Immunogenicity of Recombinant Human Erythropoietin

Tae-Hwe Heo,Young-Kwon Kim,Seung-Ju Yang,Hyun-Jeong Cho,Sung-Jo Kim 대한의생명과학회 2009 Biomedical Science Letters Vol.15 No.2

Human erythropoietin (EPO) is a glycoprotein that enhances red blood cell production by stimulating proliferation and differentiation of erythroid progenitor cells in the bone marrow. Patients with chronic kidney disease (CKD) suffer from anemia caused by reduced production of EPO in the kidney. Recombinant human EPO protein has been used successfully for the treatment of anemia associated with CKD. Recently, attention has been paid to the development of side effect of EPO, pure red cell aplasia (PRCA), in some patients with CKD. PRCA is a rare disorder of erythropoiesis that leads to a severe anemia due to an almost complete cessation of red blood cell production. EPO-related PRCA is caused by the production of EPO-neutralizing antibodies (Abs) that eliminate the biological activity of EPO as well as endogenous EPO in patients undergoing therapy. Since 1988, almost 200 cases worldwide have been reported with Ab-positive PRCA after receiving EPO therapeutics. The underlying mechanisms of the breaking of immune tolerance to self-EPO have been investigated. Modification of formulation, organic compounds of container closures, and route of administration has been suggested for the possible mechanism of increased immunogenicity of EPO. A number of assays have been used to detect Abs specific to EPO. These assays are generally grouped into two major categories: binding Ab assays and neutralizing Ab assays (bioassays). There are several types of binding Ab assays, including radioimmunoprecipitation assay, enzyme-linked immunosorbent assay, and the BIAcore biosensor assay. In vitro cellbased bioassays have been utilized for the detection of neutralizing Abs. Finally, the recent experience with anti-EPO Abs may have considerable implications for the future development and approval of EPO preparations. Also, considering that millions of patients are being treated with EPO, clinicians need to be aware of signs and consequences of this rare but severe clinical case.

Critical role of TNF inhibition in combination therapy for elderly mice with atherosclerosis

Park, Kyung-Yeon,Heo, Tae-Hwe BLACKWELLS 2017 CARDIOVASCULAR THERAPEUTICS Vol.35 No.6

In vitro and in vivo pharmacokinetic characterization of LMT-28 as a novel small molecular interleukin-6 inhibitor

Ahn, Sung-Hoon,Heo, Tae-Hwe,Jun, Hyun-Sik,Choi, Yongseok Asian Australasian Association of Animal Productio 2020 Animal Bioscience Vol.33 No.4

Objective: Interleukin-6 (IL-6) is a T cell-derived B cell stimulating factor which plays an important role in inflammatory diseases. In this study, the pharmacokinetic properties of LMT-28 including physicochemical property, in vitro liver microsomal stability and an in vivo pharmacokinetic study using BALB/c mice were characterized. Methods: LMT-28 has been synthesized and is being developed as a novel therapeutic IL-6 inhibitor. The physicochemical properties and in vitro pharmacokinetic profiles such as liver microsomal stability and Madin-Darby canine kidney (MDCK) cell permeability assay were examined. For in vivo pharmacokinetic studies, pharmacokinetic parameters using BALB/c mice were calculated. Results: The logarithm of the partition coefficient value (LogP; 3.65) and the apparent permeability coefficient values (P_app; 9.7×10^-6 cm/s) showed that LMT-28 possesses a moderate-high cell permeability property across MDCK cell monolayers. The plasma protein binding rate of LMT-28 was 92.4% and mostly bound to serum albumin. The metabolic half-life (t_1/2) values of LMT-28 were 15.3 min for rat and 21.9 min for human at the concentration 1 μM. The area under the plasma drug concentration-time curve and C_max after oral administration (5 mg/kg) of LMT-28 were 302±209 h·ng/mL and 137±100 ng/mL, respectively. Conclusion: These data suggest that LMT-28 may have good physicochemical and pharmacokinetic properties and may be a novel oral drug candidate as the first synthetic IL-6 inhibitor to ameliorate mammalian inflammation.

Quantitative analysis of specific target DNA oligomers using a DNA-immobilized packed-column system

Pack, Seung Pil,Heo, Tae-Hwe,Devarayapalli, Kamakshaiah Charyulu,Makino, Keisuke Springer-Verlag 2011 ANALYTICAL AND BIOANALYTICAL CHEMISTRY Vol.401 No.2

Direct regulation of IL-2 by curcumin

Oh, Jin-Gyo,Hwang, Da-Jeong,Heo, Tae-Hwe Elsevier 2018 Biochemical and biophysical research communication Vol.495 No.1

<P><B>Abstract</B></P> <P>Interleukin-2 (IL-2) is a crucial growth factor for both regulatory and effector T cells. Thus, IL-2 plays a critical role in the stimulation and suppression of immune responses. Recently, anti-IL-2 antibodies (Abs) have been shown to possess strong IL-2 modulatory activities by affecting the interaction between IL-2 and IL-2 receptors. In this study, we screened an herbal library to identify a compound that regulates IL-2, which resulted in the identification of curcumin as a direct binder and inhibitor of IL-2. Curcumin is a phytochemical with well-known anti-cancer properties. In this study, curcumin mimicked or altered the binding pattern of anti-IL-2 Abs against IL-2 and remarkably inhibited the interaction of recombinant IL-2 with the IL-2 receptor α, CD25. Interestingly, curcumin neutralized the biological activities of IL-2 both in vitro and in vivo. In this report, we elucidated the unsolved mechanism of the anti-cancer effect of curcumin by identifying IL-2 as a direct molecular target. Curcumin, as a small molecule IL-2 modulator, has the potential to be used to treat IL-2 related pathologic conditions.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Curcumin identified as a direct binding partner of IL-2. </LI> <LI> Binding between IL-2 and IL-2 receptor alpha is reduced by curcumin. </LI> <LI> Curcumin counteracts the side effect of IL-2, vascular leak syndrome. </LI> </UL> </P>

Gaucher병에서 resveratrol의 세포주기 조절자 p21을 통한 세포보호 효과 연구

김동현(Dong-Hyun Kim),허태회(Tae-Hwe Heo),김준범(June-Bum Kim),김성조(Sung-Jo Kim) 한국생명과학회 2010 생명과학회지 Vol.20 No.8

고셔병은 세포내의 글루코세레브로시데이즈의 결핍으로 인하여 리소좀 내의 글루코세레브로사이드가 분해되지 못하고 축적되는 질환으로 알려져 있으며, 유형의 종류에 따라 신경퇴행성 질환으로 나타나는 것으로 보고되어 있으나 아직까지 정확한 기전이 밝혀져 있지 않다. 본 논문에서는 항산화 효과 및 신경보호 효과가 있는 것으로 알려진 레스베라트롤을 고셔병 환자의 fibroblast 세포에 투여하여 세포 생존율 변화 여부 및 세포주기 조절에 관하여 분자 생물학적 기전을 알아보고자 하였다. 고셔병 세포의 p21의 mRNA 발현 수준과 단백질 발현 양상을 확인한 결과 mRNA 상의 정량적 차이는 관찰되지 않았으나 단백질 발현수준은 레스베라트롤의 농도가 높아짐에 따라 증가 되는 것을 확인하였다. 또한 세포사멸의 표지 인자 단백질로 알려진 PARP의 변화양상을 확인한 결과 레스베라트롤의 농도가 높아짐에 따라 감소하는 것을 확인 할 수 있었다. 이를 통해 폴리페놀계 천연물인 레스베라트롤이 고셔병에서 세포 손상을 치유하며, 궁극적으로 세포사멸을 억제하는 효과를 가져올 것으로 생각할 수 있으며, 본 질환에서 병증을 완화 시킬 수 있을 것으로 사료된다. Gaucher disease (GD) is caused by glucocerebrosidase functional deficiency and the most prevalent lysosomal storage disorder (LSD), with an incidence of about 1 in 20,000 new births. Resveratrol, one kind of phytoalexin, is a produced naturally by several plants and has anti-tumor, anti-aging, anti-inflammatory and neuro-protective effects. In this paper we provide the cellular protective effect of resveratrol in both type I and type II Gaucher disease cells. Resveratrol treatment did not show any significant change in the p21 and p53 mRNA expression level, however expression level of the p21 protein, a cell cycle arrest factor, shows significant increment in both types of Gaucher disease cells. These cell cycle arrest patterns were confirmed by both MTT assay measurement and microscopy detection. In comparison, expression level of poly ADP ribose polymerase (PARP), an apoptosis indicator protein, was significantly decreased in both type I and II Gaucher disease cells after treatment with resveratrol. This result indicates that resveratrol relievescellular apoptotic stress from type I and II Gaucher disease cells. Therefore, we demonstrate that resveratrol inhibits cell proliferation via p21 activity and activates cellular repair systems for Gaucher disease cells. Our results provide at least one of the molecular mechanisms of Gaucher disease and may allow the verification of potential drug targets for therapeutic trials.

Heparan sulfate regulates the antiangiogenic activity of endothelial monocyte-activating polypeptide-II at acidic pH.

Chang, Sun-Young,Ko, Hyun-Jeong,Heo, Tae-Hwe,Kang, Chang-Yuil American Society for Pharmacology and Experimental 2005 Molecular pharmacology Vol.67 No.5

<P>Endothelial monocyte-activating polypeptide-II (EMAP II) is an antiangiogenic factor for rapidly growing endothelial cells that is released from tumor cells under physiological stress such as hypoxia. We have previously shown that the interaction between EMAP II and the alpha-subunit of ATP synthase, alpha-ATP synthase, can play a regulatory function in the growth of endothelial cells. In the current study, we found that EMAP II-alpha-ATP synthase interaction could be inhibited by excess heparin, whereas the interaction could be enhanced by a low concentration of heparin. Both EMAP II and alpha-ATP synthase could specifically interact with heparin, and this interaction was increased under acidic conditions. In addition, EMAP II and alpha-ATP synthase were found to contain the heparin binding motifs determined by analysis using site-directed mutant forms. In endothelial cells, binding of EMAP II to cells was dramatically enhanced, and alpha-ATP synthase could associate with heparan sulfate at acidic pH. The inhibitory effect of EMAP II on the growth of cultured endothelial cells was also significantly enhanced at acidic pH. Analysis using mutant EMAP II proteins demonstrated that heparan sulfate was essential for the enhanced binding and EMAP II function to endothelial cells at acidic pH. Furthermore, the enhanced inhibitory effects of EMAP II could be abrogated by excess heparin or heparinase treatment. In the endothelial cell, heparan sulfate may regulate the function of EMAP II released from the tumor cell in hypoxic condition.</P>

Development of an anti-EPO antibody detection kit based on lab-on-a-chip and bridging antibody technologies

Oh, Jin-Gyo,Seong, Jihyun,Han, Sunmi,Heo, Tae-Hwe Elsevier 2018 Biologicals Vol.54 No.-

<P><B>Abstract</B></P> <P>Immunogenicity is a major concern in the use of biological drugs. In particular, antibody-mediated pure red cell aplasia (PRCA) is a rare condition that is caused by administration of recombinant erythropoietin. There are numerous assay platforms for detect EPO anti-drug antibody (ADA), and most have appropriate assay sensitivity, but in need of improvement in terms of assay turnaround time and user accessibility. Here, the new method was developed based on lab-on-a-chip technology and bridging ELISA. The FREND™ Cartridge is equipped with a microfluidic lateral flow channel, enabling easy, fast and accurate immunoassays with small sample volumes. Biotinylated EPO was immobilized on the avidin-coated solid phase of the test zone in the FREND™ cartridge. Initially, ADA in the serum sample binds to the detector conjugate (EPO-HRP-anti HRP antibody-FL bead) in the conjugation zone, and it flows into the test zone prepared with capture complex (avidin-biotinylated EPO). Unbound detector complexes are captured in the reference zone. The FREND™ system detects and quantifies the fluorescence signals in each zone and then calculates the concentration of EPO ADA in the sample. The FREND™ EPO ADA kit may be useful in local clinics as a rapid method for monitoring patients administered recombinant erythropoietin.</P>

Antiproliferative Effect of Vine Stem Extract from Spatholobus Suberectus Dunn on Rat C6 Glioma Cells Through Regulation of ROS, Mitochondrial Depolarization, and P21 Protein Expression

Kim, Hyungkuen,Yi, Sun Shin,Lee, Hak-Kyo,Heo, Tae-Hwe,Park, Sang-Kyu,Jun, Hyun Sik,Song, Ki Duk,Kim, Sung-Jo Taylor Francis 2018 Nutrition and cancer Vol.70 No.4

<P>The vine stem of Spatholobus suberectus Dunn (SS) is used as a traditional herbal medicine in China. Chinese herbal medicines are well known as natural bioactive compounds that can be used as new medicines, and their antioxidant and anticancer effects have also been reported. This study aimed to examine the anticancer effect of a high-pressure hot-water SS extract on rat C6 glioma cells. The SS extract effectively suppressed the viability and proliferation of C6 glioma cells through an antioxidant effect. Reactive oxygen species (ROS) levels in cancer cells are higher than that in normal cells. If the ROS level falls below that required for the growth of cancer cells, their rapid proliferation and growth can be suppressed. We also measured the induction of mitochondrial membrane depolarization and cell cycle arrest effect caused by the SS extract in C6 glioma cells through a FACS analysis. In addition, we observed an increase in STAT3, p53, E2F1, and p21 mRNA expression and a decrease in Bcl-2 mRNA expression by quantitative PCR. An increase in p21 protein expression of over 83% was observed through western blot analysis. All these data support the fact that the high-pressure hot-water SS extract has the potential to be used for glioma treatment.</P>

Normalization of the levels of inflammatory molecules in <i>Mycobacterium smegmatis</i> -infected U937 cells by fibrate pretreatment

Kim, Sung-Jo,Hong, Minho,Song, Ki Duk,Lee, Hak-Kyo,Ryoo, Sungweon,Heo, Tae-Hwe BioMed Central 2014 BIOLOGICAL RESEARCH Vol.47 No.1

<P><B>Background</B></P><P>Tuberculosis (TB) is a respiratory tract disease caused by <I>Mycobacterium tuberculosis</I> infection. <I>M. tuberculosis</I> exploits immune privilege to grow and divide in pleural macrophages. Fibrates are associated with the immune response and control lipid metabolism through glycolysis with β-oxidation of fatty acids.</P><P><B>Results</B></P><P>In this study, we investigated the effect of fibrate pretreatment on the immune response during <I>M. smegmatis</I> infection in U937 cells, a human leukemic monocyte lymphoma cell line. The protein expression of tumor necrosis factor α (TNF-α), an inflammatory marker, and myeloid differentiation primary response gene 88 (MyD88), a toll like receptor adaptor molecule, in the infected group increased at 1 and 6 h after <I>M. smegmatis</I> infection of U937 cells. Acetyl coenzyme A acetyl transferase-1 (ACAT-1), peroxisome proliferator-activated receptor-α (PPAR-α), TNF-α, and MyD88 decreased in U937 cells treated with fibrates at 12 and 24 h after treatment. More than a 24 h pretreatment with fibrate resulted in similar expression levels of ACAT-1 and PPAR-α between infected vehicle control and infected groups which were pretreated with fibrate for 24 h. However, upon exposure to <I>M. smegmatis,</I> the cellular expression of the TNF-α and MyD88 in the infected groups pretreated with fibrate for 24 h decreased significantly compared to that in the infected vehicle group.</P><P><B>Conclusion</B></P><P>These results suggest that fibrate pretreatment normalized the levels of inflammatory molecules in <I>Mycobacterium smegmatis</I>-infected U937 cells. Further studies are needed to confirm the findings on pathophysiology and immune defense mechanism of U937 by fibrates during <I>M. tuberculosis</I> infection.</P>