Early improvement of storage or voiding symptoms by tadalafil predicts treatment outcomes in patients with lower urinary tract symptoms from benign prostatic hyperplasia.

OBJECTIVES: To determine whether early changes in International Prostate Symptom Score predict final improvement in the quality of life and treatment satisfaction of patients with lower urinary tract symptoms from benign prostatic hyperplasia receiving tadalafil. METHODS: This post-hoc analysis of three randomized-controlled trials of tadalafil for lower urinary tract symptoms from benign prostatic hyperplasia used subpopulations based on treatment status (tadalafil, placebo) and region (Japan, Asia). Logistic regression, principal component analysis and hierarchical clustering analysis were applied to individual International Prostate Symptom Score and Patient Global Impression of Improvement response scores. RESULTS: From logistic regression, most individual International Prostate Symptom Score items predicted either International Prostate Symptom Score remitter or responder status; only International Prostate Symptom Score Quality of Life predicted Patient Global Impression of Improvement responder status in tadalafil-treated patients. Among tadalafil-treated patients, principal component analysis showewd that improvement of International Prostate Symptom Score items 2, 4, 7 and International Prostate Symptom Score Quality of Life at early visits were associated with Patient Global Impression of Improvement (final visit), whereas International Prostate Symptom Score items 1, 3, 5 and 6 at early visits were associated with total International Prostate Symptom Score at the final visit. No clear predictive items for Patient Global Impression of Improvement and total International Prostate Symptom Score existed for placebo-treated patients. Hierarchical clustering analysis showed that the closest association was between International Prostate Symptom Score Quality of Life and International Prostate Symptom Score item 7 in placebo- and tadalafil-treated patients, which supports the principal component analysis results. CONCLUSIONS: Early changes of storage and voiding symptoms by tadalafil might lead to treatment satisfaction and overall International Prostate Symptom Score improvement, respectively. This finding could help physicians predict tadalafil treatment outcomes for patients with lower urinary tract symptoms from benign prostatic hyperplasia.

Safety and effectiveness of tadalafil in pediatric patients with pulmonary arterial hypertension: a sub-group analysis based on Japan post-marketing surveillance.

OBJECTIVE: To evaluate the long-term safety and effectiveness of tadalafil in pediatric patients with pulmonary arterial hypertension (PAH) in real-world clinical practice. METHODS: This is an observational surveillance of PAH patients receiving tadalafil in the contracted sites. A sub-group analysis was performed of 391 pediatric PAH patients (<18 years) who were included from 1,704 total patients in this surveillance. Safety was assessed from the frequency of adverse drug reactions (ADRs), discontinuations due to adverse events (AEs), and serious adverse drug reactions (SADRs). Effectiveness measurements included change in World Health Organization (WHO) functional classification of PAH, cardiac catheterization (pulmonary arterial pressure: PAP), and echocardiography (tricuspid regurgitation pressure gradient: TRPG). Survival rate was also measured. RESULTS: The mean patient age was 5.7 ± 5.34 years. Associated PAH (APAH) and idiopathic PAH (IPAH) accounted for 76.0% and 17.6%, respectively, of the PAH patients. Patients were followed for up to 2 years. Among 391 patients analyzed for safety, the overall incidence rate of ADRs was 16.6%. The common ADRs (≥ 1%) were headache (2.8%), hepatic function abnormal, platelet count decreased (1.3% each), and epistaxis, (1.0%). Eleven patients (2.8%) reported 16 SADRs. Three patients died secondary to SADRs. For the effectiveness analysis, the incidence of WHO functional class improvement at 3 months, 1 year, and 2 years after the initiation of tadalafil and last observation in pediatric patients were 16.5%, 19.7%, and 16.3%, respectively. Both PAP and TRPG showed a statistically significant reduction at last observation. CONCLUSION: This manuscript reveals the use of tadalafil in the real-world pediatric population with an acceptable safety profile in Japan.

Induction of WT1-specific human CD8+ T cells from human HSCs in HLA class I Tg NOD/SCID/IL2rgKO mice.

Induction of specific immune response against therapy-resistant tumor cells can potentially improve clinical outcomes in malignancies. To optimize immunotherapy in the clinic, we aimed to create an in vivo model enabling us to analyze human cytotoxic T-lymphocyte (CTL) responses against human malignancies. To this end, we developed NOD/SCID/IL2rgKO (NSG) mice expressing the HLA class I molecules HLA-A*0201 and A*2402. In the bone marrow (BM) and spleen of HLA class I transgenic (Tg) NSG mice transplanted with cord blood hematopoietic stem cells (HSCs), we found human memory CD8(+) T cells and antigen-presenting cells. To evaluate antigen-specific human CTL responses, we immunized HLA class I Tg NSG mice using polyinosinic:polycytidylic acid mixed Wilms tumor 1 (WT1) peptides, with or without WT1 peptide-loaded autologous dendritic cells. After immunization, the frequencies of HLA-restricted WT1-specific CTLs increased significantly in the spleen. Next, we transplanted the WT1-specific T-cell receptor (WT1-TCR) gene-transduced human HSCs into HLA class I Tg NSG newborn mice. WT1 tetramer-positive CD8(+) T cells differentiated from WT1-TCR-transduced HSCs in the recipients' BM, spleen, and thymus. Upon stimulation with WT1 peptide in vitro, these CTLs produced interferon-γ and showed lytic activity against leukemia cells in an antigen-specific, HLA-restricted manner. HLA class I Tg NSG xenografts may serve as a preclinical model to develop effective immunotherapy against human malignancies.

Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia.

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34(+)CD38(+)CD19(+) and CD34(-)CD19(+) cells initiated leukemia, and in MLL-AF9 patients, CD34(-)CD19(+) cells were LICs. In MLL-ENL patients, either CD34(+) or CD34(-) cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34(+)CD38(-)CD19(-)CD33(-) cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34(+)CD38(-)CD19(-)CD33(-) cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4(+) single positive (SP), CD8(+) SP, and CD4(+)CD8(+) double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34(+)CD38(+) and CD34(-) LICs but not in CD34(+)CD38(-)CD19(-)CD33(-) HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.

Hematological and biochemical data obtained in rural northern Uganda.

Reference intervals for common hematological and clinical chemistry parameters constitute an important basis for health care. Moreover, with increasing priority in drug and vaccine development for infectious diseases in Africa, the first priority is the safety evaluation and tolerability of the candidate interventions in healthy populations. To accurately assess health status and address adverse events, clinical reference intervals in the target population are necessary. We report on hematological and biochemical indices from healthy volunteers who participated in a clinical trial in Lira, northern Uganda. Median and nonparametric 95% percentiles on five hematology and 15 biochemistry analytes are shown. Although most hematological analytes conformed to reported reference intervals and trends in Africa, literature review from different African countries highlight the need for a region-specific children reference interval that can be appropriate for the population.

Phase 1b randomized trial and follow-up study in Uganda of the blood-stage malaria vaccine candidate BK-SE36.

BACKGROUND: Up to now a malaria vaccine remains elusive. The Plasmodium falciparum serine repeat antigen-5 formulated with aluminum hydroxyl gel (BK-SE36) is a blood-stage malaria vaccine candidate that has undergone phase 1a trial in malaria-naive Japanese adults. We have now assessed the safety and immunogenicity of BK-SE36 in a malaria endemic area in Northern Uganda. METHODS: We performed a two-stage, randomized, single-blinded, placebo-controlled phase 1b trial (Current Controlled trials ISRCTN71619711). A computer-generated sequence randomized healthy subjects for 2 subcutaneous injections at 21-day intervals in Stage1 (21-40 year-olds) to 1-mL BK-SE36 (BKSE1.0) (n = 36) or saline (n = 20) and in Stage2 (6-20 year-olds) to BKSE1.0 (n = 33), 0.5-mL BK-SE36 (BKSE0.5) (n = 33), or saline (n = 18). Subjects and laboratory personnel were blinded. Safety and antibody responses 21-days post-second vaccination (Day42) were assessed. Post-trial, to compare the risk of malaria episodes 130-365 days post-second vaccination, Stage2 subjects were age-matched to 50 control individuals. RESULTS: Nearly all subjects who received BK-SE36 had induration (Stage1, n = 33, 92%; Stage2, n = 63, 96%) as a local adverse event. No serious adverse event related to BK-SE36 was reported. Pre-existing anti-SE36 antibody titers negatively correlated with vaccination-induced antibody response. At Day42, change in antibody titers was significant for seronegative adults (1.95-fold higher than baseline [95% CI, 1.56-2.43], p = 0.004) and 6-10 year-olds (5.71-fold [95% CI, 2.38-13.72], p = 0.002) vaccinated with BKSE1.0. Immunogenicity response to BKSE0.5 was low and not significant (1.55-fold [95% CI, 1.24-1.94], p = 0.75). In the ancillary analysis, cumulative incidence of first malaria episodes with ≥5000 parasites/µL was 7 cases/33 subjects in BKSE1.0 and 10 cases/33 subjects in BKSE0.5 vs. 29 cases/66 subjects in the control group. Risk ratio for BKSE1.0 was 0.48 (95% CI, 0.24-0.98; p = 0.04). CONCLUSION: BK-SE36 is safe and immunogenic. The promising potential of BK-SE36, observed in the follow-up study, warrants a double-blind phase 1/2b trial in children under 5 years. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN71619711.

A pyrrolo-pyrimidine derivative targets human primary AML stem cells in vivo.

Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

Il2rg gene-targeted severe combined immunodeficiency pigs.

A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.

Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rγKO mice.

Although physiological development of human lymphoid subsets has become well documented in humanized mice, in vivo development of human myeloid subsets in a xenotransplantation setting has remained unevaluated. Therefore, we investigated in vivo differentiation and function of human myeloid subsets in NOD/SCID/IL2rγ(null) (NSG) mouse recipients transplanted with purified lineage(-)CD34(+)CD38(-) cord blood hematopoietic stem cells. At 4-6 mo posttransplantation, we identified the development of human neutrophils, basophils, mast cells, monocytes, and conventional and plasmacytoid dendritic cells in the recipient hematopoietic organs. The tissue distribution and morphology of these human myeloid cells were similar to those identified in humans. After cytokine stimulation in vitro, phosphorylation of STAT molecules was observed in neutrophils and monocytes. In vivo administration of human G-CSF resulted in the recruitment of human myeloid cells into the recipient circulation. Flow cytometry and confocal imaging demonstrated that human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic function. Human bone marrow-derived monocytes/macrophages were further confirmed to exhibit phagocytosis and killing of Salmonella typhimurium upon IFN-γ stimulation. These findings demonstrate the development of mature and functionally intact human myeloid subsets in vivo in the NSG recipients. In vivo human myelopoiesis established in the NSG humanized mouse system may facilitate the investigation of human myeloid cell biology including in vivo analyses of infectious diseases and therapeutic interventions.

Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice.

Whereas humanized mouse models have contributed significantly to human immunology research, human T cells developing in mouse thymic environment fail to demonstrate HLA-restricted function. To achieve HLA-restricted human immune response, we created an immune-compromised non-obese diabetic/SCID/IL2rg(null) strain (NSG) with homozygous expression of HLA class I heavy chain and light chain (NSG-HLA-A2/HHD). Transplantation of purified Lin-CD34+CD38- human hematopoietic stem cells into NSG-HLA-A2/HHD newborns resulted in the development of human CD4+ and CD8+ TCR alphabeta+ T cells and CD4-CD8- and CD8+ TCR gammadelta+ cells in recipient bone marrow and spleen. Human cytotoxic T lymphocytes (CTLs) become functionally mature, as evidenced by the production of granzyme corresponding to phenotypic transition from naïve to effector memory CTLs. In these recipients, human Th17 cells developed along with Th1 and Th2 cells. Epstein-Barr virus (EBV) infection in the humanized NSG-HLA-A2/HHD recipients resulted in the formation of lymphoproliferative lesions consisting mainly of human B cells with scattered human T cells. Human CTLs developing in the recipients recognized EBV-derived peptides in an HLA-restricted manner and exerted HLA-restricted cytotoxicity against EBV-infected human B cells. The HLA-expressing humanized mouse with functional HLA-restricted T cells and consistent representation of rare T-cell subsets overcomes a major constraint in human immunology, and serves as a useful model for investigation of human immune responses against pathogens and for the development of therapeutic strategies against human diseases.

Identification of therapeutic targets for quiescent, chemotherapy-resistant human leukemia stem cells.

Human acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). Because these chemotherapy-resistant LSCs are thought to underlie disease relapse, effective therapeutic strategies specifically targeting these cells may be beneficial. Here, we report identification of a primary human LSC gene signature and functional characterization of human LSC-specific molecules in vivo in a mouse xenotransplantation model. In 32 of 61 (53%) patients with AML, either CD32 or CD25 or both were highly expressed in LSCs. CD32- or CD25-positive LSCs could initiate AML and were cell cycle-quiescent and chemotherapy-resistant in vivo. Normal human hematopoietic stem cells depleted of CD32- and CD25-positive cells maintained long-term multilineage hematopoietic reconstitution capacity in vivo, indicating the potential safety of treatments targeting these molecules. In addition to CD32 and CD25, quiescent LSCs within the bone marrow niche also expressed the transcription factor WT1 and the kinase HCK. These molecules are also promising targets for LSC-specific therapy.

Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML.

Cancer stem cells have been proposed to be important for initiation, maintenance and recurrence of various malignancies, including acute myeloid leukemia (AML). We have previously reported that CD34+CD38- human primary AML stem cells residing in the endosteal region of the bone marrow are relatively chemotherapy resistant. Using a NOD/SCID/IL2rgamma(null) mouse model of human AML, we now show that the AML stem cells in the endosteal region are cell cycle quiescent and that these stem cells can be induced to enter the cell cycle by treatment with granulocyte colony-stimulating factor (G-CSF). In combination with cell cycle-dependent chemotherapy, G-CSF treatment significantly enhances induction of apoptosis and elimination of human primary AML stem cells in vivo. The combination therapy leads to significantly increased survival of secondary recipients after transplantation of leukemia cells compared with chemotherapy alone.

Modulation of Valpha19 NKT cell immune responses by alpha-mannosyl ceramide derivatives consisting of a series of modified sphingosines.

We have demonstrated that analogues of alpha-mannosyl ceramide (alpha-ManCer) consisting of a series of immunosuppressive 2-aminoalcohol derivatives in place of sphingosine promote a greater immune response from mouse invariant Valpha19-Jalpha26 (AV19-AJ33) TCR-bearing NKT (Valpha19 NKT) cells than alpha-ManCer itself. To further characterize the immune responses of Valpha19 NKT cells to the alpha-ManCer analogues, cytokine production by the cells was examined in detail. We found that certain alpha-ManCer derivatives individually induced either Th1- or Th2-dominant cytokine production in culture. The Th1- or Th2-biased immune responses of Valpha19 NKT cells were dependent on MHC class I-like MR1, since they were induced by coculture with the MR1 transfectants previously loaded with the glycolipids and were inhibited in the presence of anti-MR1 antiserum. Presumably, the recognition of the alpha-mannosyl residue of the alpha-ManCer analogues by the invariant TCR is individually modulated, depending on the altered interaction with the groove of the antigen-presenting MR1. Priming of the Valpha19 invariant TCR-transgenic mice in vivo with these glycolipid derivatives resulted in the induction of the Th1- or Th2-biased immune responses. Thus, these alpha-ManCer derivatives are likely to be useful in immunotherapy for either Th1 or Th2 excess autoimmune diseases, modulating the function of Valpha19 NKT cells.