Method and compositions for improving allogeneic hematopoietic cell transplantation
A grafted, heteromorphic technology for use in microbial-based methods, biological agents for removal of undesirable cells, special-purpose bioreactors/fermenters, etc.
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Embodiment 1
[0055] Example 1. Examination of transplant patients
[0056] The investigation of allografts or allografts was characterized in that it was performed on 104 patients who had received non-T cell depleted allogeneic bone marrow transplants from HLA-matched siblings. Pretransplant status adjustments for these patients included butyl dimesylate treatment (n=89), total body irradiation with administration of cyclophosphamide (n=10), and cyclophosphamide (n=5). The total number of cells in the graft averaged 3 × 10 8 Cells / kg recipient body weight. CD34 + The number of cells correlated positively with an increase in survival (p=0.0004, overall survival; p=0.001, disease-free survival). The graft was additionally quantified for a cell population characterized by CD3 - , CD4 +(Hi) , CD8 - , low side scatter, here considered DCP, the number of these cells in transplanted species was inversely correlated with overall survival (OS) and disease-free survival (DFS, p=0.005). In thi...
Embodiment 2
[0057] In this way, based on the CD34 in the graft + Cell and DCP numbers describe a simple mathematical relationship (a differential equation, df) as follows: df=(CD34 + number of cells) - 2 x (number of DCPs). For a threshold of df, 1.5×10 6 Per kilogram body weight, these patients were divided into two groups: graft df greater than or equal to 1.5 × 10 6 DFS at 3 years was 67% in patients per kg body weight compared to graft df less than 1.5×10 6 34% DFS per kilogram of body weight in patients Example 2. Transplantation of selected stem cells
[0058] Donor Source: Bone marrow or peripheral blood stem cells collected from HLA-matched or mismatched donors.
[0059] Donor PBSC collection: To immobilize PBSC, G-CSF was administered daily until complete depletion of leukocytes. Donors received subcutaneous injections of G-CSF at 10 μg / kg / day according to their actual body weight. Institutional standards of donor support care must be followed during mobilization and leukap...
Embodiment 3
[0063] Example 3. Cell Sorting
[0064] General protocol for cell sorting. Cells were stained with FITC-conjugated CD34-specific monoclonal antibodies and PE-conjugated monoclonal antibodies recognizing CD4, CD123, CD36 or CD45RA (Becton-Dickinson, San Jose, CA). A directly conjugated isotype-matched irrelevant antibody was used as a reference. Cells were stained for 30 min at 4°C in a staining buffer consisting of Hank's saline buffered solution (HBSS, Mediatech, Herndon, VA) containing 2% v / v autologous human plasma or 1% human serum albumin. Excess antibody was removed by dilution with 10 volumes of staining buffer followed by centrifugation at 500 xg. Stained cells were analyzed indirectly using a FACSCAN flow cytometer (BDIS, San Jose, CA) or sorted aseptically using a FACSVANTAGE cell sorter (BDIS, San Jose, CA). Central and marginal scatter gates for viable nucleated cells were used to obtain list-mode data. Using CellQuest TM Or LYSIS-II software (BDIS, San Jose,...
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