• Toxicological Research
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• 제9권1호
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• pp.1-11
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• 1993

This study was carried out to investigate the immunopathological effects of 7, 12-Dimethylbenz[a]anthracene(DMBA) on mouse thymus. DMBA was administered subcutaneously to BALB/C mice by interscapular single injection of 50 or 100 ng/g of body weight. Each DMBA treatment group and additional corn oil control group of mice were studied on day 1, 3, 7, 14 and 21 following the injection of DMBA. DMBA treatment resulted in marked decreases in weights and cellularity of thymus. Thymus weights were decreased by 50.6 and 66.0% at 21 days, respectively, after treatment of 50ng/g and 100 ng/g DMBA.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.31-38
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• 2016

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked $IFN-{\gamma}$. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, $5{\mu}M$) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine ($5{\mu}M$) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, $20{\mu}M$) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, $10{\mu}M$) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.

• Toxicological Research
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• 제32권2호
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• pp.115-121
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• 2016

This study was conducted to examine the cellular regulatory mechanisms of sulfasalazine (SSZ) in rabbit articular chondrocytes treated with sodium nitroprusside (SNP). Cell phenotype was determined, and the MTT assay, Western blot analysis and immunofluorescence staining of type II collagen was performed in control, SNP-treated and SNP plus SSZ ($50{\sim}200{\mu}g/mL$) rabbit articular chondrocytes. Cellular proliferation was decreased significantly in the SNP-treated group compared with that in the control (p < 0.01). SSZ treatment clearly increased the SNP-reduced proliferation levels in a concentration-dependent manner (p < 0.01). SNP treatment induced significant dedifferentiation and inflammation compared with control chondrocytes (p < 0.01). Type II collagen expression levels increased in a concentration-dependent manner in response to SSZ treatment but were unaltered in SNP-treated chondrocytes (p < 0.05 and < 0.01, respectively). Cylooxygenase-2 (COX-2) expression increased in a concentration-dependent manner in response to SSZ treatment but was unaltered in SNP-treated chondrocytes (p < 0.05). Immunofluorescence staining showed that SSZ treatment increased type II collagen expression compared with that in SNP-treated chondrocytes. Furthermore, phosphorylated extracellular regulated kinase (pERK) expression levels were decreased significantly in the SNP-treated group compared with those in control chondrocytes (p < 0.01). Expression levels of pERK increased in a concentration-dependent manner by SSZ but were unaltered in SNP-treated chondrocytes. pp38 kinase expression levels increased in a concentration-dependent manner by SSZ but were unaltered in control chondrocytes (p < 0.01). In summary, SSZ significantly inhibited nitric oxide-induced cell death and dedifferentiation, and regulated extracellular regulated kinases 1 and 2 and p38 kinase in rabbit articular chondrocytes.

• The Korean Journal of Pain
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• 제11권2호
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• pp.182-193
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• 1998

Background: Ciliary neurotrophic factor (CNTF), identified as a survival factor for developing peripheral neurons is upregulated by reactive astrocytes in the traumatized tissue and in areas of terminal degeneration after a brain lesion. But in the spinal cord, CNTF is expressed in the non-astrocytic phenotypic, maybe oligodendrocytes. The present study was undertaken to determine the upregulation of CNTF expression in reactive astrocytes following spinal cord lesion in the rat. Methods: Unilateral incision of the dorsal funiculus at the thoracic level was performed and rats were sacrificed on days 3, 7, 14 and 28 postlesion. Western blot analysis, immunocytochemical analysis and double immunofluorescence for CNTF and glial fibrillary acidic protein (GFAP) were performed after spinal cord lesion. Results: A major band with 24 kDa and additional band of higher molecular weight form were detectable, and the intensity of the 24 kDa immunoreactive band increased up to 14 days postlesion and decreased toward laminectomized control values. CNTF immunoreactivity was markedly upregulated in the injured dorsal funiculus and adjacent gray matter. The time course of CNTF expression is coincident with the appearance of reactive astrocytes in the injured spinal cord. Moreover, double immunofluorescence for CNTF and glial fibrillary acidic protein (GFAP) revealed that CNTF immunoreactivity was in GFAP immunoreactive astrocytes. Conclusions: These results show that CNTF upregulation occurred in reactive astrocytes following spinal cord lesion, and suggest a role for CNTF in the regulation of astrocytic responses after spinal cord injury.

• Clinical and Experimental Reproductive Medicine
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• 제29권3호
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• pp.167-178
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• 2002

Objective : The present study was performed to investigate whether apoptosis occur in human embryos by annexin staining and detect the expression of Fas, Fas-ligand (FasL), Bax, and Bcl-2 in human fragmented embryos derived from IVF-ET by immunofluorescence and Western blot analysis. Materials and Methods: Using annexin staining, immunofluorescence and Western blot analysis on normal and fragmented embryos, we were able to detect apoptotsis and apoptotic gene products in fragmented embryos. Result: Phosphatidylserine (PS) translocation, the marker for apoptosis, were detected frequently in fragmented embryos. Bcl-2 and Bax protein were detected in both fragmented and non-fragmented embryos. When fragmented embryos compared to normal embryos, immunofluorescent intensity of Bcl-2 tended to be lower in fragmented embryos. Bax gene expression increased in the fragmented embryos compared to the normal embryos. This result supports a model in which the molar ratio of Bcl-2 to Bax determines whether apoptosis induced or inhibited in human embryo. Fas was highly expressed in human preimplantation embryos but not FasL. It suggests that embryo may undergo apoptosis by binding with FasL produced by follicular or immune cells. Conclusion: The over expression of Bax and Fas will trigger apoptosis to lead embryo fragmentation and change embryo to be nonviable.

• Journal of Ginseng Research
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• 제40권2호
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• pp.196-202
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• 2016

Background: Ginsenoside Rd (GSRd), a main component of the root of Panax ginseng, exhibits anti-inflammation functions and decreases infarct size in many injuries and ischemia diseases such as focal cerebral ischemia. M1 Macrophages are regarded as one of the key inflammatory cells having functions for disease progression. Methods: To investigate the effect of GSRd on renal ischemia/reperfusion injury (IRI) and macrophage functional status, and their regulatory role on mouse polarized macrophages in vitro, GSRd (10-100 mg/kg) and vehicle were applied to mice 30 min before renal IRI modeling. Renal functions were reflected by blood serum creatinine and blood urea nitrogen level and histopathological examination. M1 polarized macrophages infiltration was identified by flow cytometry analysis and immunofluorescence staining with $CD11b^+$, $iNOS^+$/interleukin-12/tumor necrosis factor-${\alpha}$ labeling. For the in vitro study, GSRd ($10-100{\mu}g/mL$) and vehicle were added in the culture medium of M1 macrophages to assess their regulatory function on polarization phenotype. Results: In vivo data showed a protective role of GSRd at 50 mg/kg on Day 3. Serum level of serum creatinine and blood urea nitrogen significantly dropped compared with other groups. Reduced renal tissue damage and M1 macrophage infiltration showed on hematoxylin-eosin staining and flow cytometry and immunofluorescence staining confirmed this improvement. With GSRd administration, in vitro cultured M1 macrophages secreted less inflammatory cytokines such as interleukin-12 and tumor necrosis factor-${\alpha}$. Furthermore, macrophage polarization-related pancake-like morphology gradually changed along with increasing concentration of GSRd in the medium. Conclusion: These findings demonstrate that GSRd possess a protective function against renal ischemia/reperfusion injury via downregulating M1 macrophage polarization.

• 대한수의학회지
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• 제35권2호
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• pp.317-326
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• 1995

The purpose of this study was to establish a method for in vitro culture of C parvum isolated in Korea by determination of suitable cell model to complete development of this parasite. The result obtained were summerized as follows: 1. To determine the most suitable cell line, six types of cell line were examined by microscopy. All cell lines were infected with C parvum and showed the highest infection score in HmLu cells. 2. The staining methods including DMSO-modified acid-fast(A-F) stain, hematoxylin-eosin(H & E) stain and immunofluorescence antibody(IFA) stain were applied to examine the infection of C parvum in cell culture. These staining methods were possible to examine the infection of C parvum in cell culture. The most sensitive one was IFA staining technique. 3. Developmental stages of C parvum in HmLu cell were observed. After the initial 8 hour incubation period, some trophozoites were observed. The meronts and gametes were appeared at 24-48 hour post inoculation(PI), and oocysts were observed firstly at 48-72 hour PI. 4. In H & E stain, the parasite appeared as basophilic within parasitophorous vacuole membrane(PVM) and lying in cytoplasm at near the nucleus of the host cells. It was able to distinguish the type I, type II meronts and gametes. 5. In DMSO-modified acid-fast stain, specific stained parasites were appeared firstly after 48 hour PI. The parasites were showed with different degrees of staining bright red color within PVM. 6. The endogenous stages of parasites in HmLu cell recovered at 48, 96, 120 and 144 hour after inoculation were reacted with rabbit immunized serum in immunofluorescence antibody and avidin-biotin complex peroxidase staining technique.

• BMB Reports
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• 제41권9호
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• pp.664-669
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• 2008

Znf230, the mouse homologue of the human spermatogenesis-related gene, ZNF230, has been cloned by rapid amplification of cDNA ends (RACE). This gene is expressed predominantly in testis, but its expression in different testicular cells and spermatogenic stages has not been previously analyzed in detail. In the present study, the cellular localization of the Znf230 protein in mouse testis and epididymal spermatozoa was determined by RT-PCR, immunoblotting, immunohistochemistry and immunofluorescence. It is primarily expressed in the nuclei of spermatogonia and subsequently in the acrosome system and the entire tail of developing spermatids and spermatozoa. The results indicate that Znf230 may play an important role in mouse spermatogenesis, including spermatogenic cell proliferation and sperm maturation, as well as motility and fertilization.

• Parasites, Hosts and Diseases
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• 제36권2호
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• pp.147-150
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• 1998

국내에서 사육되고 있는 개의 와포자충 감염 실태를 알아보기 위하여 1996년 1월부터 1997년 11월까지 4개 지역 (강원도 춘천, 경상남도 상주, 경기도 과천 및 성남)의 개 257두의 분변을 수집하였다 와고자충의 확인은 현재 상품화되어 판매되고 있는 CrHptosporidium 진단 킷트 (MeridianDiagnostics. Cincinnati. Ohio)를 사용하였다. 총 257두 중 25두에서 와포자충 오시스트가 확인되어 9.7%의 검출률윽 나타내었는데, 과천지역이 17 2%로 가장 높았고, 춘천지역 13.2%, 성남지역이 1.4%인 반면, 상주지역은 오시스트가 검출되지 않았다. 개의 사육형태별 검춘률은 사랍과 함께 생확하는 실내견이 13.9%로 가장 높았고, 집지키는 개가 10.1%, 그리고 잡견 (또는 사육견)이 6. 1%로 밝혀졌다. 이상의 결과로 보아 국내에 있는 여러 종류의 사육견들이 와포자충에 감염되어 있음을 화인할 수 있었으며, 특히 사람과 함께 생활하는 애완견에서 감염률이 높다는 사실은 환경의 오염에 의하여 애발견에서 사람으로 와포자충의 전파가 가능할 수도 있다는 점을 시사하는 것으로 이에 대한 대책이 필요할 것으로 생각된다.

• Parasites, Hosts and Diseases
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• 제47권4호
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• pp.353-357
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• 2009

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, $10^1$, $10^2$, and $10^3$ per $10{\mu}l$ were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < $10^2$ per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.