मोनोक्लोनल एंटीबॉडी के निष्क्रिय खिलाफ प्रशासन एच. capsulatum और फफूंद रोगज़नक़ों दूसरों
Summary
C57BL 6 / चूहों कोर्ट रोगजनन अध्ययन के लिए और सबसे अच्छा मॉडल प्रदान करने के लिए इस्तेमाल किया गया है. हम इस कवक के खिलाफ humoral रोगक्षमता के संभावित लाभ की खोज कर रहे हैं और [histone H2B और एक गर्मी झटका प्रोटीन 60kDa] कई Mabs उत्पन्न कि हम intraperitoneal प्रशासन के बाद उनके सुरक्षा प्रभावकारिता के लिए परीक्षण किया गया.
Abstract
The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.
Protocol
Discussion
यहाँ प्रस्तुत प्रोटोकॉल को दर्शाता है कि MAB एच. करने के लिए capsulatum प्रयोगात्मक murine histoplasmosis के पाठ्यक्रम को संशोधित कर सकते हैं . सेल रोगजनकों के सतह प्रतिजनों Mabs जटिल गतिशीलता है कि एक मेजबान और एक रोगज़?…
Divulgations
The authors have nothing to disclose.
Materials
| Name of Material | Company |
|---|---|
| Biological safety cabinet (BSL2) | |
| 15 mL conical tubes | Falcon, BD |
| HAM F-12 medium | Gibco |
| 37°C shaker | |
| Vortex | |
| 50 mL conical tubes | Falcon, BD |
| 26G1/2 needle | BD |
| 10 mL syringe | BD |
| 250 mL flasks | |
| FPLC (Fast protein liquid chromatography) system | GE Helthcare |
| ELISA reader | BioTek |
| Anesthesia (ketamine and Xylazine) | |
| Column stands | |
| Nylon string | |
| Heat lamp | |
| 70μm cell strainers | Falcon, BD |
| BHI agar plates | Gibco |
References
- Casadevall, A., Mukherjee, J., Scharff, M. D. Monoclonal antibody based ELISAs for cryptococcal polysaccharide. J. Immunol. Methods. 154, 27-35 (1992).
- Guimaraes, A. J., Frases, S., Gomez, F. J., Zancope-Oliveira, R. M., Nosanchuk, J. D. Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum. Infect Immun 77. , 1357-1367 (2009).
- Guimaraes, A. J., Hamilton, A. J., de M Guedes, H. L., Nosanchuk, J. D., Zancope-Oliveira, R. M. Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum. PLoS One. 3, e3449-e3449 (2008).
- Nosanchuk, J. D. Protective antibodies and endemic dimorphic fungi. Curr Mol Med. 5, 435-442 (2005).
- Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Jr, D. e. e. p. e., &, G. S., Casadevall, A. Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum. J Clin Invest. 112, 1164-1175 (2003).
- Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Deepe, G. S., , ., Casadevall, A. Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum. J. Clin. Invest. 112, 1164-1175 (2003).
- Smith, W. Responses of laboratory animals to some injectable anaesthetics. Lab Anim. 27, 30-39 (1993).
