Intrathecal Administration of High-Titer Cytomegalovirus Immunoglobulin for Cytomegalovirus Meningitis

Cytomegalovirus (CMV) central nervous system disease after hematopoietic stem cell transplantation (HSCT) is a rare but life-threatening complication. Here, we report a patient who developed CMV meningitis after HSCT and was treated with the combination therapy of intrathecal high-titer CMV immunoglobulin and antiviral drugs. A 38-year-old man with myelodysplastic syndrome received a cord blood transplant after graft failure. On day 147, he was diagnosed with CMV meningitis based on pleocytosis and CMV DNA in the cerebrospinal fluid (CSF). Intravenous ganciclovir, foscarnet, and immunoglobulin were administered; however, CMV DNA in the CSF was continuously detected. The addition of intrathecal high-titer CMV immunoglobulin resulted in CMV DNA in the CSF becoming undetectable. On day 241, CMV DNA in the CSF was detected again, but both intrathecal immunoglobulin and intravenous ganciclovir led to its disappearance. No adverse effects related to intrathecal administration were observed. The intrathecal administration of immunoglobulin may be safe and effective for CMV meningitis

Functional Analysis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor

Adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) is a promising cell-based anticancer therapy. Although clinical studies of this approach show therapeutic efficacy, additional genetic modification is necessary to enhance the efficacy and safety of CAR-T cells. For example, production of an antitumor cytokine from CAR-T cells can potentially enhance their tumor-killing activity, but there are concerns that constitutive expression of anticancer molecules will cause systemic side effects. Therefore, it is important that exogenous gene expression is confined to the tumor locality. Here, we aimed to develop an inducible promoter driven by activation signals from a CAR. Transgene expression in T cells transduced with the CD19-targeted CAR and an inducible promoter, including inducible reporter genes (CAR-T/iReporter), was only induced strongly by co-culture with CD19-positive target cells. CAR-T/iReporter cells also showed redirected cytolysis toward CD19-positive, but not CD19-negative, tumor cells. Overall, our study indicated that the inducible promoter was selectively driven by activation signals from the CAR, and transduction with the inducible promoter did not affect original effector activities including interleukin-2 and interferon-γ production and the antitumor activity of CAR-redirected cytotoxic T lymphocytes. Moreover, this inducible promoter permits visualization and quantification of the activation status in CAR-T cells. Keywords: inducible gene expression, chimeric antigen receptor, adoptive T cell therapy, B cell lymphoma, CD19, tumor targeting, in vivo imagin

Comparison of alemtuzumab, anti-thymocyte globulin, and post-transplant cyclophosphamide for graft-versus-host disease and graft-versus-leukemia in murine models.

Graft-versus-host disease is a major complication after allogeneic hematopoietic stem cell transplantation for hematological malignancies. Immunosuppressive drugs, such as anti-thymocyte globulin, alemtuzumab, and post-transplant cyclophosphamide, have been used to prevent graft-versus-host disease in HLA-mismatched haploidentical hematopoietic stem cell transplantation. Here, we investigated whether these drugs could ameliorate graft-versus-host disease without diminishing the graft-versus-leukemia effect by using a xenogeneic transplanted graft-versus-host disease/graft-versus-leukemia model. Anti-thymocyte globulin treatment diminished graft-versus-host disease symptoms, completely depleted the infiltration of inflammatory cells in the liver and intestine, and led to prolonged survival. By contrast, improvement after post-transplant cyclophosphamide treatment remained minimal. Alemtuzumab treatment modestly prolonged survival despite an apparent decrease of Tregs. In the graft-versus-leukemia model, 1.5 to 2.0 mg/kg of anti-thymocyte globulin and 0.6 to 0.9 mg/kg of alemtuzumab reduced graft-versus-host disease with minimal loss of graft-versus-leukemia effect. Mice treated with 400 mg/kg of post-transplant cyclophosphamide did not develop graft-versus-host disease or leukemia, but it was difficult to evaluate the graft-versus-leukemia effect due to the sensitivity of A20 cells to cyclophosphamide. Although the current settings provide narrow optimal therapeutic windows, further studies are warranted to maximize the benefits of each immunosuppressant

Quantitative Targeted Absolute Proteomics-Based Large-Scale Quantification of Proline-Hydroxylated α‑Fibrinogen in Plasma for Pancreatic Cancer Diagnosis

Pancreatic cancer is a devastating disease and early diagnosis and treatment are essential to improve the prognosis. We previously showed that α-fibrinogen containing hydroxylated proline residues at positions 530 and 565 is increased in plasma of pancreatic cancer patients. However, no antibody specific for hydroxylated proline-530 is available. Therefore, the purposes of this study were to develop a quantification method specific for both proline-hydroxylated α-fibrinogens by selected/multiple reaction monitoring (SRM/MRM), and to validate these modifications as pancreatic cancer markers. The target peptide for hydroxylated proline-530 contained methionine, and since variable partial oxidation of this residue would affect the quantification, hydrogen peroxide treatment was carried out to ensure complete oxidation. Quantification values of modified and unmodified α-fibrinogen were well correlated with those obtained by immunoblotting. Concentrations of modified and unmodified α-fibrinogen were quantified in 70 pancreatic cancer patients and 27 healthy controls. Percent hydroxylation of α-fibrinogen and concentration of hydroxylated α-fibrinogen were significantly greater in the plasma of patients. Furthermore, among 8 carbohydrate antigen 19-9 (CA19-9)-negative patients in stages I/II, 6 were positive for proline-hydroxylated α-fibrinogen. These results indicate that plasma concentration of proline-hydroxylated α-fibrinogen measured by SRM/MRM analysis may be a good pancreatic cancer marker, especially in CA19-9-negative patients