ABSTRACT
Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.
Subject(s)
Animals , Antibodies, Viral/blood , Base Sequence , Cell Line , Classical Swine Fever/immunology , Classical Swine Fever Virus/genetics , Cloning, Molecular , DNA, Complementary/genetics , Immunization/methods , Molecular Sequence Data , Neutralization Tests/veterinary , RNA, Viral/chemistry , Recombinant Proteins/immunology , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA , Specific Pathogen-Free Organisms , Swine , VirulenceABSTRACT
BACKGROUND: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on CD4+CD25+ T cells has not been fully examined. In this study, we produced recombinant Tim-3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to CD4+CD25+ T cells. METHODS: We isolated and cloned Tim-3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to CD4+CD25+ T cells was analyzed using flow cytometry. RESULTS: We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to CD4+CD25+ T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig. CONCLUSION: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4+CD25+ T cells.
Subject(s)
Animals , Cricetinae , Mice , Bacteria , CHO Cells , Chromatography , Clone Cells , DNA, Complementary , Flow Cytometry , Immunoglobulins , Ligands , Mucins , Mutagenesis, Site-Directed , Plasmids , Proteins , Sepharose , Staphylococcal Protein A , T-Lymphocytes , Th1 CellsABSTRACT
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-lambda1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-lambda1- immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-lambda1, 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-lambda1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-lambda1 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-lambda1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-lambda complex on HepG2 cells. CONCLUSION: Monoclonal Abs against IFN-lambda1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-lambda.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Biology , Clone Cells , Endocytosis , Escherichia coli , Fluorescence , Hep G2 Cells , Hybridomas , RhodaminesABSTRACT
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-lambda1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-lambda1- immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-lambda1, 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-lambda1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-lambda1 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-lambda1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-lambda complex on HepG2 cells. CONCLUSION: Monoclonal Abs against IFN-lambda1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-lambda.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Biology , Clone Cells , Endocytosis , Escherichia coli , Fluorescence , Hep G2 Cells , Hybridomas , RhodaminesABSTRACT
PURPOSE: Methionine synthase (MTR) and 5,10-methylenetetrahydrofolate reductase (MTHFR) are the main regulatory enzymes for homocysteine metabolism. The present case- control study was conducted to determine whether there is an association between the MTR 2756A > G or MTHFR 677C > T polymorphism and plasma homocysteine concentration in Korean subjects with ischemic stroke. MATERIALS AND METHODS: DNA samples of 237 patients who had an ischemic stroke and 223 age and sex-matched controls were studied. MTR 2756A > G and MTHFR 677C > T genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Frequencies of mutant alleles for MTR and MTHFR polymorphisms were not significantly different between the controls and cases. The patient group, however, had significantly higher homocysteine concentrations of the MTR 2756AA and MTHFR 677TT genotypes than the control group (p=0.04 for MTR, p=0.01 for MTHFR). The combined MTR 2756AA and MTHFR 677TT genotype (p= 0.04) and the homocysteine concentrations of the patient group were also higher than those of the controls. In addition, the genotype distribution was significant in the MTHFR 677TT genotype (p=0.008) and combined MTR 2756AA and MTHFR 677TT genotype (p=0.03), which divided the groups into the top 20% and bottom 20% based on their homocysteine levels. CONCLUSION: The results of the present study demonstrate that the MTR 2756A > G and MTHFR 677C > T polymorphisms interact with elevated total homocysteine (tHcy) levels, leading to an increased risk of ischemic stroke.
ABSTRACT
Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Subject(s)
Virus Replication/drug effects , Trans-Activators/antagonists & inhibitors , Immunoglobulin Variable Region/genetics , Hepatitis B virus/drug effects , Hepatitis B e Antigens/metabolism , Cell LineABSTRACT
The high level of low density lipoprotein (LDL) is a risk factor for cardiovascular disease. Apolipoprotein (apo) B is a major protein component of LDL and plays an important role in the maintenance of cholesterol homeostasis. In this study, six polymorphic sites of the apoB gene were anlaysed in 235 patients with coronary artery disease (CAD) and 216 normal control subjects. There were no significant differences in the allele frequencies of apoB polymorphisms between the control and patient groups. However, haplotype frequencies were significantly different between the CAD patients and control (p<0.05). In addition, the allelic distributions of both EcoRI and MspI polymorphisms in Koreans were similar to those in Chinese but significantly different from those in Caucasians. ApoB polymorphisms showed no association with plasma lipid levels. In conclusion, haplotype analysis of the apoB gene using multiple diallelic markers might be a useful marker for Korean CAD patients.
Subject(s)
Adult , Female , Humans , Male , Apolipoproteins B/genetics , Coronary Artery Disease/genetics , Gene Frequency , Genetic Markers , Haplotypes , Korea , Middle Aged , Polymorphism, Genetic , Genetic VariationABSTRACT
An increased risk for arterial thrombosis is associated with high plasma levels of coagulation and fibrinolytic factors such as PAI-1 and FVII. In this study, the 4G/5G polymorphism in the promoter of PAI-1 gene and Arg353-->Gln polymorphism in the FVII gene were analysed in 139 normal adults and 158 patients with coronary artery disease (CAD), and their association with plasma lipid traits was investigated. There were no significant differences in the allele frequencies of PAI-1 and FVII polymorphisms between control and patient groups. The allelic distributions of both polymorphisms in Koreans were similar to those in Japanese but significantly different from those in Caucasians. In the CAD group, the 4G homozygotes of PAI-1 polymorphism showed significantly higher levels of total (p=0.0250) and LDL cholesterol (p=0.0335) with individuals having other genotypes. However, FVII polymorphism showed no association with lipid levels. In conclusion, the 4G/5G PAI-1 promoter polymorphism and Arg353-->Gln FVII polymorphism are not major genetic risk factors for CAD in Koreans. However, 4G allele of PAI-1 polymorphism revealed to be associated with the levels of cholesterol, especially LDL cholesterol levels in CAD patients.
Subject(s)
Aged , Female , Humans , Male , Alleles , Apolipoproteins A/blood , Apolipoproteins B/blood , Coronary Disease/genetics , Coronary Disease/blood , Factor VII/genetics , Genotype , Korea , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Middle Aged , Plasminogen Activator Inhibitor 1/genetics , Polymorphism, Single Nucleotide , Promoter Regions, Genetic/genetics , Triglycerides/bloodABSTRACT
We have investigated the genetic variation in the human apo B mRNA editing protein (apobec-1) gene. Exon 3 of the apobec-1 gene was amplified by polymerase chain reaction. After detection of an additional band by single strand conformational polymorphism (SSCP) analysis, sequencing of the SSCP shift sample revealed a single-base mutation. The mutation was a CGG transversion at codon 80 resulting in a lleRMet substitution. Thes substitution was confirmed by restriction fragment length polymorphism analysis since a pvull site is abolished by the substitution. Population and family studies confirmed that the inheritance of the genotypes for apobec-1 gene polyomrphism is comtrolled by two codominant alleles (P1 and P2). A significant difference in plasma triglyceride was detected among the different apobec-1 genotypes in the CAD patients (P<0.05) Our study could provide the basis for elucidating the interaction between gentic variation of the apobec-1 gene and disorders related to lipid metabolism.
Subject(s)
Humans , Alleles , Apolipoproteins B , Codon , Exons , Genetic Variation , Genotype , Lipid Metabolism , Plasma , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Polymorphism, Single-Stranded Conformational , RNA, Messenger , Triglycerides , WillsABSTRACT
The authors have experienced a case of gonococcal conjunctivitis in 19 year-old male, who showed bilateral lid edema and copious yellowish conjunctival discharge which progress rapidly for four days. A stain of the conjunctival exudate appeared gram-negative intraCellular diplococci. This patient was treated with topical and systemic antibiotics for 7 days and topical antibiotics and corticosteroids for 12 days, with almost complete resolution. Involvement of corneal epithelium and residual punctate corneal opacity were remained slightly.
Subject(s)
Adult , Humans , Male , Young Adult , Adrenal Cortex Hormones , Anti-Bacterial Agents , Conjunctivitis , Corneal Opacity , Edema , Epithelium, Corneal , Exudates and TransudatesABSTRACT
The authors analysed clinically 44 cases of ophthalmia neonatorum from april, 1981 to march 1982. The followings are it's results; 1) Bilaterality of ophthalmia neonatorum was 52. 3%(23 cases) and unilaterality was 47.7%(21 cases), which was no significant difference in incidence between both eyes. 2) The most common causative organism was gonococcus and nasolacrimal duct obstruction was associated with 11 cases(25%) of ophthalmia neonatorum. 3) The onset of discharge varied between under 24 hours and over 21 days after birth with a mean of 5.7 days. 4) Treatment with adequate topical and systemic antibiotics were successful in eliminating the ophthalmia neonatorum.
Subject(s)
Infant, Newborn , Anti-Bacterial Agents , Incidence , Nasolacrimal Duct , Neisseria gonorrhoeae , Ophthalmia Neonatorum , ParturitionABSTRACT
We studied the relationship among hypertension and retinal and retinal vessel change of 200 hypertensive out and in patients in Seoul Red Cross Hospital. The results are: 1) About 50% of hypertensive patients fall in 2nd stage hypertensive retinopathy. Systolic blood pressure is proportion to hypertensive retinopathy. 2) Generalized narrowing accounts for 86% of retinal vessel change, focal constriction 36.5% and increased central reflex accounts for 68%. 3) Concealment account for 57.5% of A-V crossing sign and anterior displacement accounts for 34%. 4) The commonest site of A-V crossing is inferior-temporal branch and superior-temporal branch come after. The former accounts for 45.3% of A-V crossing and the latter accounts for 36.8%. 5) In grade m retinal changes, puntate hemorrhage accounts for 30. 6% of retinal hemorrhages, flame shaped hemorrhages 32.1% punctate exudates 32.1% and cotton-wool patches accounts for 39%.
Subject(s)
Humans , Blood Pressure , Constriction , Exudates and Transudates , Hemorrhage , Hypertension , Hypertensive Retinopathy , Red Cross , Reflex , Retinal Hemorrhage , Retinal Vessels , Retinaldehyde , SeoulABSTRACT
Neuromyelitis Optica(Devic's Disease) is characterized by a disease process that is relatively afebrile and strictly limited to the spinal cord and optic nerves. We experienced a case of neuromyelitis optica characterized by binocular blindness and paresthesia below the umbilicus level with paraplegia of both lower legs associated with urinary incontinence and defication difficulty. We report above case with brief review of literatures.
Subject(s)
Blindness , Leg , Neuromyelitis Optica , Optic Nerve , Paraplegia , Paresthesia , Spinal Cord , Telescopes , Umbilicus , Urinary IncontinenceABSTRACT
We experienced a case of eyelid neurofibroma in von Recklinghausen's disease, which had a typical sign, cafe-au-lait spots on generalized skin. The patient was a five year old girl who had no family history, and whose mentality was subnormal. The right eyelid, in which the neurofibroma involved, was thickened, and was ptotic. The surgical correction of the right eyelid ptosis was not satisfied.
Subject(s)
Female , Humans , Blepharoptosis , Cafe-au-Lait Spots , Eyelids , Neurofibroma , Neurofibromatosis 1 , SkinABSTRACT
This 24 yrs. old male pt. visited our hosptial on september 1971, with complaints of visual disturbance, ocular pain in Lt. eye. Behcet's disease is usually Characterized by the triad of Hypopyon, uveitis, aphthous ulcer of the mouth and genitalia. A brief review of literatures has been descrived.
Subject(s)
Humans , Male , Genitalia , Mouth , Stomatitis, Aphthous , UveitisABSTRACT
A case of bilateral congenital ptosis with strong heredity. studied and treated at eye clinic in S.R.C.H. is reported. The ptosis was associated with external ophthalmoplegia and blepharophimosis in both eyes, and right esotropia. And typically eyeballs turned media-inferiorly when lids forcibly closed (inverted Bell's phenomenon).
Subject(s)
Blepharophimosis , Esotropia , Heredity , OphthalmoplegiaABSTRACT
A case of pulsating exophthalmos is reported in a 50 years old Korean Woman. The complaints of this case were exophthalmos, chemosis on Rt. eye and intracranial bruit. The etiology of this case is considered due to toxemia of pregnancy and hypertension without trauma. A brief review of the literatures related with the etiology, symptom and therapy is made.