• IMMUNE NETWORK
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• 제9권6호
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• pp.255-264
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• 2009

Background: Chronic low grade inflammation is closely linked to type II diabetes, obesity, and atherosclerosis. Macrophages play a key role in the regulation of pro- or anti-inflammatory actions at the lesion sites of disease. Components of cordyceps militaris, cordycepin and adenosine, have been used for the modulation of inflammatory diseases. The effects of cordycepin in the modulation of macrophages have yet to be be elucidated. We investigated the effects of cordycepin and adenosine on the morphological changes of macrophages under the inflammatory condition of LPS and an anti-inflammatory condition involving high concentrations of adenosine. Methods: We confirmed the mRNA levels of the M1/M2 cytokine genes through RT-PCR and morphological change. Results: LPS-activated macrophages returned to their inactivated original shape, i.e., they looked like naive macrophages, through the treatment with high concentrations of cordycepin ($40{\mu}g/ml$). LPS and adenosine activated macrophages also returned to their original inactivated shapes after cordycepin treatment; however, at relatively higher levels of cordycepin than adenosine. This change did not occur with relatively low concentrations of cordycepin. Adenosine down-regulated the gene expression of M1 cytokines (IL-$1{\beta}$, TNF-${\alpha}$) and chemokines (CX3CR1, RANTES), such as cordycepin. Additionally, M2 cytokines (IL-10, IL-1ra, TGF-${\beta}$) were up-regulated by both cordycepin and adenosine. Conclusion: Based on these observations, both cordycepin and adenosine regulated the phenotypic switch on macrophages and suggested that cordycepin and adenosine may potentially be used as immunomodulatory agents in the treatment of inflammatory disease.

• IMMUNE NETWORK
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• 제8권1호
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• pp.7-12
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• 2008

Background: Members belonging to the interferon-lambda (IFN-${\lambda}$) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-${\lambda}$ biology, such as endocytosis of IFN-${\lambda}$, we produced monoclonal antibodies (Abs) against human IFN-${\lambda}$ and examined their usefulness. Methods: We purified recombinant human IFN-${\lambda}$1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-${\lambda}$1-immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-${\lambda}$1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-${\lambda}$ and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. Results: Four hybridoma clones secreting Abs specific to IFN-${\lambda}$1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-${\lambda}$1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-${\lambda}$1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-${\lambda}$ complex on HepG2 cells. Conclusion: Monoclonal Abs against IFN-${\lambda}$1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-${\lambda}$.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.157-163
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• 1998

흰쥐의 배아발생 동안에 피질반응 후 배아 외부에 새롭게 형성된 피질과립막 (cortical granule envelope, CGE)이 존속하는지 여부와 투명대와 배아표면의 미세구조 변화를 조사하였다. 흰쥐배아의 투명대와 배아표면의 미세구조는 주사전자현미경으로 관찰하였으며, 피질과립막 형성과 분포는 Ulex europaeus agglutinin I lectin을 표지하여 형광현미경으로 관찰하였다. 배아표면은 미수정란 과는 다르게 배아표면의 미세융모가 단축된 특징을 보였고, 8-세포기 배아에서 뚜렷하게 볼 수 있는 CGE에 의해 덮여 있다. 투명대의 구조 역시 미수정난자에 존재하는 구조와는 다른 특징을 나타냈고, 특히 투명대의 섬유성 미세공 구조가 거칠어지고 수적인 감소가 나타났다. 위란강에 피질반응에 의한 피질과립막이 형성되어 배아발생 동안에 존속하였으나 수정란보다는 엷고 국소적인 분포양상을 나타내었다. 본 연구 결과를 종합해 볼 때 흰쥐 초기 배아 발생 동안에는 배아 외부에 피질과립막이 존속하고, 수정시에는 투명대 경화 뿐만 아니라 피질반응에 의해 투명대의 미세구조와 배아표면의 구조도 변화됨을 알 수 있다.

• IMMUNE NETWORK
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• 제12권1호
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• pp.27-32
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• 2012

Background: Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. Methods: In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. Results: First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-${\beta}1$-induced germ line transcript ${\alpha}$ ($GLT{\alpha}$) and IL-4-induced $GLT{\gamma}1$ as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Conclusion: Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

• IMMUNE NETWORK
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• 제9권1호
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• pp.27-33
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• 2009

Macrophages generated in vitro using macrophage-colony stimulating factor (M-CSF) and interleukin (IL)-6 from bone marrow cells (BM-Mp) are defective in antigen presenting cell (APC) function as shown by their ability to induce the proliferation of anti-CD3 mAb-primed syngeneic T cells. However, they do express major histocompatibility (MHC) class I and II molecules. accessory molecules and intracellular adhesion molecules. Here we demonstrate that the defective APC function of macrophages is mainly due to production of $TGF-{\beta}1$ by BM-Mp. Methods: Microarray analysis showed that $TGF-{\beta}1$ was highly expressed in BM-Mp, compared to a macrophage cell line, B6D. which exerted efficient APC function. Production of $TGF-{\beta}1$ by BM-Mp was confirmed by neutralization experiments of $TGF-{\beta}1$ as well as by real time-polymerase chain reaction (PCR). Results: Addition of $anti-TGF-{\beta}1$ monoclonal antibody to cultures of BM-Mp and anti-CD3 mAb-primed syngeneic T cells efficiently induced the proliferation of syngeneic T cells. Conversely, the APC function of B6D cells was almost completely suppressed by addition of $TGF-{\beta}1$. Quantitative real time-PCR analysis also confirmed the enhanced expression of $TGF-{\beta}1$ in BM-Mp. Conclusion: The defective APC function of macrophages generated in vitro with M-CSF and IL-6 was mainly due to the production of $TGF-{\beta}1$ by macrophages.

• IMMUNE NETWORK
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• 제13권5호
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• pp.213-217
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• 2013

Enterotoxigenic Bacteroides fragilis (ETBF) is a human gut commensal bacteria that causes inflammatory diarrhea and colitis. ETBF also promotes colorectal tumorigenesis in the Min mouse model. The key virulence factor is a secreted metalloprotease called B. fragilis toxin (BFT). BFT induces E-cadherin cleavage, cell rounding, activation of the ${\beta}$-catenin pathway and secretion of IL-8 in colonic epithelial cells. However, the precise mechanism by which these processes occur and how these processes are interrelated is still unclear. E-cadherin form homophilic interactions which tethers adjacent cells. Loss of E-cadherin results in detachment of adjacent cells. Prior studies have suggested that BFT induces IL-8 expression by inducing E-cadherin cleavage; cells that do not express E-cadherin do not secrete IL-8 in response to BFT. In the current study, we found that HT29/C1cells treated with dilute trypsin solution induced E-cadherin degradation and IL-8 secretion, consistent with the hypothesis that E-cadherin cleavage causes IL-8 secretion. However, physical damage to the cell monolayer did not induce IL-8 secretion. We also show that EDTA-mediated disruption of E-cadherin interactions without E-cadherin degradation was sufficient to induce IL-8 secretion. Finally, we determined that HT29/C1 cells treated with LiCl (${\beta}$-catenin activator) induced IL-8 secretion in a dose-dependent and time-dependent manner. Taken together, our results suggest that BFT induced IL-8 secretion may occur by the following process: E-cadherin cleavage, disruption of cellular interactions, activation of the ${\beta}$-catenin pathway and IL-8 expression. However, we further propose that E-cadherin cleavage per se may not be required for BFT induced IL-8 secretion.

• IMMUNE NETWORK
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• 제13권4호
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• pp.123-132
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• 2013

Obesity is consistently increasing in prevalence and can trigger insulin resistance and type 2 diabetes. Many lines of evidence have shown that macrophages play a major role in inflammation associated with obesity. This study was conducted to determine metformin, a widely prescribed drug for type 2 diabetes, would regulate inflammation through down-regulation of scavenger receptors in macrophages from obesity-induced type 2 diabetes. RAW 264.7 cells and peritoneal macrophages were stimulated with LPS to induce inflammation, and C57BL/6N mice were fed a high-fat diet to generate obesity-induced type 2 diabetes mice. Metformin reduced the production of NO, $PGE_2$ and pro-inflammatory cytokines ($IL-1{\beta}$, IL-6 and $TNF-{\alpha}$) through down-regulation of $NF-{\kappa}B$ translocation in macrophages in a dose-dependent manner. On the other hand, the protein expressions of anti-inflammatory cytokines, IL-4 and IL-10, were enhanced or maintained by metformin. Also, metformin suppressed secretion of $TNF-{\alpha}$ and reduced the protein and mRNA expression of $TNF-{\alpha}$ in obese mice as well as in macrophages. The expression of scavenger receptors, CD36 and SR-A, were attenuated by metformin in macrophages and obese mice. These results suggest that metformin may attenuate inflammatory responses by suppressing the production of $TNF-{\alpha}$ and the expressions of scavenger receptors.

• Molecules and Cells
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• 제27권6호
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• pp.651-656
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• 2009

The formation of ${\beta}$-amyloid peptide ($A{\beta}$) is initiated from cleavage of amyloid precursor protein (APP) by a family of protease, ${\alpha}$-, ${\beta}$-, and ${\gamma}$-secretase. Sub W, a substrate peptide, consists of 10 amino acids, which are adjacent to the ${\beta}$-cleavage site of wild-type APP, and Sub M is Swedish mutant with double mutations on the left side of the ${\beta}$-cleavage site of APP. Sub W is a normal product of the metabolism of APP in the secretary pathway. Sub M is known to increase the efficiency of ${\beta}$-secretase activity, resulting in a more specific binding model compared to Sub W. Three-dimensional structures of Sub W and Sub M were studied by CD and NMR spectroscopy in water solution. On the basis of these structures, interaction models of ${\beta}$-secretase and substrate peptides were determined by molecular dynamics simulation. Four hydrogen bonds and one water-mediated interaction were formed in the docking models. In particular, the hydrogen bonding network of Sub M-BACE formed spread over the broad region of the active site of ${\beta}$-secretase (P5-P3'), and the side chain of P2- Asn formed a hydrogen bond specifically with the side chain of Arg235. These are more favorable to the cleavage of Sub M by ${\beta}$-secretase than Sub W. The two substrate peptides showed different tendency to bind to ${\beta}$-secretase and this information may useful for drug development to treat and prevent Alzheimer's disease.

• Bioinformatics and Biosystems
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• 제1권1호
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• pp.28-37
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• 2006

Recent studies in molecular biology and proteomics have identified a significant number of novel diagnostic, prognostic, and therapeutic disease markers. However, validation of these markers in clinical specimens with traditional histopathological techniques involves low throughput and is time consuming and labor intensive. Tissue microarrays (TMAs) offer a means of combining tens to hundreds of specimens of tissue onto a single slide for simultaneous analysis. This capability is particularly pertinent in the field of cancer for target verification of data obtained from cDNA micro arrays and protein expression profiling of tissues, as well as in epidemiology-based investigations using histochemical/immunohistochemical staining or in situ hybridization. In combination with automated image analysis, TMA technology can be used in the global cellular network analysis of tissues. In particular, this potential has generated much excitement in cardiovascular disease research. The following review discusses recent advances in the construction and application of TMAs and the opportunity for developing novel, highly sensitive diagnostic tools for the early detection of cardiovascular disease.

• 한국산학기술학회논문지
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• 제13권9호
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• pp.3952-3961
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• 2012

세포주기조절유전자를 식별하는 계산방법을 개발하는 것은 시스템 생물학의 중요한 주제중 하나이다. 이전 방법의 대부분은 세포주기 조절 유전자를 식별하는 표현신호의 주기적인 특성으로 간주한다. 그러나, 세포주기 조절유전자는 상대적으로 세포 네트워크를 기반으로 서로 활성화된 상대적으로 많은 상호 작용을 일으킨다고 가정한다. 본 연구에서는 세포주기 분석에 변수 위상동기화 이론을 적용하여, "통계적상 동기화를 이용한 자가조직지도 (SOMPS)", 즉, 자가조직지도와 다변수 통계 동기화 방법으로 이루어진 방법을 사용하여 여러 개의 하위집합과의 상호작용을 발생시키고자 한다. 평가방법으로 SOMPS방법 알고리즘이 세포주기조절 유전자를 방법으로 기존에 사용되는 방법들과 같이 유용할 것으로 보인다.