Liver injury induced by thirty- and fifty-nanometer-diameter silica nanoparticles.

Nano-size silica material is a promising reagent for disease diagnosis, cosmetics, and the food industry. For the successful application of nanoparticle materials in bioscience, evaluation of nano-size material toxicity is important. We previously found that nano-size silica particles caused acute liver failure in mice. However, the hepatotoxicity of nanosilica particles with the diameter of 70 nm or less is unknown. Here, we investigated the relationship between particle size and toxicity using nanosilica particles with diameters of 30, 50, and 70 nm (SP30, SP50, and SP70, respectively). We observed dose-dependent increases in hepatic injury following administration of SP50 and SP30, with SP30 causing greater acute liver injury than that seen with SP50. Smaller silica nanoparticles induced liver injury even at proportionally lower dose levels. Furthermore, we investigated the combinatorial toxicity of SP30 in the presence of chemically induced liver injury (including that caused by carbon tetrachloride, paraquat, cisplatin, and acetaminophen). We observed that particles of the smallest size tested (SP30) synergized with chemical substances in causing liver injury. These data suggest that the size (diameter) of the silica nanoparticles affects the severity of nanoparticle-induced liver injury, a finding that will be useful for future investigations in nanotechnology and nanotoxicology.

Sequential targeting of the genes encoding immunoglobulin-mu and prion protein in cattle.

Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-mu (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.

Fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (SARS) coronavirus S protein neutralizes the virus in a rhesus macaque SARS model.

BACKGROUND: There is still no effective method to prevent or treat severe acute respiratory syndrome (SARS), which is caused by SARS coronavirus (CoV). In the present study, we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS-CoV in vitro in a Rhesus macaque model of SARS. METHODS: The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichia coli-producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS. RESULTS: 5H10, which recognized the same epitope that is also a cleavage site critical for the entry of SARS-CoV into host cells, inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route. In addition, we analyzed the mode of action of 5H10, and the results suggested that 5H10 inhibited fusion between the virus envelope and host cell membrane. 5H10 has potential for use in prevention and treatment of SARS if it reemerges. CONCLUSIONS: This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner.

Revisiting tests for neglected nonlinearity using artificial neural networks.

Tests for regression neglected nonlinearity based on artificial neural networks (ANNs) have so far been studied by separately analyzing the two ways in which the null of regression linearity can hold. This implies that the asymptotic behavior of general ANN-based tests for neglected nonlinearity is still an open question. Here we analyze a convenient ANN-based quasi-likelihood ratio statistic for testing neglected nonlinearity, paying careful attention to both components of the null. We derive the asymptotic null distribution under each component separately and analyze their interaction. Somewhat remarkably, it turns out that the previously known asymptotic null distribution for the type 1 case still applies, but under somewhat stronger conditions than previously recognized. We present Monte Carlo experiments corroborating our theoretical results and showing that standard methods can yield misleading inference when our new, stronger regularity conditions are violated.

Post-translational modification of TRAIL receptor type 1 on various tumor cells and the susceptibility of tumors to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.

Production of cattle lacking prion protein.

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.

Anti-tumor effect of a recombinant Bifidobacterium strain secreting a claudin-targeting molecule in a mouse breast cancer model.

Bifidobacterium is a nonpathogenic strain of anaerobic bacteria that selectively localizes and proliferates in tumors. It has emerged as a specific carrier of anticancer proteins against malignant tumors. Claudins are tetraspanin transmembrane proteins that form tight junctions. Claudin-4 is overexpressed in certain epithelial malignant cancers. The C-terminal fragment of the Clostridium perfringens enterotoxin (C-CPE), an exotoxin without the cytotoxic domain, strongly binds to claudin-4. The C-CPE fusion toxin (C-CPE-PE23), which targets claudin-4, strongly suppresses tumor growth; however, C-CPE fusion toxins exhibit hepatic toxicity. In this study, we successfully generated a strain of Bifidobacterium longum that secreted C-CPE-PE23 (B. longum-C-CPE-PE23) and was specific to and cross reactive with human and mouse claudin-4. We evaluated the therapeutic potential of this strain against triple-negative breast cancer using a mouse model. C-CPE-PE23 decreased cell viability in a dose-dependent manner in human and mouse breast cancer cell lines. After intravenous injection, Bifidobacterium was specifically distributed in the tumors of mice bearing breast cancer tumors. Moreover, B. longum-C-CPE-PE23 significantly suppressed tumor growth in mice with breast cancer without serious side effects, such as weight loss or hepatic and renal damage. We suggest that B. longum-C-CPE-PE23 is a good candidate for breast cancer treatment. Bifidobacterium could also be used as a drug delivery system for hepatotoxic agents.

Addition of L-cysteine to the N- or C-terminus of the all-D-enantiomer [D(KLAKLAK)2] increases antimicrobial activities against multidrug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli.

BACKGROUND: Antimicrobial peptides have a broad spectrum of antimicrobial activities and are attracting attention as promising next-generation antibiotics against multidrug-resistant (MDR) bacteria. The all-d-enantiomer [D(KLAKLAK)2] has been reported to have antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa, and to be resistant to protein degradation in bacteria because it is composed of D-enantiomer compounds. In this study, we demonstrated that modification of [D(KLAKLAK)2] by the addition of an L-cysteine residue to its N- or C- terminus markedly enhanced its antimicrobial activities against Gram-negative bacteria such as MDR Acinetobacter baumannii, E. coli, and P. aeruginosa. METHODS: The peptides [D(KLAKLAK)2] (DP), DP to which L-cysteine was added at the N-terminus C-DP, and DP to which L-cysteine was added at the C-terminus DP-C, were synthesized at >95% purity. The minimum inhibitory concentrations of peptides and antibiotics were determined by the broth microdilution method. The synergistic effects of the peptides and the antibiotics against MDR P. aeruginosa were evaluated using the checkerboard dilution method. In order to assess how these peptides affect the survival of human cells, cell viability was determined using a Cell Counting Kit-8. RESULTS: C-DP and DP-C enhanced the antimicrobial activities of the peptide against MDR Gram-negative bacteria, including A. baumannii, E. coli, and P. aeruginosa. The antimicrobial activity of DP-C was greater than that of C-DP, with these peptides also having antimicrobial activity against drug-susceptible P. aeruginosa and drug-resistant P. aeruginosa overexpressing the efflux pump components. C-DP and DP-C also showed antimicrobial activity against colistin-resistant E. coli harboring mcr-1, which encodes a lipid A modifying enzyme. DP-C showed synergistic antimicrobial activity against MDR P. aeruginosa when combined with colistin. The LD50 of DP-C against a human cell line HepG2 was six times higher than the MIC of DP-C against MDR P. aeruginosa. The LD50 of DP-C was not altered by incubation with low-dose colistin. CONCLUSION: Attachment of an L-cysteine residue to the N- or C-terminus of [D(KLAKLAK)2] enhanced its antimicrobial activity against A. baumannii, E. coli, and P. aeruginosa. The combination of C-DP or DP-C and colistin had synergistic effects against MDR P. aeruginosa. In addition, DP-C and C-DP showed much stronger antimicrobial activity against MDR A. baumannii and E. coli than against P. aeruginosa.

Toxicity of Gold Nanoparticles in Mice due to Nanoparticle/Drug Interaction Induces Acute Kidney Damage.

Nanomaterials are innovative materials with many useful properties, but there is concern regarding their many unknown effects on living organisms. Gold nanoparticles are widely used as industrial materials because of their excellent properties. The potential biological hazards of gold nanoparticles are unknown, and thus, here we examined the in vivo effects of gold nanoparticles 10, 50, and 100 nm in diameter (GnP10, GnP50, and GnP100, respectively) and their interactions with drugs in mice to clarify their safety in mammals. Cisplatin, paraquat, and 5-aminosalicylic acid cause side-effect damage to the liver and kidney in mice. No hepatotoxicity or nephrotoxicity was observed when any of the gold nanoparticles alone were administered via the tail vein. In contrast, co-administration of GnP-10 with cisplatin, paraquat, or 5-aminosalicylic acid caused side-effect damage to the kidney. This suggests that gold nanoparticles with a particle size of 10 nm are potentially nephrotoxic due to their interaction with drugs.

Human monoclonal antibody GNX-8 directed to extended type 1 chain: Specific binding to human colorectal cancer.

We observed previously that two carbohydrate epitopes, extended type 1 chain Le(a)-Le(a) and Le(b)-Le(a), are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of "KM mice" with colonic cancer cell line Colo205, and with purified Le(b)-Le(a) glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le(b)-Le(a), bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le(b)-Le(a), whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-alpha-GalNAc, indicating that these epitopes are O-linked glycans.Immunohistological reactivity of GNX-8 at 1 mug/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 mug/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le(b) antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le(b)-Le(a) antigen, provides a novel direction of immunotherapy for human colorectal cancer. (c) 2009 UICC.

Human mesenchymal stem cells from bone marrow express tumor endothelial and stromal markers.

Tumor development is a complex and dynamic process that involves malignant, vascular, and stromal cells. Endosialin is a tumor endothelial marker (TEM) present in the microvasculature and stroma of human tumors. Cancer-associated fibroblasts (CAF) have been implicated in promoting tumor development and have been associated with mesenchymal stem cells (MSC). Since stem/progenitor cells recruited either from bone marrow or residing in nearby tissues can contribute to pathological processes we investigated endosialin in MSC using a novel monoclonal antibody. Endosialin is highly expressed by CAF and human bone marrow-derived MSC. MSC can form networks in a tube formation assay that is inhibited by an anti-endosialin antibody. Immunohistochemistry for human endosialin in xenograft tumors following co-injection of MSC and cancer cells identified MSC in tumor stroma. MSC are a potential target for anticancer therapeutic intervention and endosialin expression offers a new tool for the identification of MSC. Endosialin expression by both CAF and MSC further implies the potential contribution of MSC to tumor stroma via differentiation into tumor stromal fibroblasts.

Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma.

PURPOSE: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. EXPERIMENTAL DESIGN: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. RESULTS: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. CONCLUSIONS: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Endosialin/TEM 1/CD248 is a pericyte marker of embryonic and tumor neovascularization.

The formation of functional, mature blood vessels depends on the interaction between endothelial cells and pericytes. Commonality exists in the processes involved in vasculature development between tissues whether healthy or diseased. Endosialin/TEM 1 is a cell membrane protein that is expressed in blood vessels during embryogenesis and tumorigenesis but not in normal mature vessels. Antibodies developed to human endosialin were used to investigate endosialin expression and function in human prenatal brain pericytes and pericytes residing in tumors. Anti-endosialin was capable of preventing pericyte tube formation in culture and inhibited migration. Brain pericytes in culture had higher levels of endosialin/TEM 1 than TEMs-2, -3, -4, -5, -7, and -8. Immunocytochemistry revealed that endosialin was present in the cytoplasmic body and in the elongated extensions essential to pericyte function. Transgenic mice engineered to express human endosialin bred on an immunocompromised background allowed the growth of human tumor xenografts. In human colon carcinoma Colo205 and HT29 xenografts grown in human endosialin-transgenic mice, endosialin expression was largely confined to NG2-expressing perivascular cells and not CD31-positive endothelial cells. Similar methods applied to human ovarian and colon tumors confirmed endosialin expression by pericytes. The data indicate that endosialin is strongly expressed by pericytes during periods of active angiogenesis during embryonic and tumor development. Anti-endosialin antibodies may have value in identifying vasculature in malignant tissues. With the appropriate agent, targeting endosialin may interfere with blood vessel growth during tumor development.

Neutralization of hemorrhagic snake venom metalloproteinase HR1a from Protobothrops flavoviridis by human monoclonal antibody.

Human monoclonal antibodies (HuMAbs) against HR1a from Protobothrops (formerly Trimeresurus) flavoviridis venom were obtained by the fusion of SP2/0-Ag14 myeloma cells and spleen cells from KM mice immunized with purified HR1a. The ability of HuMAbs to neutralize the HR1a was determined by in vitro neutralization assay and by neutralization of the hemorrhagic activity. The initial screening of over 300 hybridoma fusion wells resulted in the establishment of 80 HR1a-reactive hybridomas. Of the reactive clones, HuMAb HR1a-7 and HR1a-18 neutralized both proteolytic and hemorrhagic activity of HR1a. Mapping of epitope recognized by the reactive clones was performed by using an ELISA that measured antibody binding to overlapping peptides (15 amino acid peptide offset frameshifted by three residues) covering the metalloproteinase domain sequence of HR1a. HuMAbs HR1a-7 and HR1a-18 neutralized HR1a by reacting with peptides of EQQRYLNNFRFIELV and IVNTLNETYRYL. The three-dimensional structure of HR1a based on a homology modeling predicted that these two epitopes are surface exposed.

[Development of an Anticancer Therapy Using Recombinant Bifidobacterium as a New Drug Delivery System (DDS)].

　Intravenously administered obligate anaerobic bacteria, such as bifidobacteria, grow specifically in tumor tissues. This specificity is attributed to the following: (1) Vascular walls in tumor tissues have nanometer- to micrometer-wide cracks, which allow the bacteria to pass through; (2) the intratumoral environment is hypoxic, due to poor vascularization, and therefore bifidobacteria can survive and proliferate in this anaerobic environment; (3) bifidobacteria cannot survive in well-oxygenated normal tissues. Moreover, unlike gram-negative bacteria, the gram-positive bifidobacteria do not produce endotoxins; therefore, there is no risk of endotoxin shock associated with their intravenous administration. Recently, the utility of bifidobacteria for specific drug delivery to tumor tissues has been highlighted. We have established a novel anti-cancer drug-delivery system using Bifidobacterium longum for the specific release of anti-tumor antibodies (e.g., antibody-drug complexes or single-chain antibodies) to targeted tumor tissues. Here, we introduce the results of our investigation.

The r40-kDa outer membrane protein human monoclonal antibody protects against Porphyromonas gingivalis-induced bone loss in rats.

BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We recently reported the construction and characterization of human immunoglobulin G isotype clones, which were specifically reactive with recombinant (r) 40-kDa outer membrane protein (OMP) of P. gingivalis. The aim of this study was to investigate the efficacy of human monoclonal antibody (hMAb) against r40-kDa OMP of P. gingivalis to the protection alveolar bone loss by P. gingivalis in rats. METHODS: The role of 40-kDa OMP in the adherence of P. gingivalis to human gingival epithelial cells (HGECs) was examined by preincubating with r40-kDa OMP hMAb before adding the HGECs. Moreover, we used a rat model to examine the effect of the anti-r40-kDa OMP hMAb in alveolar bone loss by oral infection. Forty-six days after the last infection, the periodontal bone level was assessed morphometrically on defleshed rat jaws. RESULTS: The adherence to HGECs was reduced by 84% compared to adherence levels without the antibody. P. gingivalis could not be detected from rats in a P. gingivalis-non-infected group and a group that was administered the anti-r40-kDa OMP hMAb. The bone loss in P. gingivalis-infected animals that were administered the anti-r40-kDa OMP hMAb was significantly lower than that of P. gingivalis-infected rats. CONCLUSIONS: Our results suggest that transchromosomic mouse-derived hMAb against r40-kDa OMP of P. gingivalis protects against periodontal bone loss. This newly constructed anti-r40-kDa OMP hMAb was used to protect against periodontal diseases caused by P. gingivalis infection.

Cloned transchromosomic calves producing human immunoglobulin.

Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (lambda) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78-100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.

Hepatotoxicity and Drug/Chemical Interaction Toxicity of Nanoclay Particles in Mice.

Nanomaterials are relatively new and unconventional materials with many useful properties, but their effects on biological systems are poorly understood. Nanoclay is a general term for layered mineral silicate nanoparticles that are ideally suited for use in clay-based nanocomposites. The potential biological hazards of nanoclays have not been addressed, however. Therefore, we investigated the in vivo effects and drug interactions of nanoclays. In mice, administration of nanoclay particles via the tail vein led to acute liver injury. Co-administration of nanoclay and carbon tetrachloride, paraquat, or cisplatin resulted in both liver and kidney injury. Our findings thus indicate that nanoclay particles are potentially hepato- and nephrotoxic.