The implication of serum HLA-G in angiogenesis of multiple myeloma.

BACKGROUND: Despite the advances of therapies, multiple myeloma (MM) remains an incurable hematological cancer that most patients experience relapse. Tumor angiogenesis is strongly correlated with cancer relapse. Human leukocyte antigen G (HLA-G) has been known as a molecule to suppress angiogenesis. We aimed to investigate whether soluble HLA-G (sHLA-G) was involved in the relapse of MM. METHODS: We first investigated the dynamics of serum sHLA-G, vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) in 57 successfully treated MM patients undergoing remission and relapse. The interactions among these angiogenesis-related targets (sHLA-G, VEGF and IL-6) were examined in vitro. Their expression at different oxygen concentrations was investigated using a xenograft animal model by intra-bone marrow and skin grafts with myeloma cells. RESULTS: We found that HLA-G protein degradation augmented angiogenesis. Soluble HLA-G directly inhibited vasculature formation in vitro. Mechanistically, HLA-G expression was regulated by hypoxia-inducible factor-1α (HIF-1α) in MM cells under hypoxia. We thus developed two mouse models of myeloma xenografts in intra-bone marrow (BM) and underneath the skin, and found a strong correlation between HLA-G and HIF-1α expressions in hypoxic BM, but not in oxygenated tissues. Yet when stimulated with IL-6, both HLA-G and HIF-1α could be targeted to ubiquitin-mediated degradation via PARKIN. CONCLUSION: These results highlight the importance of sHLA-G in angiogenesis at different phases of multiple myeloma. The experimental evidence that sHLA-G as an angiogenesis suppressor in MM may be useful for future development of novel therapies to prevent relapse.

PD-L1-positive circulating endothelial progenitor cells associated with immune response to PD-1 blockade in patients with head and neck squamous cell carcinoma.

A number of the inhibitors against programmed death protein 1 (PD-1) have been approved to treat recurrent or metastatic squamous cell carcinoma of head and neck (HNSCC). The interaction between PD-1 and its ligand (PD-L1) serves as an immune checkpoint that governs cytotoxic immune effectors against tumors. Numerous clinical trials of PD-1/PD-L1 inhibitors have so far been discordant about having sufficient PD-L1 expression in the tumor as a prerequisite for a successful anti-PD-1 treatment. On the other hand, vascular endothelial cells modulate immune activities through PD-L1 expression, and thus it is possible that the expressions of circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CPCs) could affect antitumor immunity as well as neoangiogenesis. Here we investigated the potential involvement of PD-L1+ CECs and PD-L1+ CPCs in PD-1 blockade treatments for HNSCC patients. We measured CD8+ T cells, CECs, and CPCs in the peripheral blood of the HNSCC patients treated by anti-PD-1 therapies. We found that their PD-L1+ CPC expression before anti-PD1 therapies was strongly correlated with treatment responses and overall survival. Moreover, if the first infusion of PD-1 inhibitors reduced ≥ 50% PD-L1+ CPCs, a significantly better outcome could be predicted. In these patients as well as in an animal model of oral cancer, Pd-l1+ CPC expression was associated with limited CD8+ T-cell infiltration into the tumors, and anti-PD-1 treatments also targeted Pd-l1+ CPCs and increased CD8+ T-cell infiltration. Our results highlight PD-L1+ CPC as a potential regulator in the anti-PD-1 treatments for HNSCC.

The Image-Histology Correlation of Subcutaneous mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cell Grafts in Nude Mice.

Previously, we have successfully used noninvasive magnetic resonance (MR) and bioluminescence imaging to detect and monitor mPEG-poly(Ala) hydrogel-embedded MIN6 cells at the subcutaneous space for up to 64 days. In this study, we further explored the histological evolution of MIN6 cell grafts and correlated it with image findings. MIN6 cells were incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) and then 5 × 106 cells in the 100 µL hydrogel solution were injected subcutaneously into each nude mouse. Grafts were removed and examined the vascularization, cell growth and proliferation with anti-CD31, SMA, insulin and ki67 antibodies, respectively, at 8, 14, 21, 29 and 36 days after transplantation. All grafts were well-vascularized with prominent CD31 and SMA staining at all time points. Interestingly, insulin-positive cells and iron-positive cells were scattered in the graft at 8 and 14 days; while clusters of insulin-positive cells without iron-positive cells appeared in the grafts at 21 days and persisted thereafter, indicating neogrowth of MIN6 cells. Moreover, proliferating MIN6 cells with strong ki67 staining was observed in 21-, 29- and 36-day grafts. Our results indicate that the originally transplanted MIN6 cells proliferated from 21 days that presented distinctive bioluminescence and MR images.

Artifact-Free Microstructures in the Interfacial Reaction between Eutectic In-48Sn and Cu Using Ion Milling.

Eutectic In-48Sn was considered a promising candidate for low-temperature solder due to its low melting point and excellent mechanical properties. Both Cu2(In,Sn) and Cu(In,Sn)2 formation were observed at the In-48Sn/Cu interface after 160 °C soldering. However, traditional mechanical polishing produces many defects at the In-48Sn/Cu interface, which may affect the accuracy of interfacial reaction investigations. In this study, cryogenic broad Ar+ beam ion milling was used to investigate the interfacial reaction between In-48Sn and Cu during soldering. The phase Cu6(Sn,In)5 was confirmed as the only intermetallic compound formed during 150 °C soldering, while Cu(In,Sn)2 formation was proven to be caused by room-temperature aging after soldering. Both the Cu6(Sn,In)5 and Cu(In,Sn)2 phases were confirmed by EPMA quantitative analysis and TEM selected area electron diffraction. The microstructure evolution and growth mechanism of Cu6(Sn,In)5 during soldering were proposed. In addition, the Young's modulus and hardness of Cu6(Sn,In)5 were determined to be 119.04 ± 3.94 GPa and 6.28 ± 0.13 GPa, respectively, suggesting that the doping of In in Cu6(Sn,In)5 has almost no effect on Young's modulus and hardness.

Highly Robust Ti Adhesion Layer during Terminal Reaction in Micro-Bumps.

The use of scaled-down micro-bumps in miniaturized consumer electronic products has led to the easy realization of full intermetallic solder bumps owing to the completion of the wetting layer. However, the direct contact of the intermetallic compounds (IMCs) with the adhesion layer may pose serious reliability concerns. In this study, the terminal reaction of the Ti adhesion layer with Cu-Sn IMCs was investigated by aging the micro-bumps at 200 °C. Although all of the micro-bumps transformed into intermetallic structures after aging, they exhibited a strong attachment to the Ti adhesion layer, which differs significantly from the Cr system where spalling of IMCs occurred during the solid-state reaction. Moreover, the difference in the diffusion rates between Cu and Sn might have induced void formation during aging. These voids progressed to the center of the bump through the depleting Cu layer. However, they neither affected the attachment between the IMCs and the adhesion layer nor reduced the strength of the bumps. In conclusion, the IMCs demonstrated better adhesive behavior with the Ti adhesion layer when compared to Cr, which has been used in previous studies.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles.

Recently, we have shown that manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) conjugated with exendin-4 (Ex4) act as a contrast agent that directly trace implanted mouse islet ß-cells by magnetic resonance imaging (MRI). Here we further advanced this technology to track implanted porcine neonatal pancreatic cell clusters (NPCCs) containing ducts, endocrine, and exocrine cells. NPCCs from one-day-old neonatal pigs were isolated, cultured for three days, and then incubated overnight with MnMEIO-Ex4 NPs. Binding of NPCCs and MnMEIO-Ex4 NPs was confirmed with Prussian blue staining in vitro prior to the transplantation of 2000 MnMEIO-Ex4 NP-labeled NPCCs beneath the left renal capsule of six nondiabetic nude mice. The 7.0 T MRI on recipients revealed persistent hypointense areas at implantation sites for up to 54 days. The MR signal intensity of the graft on left kidney reduced 62-88% compared to the mirror areas on the contralateral kidney. Histological studies showed colocalization of insulin/iron and SOX9/iron staining in NPCC grafts, indicating that MnMEIO-Ex4 NPs were taken up by mature ß-cells and pancreatic progenitors. We conclude that MnMEIO-Ex4 NPs are excellent contrast agents for detecting and long-term monitoring implanted NPCCs by MRI.

Activated naïve γδ T cells accelerate deep molecular response to BCR-ABL inhibitors in patients with chronic myeloid leukemia.

Tyrosine kinase inhibitors (TKIs) that target BCR-ABL are the frontline treatments in chronic myeloid leukemia (CML). Growing evidence has shown that TKIs also enhance immunity. Since gamma-delta T (γδT) cells possess the potent anticancer capability, here we investigated the potential involvement of γδT cells in TKI treatments for CML. We characterized γδT cells isolated from chronic-phase CML patients before and during TKI treatments. γδT expression increased significantly in CML patients who achieved major molecular response (MMR) and deep molecular response (DMR). Their Vδ2 subset of γδT also expanded, and increased expression of activating molecules, namely IFN-γ, perforin, and CD107a, as well as γδT cytotoxicity. Mechanistically, TKIs augmented the efflux of isopentenyl pyrophosphate (IPP) from CML cells, which stimulated IFN-γ production and γδT expansion. Notably, the size of the IFN-γ+ naïve γδT population in TKI-treated CML patients was strongly correlated with their rates to reach DMR and with the duration on DMR. Statistical analysis suggests that a cutoff of 7.5% IFN-γ+ naïve subpopulation of γδT in CML patients could serve as a determinant for MR4.0 sustainability. Our results highlight γδT cells as a positive regulator for TKI responses in CML patients.

Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles as a Potential Magnetic Resonance Imaging Contrast Agent for Tracking Transplanted ß-Cells.

To specifically detect and trace transplanted islet ß-cells by magnetic resonance imaging (MRI), we conjugated manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) with exendin-4 (Ex4) which specifically binds glucagon-like peptide-1 receptors on the surface of ß-cells. The size distribution of MnMEIO and MnMEIO-Ex4 NPs were 67.8 ± 1.3 and 70.2 ± 2.3 nm and zeta potential 33.3 ± 0.5 and 0.6 ± 0.1 mV, respectively. MnMEIO and MnMEIO-Ex4 NPs with iron content ≤ 40 µg/mL did not affect MIN6 ß-cell viability and insulin secretion. Positive iron staining was found in MIN6 ß-cells loaded with MnMEIO-Ex4 NPs but not in those with MnMEIO NPs. A transmission electron microscope confirmed MnMEIO-Ex4 NPs were distributed in the cytoplasm of MIN6. In vitro MR images revealed a loss of signal intensity in MIN6 ß-cells labeled with MnMEIO-Ex4 NPs but not with MnMEIO NPs. After transplantation of islets labeled with MnMEIO-Ex4, the graft under kidney capsule could be visualized on MRI as persistent hypointense areas up to 17 weeks. Moreover, histology of the islet graft showed positive staining for insulin, glucagon and iron. Our results indicate MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitoring of transplanted ß-cells by MRI.

Noninvasive Tracking of mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cells after Subcutaneous Transplantation in Mice.

Recently, we demonstrated the feasibility of subcutaneous transplantation of MIN6 cells embedded in a scaffold with poly(ethylene glycol) methyl ether (mPEG)-poly(Ala) hydrogels. In this study, we further tracked these grafts using magnetic resonance (MR) and bioluminescence imaging. After being incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles and then mixed with mPEG-poly(Ala) hydrogels, MIN6 cells appeared as dark spots on MR scans. For in vivo experiments, we transfected MIN6 cells with luciferase and/or incubated them overnight with CSPIO overnight; 5 × 106 MIN6 cells embedded in mPEG-poly(Ala) hydrogels were transplanted into the subcutaneous space of each nude mouse. The graft of CSPIO-labeled MIN6 cells was visualized as a distinct hypointense area on MR images located at the implantation site before day 21. However, this area became hyperintense on MR scans for up to 64 days. In addition, positive bioluminescence images were also observed for up to 64 days after transplantation. The histology of removed grafts showed positive insulin and iron staining. These results indicate mPEG-poly(Ala) is a suitable scaffold for ß-cell encapsulation and transplantation. Moreover, MR and bioluminescence imaging are useful noninvasive tools for detecting and monitoring mPEG-poly(Ala) hydrogel-embedded MIN6 cells at a subcutaneous site.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles in Mice.

Neonatal pancreatic cell clusters (NPCCs) are potential tissues for the treatment of diabetes. Different from adult cells, they continuously proliferate and differentiate after transplantation. In this study, we utilized magnetic resonance imaging (MRI) to detect and monitor implanted NPCCs. NPCCs were isolated from one-day-old neonatal pigs, cultured for three days, and then incubated overnight with the contrast agent chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles. In vitro, Prussian blue staining and MR scans of CSPIO-labeled NPCCs were performed. In vivo, we transplanted 2000 CSPIO-labeled NPCCs under the kidney capsule of nondiabetic nude mice. Recipients were scanned with 7.0T MRI. Grafts were removed for histology with insulin and Prussian blue staining. After being incubated overnight with CSPIO, NPCCs showed positive iron staining and appeared as dark spots on MR scans. After transplantation of CSPIO-labeled NPCCs, persistent hypointense areas were observed at recipients' implant sites for up to 54 days. Moreover, histology showed colocalization of the insulin and iron staining in 15-, 51- and 55-day NPCC grafts. Our results indicate that transplanted NPCCs survived and differentiated to ß cells after transplantation, and that MRI is a useful tool for the detection and monitoring of CSPIO-labeled NPCC grafts.

Implanted islet mass influences the effects of dipeptidyl peptidase-IV inhibitor LAF237 on transplantation outcomes in diabetic mice.

BACKGROUND: Previous studies showed inconsistent Results of the effects of dipeptidyl peptidase (DPP)-IV inhibitors on syngeneic mouse islet transplantation. We hypothesized that the implanted islet numbers are critical for the effects of DPP-IV inhibitors on the outcomes of transplantation. METHODS: One hundred and fifty or three hundred islets were syngeneically transplanted under the renal capsule of each streptozocin-diabetic C57BL/6 mouse and recipients were then treated without or with LAF237 (10 mg/kg/day, po) for 6 weeks. After transplantation, recipients' blood glucose, body weight and intraperitoneal glucose tolerance test (IPGTT) were followed-up periodically. The graft was removed for the measurement of ß-cell mass at 6 weeks. RESULTS: In recipients with 150 islets, it was not significantly different between the LAF237- treated group (n = 14) and control group (n = 14) in terms of the blood glucose, body weight, glucose tolerance at 2, 4 and 6 weeks or the graft ß-cell mass at 6 weeks. In contrast, in recipients with 300 islets, the LAF237-treated group (n = 24) did have a lower area under the curve of the IPGTT at 4 weeks (p = 0.0237) and 6 weeks (p = 0.0113) as well as more graft ß-cell mass at 6 weeks (0.655 ± 0.008 mg vs. 0.435 ± 0.006 mg, p = 0.0463) than controls (n = 24). CONCLUSIONS: Our findings revealed 6-week treatment of LAF237 improves glucose tolerance and increases graft ß-cell mass in diabetic mice transplanted with a sufficient number but not a marginal number of islets. These indicate that the effects of DPP-IV inhibitors are influenced by the implanted islet mass.

BAFF-driven NLRP3 inflammasome activation in B cells.

BAFF supports B-cell survival and homeostasis by activating the NF-κB pathway. While NF-κB is also involved in the priming signal of NLRP3 inflammasome, the role of BAFF in NLRP3 inflammasome regulation is unknown. Here we report BAFF engagement to BAFF receptor elicited both priming and activating signals for NLRP3 inflammasomes in primary B cells and B lymphoma cell lines. This induction of NLRP3 inflammasomes by BAFF led to increased NLRP3 and IL-1ß expression, caspase-1 activation, IL-1ß secretion, and pyroptosis. Mechanistically, BAFF activated NLRP3 inflammasomes by promoting the association of cIAP-TRAF2 with components of NLRP3 inflammasomes, and by inducing Src activity-dependent ROS production and potassium ion efflux. B-cell receptor (BCR) stimulation on the Lyn signaling pathway inhibited BAFF-induced Src activities and attenuated BAFF-induced NLRP3 inflammasome activation. These findings reveal an additional function of BAFF in B-cell homeostasis that is associated with BCR activities.

Porcine Neonatal Pancreatic Cell Clusters Maintain Their Multipotency in Culture and After Transplantation.

Ductal epithelium is primarily detected in porcine neonatal pancreatic cell clusters (NPCCs) bearing grafts, suggesting that transplants might exhibit progenitor-like phenotypes. Here we found that soon after NPCC isolation, PDX1+/insulin- and SOX9+ pancreatic progenitor-like cells dramatically increased while dual-hormonal progenitor-like cells were routinely observed in NPCC culture. After transplantation (Tx), insulin+ cells increased and PDX1+ and SOX9+ cells gradually decreased in both non-diabetic (NDM) and streptozotocin-induced diabetic (DM) grafts over 2 months. Strikingly, a significantly higher percentage of insulin+ cells were detected in 9-day and 16-day, but not in 23-day, 30-day and 60-day grafts implying that hyperglycemia could only facilitate NPCC-derived ß cells early post-Tx. A higher percentage of NPCC-derived ß cells in early DM grafts was determined via an enhanced neogenic differentiation based on the detection of insulin+ cells budding out from PDX1+/SOX9+ epithelium. Interestingly, a drop in SOX9+ progenitor-like cells was detected 16 days post-Tx in DM grafts whilst PDX1+ cells do not show a significant difference until 60 days post-Tx between DM and NDM grafts, demonstrating that distinct progenitor-like populations fuel new ß cells post-Tx. In conclusion, PDX1+/SOX9+ cells could be quickly activated after NPCC isolation, maintain their multipotency in culture and differentiate into new ß cell post-Tx.

NKG2A Down-Regulation by Dasatinib Enhances Natural Killer Cytotoxicity and Accelerates Effective Treatment Responses in Patients With Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by the presence of t(9;22) chromosomal translocation that results in BCR-ABL fusion gene. ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, are currently the front-line treatment options for CML. Recently, natural killer (NK) cell activation and expansion have been shown to be associated with optimal treatment responses for CML. To investigate the effects and mechanisms of these TKIs on NK cells, here we characterized activating and inhibitory NK receptors in CD3-CD16+CD56dim NK cells isolated from CML patients in chronic phase (CP). The expressions of activating NK receptors, such as NKG2D, natural cytotoxicity receptor (NCR) and DNAM-1, rebounded after successful TKI treatments for CML. In contrast, among the three surveyed inhibitory receptors (NKG2A, KIR2DL1, and KIR3DL1), only the expression of NKG2A was reverted and suppressed to a very low level by dasatinib, and not by imatinib or nilotinib. CML patients treated with dasatinib indeed expressed fewer NKG2A+ NK cells, which send negative signals for induction of NK cytotoxicity. For these dasatinib-treated patients, the duration to reach major molecular response (MMR) was shorter, and significantly correlated with individual's NKG2A+ NK cell number. This clinical relevance to NKG2A was not observed in treatments with imatinib or nilotinib. In line with dasatinib-specific down-regulation of NKG2A, NK cytotoxicity evaluated by the killing assay was also significantly higher in patients treated with dasatinib than in those treated with imatinib or nilotinib. The lower NK cytotoxicity from imatinib or nilotinib treatments could be reverted by NKG2A blockade using anti-NKG2A antibody. Further in vitro experiments revealed mechanistically that dasatinib could inactivate p38 mitogen-activated protein kinase (MAPK), and consequently affect nuclear import of GATA-3 and GATA-3 transcriptional activities for NKG2A. Our results highlight the dual effects of dasatinib in direct inhibition of ABL kinase and in immunomodulation through NKG2A down-regulation, contributing to accelerated molecular responses (MR) in CML.

Increased B cell activation is present in JAK2V617F-mutated, CALR-mutated and triple-negative essential thrombocythemia.

Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm. We have reported that increased activated B cells can facilitate platelet production mediated by cytokines regardless JAK2 mutational status in ET. Recently, calreticulin (CALR) mutations were discovered in ~30% JAK2/MPL-unmutated ET and primary myelofibrosis. Here we sought to screen for CALR mutations and to evaluate B cell immune profiles in a cohort of adult Taiwanese ET patients. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) levels, B cells toll-like receptor 4 (TLR4) expression and intracellular levels of interleukin (IL)-1ß/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF concentration was measured by ELISA. 48 healthy adults were used for comparison. 19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Compared to JAK2V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. JAK2- and CALR-mutated ET have significantly higher fraction of B cells with TLR4 expression when compared to triple-negative ET (p=0.019 and 0.02, respectively). CALR-mutated ET had significantly higher number of CD69-positive activated B cells when compared to triple-negative ET (p=0.035). In conclusion, increased B cell activation is present in ET patients across different mutational subgroups.

Eltrombopag enhances platelet adhesion by upregulating the expression of glycoprotein VI in patients with chronic immune thrombocytopenic purpura.

Eltrombopag, a thrombopoietin receptor agonist, has been approved for the treatment of patients with immune thrombocytopenia because of its abilities to enhance platelet production and reduce hemorrhage. Both platelet count and platelet adhesion are crucial to stop bleeding. Although eltrombopag is known to improve platelet counts, its effects on platelet adhesion are not yet known. This study aimed to assess the efficacy of eltrombopag on platelet production and platelet adhesive affinity. To evaluate the efficacy of low-dose eltrombopag (25 mg) for patients with chronic refractory immune thrombocytopenic purpura (ITP) and to determine the ex vivo platelet adhesion ability before and after treatment with eltrombopag, we conducted an open-label, multicenter study in which 25 Taiwanese patients with chronic ITP were enrolled. During the 6-month evaluation, the starting and maximum doses of eltrombopag were 25 and 50 mg, respectively, to maintain the platelet count of ≥50,000 per µL. Flow-based adhesion assay was used to detect the percentage of platelets adhering to immobilized von Willebrand factor-collagen on microslides. Of the enrolled patients, 48% achieved a platelet count of ≥50,000 per µL. Interestingly, 83% of all responders required 25 mg of eltrombopag daily to achieve the target platelet count. In addition, the percentage of bleeding patients was significantly reduced in both responders and nonresponders by 50% from the baseline level throughout the treatment period. The ex vivo platelet adhesion capacity was elevated after the 6-month eltrombopag treatment in both responders and nonresponders. Furthermore, glycoprotein VI (GPVI) expression was significantly upregulated after treatment with eltrombopag. Low-to-intermediate dose of eltrombopag showed good efficacy to expedite platelet production and augment platelet adhesion. These 2 factors might explain the efficacy of eltrombopag in ameliorating hemorrhage in patients with ITP.