• Animal cells and systems
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• v.8 no.2
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• pp.125-133
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• 2004

We have extended our previous work that cross-linking CD4 molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$We have extended our previous work that cross-linking CD$4^+$ molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$ T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD$4^+$T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4 molecules. The CD$4^+$cross-linking failed to induce effector cell proliferation or the transcription of TNF${\beta}$ Upregulation of TNF${\beta}$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF${\beta}$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased p$56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD4T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4molecules. The CD4 cross-linking failed to induce effector cell proliferation or the transcription of TNF$\beta$. Upregulation of TNF$\beta$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF$\beta$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased $56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.140-147
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• 2004

Urokinase is an enzyme with fibrinolytic activity (plasminogen activator) isolated from fresh urine of healthy men. Viral safety is an important prerequisite for clinical preparation of the protein from urine. In order to increase the viral safety of a high purity urokinase in regard to non-enveloped viruses, a virus removal process using a novel polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. Urokinase was able to pass through the filter with recoveries of 95% in the production scale process. No substantial changes were observed in physical and biochemical characteristics of the filtered urokinase in comparison with those of the enzyme before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production scale cartridges and tested if it could remove several experimental model viruses for human pathogenic viruses, including porcine parvovirus (PPV), human hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), bovine viral diarrhoea virus (BVDV), and bovine herpes virus (BHV). Non-enveloped viruses (PPV, HAV, and EMCV) as well as enveloped viruses (BVDV and BHV) were completely removed during filtration. The log reduction factors achieved were $\geq$4.86 for PPV, $\geq$4.60 for HAV, $\geq$6.87 for EMCV, $\geq$4.60 for BVDV, and $\geq$5.44 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of the final products.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1317-1325
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• 2008

Viral safety is an important prerequisite for clinical preparations of plasma-derived pharmaceuticals. One potential way to increase the safety of therapeutic biological products is the use of a virus-retentive filter. In order to increase the viral safety of human antihemophilic factor IX, particularly in regard to non-enveloped viruses, a virus removal process using a polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. The most critical factor affecting the filtration efficiency was operating pH and the optimum pH was 6 or 7. Flow rate increased with increasing operating pressure and temperature. Recovery yield in the optimized production-scale process was 96%. No substantial changes were observed in the physical and biochemical characteristics of the filtered factor IX in comparison with those before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production-scale cartridges and to test if it could remove several experimental model viruses for human pathogenic viruses, including human hepatitis A virus (HAV), porcine parvovirus (PPV), murine encephalomyocarditis virus (EMCV), human immunodeficiency virus type 1 (HIV), bovine viral diarrhea virus (BVDV), and bovine herpes virus (BHV). Non-enveloped viruses (HAV, PPV, and EMCV) as well as enveloped viruses (HIV, BVDV, and BHV) were completely removed during filtration. The log reduction factors achieved were $\geq$6.12 for HAV, $\geq$4.28 for PPV, $\geq$5.33 for EMCV, $\geq$5.51 for HIV, $\geq$5.17 for BVDV, and $\geq$5.75 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of factor IX.

• BMB Reports
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• v.31 no.4
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• pp.355-363
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• 1998

MHC unrestricted, antigen nonspecific killing by $CD4^+$ T-cells against virally-infected target cells was induced following cross-linking of CD4 molecules. The cytotoxicity of antibody-activated $CD4^+$ T-cells was abolished by genistein (4',5,7-trihydroxyisoflavone), a protein tyrosine kinase (PTK) inhibitor, but not by H-7, a protein kinase C (PKC) inhibitor. Genisteintreated human or bovine peripheral blood $CD4^+$ T-cells lacked PTK activity and failed to kill virally-infected target cells even after cross-linking of CD4 molecules. The cross-linking of CD4 molecules did not induce effector cell proliferation or the transcription of TNF ${\beta}$. TNF ${\beta}$ synthesis was up-regulated by incubating antibody activated effector cells with bovine herpesvirus type 1 (BHV-1) infected D17 target cells. Anti-TNF ${\beta}$ antibody partially abrogated direct effector cell-mediated antiviral cytotoxicity. On the other hand, this antibody effectively neutralized antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on effector and target cell ratio. These findings have importance to define the mechanisms of how CD4 cytotoxic cells control viral infection.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.1
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• pp.19-25
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• 2006

With particular regards to the hepatitis A virus (HAV), a terminal dry-heat treatment ($100^{\circ}C$ for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor IX concentrate. The loss of factor IX activity during dry-heat treatment was of about 3%, as estimated by a clotting assay. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor IX compared with those of the factor IX before dry-heat treatment. The dry-heat-treated factor IX was stable for up to 24 months at $4^{\circ}C$, The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV and murine encephalomyocarditis virus (EMCV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Porcine parvovirus (PPV) and bovine herpes virus (BHV) were potentially sensitive to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were ${\ge}5.60$ for HAV, ${\ge}6.08$ for EMCV, 2.64 for PPV, and 3.59 for BHV. These results indicate that dry-heat treatment improves the virus safety of factor IX concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.997-1003
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• 2008

Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment ($100^{\circ}C$ for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at $4^{\circ}C$. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were ${\geq}5.55$ for HAV, ${\geq}5.87$ for EMCV, ${\geq}5.15$ for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.

• Journal of the Korean Society of Visualization
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• v.4 no.1
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• pp.47-55
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• 2006

The left ventricular filling flow is now considered as an indicator which can be used for early diagnosing of cardiovascular diseases. Because the understanding of left ventricular flow physics is critical for this purpose, the downstream flow characteristics of the artificial heart valve are investigated using particle image velocimetry (PIV) method. In this study, we investigated the wake characteristics of flows passing through three different artificial valves (St.Jude medical bileaflet mechanical valve. Bjork-Shiley monostrut mechanical valve and St.Jude medical Biocor bio valve). The downstream flow field has remarkably altered according to the different valves. SJM MHV has the flow field similar to the pulsating circular jet and BS MHV has oblique pulsating jet. SJM BHV shows the similar flow field of clinical data of normal heart.