CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice / 한국실험동물학회지

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2(−/−)) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2(−/−) mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2(−/−) mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2(−/−) mice. These findings indicate that our RAG-2(−/−) mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

CRISPR/Cas9-mediated generation of a Plac8 knockout mouse model / 한국실험동물학회지

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice / 한국실험동물학회지

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein α (SIRPα) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout (CD47(−/−) hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, CD47(−/−) mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male CD47(−/−) mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female CD47(−/−) mice became evident at 26 weeks, but splenomegaly was identified in both genders of CD47(−/−) mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of CD47(−/−) mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our CD47(−/−) mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

Association between Homocysteine and Bone Mineral Density according to Age and Sex in Healthy Adults

BACKGROUND: There are several studies about the relationship between serum homocysteine levels and bone mineral density (BMD), but the results are varied, and the studies are limited in Korea. In our study, the relationship between serum homocysteine levels and BMD by part according to age and sex is investigated. METHODS: From March 2012 to July 2015, the 3,337 healthy adults who took a medical examination were recruited. Subjects filled in the self-recording type questionnaire and physical examination, blood test, BMD of lumbar spine and femur were measured. After sorting by aging (≤49 year old, 50-59 year old, ≥60 year old) and sex, the results were adjusted with age and body mass index (BMI) and the relationship between serum homocysteine levels and BMD by lumbar spine and femur was analyzed by multiple regression analysis. RESULTS: As results of analysis, with the adjustment with age and BMI, all age groups of men had no significant relationship between log-converted serum homocysteine levels and BMD. In women aged under 50, there were significantly negative relationships at lumbar spine (β=-0.028, P=0.038), femur neck (β=-0.062, P=0.001), and total hip (β=-0.076, P<0.001), but there was no significant relationship in other age groups (50-59 year old and ≥60 year old). CONCLUSIONS: As the serum homocysteine levels increased in women aged under 50, BMD of the lumbar spine and femur decreased, and correlations between homocysteine and BMD were different by sex and age.

Factors influencing the severity of acute viral hepatitis A / 대한간학회지

BACKGROUND/AIMS: Most patients with acute viral hepatitis A have a favorable course, but a few of them suffer from severe forms of hepatitis such as fulminant hepatitis. This study was carried out to identify the factors influencing the severity of acute viral hepatitis A. METHODS: We retrospectively reviewed the medical records of 713 patients with acute hepatitis A, who were divided into two groups: severe hepatitis A (N=87) and non-severe hepatitis A (N=626). Severe hepatitis was defined as fulminant hepatitis or prolongation of prothrombin time (INR> or =1.5). Clinical variables were compared between the two groups. RESULTS: The incidence of fulminant hepatitis was 1.4 % (10/713) in patients with acute hepatitis A. Thirty-three (4.6 %) cases exhibited HBsAg positivity. In multivariate analyses, significant alcohol intake and the presence of HBsAg were significant predictive factors of fulminant hepatitis A, and significant alcohol intake and age were significant predictive factors of severe hepatitis A. HBeAg and HBV-DNA status did not affect the clinical course of hepatitis A in chronic hepatitis B carriers. CONCLUSIONS: While most patients with acute hepatitis A have an uncomplicated clinical course, our data suggest that a more-severe clinical course is correlated with being older, significant alcohol intake, and chronic hepatitis-B-virus infection. (

A Case of Central Nervous System Actinomycosis Presenting as Brain Abscess

Actinomyces normally colonizes the mouth, colon, and vagina. Although disruption of mucosa may lead to infection at virtually any site, central nervous system actinomycosis is rare. A 45-year-old man presented with seizure and magnetic resonance imaging showed brain abscess. He was diagnosed with actinomycotic and streptococcal infection of brain by histologic and microbiologic examination. After stereotactic aspiration and biopsy, he was treated successfully by prolonged antibiotic therapy using intravenous penicillin-G and oral amoxicillin.

A Case of Central Nervous System Actinomycosis Presenting as Brain Abscess

Actinomyces normally colonizes the mouth, colon, and vagina. Although disruption of mucosa may lead to infection at virtually any site, central nervous system actinomycosis is rare. A 45-year-old man presented with seizure and magnetic resonance imaging showed brain abscess. He was diagnosed with actinomycotic and streptococcal infection of brain by histologic and microbiologic examination. After stereotactic aspiration and biopsy, he was treated successfully by prolonged antibiotic therapy using intravenous penicillin-G and oral amoxicillin.