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Prion diseases are transmissible, fatal neurologic diseases of mammals caused by the accumulation of mis-folded, disease associated prion protein (PrPd). Creutzfeldt-Jakob Disease (CJD) is the most common human prion disease and can occur by sporadic onset (sCJD) (~85% of CJD cases), genetic mutations in the prion protein gene (10–15%) or iatrogenic transmission (rare). PrPd is difficult to inactivate and many methods to reduce prion infectivity are dangerous, caustic, expensive, or impractical. Identifying viable and safe methods for decontamination of CJD exposed materials is critically important for medical facilities and research institutions. Previous research has shown that concentrated sodium hypochlorite (bleach) was effective at inactivation of CJD prions derived from brains of mice or guinea pigs. Unfortunately, human prions adapted to rodents may mis-fold differently than in humans, and the rodent adapted prions may not have the same resistance or susceptibility to inactivation present in bona fide CJD prions. To confirm that bleach was efficacious against human sourced CJD prions, we exposed different subtypes of sCJD-infected human brain homogenates to different concentrations of bleach for increasing exposure times. Initial and residual prion seeding activity following inactivation were measured using Real-Time Quaking Induced Conversion. In addition, we tested how passage of human sCJD into either transgenic mice that expressed human prion protein, or transmission of CJD to human cerebral organoids (CO), two common laboratory practices, may affect CJD prions' susceptibility to bleach inactivation. Our results show that bleach is effective against human sourced sCJD prions, and both treatment time and concentration of bleach were important factors for CJD inactivation. CJD derived from human brains, transgenic mouse brains or CO were all susceptible to inactivation with as low as a 10 percent bleach solution with a 30-minute exposure time or a 50 percent bleach solution with as little as a 1-minute exposure time. [ABSTRACT FROM AUTHOR]

Parkinson's disease is the second most common neurodegenerative disorder, affecting nearly 10 million people worldwide. Ferroptosis, a recently identified form of regulated cell death characterized by 15-lipoxygenase-mediated hydroperoxidation of membrane lipids, has been implicated in neurodegenerative disorders including amyotrophic lateral sclerosis and Parkinson's disease. Pharmacological inhibition of 15 -lipoxygenase to prevent iron- and lipid peroxidation-associated ferroptotic cell death is a rational strategy for the treatment of Parkinson's disease. We report here the characterization of PTC-041 as an anti-ferroptotic reductive lipoxygenase inhibitor developed for the treatment of Parkinson's disease. In these studies, PTC-041 potently protects primary human Parkinson's disease patient-derived fibroblasts from lipid peroxidation and subsequent ferroptotic cell death and prevents ferroptosis-related neuronal loss and astrogliosis in primary rat neuronal cultures. Additionally, PTC-041 prevents ferroptotic-mediated α-synuclein protein aggregation and nitrosylation in vitro, suggesting a potential role for anti-ferroptotic lipoxygenase inhibitors in mitigating pathogenic aspects of synucleinopathies such as Parkinson's disease. We further found that PTC-041 protects against synucleinopathy in vivo, demonstrating that PTC-041 treatment of Line 61 transgenic mice protects against α-synuclein aggregation and phosphorylation as well as prevents associated neuronal and non-neuronal cell death. Finally, we show that. PTC-041 protects against 6-hydroxydopamine-induced motor deficits in a hemiparkinsonian rat model, further validating the potential therapeutic benefits of lipoxygenase inhibitors in the treatment of Parkinson's disease. [ABSTRACT FROM AUTHOR]

Advances in fluorescence imaging technology have been crucial to the progress of neuroscience. Whether it was specific expression of indicator proteins, detection of neurotransmitters, or miniaturization of fluorescence microscopes, fluorescence imaging has improved upon electrophysiology, the gold standard for monitoring brain activity, and enabled novel methods to sense activity in the brain. Hence, we developed a lightweight and compact implantable CMOS-based lensless Ca2+ imaging device for freely moving transgenic G-CaMP mouse experiments. However, without a lens system, determination of regions of interest (ROI) has proven challenging. Localization of fluorescence activity and separation of signal from noise are difficult. In this study, we report an ROI selection method using a series of adaptive binarizations with a gaussian method and morphological image processing. The parameters for each operation such as the kernel size, sigma and footprint size were optimized. We then validated the utility of the algorithm with simulated data and freely moving nociception experiments using the lensless devices. The device was implanted in the dorsal raphe nucleus to observe pain-related brain activity following a formalin test to stimulate pain. We observed significant increases in fluorescence activity after formalin injection compared to the control group when using the ROI determination algorithm. [ABSTRACT FROM AUTHOR]

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is an X-linked disorder of glycosphingolipid metabolism caused by mutations in the GLA gene encoding alpha-galactosidase A (α-Gal). Loss of α-Gal activity leads to progressive lysosomal accumulation of α-Gal substrate, predominately globotriaosylceramide (Gb3) and its deacylated derivative globotriaosylsphingosine (lyso‐Gb3). FD manifestations include early onset neuropathic pain, gastrointestinal symptoms, and later onset life-threatening renal, cardiovascular and cerebrovascular disorders. Current treatments can preserve kidney function but are not very effective in preventing progression of cardiovascular pathology which remains the most common cause of premature death in FD patients. There is a significant need for a translational model that could be used for testing cardiac efficacy of new drugs. Two mouse models of FD have been developed. The α-Gal A-knockout (GlaKO) model is characterized by progressive tissue accumulation of Gb3 and lyso-Gb3 but does not develop any Fabry pathology besides mild peripheral neuropathy. Reports of minor cardiac function abnormalities in GlaKO model are inconsistent between different studies. Recently, G3Stg/GlaKO was generated by crossbreeding GlaKO with transgenic mice expressing human Gb3 synthase. G3Stg/GlaKO demonstrate higher tissue substrate accumulation and develop cellular and tissue pathologies. Functional renal pathology analogous to that found in early stages of FD has also been described in this model. The objective of this study is to characterize cardiac phenotype in GlaKO and G3Stg/GlaKO mice using echocardiography. Longitudinal assessments of cardiac wall thickness, mass and function were performed in GlaKO and wild-type (WT) littermate controls from 5–13 months of age. G3Stg/GlaKO and WT mice were assessed between 27–28 weeks of age due to their shortened lifespan. Several cardiomyopathy characteristics of early Fabry pathology were found in GlaKO mice, including mild cardiomegaly [up-to-25% increase in left ventricular (LV mass)] with no significant LV wall thickening. The LV internal diameter was significantly wider (up-to-24% increase at 9-months), when compared to the age-matched WT. In addition, there were significant increases in the end-systolic, end-diastolic volumes and stroke volume, suggesting volume overload. Significant reduction in Global longitudinal strain (GLS) measuring local myofiber contractility of the LV was also detected at 13-months. Similar GLS reduction was also reported in FD patients. Parameters such as ejection fraction, fractional shortening and cardiac output were either only slightly affected or were not different from controls. On the other hand, some of the cardiac findings in G3Stg/GlaKO mice were inconsistent with Fabry cardiomyopathy seen in FD patients. This could be potentially an artifact of the Gb3 synthase overexpression under a strong ubiquitous promoter. In conclusion, GlaKO mouse model presents mild cardiomegaly, mild cardiac dysfunction, but significant cardiac volume overload and functional changes in GLS that can be used as translational biomarkers to determine cardiac efficacy of novel treatment modalities. The level of tissue Gb3 accumulation in G3Stg/GlaKO mouse more closely recapitulates the level of substrate accumulation in FD patients and may provide better translatability of the efficacy of new therapeutics in clearing pathological substrates from cardiac tissues. But interpretation of the effect of treatment on cardiac structure and function in this model should be approached with caution. [ABSTRACT FROM AUTHOR]

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research. [ABSTRACT FROM AUTHOR]

Endosalpingiosis (ES) and endometriosis (EM) refer to the growth of tubal and endometrial epithelium respectively, outside of their site of origin. We hypothesize that uterine secretome factors drive ectopic growth. To test this, we developed a mouse model of ES and EM using tdTomato (tdT) transgenic fluorescent mice as donors. To block implantation factors, progesterone knockout (PKO) tdT mice were created. Fluorescent lesions were present after oviduct implantation with and without WT endometrium. Implantation was increased (p<0.05) when tdt oviductal tissue was implanted with endometrium compared to oviductal tissue alone. Implantation was reduced (p<0.0005) in animals implanted with minced tdT oviductal tissue with PKO tdT endometrium compared to WT endometrium. Finally, oviductal tissues was incubated with and without a known implantation factor, leukemia inhibitory factor (LIF) prior to and during implantation. LIF promoted lesion implantation. In conclusion, endometrial derived implantation factors, such as LIF, are necessary to initiate ectopic tissue growth. We have developed an animal model of ectopic growth of gynecologic tissues in a WT mouse which will potentially allow for development of new prevention and treatment modalities. [ABSTRACT FROM AUTHOR]

Human T-cell leukemia virus type I (HTLV-I) is an oncogenic virus whose infection can cause diverse diseases, most notably adult T-cell leukemia/lymphoma (ATL or ATLL), an aggressive and fatal malignancy of CD4 T cells. The oncogenic ability of HTLV-I is mostly attributed to the viral transcriptional transactivator Tax. Tax alone is sufficient to induce specific tumors in mice depending on the promotor used to drive Tax expression, thereby being used to understand HTLV-I tumorigenesis and model the tumor types developed in Tax transgenic mice. Tax exerts its oncogenic role predominantly by activating the cellular transcription factor NF-κB. Here, we report that genetic deletion of NF-κB1, the prototypic member of the NF-κB family, promotes adrenal medullary tumors but suppresses neurofibromas in mice with transgenic Tax driven by the HTLV-I Long Terminal Repeat (LTR) promoter. The adrenal tumors are derived from macrophages. Neoplastic macrophages also infiltrate the spleen and lymph nodes, causing splenomegaly and lymphadenopathy in mice. Nevertheless, the findings could be human relevant, because macrophages are important target cells of HTLV-I infection and serve as a virus reservoir in vivo. Moreover, the spleen, lymph nodes and adrenal glands are the most common sites of tumor cell infiltration in HTLV-I-infected patients. These data provide new mechanistic insights into the complex interaction between Tax and NF-κB, therefore improving our understanding of HTLV-I oncogenic pathogenesis. They also expand our knowledge and establish a new animal model of macrophage neoplasms and adrenal tumors. [ABSTRACT FROM AUTHOR]

Obesity is a major global health epidemic that has adverse effects on both the people affected as well as the cost to society. Several anti-obesity drugs that target GLP-1 receptors have recently come to the market. Here, we describe the effects of tesofensine, a novel anti-obesity drug that acts as a triple monoamine neurotransmitter reuptake inhibitor. Using various techniques, we investigated its effects on weight loss and underlying neuronal mechanisms in mice and rats. These include behavioral tasks, DeepLabCut videotaped analysis, electrophysiological ensemble recordings, optogenetic activation, and chemogenetic silencing of GABAergic neurons in the Lateral Hypothalamus (LH). We found that tesofensine induces a greater weight loss in obese rats than lean rats, while differentially modulating the neuronal ensembles and population activity in LH. In Vgat-ChR2 and Vgat-IRES-cre transgenic mice, we found for the first time that tesofensine inhibited a subset of LH GABAergic neurons, reducing their ability to promote feeding behavior, and chemogenetically silencing them enhanced tesofensine's food-suppressing effects. Unlike phentermine, a dopaminergic appetite suppressant, tesofensine causes few, if any, head-weaving stereotypy at therapeutic doses. Most importantly, we found that tesofensine prolonged the weight loss induced by 5-HTP, a serotonin precursor, and blocked the body weight rebound that often occurs after weight loss. Behavioral studies on rats with the tastant sucrose indicated that tesofensine's appetite suppressant effects are independent of taste aversion and do not directly affect the perception of sweetness or palatability of sucrose. In summary, our data provide new insights into the effects of tesofensine on weight loss and the underlying neuronal mechanisms, suggesting that tesofensine may be an effective treatment for obesity and that it may be a valuable adjunct to other appetite suppressants to prevent body weight rebound. [ABSTRACT FROM AUTHOR]

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "High frequency of germline recombination in Nestin-Cre transgenic mice crossed with Glucagon-like peptide 1 receptor floxed mice." The correction addresses an error in Figure 4C, specifically the incorrect number of mice. The corrected figure is provided in the notice. The article discusses the rate of germline recombination in mice and includes data on progeny genotypes. The authors of the article are listed as Yusuke Kajitani, Takashi Miyazawa, Tomoaki Inoue, Nao Kajitani, Masamichi Fujita, Yukina Takeichi, Yasutaka Miyachi, Ryuichi Sakamoto, and Yoshihiro Ogawa. [Extracted from the article]

The Cre-loxP strategy for tissue-specific gene inactivation has become a widely employed tool in several research studies. Conversely, inadequate breeding and genotyping without considering the potential for non-specific Cre-recombinase expression may lead to misinterpretations of results. Nestin-Cre transgenic mice, widely used for the selective deletion of genes in neurons, have been observed to have an incidence of Cre-line germline recombination. In this study, we attempted to generate neuron-specific Glucagon-like peptide 1 receptor (Glp1r) knock-out mice by crossing mice harboring the Nestin-Cre transgene with mice harboring the Glp1r gene modified with loxP insertion, in order to elucidate the role of Glp1r signaling in the nervous system. Surprisingly, during this breeding process, we discovered that the null allele emerged in the offspring irrespective of the presence or absence of the Nestin-Cre transgene, with a high probability of occurrence (93.6%). To elucidate the cause of this null allele, we conducted breeding experiments between mice carrying the heterozygous Glp1r null allele but lacking the Nestin-Cre transgene. We confirmed that the null allele was inherited by the offspring independently of the Nestin-Cre transgene. Furthermore, we assessed the gene expression, protein expression, and phenotype of mice carrying the homozygous Glp1r null allele generated from the aforementioned breeding, thereby confirming that the null allele indeed caused a global knock-out of Glp1r. These findings suggest that the null allele in the NestinCre-Glp1r floxed breeding arose due to germline recombination. Moreover, we demonstrated the possibility that germline recombination may occur not only during the spermatogenesis at testis but also during epididymal sperm maturation. The striking frequency of germline recombination in the Nestin-Cre driver underscores the necessity for caution when implementing precise breeding strategies and employing suitable genotyping methods. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is a genetic disorder that has been associated with priapism. The role of hydroxyurea, a common SCD therapy, in influencing the nitric oxide (NO)-cGMP pathway and its effect on priapism is unclear. To investigate the effect of hydroxyurea treatment on smooth muscle relaxation of corpus cavernosum induced by stimulation of the NO-cGMP pathway in SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice, which are used as model of priapism associated with the low bioavailability of endothelial NO. Four-month-old wild-type (WT, C57BL/6), SCD transgenic, and eNOS-/- male mice were treated with hydroxyurea (100 mg/Kg/day) or its vehicle (saline) daily for three weeks via intraperitoneal injections. Concentration-response curves for acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS) were generated using strips of mice corpus cavernosum. The SCD mice demonstrated an amplified CC relaxation response triggered by ACh, EFS, and SNP. The corpus cavernosum relaxation responses to SNP and EFS were found to be heightened in the eNOS-/- group. However, the hydroxyurea treatment did not alter these escalated relaxation responses to ACh, EFS, and SNP in the corpus cavernosum of the SCD group, nor the relaxation responses to EFS and SNP in the eNOS-/- group. In conclusion, hydroxyurea is not effective in treating priapism associated with SCD. It is likely that excess plasma hemoglobin and reactive oxygen species, which are reported in SCD, are reacting with NO before it binds to GCs in the smooth muscle of the corpus cavernosum, thus preventing the restoration of baseline NO/cGMP levels. Furthermore, the downregulation of eNOS in the penis may impair the pharmacological action of hydroxyurea at the endothelial level in SCD mice. This study emphasize the urgency for exploring alternative therapeutic avenues for priapism in SCD that are not hindered by high plasma hemoglobin and ROS levels. [ABSTRACT FROM AUTHOR]

FAM3C/ILEI is an important factor in epithelial-to-mesenchymal transition (EMT) induction, tumor progression and metastasis. Overexpressed in many cancers, elevated ILEI levels and secretion correlate with poor patient survival. Although ILEI's causative role in invasive tumor growth and metastasis has been demonstrated in several cellular tumor models, there are no available transgenic mice to study these effects in the context of a complex organism. Here, we describe the generation and initial characterization of a Tet-ON inducible Fam3c/ILEI transgenic mouse strain. We find that ubiquitous induction of ILEI overexpression (R26-ILEIind) at weaning age leads to a shortened lifespan, reduced body weight and microcytic hypochromic anemia. The anemia was reversible at a young age within a week upon withdrawal of ILEI induction. Vav1-driven overexpression of the ILEIind transgene in all hematopoietic cells (Vav-ILEIind) did not render mice anemic or lower overall fitness, demonstrating that no intrinsic mechanisms of erythroid development were dysregulated by ILEI and that hematopoietic ILEI hyperfunction did not contribute to death. Reduced serum iron levels of R26-ILEIind mice were indicative for a malfunction in iron uptake or homeostasis. Accordingly, the liver, the main organ of iron metabolism, was severely affected in moribund ILEI overexpressing mice: increased alanine transaminase and aspartate aminotransferase levels indicated liver dysfunction, the liver was reduced in size, showed increased apoptosis, reduced cellular iron content, and had a fibrotic phenotype. These data indicate that high ILEI expression in the liver might reduce hepatoprotection and induce liver fibrosis, which leads to liver dysfunction, disturbed iron metabolism and eventually to death. Overall, we show here that the novel Tet-ON inducible Fam3c/ILEI transgenic mouse strain allows tissue specific timely controlled overexpression of ILEI and thus, will serve as a versatile tool to model the effect of elevated ILEI expression in diverse tissue entities and disease conditions, including cancer. [ABSTRACT FROM AUTHOR]

In a short time, several types of injectable and oral therapeutics have been developed and used to effectively manage patients with coronavirus disease 2019 (COVID-19). BEN815 is an improved mixture of three extracts (Psidium guajava, Camellia sinensis, and Rosa hybrida) recognized by the Ministry of Food and Drug Safety of Korea as a health food ingredient that alleviates allergic rhinitis. The current animal efficacy study was performed to assess its probability of improving COVID-19 symptoms. BEN815 treatment significantly increased the survival of K18-hACE2 transgenic mice and reduced viral titers in the lungs at 5 days post infection (DPI). Furthermore, the lungs of the treated mice showed mild tissue damage at 5 DPI and nearly complete recovery from COVID-19 at 14 DPI. BEN815 appears to be an effective and minimally toxic anti-SARS-CoV-2 agent in mice and has potential for clinical applications. [ABSTRACT FROM AUTHOR]

Long-term memory tests are commonly used to facilitate the diagnosis of hippocampal-related neurological disorders such as Alzheimer's disease due to their relatively high specificity and sensitivity to damage to the medial temporal lobes compared to standard commonly used clinical tests. Pathological changes in Alzheimer's disease start years before the formal diagnosis is made, partially due to testing too late. This proof-of-concept exploratory study aimed to assess the feasibility of introducing an unsupervised digital platform for continuous testing of long-term memory over long periods outside the laboratory environment. To address this challenge, we developed a novel digital platform, hAge ('healthy Age'), which integrates double spatial alternation, image recognition and visuospatial tasks for frequent remote unsupervised assessment of spatial and non-spatial long-term memory carried out continuously over eight week period. To demonstrate the feasibility of our approach, we tested whether we could achieve sufficient levels of adherence and whether the performance on hAge tasks is comparable to the performance observed in the analogous standard tests measured in the controlled laboratory environments.191 healthy adults (67% females, 18-81 years old) participated in the study. We report an estimated 42.4% adherence level with minimal inclusion criteria. In line with findings using standard laboratory tests, we showed that performance on the spatial alternation task negatively correlated with inter-trial periods and the performance levels on image recognition and visuospatial tasks could be controlled by varying image similarity. Importantly, we demonstrated that frequent engagement with the double spatial alternation task leads to a strong practice effect, previously identified as a potential measure of cognitive decline in MCI patients. Finally, we discuss how lifestyle and motivation confounds may present a serious challenge for cognitive assessment in real-world uncontrolled environments. [ABSTRACT FROM AUTHOR]

5xFAD transgenic (TG) mice are used widely in AD preclinical trials; however, data on sample sizes are largely unaddressed. We therefore performed estimates of sample sizes and effect sizes for typical behavioural and neuropathological outcome measures in TG 5xFAD mice, based upon data from single-sex (female) groups. Group-size estimates to detect normalisation of TG body weight to WT littermate levels at 5.5m of age were N = 9–15 depending upon algorithm. However, by 1 year of age, group sizes were small (N = 1 –<6), likely reflecting the large difference between genotypes at this age. To detect normalisation of TG open-field hyperactivity to WT levels at 13-14m, group sizes were also small (N = 6–8). Cued learning in the Morris water maze (MWM) was normal in Young TG mice (5m of age). Mild deficits were noted during MWM spatial learning and memory. MWM reversal learning and memory revealed greater impairment, and groups of up to 22 TG mice were estimated to detect normalisation to WT performance. In contrast, Aged TG mice (tested between 13 and 14m) failed to complete the visual learning (non-spatial) phase of MWM learning, likely due to a failure to recognise the platform as an escape. Estimates of group size to detect normalisation of this severe impairment were small (N = 6–9, depending upon algorithm). Other cognitive tests including spontaneous and forced alternation and novel-object recognition either failed to reveal deficits in TG mice or deficits were negligible. For neuropathological outcomes, plaque load, astrocytosis and microgliosis in frontal cortex and hippocampus were quantified in TG mice aged 2m, 4m and 6m. Sample-size estimates were ≤9 to detect the equivalent of a reduction in plaque load to the level of 2m-old TG mice or the equivalent of normalisation of neuroinflammation outcomes. However, for a smaller effect size of 30%, larger groups of up to 21 mice were estimated. In light of published guidelines on preclinical trial design, these data may be used to provide provisional sample sizes and optimise preclinical trials in 5xFAD TG mice. [ABSTRACT FROM AUTHOR]

In studies exploring the potential for nanosecond duration electric pulses to serve as a novel modality for neuromodulation, we found that a 5 ns pulse triggers an immediate rise in [Ca2+]i in isolated bovine adrenal chromaffin cells. To facilitate ongoing efforts to understand underlying mechanisms and to work toward carrying out investigations in cells in situ, we describe the suitability and advantages of using isolated murine adrenal chromaffin cells expressing, in a Cre-dependent manner, the genetically-encoded Ca2+indicator GCaMP6f. Initial experiments confirmed that Ca2+ responses evoked by a 5 ns pulse were similar between fluorescent Ca2+ indicator-loaded murine and bovine chromaffin cells, thereby establishing that 5 ns-elicited excitation of chromaffin cells occurs reproducibly across species. In GCaMP6f-expressing murine chromaffin cells, spontaneous Ca2+ activity as well as nicotinic receptor agonist- and 5 ns evoked-Ca2+ responses consistently displayed similar kinetic characteristics as those in dye-loaded cells but with two-twentyfold greater amplitudes and without photobleaching. The high signal-to-noise ratio of evoked Ca2+ responses as well as spontaneous Ca2+ activity was observed in cells derived from Sox10-Cre, conditional GCaMP6f mice or TH-Cre, conditional GCaMP6f mice, although the number of cells expressing GCaMP6f at sufficiently high levels for achieving high signal-to-noise ratios was greater in Sox10-Cre mice. As in bovine cells, Ca2+ responses elicited in murine GCaMP6f-expressing cells by a 5 ns pulse were mediated by the activation of voltage-gated Ca2+ channels but not tetrodotoxin-sensitive voltage-gated Na+ channels. We conclude that genetically targeting GCaMP6f expression to murine chromaffin cells represents a sensitive and valuable approach to investigate spontaneous, receptor agonist- and nanosecond electric pulse-induced Ca2+ responses in vitro. This approach will also facilitate future studies investigating the effects of ultrashort electric pulses on cells in ex vivo slices of adrenal gland, which will lay the foundation for using nanosecond electric pulses to stimulate neurosecretion in vivo. [ABSTRACT FROM AUTHOR]

Aims: Accumulating data demonstrates that new adult cardiomyocytes (CMs) are generated throughout life from pre-existing CMs, although the absolute magnitude of CM self-renewal is very low. Modifying epigenetic histone modifications or activating the Hippo-Yap pathway have been shown to promote adult CM cycling and proliferation. Whether these interventions work through common pathways or act independently is unknown. For the first time we have determined whether lysine demethylase 4D (KDM4D)-mediated CM-specific H3K9 demethylation and Hippo pathways inhibition have additive or redundant roles in promoting CM cell cycle re-entry. Methods and results: We found that activating Yap1 in cultured neonatal rat ventricular myocytes (NRVM) through overexpressing Hippo pathway inhibitor, miR-199, preferentially increased S-phase CMs, while H3K9me3 demethylase KDM4D preferentially increased G2/M markers in CMs. Together KDM4D and miR-199 further increased total cell number of NRVMs in culture. Inhibition of Hippo signaling via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1) also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducible activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while miR-199 and si-Sav1 preferentially up-regulated genes involved in G1/S phase. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Using transgenic mice over-expressing KDM4D together with Myc, we demonstrated that KDM4D/Myc significantly increased CM cell cycling but did not affect cardiac function. Conclusions: KDM4D effects on CM cell cycle activity are additive with the Hippo-Yap1 pathway and appear to preferentially regulate different cell cycle regulators. This may have important implications for strategies that target cardiac regeneration in treating heart disease [ABSTRACT FROM AUTHOR]

C57BL/6J (B6J) and C57BL/6N (B6N) mice are the most frequently used substrains in C57BL/6 (B6) inbred mice, serving as physiological models for in vivo studies and as background strains to build transgenic mice. However, the differences in metabolic phenotypes between B6J and B6N mice are not coherent, and genotypic differences in metabolically important tissues have not been well studied. The phenotypic differences between B6J and B6N substrains have often been attributed to the role of the nicotinamide nucleotide transhydrogenase (Nnt) gene, whereby B6J has a spontaneous missense mutation of Nnt. Nevertheless, phenotypic differences between the two cannot be explained by Nnt mutations alone, especially in metabolic traits. Therefore, we aimed to investigate the genetic cause of the phenotypic differences between B6J and B6N mice. Determining consistent genetic differences across multiple tissues involved in metabolic traits such as subcutaneous and visceral white adipose tissues, brown adipose tissue, skeletal muscle, liver, hypothalamus, and hippocampus, may help explain phenotypic differences in metabolism between the two substrains. We report candidate genes along with comparative data on body weight, tissue weight, blood components involved in metabolism, and energy balance of B6J and B6N mice. Insulin degrading enzyme, adenylosuccinate synthase 2, and ectonucleotide triphosphate diphosphohydrolase 4 were highly expressed in B6J mice compared with those in B6N mice, and Nnt, WD repeat and FYVE domain containing 1, and dynein light chain Tctex-type 1 were less expressed in B6J mice compared with those in B6N mice in all seven tissues. Considering the extremely wide use of both substrains and their critical importance in generating transgenic and knock-out models, these findings guide future research across several interrelated fields. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a common disorder and a major cause of disability in the elderly population. WNT16 has been suggested to play important roles in joint formation, bone homeostasis and OA development, but the mechanism of action is not clear. Transgenic mice lacking Wnt16 expression (Wnt16-/-) have a more severe experimental OA than control mice. In addition, Wnt16-/- mice have a reduced cortical thickness and develop spontaneous fractures. Herein, we have used Cre-Wnt16flox/flox mice in which Wnt16 can be conditionally ablated at any age through tamoxifen-inducible Cre-mediated recombination. Wnt16 deletion was induced in 7-week-old mice to study if the Cre-Wnt16flox/flox mice have a more severe OA phenotype after destabilizing the medial meniscus (DMM surgery) than littermate controls with normal Wnt16 expression (Wnt16flox/flox). WNT16 deletion was confirmed in articular cartilage and cortical bone in Cre-Wnt16flox/flox mice, shown by immunohistochemistry and reduced cortical bone area compared to Wnt16flox/flox mice. After DMM surgery, there was no difference in OA severity in the articular cartilage in the knee joint between the Cre-Wnt16flox/flox and Wnt16flox/flox mice in neither female nor male mice. In addition, there was no difference in osteophyte size in the DMM-operated tibia between the genotypes. In conclusion, inactivation of Wnt16 in adult mice do not result in a more severe OA phenotype after DMM surgery. Thus, presence of WNT16 in adult mice does not have an impact on experimental OA development. Taken together, our results from Cre-Wnt16flox/flox mice and previous results from Wnt16-/- mice suggest that WNT16 is crucial during synovial joint establishment leading to limited joint degradation also later in life, after onset of OA. This may be important when developing new therapeutics for OA treatment. [ABSTRACT FROM AUTHOR]

Skeletal alterations in the head and neck region, such as midfacial hypoplasia, foramen magnum stenosis and spinal canal stenosis, are commonly observed in patients with mucopolysaccharidosis (MPS). However, enzyme replacement therapy (ERT), one of the major treatment approaches for MPS, shows limited efficacy for skeletal conditions. In this study, we analysed the craniofacial morphology of mice with MPS type VII, and investigated the underlying mechanisms promoting jaw deformities in these animals. Furthermore, we investigated the effects of C-type natriuretic peptide (CNP), a potent endochondral ossification promoter, on growth impairment of the craniofacial region in MPS VII mice when administered alone or in combination with ERT. MPS VII mice exhibited midfacial hypoplasia caused by impaired endochondral ossification, and histological analysis revealed increased number of swelling cells in the resting zone of the spheno-occipital synchondrosis (SOS), an important growth centre for craniomaxillofacial skeletogenesis. We crossed MPS VII mice with transgenic mice in which CNP was expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP. The maxillofacial morphological abnormalities associated with MPS VII were ameliorated by CNP expression, and further prevented by a combination of CNP and ERT. Histological analysis showed that ERT decreased the swelling cell number, and CNP treatment increased the width of the proliferative and hypertrophic zones of the SOS. Furthermore, the foramen magnum and spinal stenoses observed in MPS VII mice were significantly alleviated by CNP and ERT combination. These results demonstrate the therapeutic potential of CNP, which can be used to enhance ERT outcome for MPS VII-associated head and neck abnormalities. [ABSTRACT FROM AUTHOR]

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7–iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild–type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7–ChR2 mice. [ABSTRACT FROM AUTHOR]

Recurrence of proteinuria after kidney transplantation in primary focal segmental glomerulosclerosis (FSGS) is unpredictable. Several putative circulating permeability factors (CPFs) have been suggested, but none have been validated. A clinically relevant experimental model is required that demonstrates the presence of CPF(s) in patient material, to study CPF(s) and possibly predict recurrence in patients. We aimed to develop a FSGS-prone Thy-1.1 transgenic mouse model with accelerated proteinuria after injection of samples from patients with FSGS. The Thy-1.1 transgene was backcrossed into 5 mouse strains. The age of onset and severity of spontaneous proteinuria varied between the different genetic backgrounds. 129X1/SvThy-1.1 and 129S2/SvPasThy-1.1 mice displayed proteinuria at 4 weeks, whereas Balb/cThy-1.1 and C57BL/6JThy-1.1 mice developed proteinuria from 6 weeks, and were used further. We determined the maximum protein dose that could be injected without causing protein overload in each background. Balb/cThy-1.1 and C57BL/6JThy-1.1 males and females were injected with presumably CPF-containing plasmapheresis effluent from 6 FSGS patients, which induced albuminuria particularly in Balb/cThy-1.1 males. Unfortunately, no response could be detected when using sera instead of plasmapheresis effluent, serum being more clinically relevant in the context of predicting FSGS recurrence. Considering the differences between responses elicited by serum and plasmapheresis effluent, simultaneously collected serum, plasma, and plasmapheresis effluent were tested. Whereas we could detect responses using a validated in vitro model, none of these presumably CPF-containing samples induced proteinuria in Balb/cThy-1.1 males. Thus, we have extensively tested the Thy-1.1 mouse model on different genetic backgrounds with proteinuria after injection of FSGS patient material as clinically relevant readout. The Balb/cThy-1.1 male mouse strain demonstrated the most promising results, but to detect CPF activity in FSGS serum e.g. prior to kidney transplantation, this strain clearly lacks sensitivity and is therefore not yet clinically applicable. It could, however, still be used as research tool to study CPFs in patient samples that did induce proteinuria. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disorder. Although most cases of ALS are sporadic, 5–10% of cases are familial, with mutations associated with over 40 genes. There is variation of ALS symptoms within families carrying the same mutation; the disease may develop in one sibling and not in another despite the presence of the mutation in both. Although the cause of this phenotypic variation is unknown, it is likely related to genetic modifiers of disease expression. The identification of ALS causing genes has led to the development of transgenic mouse models of motor neuron disease. Similar to families with familial ALS, there are background-dependent differences in disease phenotype in transgenic mouse models of ALS suggesting that, as in human ALS, differences in phenotype may be ascribed to genetic modifiers. These genetic modifiers may not cause ALS rather their expression either exacerbates or ameliorates the effect of the mutant ALS causing genes. We have reported that in both the G93A-hSOD1 and G59S-hDCTN1 mouse models, SJL mice demonstrated a more severe phenotype than C57BL6 mice. From reciprocal intercrosses between G93A-hSOD1 transgenic mice on SJL and C57BL6 strains, we identified a major quantitative trait locus (QTL) on mouse chromosome 17 that results in a significant shift in lifespan. In this study we generated reciprocal intercrosses between transgenic G59S-hDCTN1 mice on SJL and C57BL6 strains and identified survival QTLs on mouse chromosomes 17 and 18. The chromosome 17 survival QTL on G93A-hSOD1 and G59S-hDCTN1 mice partly overlap, suggesting that the genetic modifiers located in this region may be shared by these two ALS models despite the fact that motor neuron degeneration is caused by mutations in different proteins. The overlapping region contains eighty-seven genes with non-synonymous variations predicted to be deleterious and/or damaging. Two genes in this segment, NOTCH3 and Safb/SAFB1, have been associated with motor neuron disease. The identification of genetic modifiers of motor neuron disease, especially those modifiers that are shared by SOD1 and dynactin-1 transgenic mice, may result in the identification of novel targets for therapies that can alter the course of this devastating illness. [ABSTRACT FROM AUTHOR]

The key role played by host-microbiota interactions on human health, disease onset and progression, and on host response to treatments has increasingly emerged in the latest decades. Indeed, dysbiosis has been associated to several human diseases such as obesity, diabetes, cancer and also neurodegenerative disease, such as Parkinson, Huntington and Alzheimer's disease (AD), although whether causative, consequence or merely an epiphenomenon is still under investigation. In the present study, we performed a metabologenomic analysis of stool samples from a mouse model of AD, the 3xTgAD. We found a significant change in the microbiota of AD mice compared to WT, with a longitudinal divergence of the F/B ratio, a parameter suggesting a gut dysbiosis. Moreover, AD mice showed a significant decrease of some amino acids, while data integration revealed a dysregulated production of desaminotyrosine (DAT) and dihydro-3-coumaric acid. Collectively, our data show a dysregulated gut microbiota associated to the onset and progression of AD, also indicating that a dysbiosis can occur prior to significant clinical signs, evidenced by early SCFA alterations, compatible with gut inflammation. [ABSTRACT FROM AUTHOR]

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light. [ABSTRACT FROM AUTHOR]

Coronavirus disease (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is currently spreading globally. To overcome the COVID-19 pandemic, preclinical evaluations of vaccines and therapeutics using K18-hACE2 and CAG-hACE2 transgenic mice are ongoing. However, a comparative study on SARS-CoV-2 infection between K18-hACE2 and CAG-hACE2 mice has not been published. In this study, we compared the susceptibility and resistance to SARS-CoV-2 infection between two strains of transgenic mice, which were generated in FVB background mice. K18-hACE2 mice exhibited severe weight loss with definitive lethality, but CAG-hACE2 mice survived; and differences were observed in the lung, spleen, cerebrum, cerebellum, and small intestine. A higher viral titer was detected in the lungs, cerebrums, and cerebellums of K18-hACE2 mice than in the lungs of CAG-hACE2 mice. Severe pneumonia was observed in histopathological findings in K18-hACE2, and mild pneumonia was observed in CAG-hACE2. Atrophy of the splenic white pulp and reduction of spleen weight was observed, and hyperplasia of goblet cells with villi atrophy of the small intestine was observed in K18-hACE2 mice compared to CAG-hACE2 mice. These results indicate that K18-hACE2 mice are relatively susceptible to SARS-CoV-2 and that CAG-hACE2 mice are resistant to SARS-CoV-2. Based on these lineage-specific sensitivities, we suggest that K18-hACE2 mouse is suitable for highly susceptible model of SARS-CoV-2, and CAG-hACE2 mouse is suitable for mild susceptible model of SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Tyrosine-protein kinase receptor Tie2, also known as Tunica interna Endothelial cell Kinase or TEK plays a prominent role in endothelial responses to angiogenic and inflammatory stimuli. Here we generated a novel inducible Tie2 knockout mouse model, which targets mature (micro)vascular endothelium, enabling the study of the organ-specific contribution of Tie2 to these responses. Mice with floxed Tie2 exon 9 alleles (Tie2floxed/floxed) were crossed with end-SCL-Cre-ERT transgenic mice, generating offspring in which Tie2 exon 9 is deleted in the endothelial compartment upon tamoxifen-induced activation of Cre-recombinase (Tie2ΔE9). Successful deletion of Tie2 exon 9 in kidney, lung, heart, aorta, and liver, was accompanied by a heterogeneous, organ-dependent reduction in Tie2 mRNA and protein expression. Microvascular compartment-specific reduction in Tie2 mRNA and protein occurred in arterioles of all studied organs, in renal glomeruli, and in lung capillaries. In kidney, lung, and heart, reduced Tie2 expression was accompanied by a reduction in Tie1 mRNA expression. The heterogeneous, organ- and microvascular compartment-dependent knockout pattern of Tie2 in the Tie2floxed/floxed;end-SCL-Cre-ERT mouse model suggests that future studies using similar knockout strategies should include a meticulous analysis of the knockout extent of the gene of interest, prior to studying its role in pathological conditions, so that proper conclusions can be drawn. [ABSTRACT FROM AUTHOR]

Background: Children and adult with sickle cell disease (SCD) display priapism associated with low nitric oxide (NO) bioavailability and oxidative stress in penis. Aim: This study aimed to evaluate the effects of hybrid compound RVT-FxMe, derived from resveratrol bearing a NO-donor subunit, on two murine model that display priapism phenotype, SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice. Methods: Wild-type, SCD, and eNOS-/- mice were treated with RVT-FxMe (25 mg/kg/d, 2 weeks). Outcomes: Hematological parameters, concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as to electrical field stimulation (EFS), were obtained in mice corpus cavernosum strips. Results: Corpus cavernosum relaxations to SNP and EFS were increased in eNOS-/- group, which were normalized by RVT-FxMe treatment. SCD mice exhibited an excessive CC relaxant response induced by ACh, EFS and SNP RVT-FxMe treatment did not change the increased relaxant responses to ACh, EFS and SNP in corpus cavernosum from SCD group. Clinical translation: Excess of plasma hemoglobin in SCD may interfere in pharmacological activity of NO donors compounds. Strength/Limitations: While mechanistic data with promising potential is showed, the current study is not without limitations. RVT-FxMe effects in the mid- and long-term warrant complementary studies. Conclusion: Treatment with RVT-FxMe reversed the enhanced NO-cGMP-mediated CC relaxations in eNOS-/- mice, but not in SCD mice; it is likely that excess of plasma hemoglobin in SCD mice act to inactivate NO before it reaches soluble guanylyl cyclase, avoiding restoration of NO bioavailability in penis. [ABSTRACT FROM AUTHOR]

The SIRPa-CD47 axis plays an important role in T cell recruitment to sites of immune reaction and inflammation but its role in T cell antigen priming is incompletely understood. Employing OTII TCR transgenic mice bred to Cd47-/- (Cd47KO) or SKI mice, a knock-in transgenic animal expressing non-signaling cytoplasmic-truncated SIRPa, we investigated how the SIRPa-CD47 axis contributes to antigen priming. Here we show that adoptive transfer of Cd47KO or SKI Ova-specific CD4+ T cells (OTII) into Cd47KO and SKI recipients, followed by Ova immunization, elicited reduced T cell division and proliferation indices, increased apoptosis, and reduced expansion compared to transfer into WT mice. We confirmed prior reports that splenic T cell zone, CD4+ conventional dendritic cells (cDCs) and CD4+ T cell numbers were reduced in Cd47KO and SKI mice. We report that in vitro derived DCs from Cd47KO and SKI mice exhibited impaired migration in vivo and exhibited reduced CD11c+ DC proximity to OTII T cells in T cell zones after Ag immunization, which correlates with reduced TCR activation in transferred OTII T cells. These findings suggest that reduced numbers of CD4+ cDCs and their impaired migration contributes to reduced T cell-DC proximity in splenic T cell zone and reduced T cell TCR activation, cell division and proliferation, and indirectly increased T cell apoptosis. [ABSTRACT FROM AUTHOR]

Introduction: Because of the strong correlation between the blood concentration of circulating resistin and the illness severity of septic patients, resistin has been proposed as a mediator of sepsis pathophysiology. In vitro data indicate that human resistin directly impairs neutrophil migration and intracellular bacterial killing, although the significance of these findings in vivo remain unclear. Objective: The objectives of the present study were: (1) to validate the expression of human resistin in a clinically relevant, murine model of surgical sepsis, (2) to assess how sepsis-induced changes in resistin correlate with markers of infection and organ dysfunction, and (3) to investigate whether the expression of human resistin alters immune function or disease outcomes in vivo. Methods: 107 male, C57BL/6 mice transgenic for the human resistin gene and its promoter elements (Retn+/−/−, or Retn+) were generated on a Retn−/− (mouse resistin knockout, or Rko) background. Outcomes were compared between age-matched transgenic and knockout mice. Acute sepsis was defined as the initial 24 h following cecal ligation and puncture (CLP). Physiologic and laboratory parameters correlating to the human Sequential Organ Failure Assessment (SOFA) Score were measured in mice, and innate immune cell number/function in the blood and peritoneal cavity were assessed. Results: CLP significantly increased circulating levels of human resistin. The severity of sepsis-induced leukopenia was comparable between Retn+ and Rko mice. Resistin was associated with increased production of neutrophil reactive oxygen species, a decrease in circulating neutrophils at 6 h and an increase in peritoneal Ly6Chi monocytes at 6 h and 24 h post-sepsis. However, intraperitoneal bacterial growth, organ dysfunction and mouse survival did not differ with resistin production in septic mice. Significance: Ex vivo resistin-induced impairment of neutrophil function do not appear to translate to increased sepsis severity or poorer outcomes in vivo following CLP. [ABSTRACT FROM AUTHOR]

Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products. [ABSTRACT FROM AUTHOR]

Background: Transgenic overexpression of apolipoprotein A-I (apoA1) has been shown to delay atherosclerosis lesion progression and promote lesion regression in mouse models; however, apoA1 is subject to oxidation by myeloperoxidase (MPO) and loss of function. The activity of oxidant resistant human apoA1 was compared to unmodified human apoA1 in mouse models of atherosclerosis progression and regression. Methods and results: Human apoA1 and the MPO oxidant resistant 4WF isoform transgenic mice were bred to LDL receptor deficient (LDLr KO) mice and fed a western-type diet. High level expression of these human apoA1 isoforms did not lead to increased HDL-cholesterol levels on the LDLr KO background. In males and females, lesion progression was studied over time, and both apoA1 and 4WF transgenic mice vs. LDLr KO mice had significant and similar delayed lesion progression and reduced non-HDL cholesterol. Using time points with equivalent lesion areas, lesion regression was initiated by feeding the mice a low-fat control diet containing a microsomal triglyceride transfer protein inhibitor for 7 weeks. Lesions regressed more in the male apoA1 and 4WF transgenics vs. the LDLr KO, but the 4WF isoform was not superior to the unmodified isoform in promoting lesion regression. Conclusions: Both human apoA1 and the 4WF MPO oxidant resistant apoA1 isoform delayed lesion progression and promoted lesion regression in LDLr KO mice, with more pronounced effects in males than females; moreover, the 4WF isoform functioned similarly to the unmodified human apoA1 isoform. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the leading cause of dementia in the elderly, but therapeutic options are lacking. Despite long being able to effectively treat the ill-effects of pathology present in various rodent models of AD, translation of these strategies to the clinic has so far been disappointing. One potential contributor to this situation is the fact that the vast majority of AD patients have other dementia-contributing comorbid pathologies, the most common of which are vascular in nature. This situation is modeled relatively infrequently in basic AD research, and almost never in preclinical studies. As part of our efforts to develop small molecule, anti-inflammatory therapeutics for neurological injury and disease, we have recently been exploring potentially promising treatments in preclinical multi-morbidity contexts. In the present study, we generated a mouse model of mixed amyloid and hyperhomocysteinemia (HHcy) pathology in which to test the efficacy of one of our anti-inflammatory compounds, MW151. HHcy can cause cerebrovascular damage and is an independent risk factor for both AD dementia and vascular contributions to cognitive impairment and dementia. We found that MW151 was able to partially rescue hippocampal-dependent spatial memory and learning deficits in this comorbidity context, and further, that the benefit is associated with a normalization of hippocampal metabolites detectable via magnetic resonance spectroscopy. These findings provide evidence that MW151 in particular, and potentially anti-inflammatory treatment more generally, may be beneficial in AD patients with comorbid vascular pathology. [ABSTRACT FROM AUTHOR]

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure in humans. Among the various PKD-related molecules, PKD2L1 forms cation channels, but its physiological importance is obscure. In the present study, we established a transgenic mouse line by overexpressing the dominant-negative form of the mouse PKD2L1 gene (i.e., lacking the pore-forming domain). The resulting PKD2L1del-Tg mice exhibited supraventricular premature contraction, as well as enhanced sensitivity to β-adrenergic stimulation and unstable R-R intervals in electrocardiography. During spontaneous atrial contraction, PKD2L1del-Tg atria showed enhanced sensitivity to isoproterenol, norepinephrine, and epinephrine. Action potential recording revealed a shortened action potential duration in PKD2L1del-Tg atria in response to isoproterenol. These findings indicated increased adrenergic sensitivity in PKD2L1del-Tg mice, suggesting that PKD2L1 is involved in sympathetic regulation. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease (ALD) is one of the leading causes of morbidity and mortality from hepatic complications. C1q/TNF-related protein 3 (CTRP3) is an adiponectin paralog and, in male mice, increased levels of circulating CTRP3 prevents ALD. Therefore, the purpose of this study was to replicate the observed hepatoprotective effect of elevated circulating CTRP3 levels in female mice. Twelve-week-old female wildtype and CTRP3 overexpressing transgenic mice were fed the Lieber-DeCarli alcohol-containing liquid diet (5% vol/vol) for 6 weeks. Unlike the previous study with male mice, CTRP3 overexpression provided no attenuation to alcohol-induced hepatic lipid accumulation, cytokine production, or overall mortality. In conclusion, there appears to be a clear sex-specific effect of CTRP3 in response to alcohol consumption that needs to be explored further. [ABSTRACT FROM AUTHOR]

During pregnancy in placental mammals, small numbers of maternal cells (maternal microchimeric cells, or MMc cells) migrate into the fetus and persist decades, or perhaps for the rest of their lives, and higher frequencies of MMc cells are reported to correlate with variety of phenomena, such as immune tolerance, tissue repair, and autoimmune diseases. While detection of these MMc cells is considered in all pregnancies, their frequency differs largely according to tissue type and disease cases, and it remains unclear whether the number of MMc cells differs significantly among embryos in normal pregnancies. Here, for the first time, we developed a whole embryonic detection method for MMc cells using transgenic mice and counted live MMc cells in each individual embryo. Using this technique, we found that the number of MMc cells was comparable in most of the analyzed embryos; however, around 500 times higher number of MMc cells was detected in one embryo at the latest stage. This result suggests that the number of MMc cells could largely differ in rare cases with unknown underlying mechanisms. Our methodology provides a basis for testing differences in the numbers of MMc cells among individual embryos and for analyzing differences in MMc cell type repertoires in future studies. These data could provide a hint toward understanding the mechanisms underlying the variety of apparently inconsistent MMc-related phenomena. [ABSTRACT FROM AUTHOR]

Ac3IV (Ac-CYIQNCPRG-NH2) is an enzymatically stable vasopressin analogue that selectively activates Avpr1a (V1a) and Avpr1b (V1b) receptors. In the current study we have employed streptozotocin (STZ) diabetic transgenic Ins1Cre/+;Rosa26-eYFP and GluCreERT2;Rosa26-eYFP mice, to evaluate the impact of sustained Ac3IV treatment on pancreatic islet cell morphology and transdifferentiation. Twice-daily administration of Ac3IV (25 nmol/kg bw) to STZ-diabetic Ins1Cre/+;Rosa26-eYFP mice for 12 days increased pancreatic insulin (p<0.01) and significantly reversed the detrimental effects of STZ on pancreatic islet morphology. Such benefits were coupled with increased (p<0.01) beta-cell proliferation and decreased (p<0.05) beta-cell apoptosis. In terms of islet cell lineage tracing, induction of diabetes increased (p<0.001) beta- to alpha-cell differentiation in Ins1Cre/+;Rosa26-eYFP mice, with Ac3IV partially reversing (p<0.05) such transition events. Comparable benefits of Ac3IV on pancreatic islet architecture were observed in STZ-diabetic GluCreERT2;ROSA26-eYFP transgenic mice. In this model, Ac3IV provoked improvements in islet morphology which were linked to increased (p<0.05-p<0.01) transition of alpha- to beta-cells. Ac3IV also increased (p<0.05-p<0.01) CK-19 co-expression with insulin in pancreatic ductal and islet cells. Blood glucose levels were unchanged by Ac3IV in both models, reflecting the severity of diabetes induced. Taken together these data indicate that activation of islet receptors for V1a and V1b positively modulates alpha- and beta-cell turnover and endocrine cell lineage transition events to preserve beta-cell identity and islet architecture. [ABSTRACT FROM AUTHOR]

Inactivation of the tumor suppressor p53 (encoded by the Trp53 gene) is relevant for development and growth of different cancers, including osteosarcoma, a primary bone tumor mostly affecting children and young adolescents. We have previously shown that deficiency of the ribosomal S6 kinase 2 (Rsk2) limits osteosarcoma growth in a transgenic mouse model overexpressing the proto-oncogene c-Fos. Our initial aim for the present study was to address the question, if Rsk2 deficiency would also influence osteosarcoma growth in another mouse model. For that purpose, we took advantage of Trp53fl/fl mice, which were crossed with Runx2Cre transgenic mice in order to inactivate p53 specifically in osteoblast lineage cells. However, since we unexpectedly identified Runx2Cre-mediated recombination also in the thymus, the majority of 6-month-old Trp53fl/fl;Runx2-Cre (thereafter termed Trp53Cre) animals displayed thymic lymphomas, similar to what has been described for Trp53-deficient mice. Since we did not detect osteosarcoma formation at that age, we could not follow our initial aim, but we studied the skeletal phenotype of Trp53Cre mice, with or without additional Rsk2 deficiency. Here we unexpectedly observed that Trp53Cre mice display a unique accumulation of trabecular bone in the midshaft region of the femur and the humerus, consistent with its previously established role as a negative regulator of osteoblastogenesis. Since this local bone mass increase in Trp53Cre mice was significantly reduced by Rsk2 deficiency, we isolated bone marrow cells from the different groups of mice and analyzed their behavior ex vivo. Here we observed a remarkable increase of colony formation, osteogenic differentiation and proliferation in Trp53Cre cultures, which was unaffected by Rsk2 deficiency. Our data thereby confirm a critical and tumorigenesis-independent function of p53 as a key regulator of mesenchymal cell differentiation. [ABSTRACT FROM AUTHOR]

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas. [ABSTRACT FROM AUTHOR]

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases. [ABSTRACT FROM AUTHOR]

Arrhythmogenic right ventricular cardiomyopathy is a hereditary, rare disease with an increased risk for sudden cardiac death. The disease-causing mutations are located within the desmosomal complex and the highest incidence is found in plakophilin2. However, there are other factors playing a role for the disease progression unrelated to the genotype such as inflammation or exercise. Competitive sports have been identified as risk factor, but the type and extend of physical activity as cofactor for arrhythmogenesis remains under debate. We thus studied the effect of light voluntary exercise on cardiac health in a mouse model. Mice with a heterozygous PKP2 loss-of-function mutation were given the option to exercise in a running wheel which was monitored 24 h/d. We analyzed structural and functional development in vivo by echocardiography which revealed that neither the genotype nor the exercise caused any significant structural changes. Ejection fraction and fractional shortening were not influenced by the genotype itself, but exercise did cause a drop in both parameters after 8 weeks, which returned to normal after 16 weeks of training. The electrophysiological analysis revealed that the arrhythmogenic potential was slightly higher in heterozygous animals (50% vs 18% in wt littermates) and that an additional stressor (isoprenaline) did not lead to an increase of arrhythmogenic events pre run or after 8 weeks of running but the vulnerability was increased after 16 weeks. Exercise-induced alterations in Ca handling and contractility of isolated myocytes were mostly abolished in heterozygous animals. No fibrofatty replacements or rearrangement of gap junctions could be observed. Taken together we could show that light voluntary exercise can cause a transient aggravation of the mutation-induced phenotype which is abolished after long term exercise indicating a beneficial effect of long term light exercise. [ABSTRACT FROM AUTHOR]

Early life respiratory insults, such as viral infections or hyperoxia, often increase asthma susceptibility later in life. The mechanisms underlying this increased susceptibility are not fully understood. IL-33 has been shown to be critically involved in allergic airway diseases. IL-33 expression in the neonatal lung can be increased by various respiratory insults associated with asthma development. Therefore, we investigated whether and how early life increases in IL-33 impact allergic airway responses later in life. Using a novel IL-33 transgenic mouse model, in which full-length IL-33 was inducible overexpressed in lung epithelial cells, we transiently upregulated lung IL-33 expression in neonatal mice for one week. After resting for 4–6 weeks, mice were intranasally exposed to a single-dose of recombinant IL-33 or the airborne allergen Alternaria. Alternatively, mice were exposed to Alternaria and ovalbumin multiple times for one month. We found that a transient increase in IL-33 expression during the neonatal period promoted IL-5 and IL-13 production when mice were later exposed to a single-dose of IL-33 or Alternaria in adulthood. However, increased IL-33 expression during the neonatal period did not affect airway inflammation, type 2 cytokine production, lung mucus production, or antigen-specific antibody responses when adult mice were exposed to Alternaria and ovalbumin multiple times. These results suggest that transient increased IL-33 expression early in life may have differential effects on allergic airway responses in later life, preferentially affecting allergen-induced acute type 2 cytokine production. [ABSTRACT FROM AUTHOR]

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect. [ABSTRACT FROM AUTHOR]

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1tm1Mmt/+; Ddx4-CreTr/+ (ΔCtnnb1) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. ΔCtnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro, Thy1-positive undifferentiated spermatogonia from ΔCtnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance (Bcl6b, Gfra1, Plzf) and increased levels for markers associated with progenitor and differentiating spermatogonia (Kit, Rarg, Sohlh1) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal. [ABSTRACT FROM AUTHOR]

The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals. [ABSTRACT FROM AUTHOR]

Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before. [ABSTRACT FROM AUTHOR]

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis (MS). Inflammatory CD4+ T cell responses directed against CNS antigens, including myelin proteolipid protein (PLP), are key mediators of EAE. Dendritic cells (DCs) are critical for the induction of T cell responses against infectious agents. However, the importance of DCs in priming self-reactive CD4+ T cells in autoimmune disease such as MS has been unclear. To determine the requirement of DCs in PLP-specific CD4+ T cell responses and EAE, we genetically deleted CD11c+ DCs in PLP T cell receptor (TCR) transgenic SJL mice constitutively. DC deficiency did not impair the development, selection or the pathogenic function of PLP-specific CD4+ T cells in these mice, and resulted in accelerated spontaneous EAE compared to DC sufficient controls. In addition, using a genetic approach to ablate DCs conditionally in SJL mice, we show that CD11c+ DCs were dispensable for presenting exogenous or endogenous myelin antigen to PLP-specific T cells and for promoting pro-inflammatory T cell responses and severe EAE. Our findings demonstrate that constitutive or conditional ablation of CD11c+ DCs diminished self-tolerance to PLP autoantigen. They further show that in the absence of DCs, non-DCs can efficiently present CNS myelin antigens such as PLP to self-reactive T cells, resulting in accelerated onset of spontaneous or induced EAE. [ABSTRACT FROM AUTHOR]