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Compositions and methods for preventing transepithelial transmission of hiv

A composition, epithelial technology, applied in the field of application of ICAM-1 agonist/antagonist to prevent HIV from spreading through mucosal surfaces

Inactive Publication Date: 2010-12-15
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of ICAM-1, other than interaction with LFA-1, in viral transmission at sites of HIV exposure or at sites where the virus enters the body has not been addressed until now

Method used

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  • Compositions and methods for preventing transepithelial transmission of hiv
  • Compositions and methods for preventing transepithelial transmission of hiv
  • Compositions and methods for preventing transepithelial transmission of hiv

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Isolation of cell-free HIV and HIV-infected peripheral blood lymphocytes and monocytes

[0082] HIV-1 Ba-L (Advanced Biotechnologies Inc., Columbia, MD), a macrophage-tropic, NSI, CCR5-applied variant of HIV, is commercially available as 1.0 ml aliquots (1 x 10 6 50% tissue culture infectious dose (TCID 50 ) / ml) and stored in liquid nitrogen. Virus stocks were freshly thawed before application to cultures.

[0083] Human PBMCs were isolated by centrifugation of leukapheresis-processed blood on Ficoll Hypaque (Pharmacia, Piscataway, NJ). Total PBMCs were treated with RPMI-1640 supplemented with 100U / ml penicillin / 100mcg streptomycin, 10ng / ml gentamycin, and 2mM L-glutamine (medium and all supplements were from Life Technologies, Grand Island, NY) diluted to 1×10 7 / ml, then plated at 75 or 150cm 2 Tissue culture flasks (Corning Scientific Products, Inc., Cambridge, MA). During the first 12 hours of culture, cells were resuspended in 20% heat-inactivated FCS (56°...

Embodiment 2

[0087] Transwell culture of human cervical epithelial cells

[0088] The spontaneously transformed human cervical epithelial cell line ME-180 (ATCC, Rockville, MD) was cultured in a 75 cm 2 Cells were routinely passaged every 3 days by treating them with 0.05% trypsin-EDTA (Life Technologies) in cRPMI-10% FCS in flasks. ME-180 cells in 2 × 10 5 / 0.1 ml cRPMI-10% FCS or -10% HS / 5.0 μm was plated on a 12 mm diameter transwell insert (Corning Scientific Products, Inc.). Maintain ME-180 cells in trans wells at 37 °C, 5% CO 2 Down. Change the medium every 2-3 days. After 7 days, cells formed a polarized complete monolayer on the transwell insert, which was confirmed by monitoring the impedance across the epithelium using an ohmmeter (Millipore) and staining with a fluorochrome-labeled anti-ZO1 antibody, where ZO1 is a protein found in tight junctions of epithelial cells (20) (1:50 dilution, Zymed, San Francisco, CA). The ME-180 monolayer on the insert is ready to use for ex...

Embodiment 3

[0090] Transepithelial migration and HIV-1 transmission

[0091] Transepithelial migration assays were performed as described by Bomsel with several modifications. In short, the 1×10 6 HIV-1 Ba-L Infected monocytes, 1×10 6 HIV-1 Ba-L Infected PBLs, or monocytes or cell-free HIV-1 in PBL culture supernatants were added to the apical side of the epithelial cell monolayer. A time-course analysis was performed, with media collected on the apical and basal sides between 1-48 hours after addition of infected cells to the inserts. Spread across the monolayer approaches a maximum at 24 hours, the time point chosen for all subsequent experiments. The viability of monocytes and PBLs was assessed by trypan blue (Sigma) exclusion before adding cells to transwells and results were always >90% after 24 hours of propagation. The amount of HIV-1 p24 antigen in the apical and basolateral supernatants was quantified by HIV-1 p24 antigen ELISA assay (Dupont NEN) (sensitivity 12.5 pg / ml-350...

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Abstract

Methods, compositions and articles for blocking transepithelial transmission of HIV with ICAM-1 agonist / antagonists are provided. The ICAM-1 agonist / antagonists block viral transmission at the site of exposure to the virus. Blocking viral transmission does not require blocking the binding of ICAM-1 to LFA-1. The ICAM-1 agonist / antagonists may be applied to mucosal surfaces, on or in contraceptive devices or in oral solutions such as breast milk supplements and infant formula.

Description

[0001] This application is the filing date of June 23, 2000, the application number is 00811362.9 (international application number is PCT / US00 / 17293), and the invention title is "composition and method for preventing HIV transepithelial transmission" Divisional application. [0002] The US Government has certain rights in this invention under Grant Nos. DA 09717, DA 09973, DA 05972, and AI 07417 awarded by NIDA and NIAID, respectively. This application claims priority to US Provisional Patent Application 60 / 140,698, filed June 24, 1999, which is incorporated herein by reference in its entirety. field of invention [0003] The present invention relates to methods, compositions and articles of manufacture for preventing the spread of the human immunodeficiency virus ("HIV"). In a particularly preferred embodiment, the invention relates to ICAM-1 agonists / antagonists and their use in preventing HIV transmission across mucosal surfaces. [0004] refer to [0005] This article ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61K39/00A61K9/02A61K9/00A61K9/08A61K9/12A61P31/18A23L1/29A61F6/00A61F6/04A23L33/00A61K9/06A61K9/10A61K9/20C07K16/28C12N5/08
CPCA61K2039/505C07K16/2821A61P31/18
Inventor 理查德·B·马卡姆克里斯滕·V·卡纳
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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