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In JoVE (1)
Other Publications (8)
Articles by Monica S. Thakar in JoVE
JoVE Immunology and Infection
High-throughput Detection Method for Influenza Virus
1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine, 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin, 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin
This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens.
Other articles by Monica S. Thakar on PubMed
Biodistributions, Myelosuppression, and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the Alpha-emitting Radionuclides Bismuth-213 or Astatine-211
We previously investigated the potential of targeted radiotherapy using a bismuth-213 ((213)Bi)-labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. Although this approach allowed sustained marrow engraftment, limited availability, high cost, and short half-life of (213)Bi induced us to investigate an alternative alpha-emitting radionuclide, astatine-211 ((211)At), for the same application. Biodistribution and toxicity studies were conducted with conjugates of the anti-murine CD45 antibody 30F11 with either (213)Bi or (211)At. Mice were injected with 2 to 50 muCi on 10 microg or 20 muCi on 2 or 40 microg of 30F11 conjugate. Biodistribution studies showed that the spleen contained the highest concentration of radioactivity, ranging from 167 +/- 23% to 417 +/- 109% injected dose/gram (% ID/g) after injection of the (211)At conjugate and 45 +/- 9% to 166 +/- 11% ID/g after injection of the (213)Bi conjugate. The higher concentrations observed for (211)At-labeled 30F11 were due to its longer half-life, which permitted better localization of isotope to the spleen before decay. (211)At was more effective at producing myelosuppression for the same quantity of injected radioactivity. All mice injected with 20 or 50 muCi (211)At, but none with the same quantities of (213)Bi, had lethal myeloablation. Severe reversible acute hepatic toxicity occurred with 50 muCi (213)Bi, but not with lower doses of (213)Bi or with any dose of (211)At. No renal toxicity occurred with either radionuclide. The data suggest that smaller quantities of (211)At-labeled anti-CD45 antibody are sufficient to achieve myelosuppression and myeloablation with less nonhematologic toxicity compared with (213)Bi-labeled antibody.
Transmission and Expansion of HOXB4-induced Leukemia in Two Immunosuppressed Dogs: Implications for a New Canine Leukemia Model
There are currently no large animal models to study the biology of leukemia and development of novel antileukemia therapies. We have previously shown that dogs transplanted with homeobox B4 (HOXB4)-transduced autologous CD34(+) cells developed myeloid leukemia associated with HOXB4 overexpression. Here we describe the transmission, engraftment, and expansion of these canine leukemia cells into two genetically unrelated, immunosuppressed dogs.
Reagents for Astatination of Biomolecules. 4. Comparison of Maleimido-closo-decaborate(2-) and Meta-[(211)At]astatobenzoate Conjugates for Labeling Anti-CD45 Antibodies with [(211)At]astatine
An investigation was conducted to compare the in vivo tissue distribution of a rat antimurine CD45 monoclonal antibody (30F11) and an irrelevant mAbs (CA12.10C12) labeled with (211)At using two different labeling methods. In the investigation, the mAbs were also labeled with (125)I to assess the in vivo stability of the labeling methods toward deastatination. One labeling method employed N-hydroxysuccinimidyl meta-[(211)At]astatobenzoate, [(211)At]1c, and N-hydroxysuccinimidyl meta-[(125)I]iodobenzoate, [(125)I]1b, in conjugation reactions to obtain the radiolabeled mAbs. The other labeling method involved conjugation of a maleimido-closo-decaborate(2-) derivative, 2, with sulfhydryl groups on the mAbs, followed by labeling of the mAb-2 conjugates using Na[(211)At]At or Na[(125)I]I and chloramine-T. Concentrations of the (211)At/(125)I pair of radiolabeled mAbs in selected tissues were examined in BALB/c mice at 1, 4, and 24 h post injection (pi). The co-injected anti-CD45 mAb, 30F11, labeled with [(125)I]1b and [(211)At]1c targeted the CD45-bearing cells in the spleen with the percent injected dose (%ID) of (125)I in that tissue being 13.31 ± 0.78; 17.43 ± 2.56; 5.23 ± 0.50; and (211)At being 6.56 ± 0.40; 10.14 ± 1.49; 7.52 ± 0.79 at 1, 4, and 24 h pi (respectively). However, better targeting (or retention) of the (125)I and (211)At was obtained for 30F11 conjugated with the closo-decaborate(2-), 2. The %ID in the spleen of (125)I (i.e., [(125)I]30F11-2) being 21.15 ± 1.33; 22.22 ± 1.95; 12.41 ± 0.75; and (211)At (i.e., [(211)At]30F11-2) being 22.78 ± 1.29; 25.05 ± 2.35; 17.30 ± 1.20 at 1, 4, and 24 h pi (respectively). In contrast, the irrelevant mAb, CA12.10C12, labeled with (125)I or (211)At by either method had less than 0.8% ID in the spleen at any time point, except for [(211)At]CA12.10C12-1c, which had 1.62 ± 0.14%ID and 1.21 ± 0.08%ID at 1 and 4 h pi. The higher spleen concentrations in that conjugate appear to be due to in vivo deastatination. Differences in (125)I and (211)At concentrations in lung, neck, and stomach indicate that the meta-[(211)At]benzoyl conjugates underwent deastatination, whereas the (211)At-labeled closo-decaborate(2-) conjugates were very stable to in vivo deastatination. In summary, using the closo-decaborate(2-) (211)At labeling approach resulted in higher concentrations of (211)At in target tissue (spleen) and higher stability to in vivo deastatination in this model. These findings, along with the simpler and higher-yielding (211)At-labeling method, provide the basis for using the closo-decaborate(2-) labeling reagent, 2, in our continued studies of the application of (211)At-labeled mAbs for conditioning in hematopoietic cell transplantation.
ASH Evidence-based Guidelines: is There a Role for Second Allogeneic Transplant After Relapse?
A 35-year-old male with a FLT3(+) AML underwent allogeneic peripheral blood stem cell transplant using a myeloablative non-total body irradiation (TBI) conditioning regimen from his HLA-matched sibling donor. Following transplantation, he developed grade II acute graft-versus-host disease (GVHD) that resolved with increasing immunosuppression. The medications were subsequently discontinued, and he did not develop any evidence of chronic GVHD. Eighteen months after transplant, while off all immunosuppression, he developed fatigue and a blood count showed circulating blasts consistent with relapse of his disease. Among the various therapeutic questions is whether there is a role for a second allogeneic transplant to treat his disease and if so, at what time, with what conditioning, and with which type of donor.
Plerixafor-moblized Stem Cells Alone Are Capable of Inducing Early Engraftment Across the MHC-haploidentical Canine Barrier
Evaluation of Posttransplant Methotrexate to Facilitate Engraftment in the Canine Major Histocompatibility Complex-haploidentical Nonmyeloablative Transplant Model
Posttransplant cyclophosphamide has been shown to control graft-versus-host disease and facilitate engraftment in the major histocompatibility complex-haploidentical transplant setting. Here, we hypothesized that methotrexate (MTX) could be used in a similar fashion. In patients with genetic diseases, the use of MTX rather than an alkylating agent such as cyclophosphamide would be preferable due to its reduced risk of promoting secondary malignancies.
Transforming Growth Factor-beta-activated Kinase 1 Regulates Natural Killer Cell-mediated Cytotoxicity and Cytokine Production
Carma1, a caspase recruitment domain-containing membrane-associated guanylate kinase, initiates a unique signaling cascade via Bcl10 and Malt1 in NK cells. Carma1 deficiency results in reduced phosphorylation of JNK1/2 and activation of NF-κB that lead to impaired NK cell-mediated cytotoxicity and cytokine production. However, the precise identities of the downstream signaling molecules that link Carma1 to these effector functions were not defined. Here we show that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is abundantly present in NK cells, and activation via NKG2D results in its phosphorylation. Lack of Carma1 considerably reduced TAK1 phosphorylation, demonstrating the dependence of TAK1 on Carma1 in NKG2D-mediated NK cell activations. Pharmacological inhibitor to TAK1 significantly reduced NK-mediated cytotoxicity and its potential to generate IFN-γ, GM-CSF, MIP-1α, MIP-1β, and RANTES. Conditional in vivo knockdown of TAK1 in NK cells from Mx1Cre(+)TAK1(fx/fx) mice resulted in impaired NKG2D-mediated cytotoxicity and cytokine/chemokine production. Inhibition or conditional knockdown of TAK1 severely impaired the NKG2D-mediated phosphorylation of ERK1/2 and JNK1/2 and activation of NF-κB and AP1. Our results show that TAK1 links Carma1 to NK cell-mediated effector functions.
Immunomodulatory Effects Induced by Cytotoxic T Lymphocyte Antigen 4 Immunoglobulin with Donor Peripheral Blood Mononuclear Cell Infusion in Canine Major Histocompatibility Complex-haplo-identical Non-myeloablative Hematopoietic Cell Transplantation
BACKGROUND AIMS. Previously, cytotoxic T lymphocyte antigen 4 (CTLA4) immunoglobulin (Ig) has been shown to allow sustained engraftment in dog leukocyte antigen (DLA)-identical hematopoietic cell transplant (HCT) after non-myeloablative conditioning with 100 cGy total body irradiation (TBI). In the current study, we investigated the efficacy of pre-transplant CTLA4-Ig in promoting engraftment across a DLA-mismatched barrier after non-myeloablative conditioning. METHODS. Eight dogs were treated with CTLA4-Ig and donor peripheral blood mononuclear cells (PBMC) prior to receiving 200 cGy TBI followed by transplantation of granulocyte-colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells from DLA haplo-identical littermates with post-grafting immunosuppression. A control group of six dogs was conditioned with 200 cGy only and transplanted with grafts from DLA haplo-identical littermates followed by post-grafting immunosuppression. RESULTS. In vitro and in vivo donor-specific hyporesponsiveness was demonstrated on day 0 before TBI in eight dogs that received CTLA4-Ig combined with donor PBMC infusions. Four of five dogs treated with increased doses of CTLA4-Ig achieved initial engraftment but eventually rejected, with a duration of mixed chimerism ranging from 12 to 22 weeks. CTLA4-Ig did not show any effect on host natural killer (NK) cell function in vitro or in vivo. No graft-versus-host disease (GvHD) was observed in dogs receiving CTLA4-Ig treatment. CONCLUSIONS. Non-myeloablative conditioning with 200 cGy TBI and CTLA4-Ig combined with donor PBMC infusion was able to overcome the T-cell barrier to achieve initial engraftment without GvHD in dogs receiving DLA haplo-identical grafts. However, rejection eventually occurred; we hypothesize because of the inability of CTLA4-Ig to abate natural killer cell function.
