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The function of mucosal-associated invariant T (MAIT) cells, a burgeoning member of innate-like T cells abundant in humans and implicated in many diseases, remains obscure. To explore this, mice with a rearranged T cell receptor (TCR) α or β locus, specific for MAIT cells, were generated via induced pluripotent stem cells derived from MAIT cells and were designated Vα19 and Vβ8 mice, respectively. Both groups of mice expressed large numbers of MAIT cells. The MAIT cells from these mice were activated by cytokines and an agonist to produce IFN-γ and IL-17. While Vβ8 mice showed resistance in a cancer metastasis model, Vα19 mice did not. Adoptive transfer of MAIT cells from the latter into the control mice, however, recapitulated the resistance. These mice present an implication for understanding the role of MAIT cells in health and disease and in developing treatments for the plethora of diseases in which MAIT cells are implicated. [ABSTRACT FROM AUTHOR]

Predicting plasma concentration-time profiles of disproportionate metabolites in humans is crucial for evaluating metabolites according to the Safety Testing guidelines. We evaluated Css-MRTpo, an empirical method, using chimeric mice with humanised livers capable of generating human-disproportionate metabolites. Azilsartan and AZ-M2 were administered to humanised chimeric mice, and pharmacokinetic parameters were obtained. Pharmacokinetic data for DS-1971a and DS-M1 in humanised chimeric mice were obtained from the literature. The human plasma concentration-time profiles of these compounds were simulated using the Css-MRTpo method. Azilsartan, DS-1971a, and PF-04937319 produced human disproportionate metabolites, AZ-M2, DS-M1, and PF-M1, respectively. The predicted human pharmacokinetic profiles of PF-04937319 and PF-M1 were obtained from a previous study, and their outcomes were re-evaluated. Our findings revealed that the plasma concentrations of the three metabolites were unexpectedly underpredicted, whereas the three unchanged drugs were reasonably predicted. Further, the introduction of the empirical scaling factor of 3, obtained from six model compounds, improved the predictability of metabolites, suggesting the potential usefulness of the Css-MRTpo method in combination with humanised chimeric mice for predicting the pharmacokinetic profiles of disproportionate metabolites at the early stage of new drug development. [ABSTRACT FROM AUTHOR]

Primary human hepatocytes (PHHs) have been commonly used as the gold standard in many drug metabolism studies, regardless of having large inter-individual variation. These inter-individual variations in PHHs arise primarily from genetic polymorphisms, as well as from donor health conditions and storage conditions prior to cell processing. To equalize the effects of the latter two factors, PHHs were transplanted to quality-controlled mice providing human hepatocyte proliferation niches, and engrafted livers were generated. Cells that were harvested from engrafted livers, call this as experimental human hepatocytes (EHH; termed HepaSH cells), were stably and reproducibly produced from 1014 chimeric mice produced by using 17 different PHHs. Expression levels of acute phase reactant (APR) genes as indicators of a systemic reaction to the environmental/inflammatory insults of liver donors varied widely among PHHs. In contrast to PHHs, the expression of APR genes in HepaSH cells was found to converge within a narrower range than in donor PHHs. Further, large individual differences in the expression levels of drug metabolism-related genes (28 genes) observed in PHHs were greatly reduced among HepaSH cells produced in a unified in vivo environment, and none deviated from the range of gene expression levels in the PHHs. The HepaSH cells displayed a similar level of drug-metabolizing enzyme activity and gene expression as the average PHHs but retained their characteristics for drug-metabolizing enzyme gene polymorphisms. Furthermore, long-term 2D culture was possible and HBV infection was confirmed. These results suggest that the stably and reproducibly providable HepaSH cells with lesser inter-individual differences in drug-metabolizing properties, may have a potential to substitution for PHH as practical standardized human hepatocytes in drug discovery research. [Display omitted] • HepaSH cells were prepared from 1014 chimeric mice engrafted with human hepatocytes. • HepaSH cells showed several (108 cells/mouse) high-quality cells (95% human cells). • HepaSH cells had a stable inflammatory index regardless of donor inflammatory status. • HepaSH cells exhibit robust and reproducible activity of drug-metabolizing enzymes. • HepaSH cells are experimental human hepatocytes used in drug development. [ABSTRACT FROM AUTHOR]

The function of mucosal-associated invariant T (MAIT) cells, a burgeoning member of innate-like T cells abundant in humans and implicated in many diseases, remains obscure. To explore this, mice with a rearranged T cell receptor (TCR) α or β locus, specific for MAIT cells, were generated via induced pluripotent stem cells derived from MAIT cells and were designated Vα19 and Vβ8 mice, respectively. Both groups of mice expressed large numbers of MAIT cells. The MAIT cells from these mice were activated by cytokines and an agonist to produce IFN-γ and IL-17. While Vβ8 mice showed resistance in a cancer metastasis model, Vα19 mice did not. Adoptive transfer of MAIT cells from the latter into the control mice, however, recapitulated the resistance. These mice present an implication for understanding the role of MAIT cells in health and disease and in developing treatments for the plethora of diseases in which MAIT cells are implicated.

Pluripotent stem cells (PSCs) hold great promise for cell‐based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light‐inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease‐deficient CRISPR‐associated protein 9 for induced PSCs reprogramming. This system enables remote, non‐invasive optogenetical regulation of endogenous Sox2 and Oct4 loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSCLIRE) under light‐emitting diode‐based illumination. iPSCLIRE cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSCLIRE cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non‐invasive control of user‐defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non‐invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications. [ABSTRACT FROM AUTHOR]

Background: Psoriasis is characterized by aberrant activation of several pro‐inflammatory circuits as well as abnormal hyperproliferation and dysregulated apoptosis of keratinocytes (KCs). Most currently available therapeutic options primarily target psoriasis‐associated immunological defects rather than epidermal abnormalities. Objective: To investigate the efficacy of the histone deacetylase (HDAC) inhibitor, Vorinostat, in targeting hyperproliferation and impaired apoptosis in psoriatic skin. Methods: Vorinostat effect was investigated in primary KCs cell cultures using cell cycle analysis by flow cytometry, apoptosis assays (Annexin V‐FICH and caspase‐3/7) and antibody arrays, qRT‐PCR and immunohistochemistry. Vorinostat impact on clinical manifestations of psoriasis was investigated in a chimeric mouse model. Results: Vorinostat was found to inhibit KCs proliferation and to induce their differentiation and apoptosis. Using a chimeric mouse model, vorinostat was found to result in marked attenuation of a psoriasiform phenotype with a significant decrease in epidermal thickness and inhibition of epidermal proliferation. Conclusions: Our results support the notion that vorinostat, a prototypic HDAC inhibitor, may be of potential use in the treatment of psoriasis and other hyperproliferative skin disorders. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity.

Adeno-associated virus (AAV) mediated gene therapy has been successfully applied in clinical trials, including hemophilia. Novel AAV vectors have been developed with enhanced transduction and specific tissue tropism. Considering the difference in efficacy of AAV transduction between animal models and patients, the chimeric xenograft mouse model with human hepatocytes has unique advantages of studying AAV transduction efficiency in human hepatocytes. However, it is unclear whether the results in humanized mice can predict AAV transduction efficiency in human hepatocytes. To address this issue, we studied the AAV transduction efficacy in canine hepatocytes in both canine hepatocyte xenografted mice and real dogs. After administration of AAV vectors from different serotypes into canine hepatocyte xenograft mice, AAV8 induced the best canine hepatocyte transduction followed by AAV9, then AAV3, 7, 5 and 2. After administration of AAV/cFIX (cFIX-opt-R338L) vectors in hemophilia B dogs, consistent with the result in chimeric mice, AAV8 induced the highest cFIX protein expression and function, followed by AAV9 and then AAV2. These results suggest that mice xenografted with hepatocytes from different species could be used to predict the AAV liver transduction in real species and highlight this potential platform to explore novel AAV variants for future clinical applications.

Adeno-associated virus (AAV) mediated gene therapy has been successfully applied in clinical trials, including hemophilia. Novel AAV vectors have been developed with enhanced transduction and specific tissue tropism. Considering the difference in efficacy of AAV transduction between animal models and patients, the chimeric xenograft mouse model with human hepatocytes has unique advantages of studying AAV transduction efficiency in human hepatocytes. However, it is unclear whether the results in humanized mice can predict AAV transduction efficiency in human hepatocytes. To address this issue, we studied the AAV transduction efficacy in canine hepatocytes in both canine hepatocyte xenografted mice and real dogs. After administration of AAV vectors from different serotypes into canine hepatocyte xenograft mice, AAV8 induced the best canine hepatocyte transduction followed by AAV9, then AAV3, 7, 5 and 2. After administration of AAV/cFIX (cFIX-opt-R338L) vectors in hemophilia B dogs, consistent with the result in chimeric mice, AAV8 induced the highest cFIX protein expression and function, followed by AAV9 and then AAV2. These results suggest that mice xenografted with hepatocytes from different species could be used to predict the AAV liver transduction in real species and highlight this potential platform to explore novel AAV variants for future clinical applications. [ABSTRACT FROM AUTHOR]

Abstract We have succeeded in stable mass production of chimeric PXB-mice, whose liver is repopulated by human hepatocytes at a ratio of more than 70%, and we are providing these mice to academia and pharmaceutical companies to support the development of new drugs or studies of liver function. Furthermore, we isolated human hepatocytes, called PXB-cells, from the chimeric mice, and provide them for clients weekly for in vitro studies. In this review, we summarize the existing characterizations of PXB-mice and PXB-cells and their present and future applications.

p-Toluic acid, a metabolite of organic solvent xylene, has a high reported no‐observed‐effect level (NOEL, 1000 mg/kg) in rats, possibly because of direct glycine conjugation to methylhippuric acid. In this study, plasma levels of p-toluic acid and its glycine conjugate in mice and humanised-liver mice were evaluated after oral administrations. Although rapid conversion of p-toluic acid to its glycine conjugate was evident from mouse plasma concentrations, the biotransformation of p-toluic acid was slower in humanised-liver mice. The input parameters for physiologically based pharmacokinetic (PBPK) models were determined using fitting procedures to create PBPK-generated plasma concentration curves. The PBPK-modelled hepatic concentrations of p-toluic acid in humanised-liver mice were higher than those observed in plasma. PBPK-modelled hepatic and plasma concentrations of p-toluic acid also indicated slow elimination in humans. These results suggest that rapid conjugations of p-toluic acid reportedly observed in rats could result in overestimation of NOELs for conjugatable chemicals when extrapolated to humanised-liver mice or humans. [ABSTRACT FROM AUTHOR]

Careful selection of the host embryo is critical to the efficient production of knockout (KO) mice when injecting mouse embryonic stem (mES) cells into blastocysts. B6(Cg)-Tyrc−2j/J (B6 albino) and C57BL/6NTac (B6NTac) strains of mice are widely used to produce host blastocysts for such procedures. Here, we tested these two strains to identify an appropriate match for modified agouti C57BL/6N (JM8A3.N1) mES cells. When comparing blastocyst yield, super-ovulated B6NTac mice produced more injectable blastocysts per female than B6 albino mice (8.2 vs. 5.4). There was no significant difference in birth rate when injected embryos were transferred to the same pseudopregnant recipient strain. However, the live birth rate was significantly higher for B6NTac blastocysts than B6 albino blastocysts (62.7% vs. 50.2%). In addition, the proportion of pups exhibiting high-level and complete chimerism, as identified by coat color, was also significantly higher in the B6NTac strain. There was no obvious difference in the efficiency of germline transmission (GLT) when compared between B6NTac and B6 albino host embryos (61.5% vs. 63.3% for mES clones; 64.5% vs. 67.9% for genes, respectively), thus suggesting that an equivalent GLT rate could be obtained with only a few blastocyst injections for B6NTac embryos. In conclusion, our data indicate that B6NTac blastocysts are a better choice for the microinjection of JM8A3.N1 mES cells than B6 albino blastocysts. [ABSTRACT FROM AUTHOR]

Many nongenotoxic chemicals have been shown to produce liver tumors in mice and/or rats by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). Studies with phenobarbital (PB) and other compounds have identified the key events for this MOA: CAR activation; increased hepatocellular proliferation; altered foci formation; and ultimately the development of adenomas/carcinomas. In terms of human relevance, the pivotal species difference is that CAR activators are mitogenic agents in mouse and rat hepatocytes, but they do not stimulate increased hepatocellular proliferation in humans. This conclusion is supported by substantial in vitro studies with cultured rodent and human hepatocytes and also by in vivo studies with chimeric mice with human hepatocytes. Examination of the literature reveals many similarities in the hepatic effects and species differences between activators of rodent CAR and the peroxisome proliferator-activated receptor alpha (PPARα), with PPARα activators also not being mitogenic agents in human hepatocytes. Overall, a critical analysis of the available data demonstrates that the established MOA for rodent liver tumor formation by PB and other CAR activators is qualitatively not plausible for humans. This conclusion is supported by data from several human epidemiology studies. [ABSTRACT FROM AUTHOR]

Central nervous system (CNS)-border associated macrophages (BAMs) maintain their steady-state population during adulthood and are not replaced by circulating monocytes under physiological conditions. Their roles in CNS integrity and functions under pathological conditions remain largely unknown. Until recently, BAMs and microglia could not be unequivocally distinguished due to expression of common macrophage markers. We investigated the transcriptional profiles of immunosorted BAMs from rat sham-operated and ischemic brains using RNA sequencing. We found that BAMs express the distinct transcriptional signature than microglia and infiltrating macrophages. The enrichment of functional groups associated with the cell cycle in CD163+ cells isolated 3 days after the ischemic injury indicates the proliferative capacity of these cells. The increased number of CD163+ cells 3 days post-ischemia was corroborated by flow cytometry and detecting the increased number of CD163+ cells positive for a proliferation marker Ki67 at perivascular spaces. CD163+ cells in the ischemic brains up-regulated many inflammatory genes and parenchymal CD163+ cells expressed iNOS, which indicates acquisition of a pro-inflammatory phenotype. In mice, BAMs typically express CD206 and we found a subset of these cells expressing CD169. Chimeric mice generated by transplanting bone marrow of donor Cx3cr1gfpCCR2rfp mice to wild type hosts showed an increased number of CX3CR1+CD169+ perivascular macrophages 3 days post-ischemia. Furthermore, these cells accumulated in the brain parenchyma and we detected replacement of perivascular macrophages by peripheral monocytic cells in the sub-acute phase of stroke. In line with the animal results, post-mortem brain samples from human ischemic stroke cases showed time-dependent accumulation of CD163+ cells in the ischemic parenchyma. Our findings indicate a unique transcriptional signature of BAMs, their local proliferation and migration of inflammatory BAMs to the brain parenchyma after stroke in animal models and humans.

Immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/ SzJ (NSG) mice are a valuable resource to study human hematopoietic stem cells. Prolonged multilineage hematopoiesis indi- cates stem cell engraftment and generally is measured by flow cytometry. In this study, we took advantage of the multi-parameter detec- tion afforded by modern flow cytometers to optimize detection of human hematopoiesis in NSG mice. Antigens widely expressed by mouse or human cells were evaluated as mark- ers to distinguish mixtures of these cells to optimize and test the limits of chimerism detection. The bone marrow, spleen, and liver of NSG mice transplanted with human hematopoietic cells were analyzed for evidence of engraftment.Mouse bone marrow cells were best marked for exclusion by staining with a combination of CD45, TER-119, and anti-H-2Kd monoclonal antibodies, whereas live human cells were most accurately identified by elimination of cell doublets and positive staining for CD59. Human stem cells (CD34++CD133+CD38low) and progenitors were detected in the bone marrow and liver, but not in the spleen. An unusual pattern of myeloid antigen expression was detected in the bone marrow and CD3+CD4+CD8+ T-cells were detected in the spleen. We concluded that multicolor flow cyto- metric analysis that clearly distinguishes mouse and human cells offers accurate detec- tion of human chimerism in NSG mice. Human hematopoiesis can be detected in the bone marrow and liver of NSG mice with T- lymphopoiesis, possibly occurring in the spleen.

Obeticholic acid (OCA) is a selective farnesoid X receptor (FXR) agonist that regulates bile acid and lipid metabolism. FXR activation induces distinct changes in circulating cholesterol among animal models and humans. The mechanistic basis of these effects has been elusive because of difficulties in studying lipoprotein homeostasis in mice, which predominantly package circulating cholesterol in HDLs. Here, we tested the effects of OCA in chimeric mice whose livers are mostly composed (≥80%) of human hepatocytes. Chimeric mice exhibited a human-like ratio of serum LDL cholesterol (LDL-C) to HDL cholesterol (HDL-C) at baseline. OCA treatment in chimeric mice increased circulating LDL-C and decreased circulating HDL-C levels, demonstrating that these mice closely model the cholesterol effects of FXR activation in humans. Mechanistically, OCA treatment increased hepatic cholesterol in chimeric mice but not in control mice. This increase correlated with decreased SREBP-2 activity and target gene expression, including a significant reduction in LDL receptor protein. Cotreatment with atorvastatin reduced total cholesterol, rescued LDL receptor protein levels, and normalized serum LDL-C. Treatment with two clinically relevant nonsteroidal FXR agonists elicited similar lipoprotein and hepatic changes in chimeric mice, suggesting that the increase in circulating LDL-C is a class effect of FXR activation.

Abstract It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a “dose effect”. Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.

Embryonic stem cells are commonly used for generation of transgenic mice. Embryonic stem cells could participate in the development of chimeric animals after injection into a blastocyst. Injection of genetically modified embryonic stem cells could lead to germ line transmission of a transgene or genomic modification in chimeric mice. Such founders are used to produce transgenic lines of mice. There are several projects dedicated to production of knock-out mouse lines (KOMP Repository, EUCOMM, Lexicon Genetics). Never-theless, there is a need for complex genome modifications, such as large deletions, reporter genes insertion into the 3’ gene regulatory sequence, or site-specific modifications of the genome. To do that, researchers need an embryonic stem cell line that is able to participate in chimeric animal formation even after prolonged culture in vitro. Several lines of mouse embryonic stem cells were produced in the Laboratory of Developmental Genetics of the Institute of Cytology and Genetics SB RAS. We tested DGES1 cell line (2n = 40, XY) (129S2/SvPasCrl genetic background) for chimeric mice production at the Center for Genetic Resources of Laboratory Animals at ICG SB RAS. Embryonic stem cells were injected into 136 blastocysts (B6D2F1 genetic background), which were transplanted into CD-1 mice. Among 66 progeny, 15 were chimeric, 4 of which were more than 80 % chimeric judged by coat color. All chimeras were males without developmental abnormalities. 10 of 15 males were fertile. Microsatellite analysis of the progeny of chimeric mice revealed embryonic stem cell line DGES1 contribution to the gamete formation. Thus, a novel DGES1 embryonic stem cell line could be efficiently used for transgenic mouse production using B6D2F1 blastocysts and CD-1 recipients.

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a “dose effect”. Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus infection cannot be cured by current therapies, so new treatments are urgently needed. We recently identified novel inhibitors of the hepatitis B virus ribonuclease H that suppress viral replication in cell culture. Here, we employed immunodeficient FRG KO mice whose livers had been engrafted with primary human hepatocytes to ask whether ribonuclease H inhibitors can suppress hepatitis B virus replication in vivo . Humanized FRG KO mice infected with hepatitis B virus were treated for two weeks with the ribonuclease H inhibitors #110, an α-hydroxytropolone, and #208, an N-hydroxypyridinedione. Hepatitis B virus viral titers and S and e antigen plasma levels were measured. Treatment with #110 and #208 caused significant reductions in plasma viremia without affecting hepatitis B virus S or e antigen levels, and viral titers rebounded following treatment cessation. This is the expected pattern for inhibitors of viral DNA synthesis. Compound #208 suppressed viral titers of both hepatitis B virus genotype A and C isolates. These data indicate that Hepatitis B virus replication can be suppressed during infection in an animal by inhibiting the viral ribonuclease H, validating the ribonuclease H as a novel target for antiviral drug development. [ABSTRACT FROM AUTHOR]

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion : mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines. [ABSTRACT FROM AUTHOR]

Embryonic stem cells are commonly used for generating transgenic mice. Embryonic stem cells could participate in the development of chimeric animals after being injected into a blastocyst. Injection of genetically modified embryonic stem cells could lead to the transmission of the germ lines of a transgene or genomic modification in chimeric mice. Such founders are used to produce transgenic lines of mice. There are several projects dedicated to producing knock-out mouse lines (KOMP Repository, EUCOMM, Lexicon Genetics). Nevertheless, there is a need for complex genome modifications, such as large deletions, reporter genes' insertion into the 3' gene regulatory sequence, or site-specific modifications of the genome. To do this, researchers need an embryonic stem cell line that is able to participate in the formation of chimeric animals even after prolonged cultivation in vitro. Several lines of embryonic stem cells of mice were produced in the Laboratory of Developmental Genetics of the Institute of Cytology and Genetics (ICG), Siberian Branch, Russian Academy of Sciences (SB RAS). We tested the DGES1 cell line (2 n = 40, XY) (of the 129S2/SvPasCrl genetic background) for chimeric mice production at the Center for Genetic Resources of Laboratory Animals at ICG SB RAS. Embryonic stem cells were injected into 136 blastocysts (of the B6D2F1 genetic background), which were transplanted into CD-1 mice. Among 66 progeny, 15 were chimeric, 4 of which were more than 80% chimeric judged by the coat color. All chimeras were males without developmental abnormalities. 10 of 15 males were fertile. Microsatellite analysis of the progeny of chimeric mice revealed the contribution of the embryonic stem cell line DGES1 to the gamete formation. Thus, a novel DGES1 embryonic stem cell line could be efficiently used for transgenic mouse production using the B6D2F1 blastocysts and CD-1 recipients. [ABSTRACT FROM AUTHOR]

We describe a novel device that can be used for the transcervical transfer of embryos into pseudopregnant female mice. This nonsurgical embryo transfer (NSET) device is as efficient as standard surgical embryo transfer in the production of transgenic mice, and can also be used for the transfer of embryonic stem cell–containing chimeric blastocysts and cryopreserved embryos. In addition to the elimination of surgery, recipient females do not have to be anesthetized. The NSET device eliminates a painful surgical procedure as well as potential complications associated with anesthesia/post-operative care, reduces the technical expertise and equipment needed for surgical transfer, and represents substantial cost savings and regulatory reduction. NSET technology provides an easy and rapid alternative to surgical embryo transfer.

In the 1970s and 1980s it became increasingly clear that blood transfusions could induce a form of chronic hepatitis that could not be ascribed to any of the viruses known to cause liver inflammation. In 1989, the hepatitis C virus (HCV) was discovered and found to be the major causative agent of these infections. Because of its narrow ropism, the in vivo study of this virus was, especially in the early days, limited to the chimpanzee. In the past decade, several alternative animal models have been created. In this review we review these novel animal models and their contribution to our current understanding of the biology of HCV.

Embryonic stem (ES) cells from a C57BL/6N (B6N) background injected into B6(Cg)-Tyrc-2J/J (B6-albino) recipient blastocysts are commonly used for generating genetically modified mouse models. To understand the influence of the recipient blastocyst strain on germline transmission, BALB/cAnNTac and B6-albino germline transmission rates were compared using the C57BL6/N-derived C2 ES cell line. A total of 92 ES cell clones from 27 constructs were injected. We compared blastocyst yield, birth rate, chimera formation rate, and high-percentage (>50 %) male chimera formation rate. For germline transmission, we analyzed 24 clones from 19 constructs, which generated high-percentage male chimeras from both donor strains. B6-albino hosts resulted in higher mean blastocyst yields per donor than did BALB/c ones (3.6 vs. 2.5). However, BALB/c hosts resulted in a higher birth rate than B6-albino ones (36 vs. 27 %), a higher chimera formation rate (50 vs. 42 %), a higher high-percentage male chimera rate (10 vs. 8 %), and a higher germline transmission rate (65 vs. 49 %), respectively. Our data suggest that BALB/c is a suitable blastocyst host strain for C2 ES cells and has an advantage over the B6-albino strain for receiving the injection of C2 ES cells. [ABSTRACT FROM AUTHOR]

Aim We identified four cases of infection with hepatitis B virus genotype G and A2 recombinant (HBV/G/A2) strains, which were initially overlooked by enzyme immunoassay-based genotyping. The patients were all men who have sex with men (MSM) and inhabited several metropolitan areas of Japan, suggesting that the recombinant strains may be circulating among high-risk groups such as MSM. Here, we investigated the genomic structure and virological properties of the HBV/G/A2 strains. Methods Complete genome sequences of the isolates were determined and phylogenetically analyzed. Replication efficiency of HBV/G/A2 was investigated by transfecting plasmids containing 1.24-fold viral genome. The in vivo viral kinetics of HBV/G/A2 were investigated using chimeric mice with humanized livers. Results Phylogenetic analysis revealed that the four strains were almost identical (>99.7% homologous). The preS2/S region of these strains was highly homologous to that of genotype A2 and the remaining region was almost identical to that of genotype G, reflecting inter-genotypic recombination. Interestingly, in all four cases, genotype A was co-infected as a minor population. In vitro analysis revealed that HBV/G/A2 had a low replication rate. Although detectable viremia was not measurable following the inoculation of HBV/G/A2 into chimeric mice, subsequent superinfection of HBV genotype A greatly enhanced HBV/G/A2 replication and viral spread. Conclusion We found that four cases of HBV/G/A2 recombinant among MSM patients in the metropolitan areas of Japan, and HBV/A co-infections are required for its efficient replication. High-risk groups such as MSM should be carefully tested for infection of genotype G-derived variants. [ABSTRACT FROM AUTHOR]

Purpose: The overall objective was to elucidate cellular mechanisms by which G-CSF enhances recovery from traumatic brain injury in a hippocampal-dependent learning task. Methods: Chimeric mice were prepared by transplanting bone marrow cells that express green fluorescent protein (GFP+) from a transgenic "green" mice into C57BL/6 mice. Two months later, the animals sustained mild controlled cortical impact (CCI) to the right frontal-parietal cortex, followed by G-CSF (100 μg/kg) treatment for 3 consecutive days. The primary behavioral end-point was performance on the radial arm water maze (RAWM) assessed before and after CCI (days 7 and 14). Secondary endpoints included a), motor performance on a rotating cylinder (rotarod), b) measurement of microglial and astroglial response, c) hippocampal neurogenesis, and d) measures of neurotrophic factors (BDNF, GDNF) in brain homogenates. Results: G-CSF treatment resulted in significantly better performance on the rotorod at one week, and in the RAWMafter one and two weeks. The cellular changes found 2 wks after CCI in the G-CSF group included increased numbers of hippocampal newborn neurons as well as astrocytosis and microgliosis in striatum and frontal cortex on both sides of brain. GFP+ cells that co-labeled with Iba1 (microglial marker) comprised a significant proportion of striatal microglia in G-CSF treated animals, indicating the capacity of G-CSF to increase microglial recruitment to the site of injury. Neurotrophic factors GDNF and BDNF, elaborated by activated microglia and astrocytes, were increased in G-CSF treated mice. Conclusions: G-CSF serves as a neurotrophic factor that increases hippocampal neurogenesis (or enhances survival of new-born neurons), and activates astrocytes and microglia. In turn, these activated glia release a plethora of cytokines and neurotrophic factors that contribute, in a poorly understood cascade, to the brain's repair response. G-CSF also acts directly on bone marrow-derived cells to enhance recruitment of microglia to the site of CCI from circulating monocytes to the site of CCI. [ABSTRACT FROM AUTHOR]

The central nervous system (CNS) is a very unique system with multiple features that differentiate it from systemic tissues. One of the most captivating aspects of its distinctive nature is the presence of the blood brain barrier (BBB), which seals it from the periphery. Therefore, to preserve tissue homeostasis, the CNS has to rely heavily on resident cells such as microglia. These pivotal cells of the mononuclear lineage have important and dichotomous roles according to various neurological disorders. However, certain insults can overwhelm microglia as well as compromising the integrity of the BBB, thus allowing the infiltration of bone marrow-derived macrophages (BMDMs). The use of myeloablation and bone marrow transplantation allowed the generation of chimeric mice to study resident microglia and infiltrated BMDM separately. This breakthrough completely revolutionized the way we captured these 2 types of mononuclear phagocytic cells. We now realize that microglia and BMDM exhibit distinct features and appear to perform different tasks. Since these cells are central in several pathologies, it is crucial to use chimeric mice to analyze their functions and mechanisms to possibly harness them for therapeutic purpose. This review will shed light on the advent of this methodology and how it allowed deciphering the ontology of microglia and its maintenance during adulthood. We will also compare the different strategies used to perform myeloablation. Finally, we will discuss the landmark studies that used chimeric mice to characterize the roles of microglia and BMDM in several neurological disorders. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger. [ABSTRACT FROM AUTHOR]

Background: Cranial radiotherapy is used to treat tumors of the central nervous system (CNS), as well as non-neoplastic conditions such as arterio-venous malformations; however, its use is limited by the tolerance of adjacent normal CNS tissue, which can lead to devastating long-term sequelae for patients. Despite decades of research, the underlying mechanisms by which radiation induces CNS tissue injury remain unclear. Neuroinflammation and immune cell infiltration are a recognized component of the CNS radiation response; however, the extent and mechanisms by which bone marrow-derived (BMD) immune cells participate in late radiation injury is unknown. Thus, we set out to better characterize the response and tested the hypothesis that C-C chemokine receptor type 2 (CCR2) signaling was required for myeloid cell recruitment following brain irradiation.Methods: We used young adult C57BL/6 male bone marrow chimeric mice created with donor mice that constitutively express enhanced green fluorescent protein (eGFP). The head was shielded to avoid brain radiation exposure during chimera construction. Radiation dose and time response studies were conducted in wild-type chimeras, and additional experiments were performed with chimeras created using donor marrow from CCR2 deficient, eGFP-expressing mice. Infiltrating eGFP+ cells were identified and quantified using immunofluorescent microscopy.Results: Brain irradiation resulted in a dose- and time-dependent infiltration of BMD immune cells (predominately myeloid) that began at 1 month and persisted until 6 months following ≥15 Gy brain irradiation. Infiltration was limited to areas that were directly exposed to radiation. CCR2 signaling loss resulted in decreased numbers of infiltrating cells at 6 months that appeared to be restricted to cells also expressing major histocompatibility complex class II molecules.Conclusions: The potential roles played by infiltrating immune cells are of current importance due to increasing interest in immunotherapeutic approaches for cancer treatment and a growing clinical interest in survivorship and quality of life issues. Our findings demonstrate that injury from brain radiation facilitates a dose- and time-dependent recruitment of BMD cells that persists for at least 6 months and, in the case of myeloid cells, is dependent on CCR2 signaling. [ABSTRACT FROM AUTHOR]

Most of what we know about a drug prior to human clinical studies is derived from animal testing. Because animals and humans have substantial differences in their physiology and in their drug metabolism pathways, we do not know very much about the pharmacokinetic and pharmacodynamic behavior of a drug in humans until after it is administered to many people. Hence, drug-induced liver injury has become a significant public health problem, and we have a very inefficient drug development process with a high failure rate. Because the human liver is at the heart of these problems, chimeric mice with humanized livers could be used to address these issues. We examine recent evidence indicating that drug testing in chimeric mice could provide better information about a drug's metabolism, disposition, and toxicity (i.e., its 'behavior') in humans and could aid in developing personalized medicine strategies, which would improve drug efficacy and safety. [ABSTRACT FROM AUTHOR]

The main pathogenic event caused by Schistosoma mansoni infection is characterized by a granulomatous inflammatory reaction around parasite eggs and fibrosis in the liver. We have previously shown that transplantation of bone marrow cells (BMC) promotes a reduction in liver fibrosis in chronically S. mansoni -infected mice. Here we investigated the presence and phenotype of bone marrow-derived cells in livers of S. mansoni -infected mice. During the chronic phase of infection, C57BL/6 mice had an increased number of circulating mesenchymal stem cells and endothelial progenitor cells in the peripheral blood when compared to uninfected controls. In order to investigate the fate of BMC in the liver, we generated bone marrow chimeric mice by transplanting BMC from transgenic green fluorescent protein (GFP) mice into lethally irradiated wild-type C57BL/6 mice. S. mansoni -infected chimeric mice did not demonstrate increased mortality and developed similar liver histopathological features, when compared to wild-type S. mansoni -infected mice. GFP + bone marrow-derived cells were found in the liver parenchyma, particularly in periportal regions. CD45 + GFP + cells were found in the granulomas. Flow cytometry analysis of digested liver tissue characterized GFP + cells as lymphocytes, myeloid cells and stem cells. GFP + cells were also found in areas of collagen deposition, although rare GFP + cells expressed the myofibroblast cell marker α-SMA. Additionally GFP + endothelial cells (co-stained with von Willebrand factor) were frequently observed, while BMC-derived hepatocytes (GFP + albumin + cells) were sparsely found in the liver of chimeric mice chronically infected with S. mansoni . In conclusion, BMC are recruited to the liver during chronic experimental infection with S. mansoni and contribute to the generation of different cell types involved, not only in disease pathogenesis, but possibly in liver regeneration and repair. [ABSTRACT FROM AUTHOR]

Objective: During respiratory inflammation, it is generally assumed that dendritic cells differentiating from the bone marrow are immunogenic rather than immunoregulatory. Using chimeric mice, the outcomes of airways inflammation on bone marrow progenitor cells were studied. Methods: Immune responses were analyzed in chimeric mice engrafted for >16 weeks with bone marrow cells from mice with experimental allergic airways disease (EAAD). Results: Responses to sensitization and challenge with the allergen causing inflammation in the bone marrow-donor mice were significantly reduced in the chimeric mice engrafted with bone marrow cells from mice with EAAD (EAAD-chimeric). Responses to intranasal LPS and topical fluorescein isothiocyanate (non-specific challenges) were significantly attenuated. Fewer activated dendritic cells from the airways and skin of the EAAD-chimeric mice could be tracked to the draining lymph nodes, and may contribute to the significantly reduced antigen/chemical-induced hypertrophy in the draining nodes, and the reduced immune responses to sensitizing allergens. Dendritic cells differentiating in vitro from the bone marrow of >16 weeks reconstituted EAAD-chimeric mice retained an ability to poorly prime immune responses when transferred into naïve mice. Conclusions: Dendritic cells developing from bone marrow progenitors during airways inflammation are altered such that daughter cells have reduced antigen priming capabilities. [ABSTRACT FROM AUTHOR]

1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of14C-labelled ASP015K were examined using a liquid chromatography–radiometric detector and mass spectrometer (LC–RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigatedviaLC–MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring andN-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates. [ABSTRACT FROM PUBLISHER]

The role of bone marrow-derived cells in gastric cancer formation was not fully understood. In this study, bone marrow from female green fluorescent protein transgenic mice was transplanted into male wild-type mice to generate sex-mismatched chimeric mice. The chimeric mice were treated with carcinogen to induce gastric cancer. At time of sacrifice, 18.2 % (2/11) of mice showed severe dysplasia and 25 % (3/12) of mice successfully induced with cancer. Fluorescence in situ hybridization results showed that bone marrow-derived cells participated in renewal of gastric mucosa and cell fusion was observed in both precancerous lesions and adenocarcinoma, but no sign of fusion was observed in squamous cell carcinoma. Our findings suggest that bone marrow-derived cells participate in renewal of gastric mucosa during chronic damage and might have acquired the phenotype of gastric epithelial cells through cell fusion. Fusion between gastric epithelial cells and bone marrow-derived cells was involved in increased carcinogenesis. [ABSTRACT FROM AUTHOR]

Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as "colony-forming units in spleen (CFU-s)", we hypothesize that "colony-forming units in liver (CFU-L)" serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of "hepatocytes that engraft and proliferate in vivo" as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells., (Copyright © 2023. Published by Elsevier Inc.)

Infection by Trypanosoma cruzi, the aetiological agent of Chagas disease, causes an intense inflammatory reaction in several tissues, including the myocardium. We have previously shown that transplantation of bone marrow cells ( BMC) ameliorates the myocarditis in a mouse model of chronic Chagas disease. We investigated the participation of BMC in lesion repair in the heart and skeletal muscle, caused by T. cruzi infection in mice. Infection with a myotropic T. cruzi strain induced an increase in the percentage of stem cells and monocytes in the peripheral blood, as well as in gene expression of chemokines SDF-1, MCP1, 2, and 3 in the heart and skeletal muscle. To investigate the fate of BMC within the damaged tissue, chimeric mice were generated by syngeneic transplantation of green fluorescent protein ( GFP+) BMC into lethally irradiated mice and infected with Trypanosoma cruzi. Migration of GFP+ BMC to the heart and skeletal muscle was observed during and after the acute phase of infection. GFP+ cardiomyocytes and endothelial cells were present in heart sections of chimeric chagasic mice. GFP+ myofibres were observed in the skeletal muscle of chimeric mice at different time points following infection. In conclusion, BMC migrate and contribute to the formation of new resident cells in the heart and skeletal muscle, which can be detected both during the acute and the chronic phase of infection. These findings reinforce the role of BMC in tissue regeneration. [ABSTRACT FROM AUTHOR]

The proinflammatory cytokine interleukin-1β (IL-1β) plays a major role in the signal transduction of immune stimuli from the periphery to the central nervous system, and has been shown to be an important mediator of the immune-induced stress hormone release. The signaling pathway by which IL-1β exerts this function involves the blood-brain-barrier and induced central prostaglandin synthesis, but the identity of the blood-brain-barrier cells responsible for this signal transduction has been unclear, with both endothelial cells and perivascular macrophages suggested as critical components. Here, using an irradiation and transplantation strategy, we generated mice expressing IL-1 type 1 receptors (IL-1R1) either in hematopoietic or non-hematopoietic cells and subjected these mice to peripheral immune challenge with IL-1β. Following both intraperitoneal and intravenous administration of IL-1β, mice lacking IL-1R1 in hematopoietic cells showed induced expression of the activity marker c-Fos in the paraventricular hypothalamic nucleus, and increased plasma levels of ACTH and corticosterone. In contrast, these responses were not observed in mice with IL-1R1 expression only in hematopoietic cells. Immunoreactivity for IL-1R1 was detected in brain vascular cells that displayed induced expression of the prostaglandin synthesizing enzyme cyclooxygenase-2 and that were immunoreactive for the endothelial cell marker CD31, but was not seen in cells positive for the brain macrophage marker CD206. These results imply that activation of the HPA-axis by IL-1β is dependent on IL-1R1s on non-hematopoietic cells, such as brain endothelial cells, and that IL-1R1 on perivascular macrophages are not involved. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Diseases such as temporal lobe epilepsy, brain trauma and stroke can induce endothelial cell proliferation and angiogenesis in specific brain areas. During status epilepticus (SE), bone marrow-derived cells are able to infiltrate and proliferate, dramatically increasing at the site of injury. However, it is still unclear whether these cells directly participate in vascular changes induced by SE. Method: To investigate the possible role of bone marrow-derived cells in angiogenesis after seizures, we induced SE by pilocarpine injection in previously prepared chimeric mice. Mice were euthanized at 8h, 7d or 15d after SE onset. Results: Our results indicated that SE modified hippocampal vascularization and induced angiogenesis. Further, bone marrow-derived GFP+ cells penetrated through the parenchyma and participated in the formation of new vessels after SE. We detected bone marrow-derived cells closely associated with vessels in the hippocampus, increasing the density of blood vessels that had decreased immediately after pilocarpine-induced SE. Conclusion: We conclude that epileptic seizures directly affect vascularization in the hippocampus mediated by bone marrow-derived cells in a time-dependent manner. [Copyright &y& Elsevier]

ABSTRACT Preclinical studies in animal models are used routinely during drug development, but species differences of pharmacokinetics (PK) between animals and humans have to be taken into account in interpreting the results. Human hepatocytes are also widely used to examine metabolic activities mediated by cytochrome P450 (P450) and other enzymes, but such in vitro metabolic studies also have limitations. Recently, chimeric mice with humanized liver (h-chimeric mice), generated by transplantation of human donor hepatocytes, have been developed as a model for the prediction of metabolism and PK in humans, using both in vitro and in vivo approaches. The expression of human-specific metabolic enzymes and metabolic activities was confirmed in humanized liver of h-chimeric mice with high replacement ratios, and several reports indicate that the profiles of P450 and non-P450 metabolism in these mice adequately reflect those in humans. Further, the combined use of h-chimeric mice and r-chimeric mice, in which endogenous hepatocytes are replaced with rat hepatocytes, is a promising approach for evaluation of species differences in drug metabolism. Recent work has shown that data obtained in h-chimeric mice enable the semi-quantitative prediction of not only metabolites, but also PK parameters, such as hepatic clearance, of drug candidates in humans, although some limitations remain because of differences in the metabolic activities, hepatic blood flow and liver structure between humans and mice. In addition, fresh h-hepatocytes can be isolated reproducibly from h-chimeric mice for metabolic studies. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

1. The biotransformation, hepatic transporter and blood chemistry effects of troglitazone were investigated following 7 days of dosing at 600 mg/kg/day to chimeric murinized or humanized FRG mice, Mo-FRG and Hu-FRG mice, respectively. 2. Clinical chemistry and histopathology analysis revealed a significant drop in humanization over the time course of the study for the Hu-FRG mice but no significant changes associated with troglitazone treatment in either the Mo-FRG or the Hu-FRG models. No changes in transporter expression in livers of these mice were observed. Oxidative and conjugative metabolic pathways were identified with a 15- to 18-fold increase in formation of troglitazone sulfate in the Hu-FRG mice compared with the Mo-FRG mice in blood and bile, respectively. This resembles the troglitazone metabolism in human and these data are comparable with the formation of this metabolite in the chimeric uPA+/+/SCID mice. 3. However, larger amounts of troglitazone glucuronide were also observed in the Hu-FRG mouse compared with the Mo-FRG mouse which may be an effect of the drop in humanization of the Hu-FRG mouse during the study. 4. Highly humanized mice have a considerable potential in providing a useful first insight into circulating human metabolites of candidate drugs metabolized in the liver. [ABSTRACT FROM AUTHOR]

Background: Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-kB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-kB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated. Methods: To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IkB kinase in the myeloid cells (IKKβΔmye), IL-6-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr. WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI. Results: Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβΔmye mice as well as in IL6-/- to WT chimeric mice. Conclusion: Given that IKKβΔmye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-kB-IL-6 signaling pathways induce inflammation, contributing to VILI, and IkB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury. [ABSTRACT FROM AUTHOR]

1-Pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122) is an agonist of the cannabinoid receptors CB and CB. In this study, the phase I and phase II metabolisms of JWH-122 were investigated using two models. In vitro studies using incubations of JWH-122 with human liver microsomes were performed to obtain metabolites of the drug at the initial step; 11 classes of metabolites were found and analyzed by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hydroxylation(s) on the naphthalene moiety and/or the indole moiety of the molecule took place as such or in combination with dehydrogenation or cleavage of the N-pentyl side chain. Furthermore, dihydrodiol metabolites were formed probably via epoxide formation on the naphthalene moiety, irrespective of the combination with hydroxylation(s). A metabolite carrying a carboxyl group on the N-pentyl side chain was also detected. As the second step of the study, in vivo experiments using chimeric mice were performed; the mice were orally administered JWH-122, and their urine samples were collected, subjected to enzymatic hydrolysis, and analyzed by LC-MS and LC-MS-MS. The urine samples without hydrolysis were also analyzed for their molecular formulae in the conjugated forms by LC-high resolution MS. The in vivo model using chimeric mice confirmed most metabolite classes and clarified the phase II metabolism of JWH-122. It was concluded that all metabolites formed in vivo were excreted conjugated as glucuronide or sulfate, with conjugation rates above 50 %. [ABSTRACT FROM AUTHOR]

Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical com- ponent for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease. [ABSTRACT FROM AUTHOR]

Chimeric mice, which have human hepatocytes engrafted in their liver, have been used to study human drug metabolism and pharmacodynamic responses for nearly 20 years. However, there are very few examples where their use has prospectively impacted the development of a candidate medication. Here, three different chimeric mouse models and their utility for pharmacology studies are evaluated. Several recent studies indicate that using these chimeric mouse models could help to overcome traditional (predicting human-specific metabolites and toxicities) and 21st century problems (strategies for personalized medicine and selection of optimal combination therapies) in drug development. These examples suggest that there are many opportunities in which the use of chimeric mice could significantly improve the quality of preclinical drug assessment. [Copyright &y& Elsevier]

Purpose: Optic neuritis occurring in multiple sclerosis (MS) is a disease characterized by chronic inflammation and demyelination in the optic nerve. Although it has been well appreciated that leukocyte infiltration into the optic nerve is an early event during the course of the disease, there has been no study on visualizing and quantifying leukocyte trafficking in the retina during the progression of MS., Methods: In this study, we generated green fluorescent protein (GFP) + bone marrow chimeric mice, in which GFP-labeled leukocytes facilitate the visualization of their trafficking in the retina. This reporter was then integrated with a well-established rodent model for MS-experimental autoimmune encephalomyelitis (EAE), allowing high resolution in vivo scanning laser ophthalmoscopy (SLO) to track leukocyte movement in the retina in real time. Quantification of leukocyte trafficking was accomplished through Imaris software., Results: Through SLO, we were able to localize the GFP signal, allowing us to clearly identify leukocytes within the vascular space. We observed more intense leukocyte migration in the retina of EAE mice, exhibiting three distinct movement behaviors: flowing, rolling/crawling and adherent. There was a marked increase in leukocyte rolling and adhesion in retinal vasculature, particularly in the veins and capillaries after induction of EAE. The velocity of rolling leukocytes ranged from 12.0 to 1065.0 μm/sec in the veins as compared to 14.1 to 942.0 in the capillaries. Furthermore, focal areas of recurrent leukocyte adhesion to endothelial surfaces were observed in EAE retinas., Conclusion: We generated a novel model that makes it possible to non-invasively track leukocyte trafficking in the retina of EAE mice. Our study demonstrates that leukocyte migration in an MS model is distinctly different from the control, suggesting that leukocytes may play a key role in the development of retinal vascular inflammation and optic neuritis during MS, warranting further investigation of the pathological roles of leukocytes in the disease onset and progression., Competing Interests: Conflict of Interest Statement There is no financial interest or conflict of interest.