Programmed cell death 1‐expressing CD56‐negative natural killer (NK) cell expansion is a hallmark of chronic NK cell activation during dasatinib treatment

Dasatinib treatment markedly increases the number of large granular lymphocytes including natural killer (NK) cells in a proportion of Ph⁺ leukemia patients, which associates with a better prognosis. In‐depth immune profiling of NK cells can predict therapeutic response in these patients. In the present study, we showed that CD56‐negative (CD56^neg) NK cells increased exclusively in cytomegalovirus‐seropositive (CMV⁺) patients treated with dasatinib. The increase longitudinally paralleled with progressive differentiation of CD56^dim NK cells during dasatinib therapy driven by CMV reactivation as shown by principal component analysis on 19 NK cell markers. The CD56^neg NK cells showed downregulation of NK‐activating receptors, upregulation of PD‐1, and lower cytotoxicity and cytokine production, indicating that these cells are anergic and dysfunctional as seen in chronic infections with HIV‐1 or hepatitis C virus. Moreover, cytolytic activity of CD56^dim and CD56^neg NK cells against leukemia cells was partially restored by nivolumab in proportion to the frequency of PD‐1⁺ NK cells. The proportion of patients who achieved deep molecular responses at 2 years was significantly higher in dasatinib‐treated patients with ≥3% CD56^neg NK cells than in those with fewer CD56^neg NK cells (54.5% vs 15.8%, P = .0419). These findings suggest that CD56^neg NK cells may be an exhausted population induced by chronic activation through CMV reactivation during dasatinib therapy. Expansion of CD56^neg NK cells is a hallmark of chronic NK cell activation in patients treated with dasatinib and may predict a better clinical outcome. Furthermore, PD‐1 blockade may enhance anti‐leukemia responses of such NK cells.     

Thrombopoietin Induces Association of Crkl With STAT5 But Not STAT3 in Human Platelets

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, isconstitutively tyrosine phosphorylated in hematopoietic cells fromchronic myelogenous leukemia (CML) patients. We recently reported thatthrombopoietin induces tyrosine phosphorylation of Crkl in normalplatelets. In this study, we demonstrate that thrombopoietin inducesassociation of Crkl with a tyrosine phosphorylated 95- to 100-kDprotein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, wedemonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. Thecoimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizingpeptide for Crkl antisera or phenyl phosphate (20 mmol/L). Afterdenaturing of Crkl immunoprecipitates, Crkl was stillimmunoprecipitated by Crkl antisera. However, coimmunoprecipitation ofSTAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity asdemonstrated by electrophoretic mobility shift assays (EMSA). Using a-casein promoter STAT5 binding site as a probe, we have alsodemonstrated that Crkl antisera supershift the STAT5-DNA complex,suggesting that Crkl is a component of the complex in the nucleus.Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in astimulation-dependent manner. In vitro, glutathione S-transferase(GST)-Crkl bound to STAT5 inducibly through its SH2 domain. Theseresults indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietincommonly induce association of STAT5 and Crkl and that the complextranslocates to the nucleus and binds to DNA. Interestingly, suchassociation between STAT5 and Crkl was not observed incytokine-stimulated murine cells, suggesting an intriguing possibilitythat components of the human STAT5-DNA complex may be different fromthose of the murine counterpart.     

Regulation of human B lymphopoiesis by the transforming growth factor-beta superfamily in a newly established coculture system using human mesenchymal stem cells as a supportive microenvironment

OBJECTIVES: To characterize and evaluate the validity of a novel coculture system for studying human B-lymphocyte developmental biology. MATERIALS AND METHODS: We developed a long-term culture system to produce B lymphocytes from human CD34(+) cells purified from umbilical cord blood using human mesenchymal stem cells (hMSC) as stroma. We evaluated the effects of several low molecular weight inhibitors, recombinant proteins, and neutralizing antibodies (Abs) as potential regulators of B-lymphocyte development. RESULTS: Our cocultures of 2000 CD34(+) cells in the presence of stem cell factor and Flt3-ligand produced 1-5 x 10(5) CD10(+) cells after 4 weeks of culture. Surface IgM(+) immature B cells began to appear after 4 weeks. We evaluated the negative-regulatory effects of the transforming growth factor (TGF)-beta superfamily on human B lymphopoiesis, and found that adding an anti-activin A antibody enhanced generation of CD10(+) cells two- to three-fold. As well, the proportion of CD10(+) cells in the generated cells increased markedly, indicating that activin A downregulated B lymphopoiesis more efficiently than myelopoiesis. Addition of TGF-beta1 suppressed B-lymphocyte production by 20% to 30%, while addition of an anti-bone morphogenetic protein (BMP)-4 antibody or recombinant BMP-4 had no effect. Therefore, the strength of ability to suppress human B lymphopoiesis seemed to be activin A > TGF-beta1 > BMP-4. None of these three factors influenced the emergence of IgM(+) cells. CONCLUSIONS: hMSC coculture supported human B lymphopoiesis. Activin A selectively suppressed B lymphocyte production.     

A newly developed double lumen microballoon catheter with a side hole: initial experience of intraarterial infusion chemotherapy and/or embolization

Purpose

To introduce a newly developed double lumen microballoon catheter with a side hole for intraarterial infusion chemotherapy and/or embolization.

Methods and materials

Seven patients with malignant tumors, for whom superselective catheterization was considered difficult or had failed, underwent intraarterial infusion chemotherapy and/or embolization with the 3.3-Fr microballoon catheter. The catheter has a double lumen and a side hole to facilitate infusion from the proximal end of the balloon. The balloon was placed on the distal side of the target artery branching site. Inflation of the balloon and occlusion of the main lumen with the tip of the occlusion device allowed for intraarterial infusion chemotherapy and/or embolization of the target artery via the side hole.

Results

Successful intraarterial infusion chemotherapy and/or embolization with the microballoon catheter was performed in all patients with no complications.

Conclusions

The newly developed microballoon catheter achieves intraarterial infusion chemotherapy and/or embolization without the need for superselective catheterization.     

Favorable effects of egg white protein on lipid metabolism in rats and mice

Egg is a cholesterol-rich food and has a strong hyper-cholesterolemic action. However, all the cholesterol is in egg yolk and egg white is cholesterol-free. The effect of egg white protein and its hydrolysates on the serum lipids were compared with casein and soybean protein in rats and mice. The animals were given 30% casein diet (Ca group) or diets of 15% casein plus 15% soybean protein isolate (SPI group), egg white protein (EW group) or egg white protein hydrolysates (EW-P group) for 3 (rats) or 2 (mice) weeks. Food intake and growth were very similar among the different dietary groups. Hypocholesterolemic effect was observed in SP, EW and EW-P groups in rats and EW group in mice. Prevention of the reduction of HDL-cholesterol was found in EW and EW-P groups in rats and EW-P group in mice. The result suggests the possibility of the use of egg white for the prevention and treatment of hyper-cholesteremia.     

The relationship between 67Ga accumulation and ATP metabolism in tumor cells in vitro

Previously, we reported that the deposition of ⁶⁷Ga into malignant tumors may be a sensitive index of proliferative activity in tumor cells. For the purpose of elucidation of this hypothesis, we investigated the relationship between the accumulation of ⁶⁷Ga into malignant tumor cells and the intra cellular ATP metabolism in vitro.The uptake of ⁶⁷Ga into tumor cells was inhibited by adding NaF which is an inhibitor of ATP production. Furthermore, the uptake of ⁶⁷Ga into tumor cells was strongly inhibited by adding ouabain which is a specific inhibitor of Na⁺–K⁺-ATPase. From these in vitro results, it was concluded that there is a correlation between ⁶⁷Ga uptake and intra cellular ATP metabolism in tumor cells.     

The relationship between Ga-67 accumulation and cell cycle in malignant tumor cells in vitro

Previously we reported that the deposition of ⁶⁷Ga into malignant tumor may be a sensitive index of proliferative activity in tumor cells. For the purpose of elucidation of this hypothesis, we investigated the relationship between the accumulation of ⁶⁷Ga into malignant tumor cells and the cell cycle in vitro. We discovered that the uptake of both ⁶⁷Ga and ⁵⁹Fe into synchronized mouse tumor cells reaches a peak at the G2 stage which precedes cellular proliferation. Both iron and transferrin are specifically required by cells in culture for cell division, and the fact that ⁶⁷Ga and ⁵⁹Fe uptake into tumor cells peaks at the G2 stage of the cell cycle suggests that there is a correlation between ⁶⁷Ga uptake and the rate of cellular proliferation in malignant tumor cells.