Naomi Shimizu

Critical Roles of Muscle-Secreted Angiogenic Factors in Therapeutic Neovascularization

The discovery of bone marrow–derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 “no-option” patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1&bgr; (IL-1&bgr;). We then examined an experimental model of limb ischemia using IL-1&bgr;–deficient mice. Implantation of IL-1&bgr;–deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1&bgr;–deficient mononuclear cells increased expression of IL-1&bgr; and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1&bgr; markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.

A Phase I Study of In vitro Expanded Natural Killer T Cells in Patients with Advanced and Recurrent Non–Small Cell Lung Cancer

Purpose: Human Vα24 natural killer T (Vα24 NKT) cells bearing an invariant Vα24JαQ antigen receptor are activated by a glicolipid ligand α-galactosylceramide (αGalCer; KRN7000) in a CD1d-dependent manner. The human Vα24 NKT cells activated with αGalCer and interleukin-2 have been shown to produce large amounts of cytokines, such as IFN-γ, and also exerting a potent killing activity against various tumor cell lines. We did a phase I study with autologous activated Vα24 NKT cell therapy. Experimental Design: Patients with advanced or recurrent non–small cell lung cancer received i.v. injections of activated Vα24 NKT cells (level 1: 1 × 107/m2 and level 2: 5 × 107/m2) to test the safety, feasibility, and clinical response of this therapeutic strategy. Immunomonitoring was also done in all cases. Results: Six patients were enrolled in this study. No severe adverse events were observed during this study in any patients. After the first and second injection of activated Vα24 NKT cells, an increased number of peripheral blood Vα24 NKT cells was observed in two of three cases receiving a level 2 dose of activated Vα24 NKT cells. The number of IFN-γ-producing cells in peripheral blood mononuclear cells increased after the administration of activated Vα24 NKT cells in all three cases receiving the level 2 dose. No patient was found to meet the criteria for either a partial or a complete response. Conclusions: The clinical trial with activated Vα24 NKT cell administration was well tolerated and carried out safely with minor adverse events even in patients with advanced diseases.

A Phase I-II Study of α-Galactosylceramide-Pulsed IL-2/GM-CSF-Cultured Peripheral Blood Mononuclear Cells in Patients with Advanced and Recurrent Non-Small Cell Lung Cancer

To evaluate the safety, immune responses, and antitumor responses after the administration of α-galactosylceramide (αGalCer) KRN7000-pulsed PBMC cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs), a phase I-II study in patients with non-small cell lung cancer was conducted. Patients with advanced non-small cell lung cancer or recurrent lung cancer refractory to the standard therapy were eligible. αGalCer-pulsed IL-2/GM-CSF-cultured PBMCs (1 × 109/m2) were i.v. administered four times. Immune responses were monitored weekly. Twenty-three patients were enrolled in this study and 17 cases (73.9%) completed. No severe adverse event related to the treatment was observed. After the injection of αGalCer-pulsed IL-2/GM-CSF-cultured PBMCs, an increased number of IFN-γ-producing cells in the peripheral blood were detected in 10 patients (58.8%). Five cases remained as stable disease, and the remaining 12 cases were evaluated as progressive disease. The estimated median survival time (MST) of the 17 cases was 18.6 mo (range, 3.8 to 36.3 mo). Ten patients who displayed increased IFN-γ-producing cells (≥2-fold) showed prolonged MST (31.9 mo; range, 14.5 to 36.3 mo) as compared with poor-responder patients (n = 7) MST (9.7 mo; range, 3.8 to 25.0 mo) (log-rank test, p = 0.0015). The administration of αGalCer-pulsed IL-2/GM-CSF-cultured PBMCs was well tolerated and was accompanied by the successful induction of NKT cell-dependent immune responses. The increased IFN-γ-producing cells that result from αGalCer stimulation in PBMCs were significantly associated with prolonged MST. These results are encouraging and warrant further evaluation for survival benefit of this immunotherapy.

Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy

Vα24 natural killer T (NKT) cells have potent anti-tumor activity. We performed a phase II clinical study in patients with head and neck squamous cell carcinoma (HNSCC) using ex vivo expanded Vα24 NKT cells and α-galactosylceramide (αGalCer; KRN7000)-pulsed antigen-presenting cells (APCs) to investigate the efficacy and induction of NKT cell-specific immune responses. The subjects were 10 patients with locally recurrent and operable HNSCC. One course of nasal submucosal administration of αGalCer-pulsed APCs and intra-arterial infusion of activated NKT cells via tumor-feeding arteries was given before salvage surgery. Anti-tumor effects, NKT cell-specific immune responses in extirpated cancer tissue and peripheral blood, safety, and pathological effects were evaluated. Five cases achieved objective tumor regression. The number of NKT cells increased in cancer tissues in 7 cases and was associated with tumor regression. The combination therapy induced NKT cell-specific immune responses in cancer tissues that were associated with beneficial clinical effects.

Contribution of Anaphylatoxin C5a to Late Airway Responses After Repeated Exposure of Antigen to Allergic Rats

We attempted to elucidate the contribution of complement to allergic asthma. Rat sensitized to OVA received repeated intratracheal exposures to OVA for up to 3 consecutive days, and pulmonary resistance was then estimated for up to 6 h after the last exposure. Whereas the immediate airway response (IAR) in terms of RL tended to decrease in proportion to the number of OVA exposures, late airway response (LAR) became prominent only after three. Although premedication with two kinds of complement inhibitors, soluble complement receptor type 1 (sCR1) or nafamostat mesylate, resulted in inhibition of the IAR after either a single or a double exposure, the LAR was inhibited after the triple. Premedication with a C5a receptor antagonist (C5aRA) before every exposure to OVA also inhibited the LAR after three. Repeated OVA exposure resulted in eosinophil and neutrophil infiltration into the bronchial submucosa which was suppressed by premedication with sCR1 or C5aRA. Up-regulation of C5aR mRNA was shown in lungs after triple OVA exposure, but almost no up-regulation of C3aR. Pretreatment with sCR1 or C5aRA suppressed the up-regulation of C5aR expression as well as cytokine messages in the lungs. The suppression of LAR by pretreatment with sCR1 was reversed by intratracheal instillation of rat C5a desArg the action of which was inhibited by C5aRA. In contrast, rat C3a desArg or cytokine-induced neutrophil chemoattractant-1 induced cellular infiltration into the bronchial submucosa by costimulation with OVA, but these had no influence on the LAR. These differences might be explained by the fact that costimulation with OVA and C5a synergistically potentiated IAR, whereas that with OVA and either C3a or cytokine-induced neutrophil chemoattractant-1 did not. C5a generated by Ag-Ab complexes helps in the production of cytokines and contributes to the LAR after repeated exposure to Ag.

Long-term outcome of therapeutic neovascularization using peripheral blood mononuclear cells for limb ischemia

Background—Injection of bone marrow mononuclear cells has been reported to promote neovascularization of ischemic tissues effectively. We found that peripheral blood mononuclear cells were as efficient as bone marrow mononuclear cells for the treatment of limb ischemia in animals and showed that this treatment was feasible and safe in no-option patients with limb ischemia. However, the long-term outcome of such therapy has not been investigated. Methods and Results—We retrospectively analyzed the data for 42 patients who were treated between July 2002 and December 2005 by using the log-rank test, the Kaplan-Meier method, and the Cox proportional hazard model. Improvement of ischemic symptoms was observed in 60% to 70% of the patients. The annual rate of major amputation was decreased markedly by treatment. Improvement of ischemic symptoms was less marked in arteriosclerosis obliterans (ASO) patients on dialysis compared with nonhemodialysis ASO or thromboangiitis obliterans patients. Indeed, the survival rate of these patients was lower than that of nonhemodialysis ASO or thromboangiitis obliterans patients. Major adverse events such as death, major amputation, and cardiovascular events occurred mostly in ASO patients, and most of them were on dialysis. There was no significant difference in the cardiovascular event-free rate between responders and nonresponders. The survival rate of younger responders was better than that of nonresponders. Conclusions—Although this study was not placebo-controlled and these initial results were from a retrospective analysis, injection of peripheral blood mononuclear cells might be safe and potentially effective for the treatment of limb ischemia, but caution is needed when managing ASO patients on dialysis.

Successful combination treatment with bevacizumab, thalidomide and autologous PBSC for severe POEMS syndrome

Successful combination treatment with bevacizumab, thalidomide and autologous PBSC for severe POEMS syndrome

Restrictive usage of monoclonal immunoglobulin λ light chain germline in POEMS syndrome

POEMS syndrome is a rare plasma cell disorder characterized by peripheral neuropathy, monoclonal gammopathy, and high levels of serum vascular endothelial growth factor, the pathogenesis of which remains unclear. A unique feature of this syndrome is that the proliferating monoclonal plasma cells are essentially lambda-restricted. Here we determined complete nucleotide sequences of monoclonal immunoglobulin lambda light chain (IGL) variable regions in 11 patients with POEMS syndrome. The V-region of the Ig lambda gene of all 11 patients was restricted to the V lambda 1 subfamily. Searching for homologies with IGL germlines revealed that 2 germlines, IGLV1-44*01 (9/11) and IGLV1-40*01 (2/10), were identified, with an average homology of 91.1%. The IGLJ3*02 gene was used in 11 of 11 re-arrangements with an average homology of 92.2%. These data suggest that the highly restricted use of IGL V lambda 1 germlines plays an important role in the pathogenesis of POEMS syndrome.

Zoledronate has an antitumor effect and induces actin rearrangement in dexamethasone-resistant myeloma cells

New strategies are needed to overcome the resistance of multiple myeloma (MM) to dexamethasone (Dex). Several recent in vitro studies demonstrated the antitumor effect of nitrogen‐containing amino‐bisphosphonates (N‐BPs) in various tumor cell lines. Inhibition of the prenylation of small G proteins is assumed to be one of the principal mechanisms by which N‐BPs exert their effects. There have been few reports on N‐BP treatment of MM cells that are resistant to Dex. Additionally, it is not known how small G proteins are altered in N‐BP‐treated MM cells. In this study, we evaluated the effect of the most potent N‐BP, zoledronate (ZOL), on a Dex‐resistant human MM cell subline (Dex‐R) that we established from the well‐documented RPMI8226 cell line. ZOL reduced the viability and induced apoptosis of Dex‐R cells. Some of the ZOL‐treated RPMI8226 cells and ZOL‐treated Dex‐R cells were elongated; however, elongated cells were not seen among the Dex‐treated RPMI8226 cells. Furthermore, we found that portions of the small G proteins, Rho and Rap1A, were unprenylated in the ZOL‐treated MM cells. Geranylgeraniol reduced the above‐mentioned ZOL‐induced effects. These findings suggest that ZOL may be beneficial for the treatment of Dex‐resistant MM by suppressing the processing of RhoA and Rap1A.

Bortezomib-induced neuropathy: Axonal membrane depolarization precedes development of neuropathy

OBJECTIVE Bortezomib is a proteasome inhibitor with high efficacy for multiple myeloma but with severe peripheral neurotoxicity, leading to dose modification and severe neurological disability. This study aimed to investigate the pathophysiology of bortezomib-induced neuropathy. METHODS Threshold tracking was used to assess the excitability of sensory and motor axons. Measurements were sequentially performed before and after bortezomib treatment in nine patients with newly diagnosed multiple myeloma. RESULTS In total, 67% of patients finally developed symptomatic neuropathy. Changes in sensory axonal excitability indices readily occurred after the first course of administration. Patterns of changes in excitability indices suggest membrane depolarization (decreased superexcitability, P<0.001; decreased depolarizing threshold electrotonus 90-100ms, P=0.02). Abnormalities in nerve conduction parameters suggestive of axonal degeneration appeared after the second course of treatment. CONCLUSIONS Bortezomib induces a depolarizing shift in resting membrane potential prior to the development of neuropathy. Membrane depolarization could be associated with impairment of electrogenic Na(+)-K(+)-ATPase-dependent pump caused by toxic effects of bortezomib on mitochondria. SIGNIFICANCE Axonal depolarization and hyperexcitability might enhance neurodegeneration in bortezomib-induced neuropathy.