined the T cells expressing cytotoxic T lymphocyte antigen-4 (CTLA-4), which is known to suppress T cell activation, and found a higher frequency of CTLA-4+ regulatory T cells in draining lymph node cells in AD than in non-AD mice. Furthermore, the blockade of CTLA-4 using a monoclonal antibody eliminated the difference in ear swelling between non-AD and AD mice. These findings suggested that CTLA-4+T cells may contribute to suppressing the CHS responses in AD mice.

Adjusting Heterodimeric Coiled-Coils (K/E Zipper) to Connect Autophagy-Inducing Peptide with Cell-Penetrating Peptide.

A connection of a functional peptide with a cell-penetrating peptide (CPP) used a heterodimeric coiled-coil as a molecular zipper can improve the intracellular delivery and activity of the functional peptide. However, the chain length of the coiled coil required for functioning as the molecular zipper is unknown at present. To solve the problem, we prepared an autophagy-inducing peptide (AIP) that conjugates with the CPP via heterodimeric coiled-coils consisting of 1 to 4 repeating units (K/E zipper; AIP-Kn and En-CPP), and we investigated the optimum length of the K/E zipper for effective intracellular delivery and autophagy induction. Fluorescence spectroscopy showed that K/E zippers with n = 3 and 4 formed a stable 1:1 hybrid (AIP-K3/E3-CPP and AIP-K4/E4-CPP, respectively). Both AIP-K3 and AIP-K4 were successfully delivered into cells by the corresponding hybrid formation with K3-CPP and K4-CPP, respectively. Interestingly, autophagy was also induced by the K/E zippers with n = 3 and 4, more intensively by the former than by the latter. The peptides and K/E zippers used in this study did not show significant cytotoxicity. These results indicate that the effective induction of autophagy occurs via an exquisite balance of the association and dissociation of the K/E zipper in this system.

Applying a next generation risk assessment framework for skin sensitisation to inconsistent new approach methodology information.

Cosmetic products must be safe for their intended use. Regulatory bans on animal testing for new ingredients have resulted in a shift towards the use of new approach methodologies (NAMs) such as in silico predictions and in chemico / in vitro data. Defined approaches (DAs) have been developed to interpret combinations of NAMs to provide information on skin sensitization hazard and potency, three having been adopted within OECD Test Guideline 497. However, the challenge remains as to how DAs can be used to derive a quantitative point of departure for use in next generation risk assessment (NGRA). Here we provide an update to our previously published NGRA framework and present two hypothetical consumer risk assessment scenarios (rinse-off and leave-on) on one case study ingredient. Diethanolamine (DEA) was selected as the case study ingredient based on the existing NAM information demonstrating differences with respect to the outcomes from in silico predictions and in chemico / in vitro data. Seven DAs were applied, and these differences resulted in divergent DA outcomes and reduced confidence with respect to the hazard potential and potency predictions. Risk assessment conclusion for the rinse-off exposure led to an overall decision of safe for all applied DAs. Risk assessment conclusion for the higher leave-on exposure was safe when based on some DAs but unsafe based on others. The reasons for this were evaluated as well as the inherent uncertainty from the use of each NAM and DA in the risk assessment, enabling further refinement of our NGRA framework.

Incorporating integrated testing strategy (ITSv1) defined approach into read-across (RAx) in predicting skin sensitization potency: ITSv1-based RAx.

Recently, due to regulatory and ethical demands, new approach methodologies (NAMs), defined approaches (DAs), and read-across (RAx) have been used in the risk assessment of skin sensitization. Integrated testing strategy (ITS)v1 DA, adopted in OECD Guideline No. 497, can be used for skin sensitization potency categorization. However, ITSv1 DA alone is not used for further refinement of the potency prediction based on EC3 (the estimated concentration that produces a stimulation index of 3 in murine local lymph node assay) values. Moreover, there is no explicit approach to incorporating NAM/DA data into RAx to fill the data gap of EC3 values with high confidence. This study developed a strategy incorporating ITSv1 DA into RAx to predict skin sensitization potency: ITSv1-based RAx. To examine the reliability of this novel strategy, a case study with lilial, a fragrance material, was performed. Based on ITSv1-based RAx, the skin sensitization potency of lilial was determined by extrapolating the EC3 value of 9.5% for the suitable analogue bourgeonal, which was close to the historical EC3 value of 8.6%. The result suggested that the strategy can refine the prediction of EC3 values with high confidence and be useful for the risk assessment of skin sensitization.

Evaluating the applicability of the Ames test for cosmetic packaging assessment by comparing carcinogenic risk levels of migrants from plastics with biological detection limits.

Risk assessments for cosmetic packaging are required according to the EU Cosmetics Regulation (EC) No. 1223/2009, however, the assessment method is well-established for food packaging but limited for cosmetic packaging. In food packaging assessments, Cramer class III TTC (90 µg/day) is applied as the threshold for systemic toxicity when the Ames test including the process of sample concentration steps provides the negative results. However, the human health risks of mutagenic and carcinogenic migrants at exposure levels where the Ames test with the concentrated samples cannot detect are unclear. In the present study, to confirm the applicability of the Ames test for cosmetic packaging assessments, the toxicological data on 37 candidate migrants with Ames test-positive results was collected. For these migrants, the carcinogenic risk levels through cosmetics use were compared to the detection levels of the Ames test for concentrated samples. Regarding at least 32 migrants, the case study showed the negative result from the Ames test incorporating the sample concentration process would indicate negligible mutagenic and carcinogenic risks of packaging extracts. Therefore, application of the Ames test to cosmetic packaging assessments would be helpful to ensure the safety for mutagenicity and carcinogenicity as well as use Cramer-TTC for systemic toxicity.

Importance of tenosynovitis in preventing the progression through rheumatoid arthritis continuum.

Rheumatoid arthritis (RA) has long been characterized by synovitis and bone erosions typically developing symmetrically in small joints. However, recent advances in imaging modalities have indicated frequent association of tenosynovitis with RA, and some consider tenosynovitis to be not just a complication but a major trait of RA. Furthermore, as there are cases with tenosynovitis preceding the clinical detection of inflammatory arthritis in predisposed individuals, tenosynovitis may constitute an important biomarker in defining the pre-RA phase of disease development. Tenosynovitis itself must be treated as it causes functional impairment and physical as well as socioeconomic burden, and its treatment may result in effective prevention of RA development at a pre-arthritic stage. Thus, further efforts need to be taken in detecting and treating tenosynovitis in the pre-RA stage, which can be facilitated by ultrasonography and magnetic resonance imaging.

Expansion of the Cosmetics Europe skin sensitisation database with new substances and PPRA data.

The assessment of skin sensitisation is a key requirement in all regulated sectors, with the European Union's regulation of cosmetic ingredients being most challenging, since it requires quantitative skin sensitisation assessment based on new approach methodologies (NAMs). To address this challenge, an in-depth and harmonised understanding of NAMs is fundamental to inform the assessment. Therefore, we compiled a database of NAMs, and in vivo (human and local lymph node assay) reference data. Here, we expanded this database with 41 substances highly relevant for cosmetic industry. These structurally different substances were tested in six NAMs (Direct Peptide Reactivity Assay, KeratinoSens™, human Cell Line Activation Test, U-SENS™, SENS-IS, Peroxidase Peptide Reactivity Assay). Our analysis revealed that the substances could be tested without technical limitations, but were generally overpredicted when compared to reference results. Reasons for this reduced predictivity were explored through pairwise NAM comparisons and association of overprediction with hydrophobicity. We conclude that more detailed understanding of how NAMs apply to a wider range of substances is needed. This would support a flexible and informed choice of NAMs to be optimally applied in the context of a next generation risk assessment framework, ultimately contributing to the characterisation and reduction of uncertainty.

Implementation of a dermal sensitization threshold (DST) concept for risk assessment: structure-based DST and in vitro data-based DST.

Skin sensitization resulting in allergic contact dermatitis represents an important toxicological endpoint as part of safety assessments. When available substance-specific sensitization data are inadequate, the dermal sensitization threshold (DST) concept has been proposed to set a skin exposure threshold to provide no appreciable risk of skin sensitization. Structure-based DSTs, which include non-reactive, reactive, and high potency category (HPC) DSTs, can be applied to substances with an identified chemical structures. An in vitro data-based "mixture DST" can be applied to mixtures based on data from in vitro test methods, such as KeratinoSens™ and the human Cell Line Activation Test. The purpose of this review article is to discuss the practical use of DSTs for conducting sound sensitization risk assessments to assure the safety of consumer products. To this end, several improvements are discussed in this review. For application of structure-based DSTs, an overall structural classification workflow was developed to exclude the possibility that "HPC but non-reactive" chemicals are misclassified as "non-reactive", because such chemicals should be classified as HPC chemicals considering that HPC rules have been based on the chemical structure of high potency sensitizers. Besides that, an extended application of the mixture DST principle to mixtures that either is cytotoxic or evaluated as positive was proposed. On a final note, we also developed workflows that integrate structure-based and in vitro-based mixture DST. The proposed workflows enable the application of the appropriate DST, which serves as a point of departure in the quantitative sensitization risk assessment.

Distinctive High Expression of Antiretroviral APOBEC3 Protein in Mouse Germinal Center B Cells.

Tissue and subcellular localization and its changes upon cell activation of virus-restricting APOBEC3 at protein levels are important to understanding physiological functions of this cytidine deaminase, but have not been thoroughly analyzed in vivo. To precisely follow the possible activation-induced changes in expression levels of APOBEC3 protein in different mouse tissues and cell populations, genome editing was utilized to establish knock-in mice that express APOBEC3 protein with an in-frame FLAG tag. Flow cytometry and immunohistochemical analyses were performed prior to and after an immunological stimulation. Cultured B cells expressed higher levels of APOBEC3 protein than T cells. All differentiation and activation stages of freshly prepared B cells expressed significant levels of APOBEC3 protein, but germinal center cells possessed the highest levels of APOBEC3 protein localized in their cytoplasm. Upon immunological stimulation with sheep red blood cells in vivo, germinal center cells with high levels of APOBEC3 protein expression increased in their number, but FLAG-specific fluorescence intensity in each cell did not change. T cells, even those in germinal centers, did not express significant levels of APOBEC3 protein. Thus, mouse APOBEC3 protein is expressed at distinctively high levels in germinal center B cells. Antigenic stimulation did not affect expression levels of cellular APOBEC3 protein despite increased numbers of germinal center cells.

Anti-prion activity of cellulose ether is impaired in mice lacking pre T-cell antigen receptor α, T-cell receptor δ, or lytic granule function.

The anti-prion activity of cellulose ether (CE) has been reported in rodents, but the mechanism of action is not well understood. As defects in early T-cell development have been reported in Tga20 mice which show only a slight effect of CE administration, we investigated the involvement of immune functions in the CE action. We confirmed an insertion of the prion protein transgene into the pre T-cell antigen receptor α gene of Tga20 mice, and its impaired expression in the thymus and other tissues. The influence of immune suppression on the CE effect was then examined in high CE-responder mice treated with immunosuppressive agents or neonatal thymectomy. As neonatal thymectomy significantly reduced the CE effect, we compared the influence of various T-cell defects in mice with similar genetic backgrounds. The CE effect was increased or unchanged in mice with defects in the αß T-cell lineage, whereas it was abolished in T-cell receptor δ deficient mice. Further, when other immune defects were examined, the CE effect was reduced in mice with lysosomal trafficking dysfunction, but was unchanged in mice deficient in B-cell differentiation or toll-like receptor 4 signaling. These findings collectively suggest that the mechanism of CE action may involve γδ T cells and lytic granule function, as well as immune factors like natural killer T cells which are lacking in pre T-cell antigen receptor α deficient mice and neonatally thymectomized mice.