Augmented Serum Amyloid A1/2 Mediated by TNF-induced NF-kB in Human Serous Ovarian Epithelial Tumors

최형좌,Rosa Mistica C. Ignacio,이은숙,Andrew J. Wilson,Dineo Khabele,손덕수 대한면역학회 2017 Immune Network Vol.17 No.2

Tumor necrosis factor-a (TNF) is well known to be involved in the immune system and ovarian inflammation. Ovarian cancer is an inflammation-related malignancy that lacks early screening strategies, resulting in late diagnosis followed by high mortality. Based on our previous data, TNF induced abundant serum amyloid A (SAA), an acute phase protein linked to inflammation, in ovarian granulosal cells. To date, the regulation and expression of SAA in ovarian cancer is not fully elucidated. Here, we investigated the relationship between TNF and SAA by comparing human normal ovarian tissues and serous ovarian tumors. We found that SAA1/2 was significantly expressed in tumor tissues, but no or trace expression levels in normal tissues. TNF was also significantly upregulated in ovarian tumor tissues compared to normal tissues. Moreover, TNF significantly increased SAA1/2 levels in human ovarian cancer cell lines, OVCAR-3 and SKOV-3, in a time-dependent manner. Since the SAA1 promoter contains two nuclear factor (NF)-kB sites, we examined whether TNF regulates SAA1 promoter activity. Deletion analysis revealed that the proximal NF-kB site (–95/–85) played a critical role in regulating TNF-induced SAA1 promoter activity. Within 2 h after intraperitoneal injection of lipopolysaccharide, a product known to stimulate release of TNF, SAA preferably localized to ovarian epithelial cells and the thecal-interstitial layers compared to granulosal cell layers. Based on Gene Expression Omnibus (GEO) database, SAA1/2 and TNF were dominantly expressed in advanced grade ovarian cancer. Taken together, the accumulation of SAA1/2 in ovarian cancer could be mediated by TNF-induced NF-kB activation.

Localization of Serum Amyloid A3 in the Mouse Ovary

최형좌,Rosa Mistica C. Ignacio,이은숙,Katherine F. Roby,Paul F. Terranova,손덕수 대한면역학회 2017 Immune Network Vol.17 No.4

Tumor necrosis factor-α (TNF-α) induces serum amyloid A (SAA) 3 among acute-phase proteins in mouse granulosa cells by activating NF-κB signaling via p55 TNF-α receptor type 1. However, the localization of SAA3 within the ovary is unknown. Here we investigated ovarian localization of SAA3 in a mouse ovulation model and in response to IL-1β, a proinflammatory mediator. For the ovulation model, equine chorionic gonadotropin (eCG; 2.5 IU) was administered to mice subcutaneously (sc) to stimulate follicular development on day 25 of age and then 50 h after eCG, human chorionic gonadotropin (hCG; 2.5 IU) was administered sc to induce ovulation. The mouse ovulation model was characterized by the localization of CYP19 mRNA expression to granulosa layers of larger follicles. SAA3 mRNA, determined by in situ hybridization, was broadly expressed throughout the whole ovary. Granulosa layers and small follicles expressed higher SAA3 mRNA compared to thecal-interstitial layers and large follicles, respectively. Interestingly, atretic follicles contained cells expressing intense SAA3 mRNA. After ovulation, SAA3 mRNA expression was intensely evident in ruptured follicles and corpora lutea (CL). The intraperitoneal administration of IL-1β revealed the intense and extensive appearance of specific cells expressing SAA3 mRNA around follicles and in CL. In addition, Gene Expression Omnibus (GEO) database analysis supported expression pattern of SAA3 mRNA observed in mouse ovulation model. Taken together, SAA3 was broadly distributed through the whole ovary, but intensely expressed in atretic follicles and CL. Furthermore, proinflammatory mediators could trigger the intense appearance of SAA3 around follicles and in CL.

원심분리법을 이용한 혈청 내 내독소의 개선된 측정방법 연구

최형좌 ( Hyeong Jwa Choi ),임유정 ( Yoo Jung Lim ),이은희 ( Eun Hee Lee ),박진연 ( Jin Yeon Park ),미글레나 ( Miglena G. Prabagar ),박형순 ( Hyung Soon Park ),강영선 ( Young Sun Kang ) 한국미생물생명공학회(구 한국산업미생물학회) 2013 한국미생물·생명공학회지 Vol.41 No.2

Endotoxins are part of the outer membrane of the cell wall of gram-negative bacteria and are continuously released during bacterial growth. Endotoxins typically induce severe sepsis and septic shock, which cause more than 50% of mortalities. Endotoxins are easily measured in the serum by the limulus amebocyte lysate (LAL) test. However, a nonspecific result is obtained, because the high concentration of serum proteins disturbs the enzyme reaction of the LAL test. In order to solve this problem, the LAL test was performed in this study after the centrifugation of the boiled serum samples to remove the impurities. As a result, among the various conditions examined, endotoxin measurement with the LAL test was the most accurate and repeatable after centrifugation of the boiled serum at 100oC. Moreover, the endotoxin was accurately and repeatedly measured from the prepared sera of mice that had been administered an intraperitoneal injection of purified lipopolysaccharides (LPS) or E. coli. Therefore, the application of centrifugation to remove impurities from boiled serum gives an accurate measurement of endotoxins in the sera of normal subjects or patients, and this will lead to the improved diagnosis and prevention of diseases caused by endotoxins. In addition, the centrifugation of boiled serum samples should be considered and included in the development of endotoxin test kits.

Humanizing NOD/SCID/IL-2Rnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34+ cells

강영경,고윤미,최애리,최형좌,서진희,이민영,이준아 대한혈액학회 2016 Blood Research Vol.51 No.1

BackgroundHumanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function.MethodsFourteen NOD/SCID/IL-2Rnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. ResultsMost of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percent-age of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, re-spectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice.ConclusionWe adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any sig-nificant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.

Humanizing NOD/SCID/IL-2Rnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34+ cells

강영경,고윤미,최애리,최형좌,서진희,이민영,이준아 대한혈액학회 2016 Blood Research Vol.51 No.1

BackgroundHumanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function.MethodsFourteen NOD/SCID/IL-2Rnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. ResultsMost of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percent-age of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, re-spectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice.ConclusionWe adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any sig-nificant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.