Photoactivatable Materials for Versatile Single-Cell Patterning Based on the Photocaging of Cell-Anchoring Moieties through Lipid Self-Assembly.

Versatile methods for patterning multiple types of cells with single-cell resolution have become an increasingly important technology for cell analysis, cell-based device construction, and tissue engineering. Here, we present a photoactivatable material based on poly(ethylene glycol) (PEG)-lipids for patterning a variety of cells, regardless of their adhesion abilities. In this study, PEG-lipids bearing dual fatty acid chains were first shown to perfectly suppress cell anchoring on their coated substrate surfaces whereas those with single-chain lipids stably anchored cells through lipid-cell membrane interactions. From this finding, a PEG-lipid with one each of both normal and photocleavable fatty acid chains was synthesized as a material that could convert the chain number from two to one by exposure to light. On the photoconvertible PEG-lipid surface, cell anchoring was activated by light exposure. High-speed atomic force microscopy measurements revealed that this photocaging of the lipid-cell membrane interaction occurs because the hydrophobic dual chains self-assemble into nanoscale structures and cooperatively inhibit the anchoring. Light-induced dissociation of the lipid assembly achieved the light-guided fine patterning of multiple cells through local photoactivation of the anchoring interactions. Using this surface, human natural killer cells and leukemia cells could be positioned to interact one-by-one. The cytotoxic capacity of single immune cells was then monitored via microscopy, showing the proof-of-principle for applications in the high-throughput analysis of the heterogeneity in individual cell-cell communications. Thus, the substrate coated with our photoactivatable material can serve as a versatile platform for the accurate and rapid patterning of multiple-element cells for intercellular communication-based diagnostics.

Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients.

The expression of classical human leukocyte antigen class I antigens (HLA-I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA-I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA-I and non-classical HLA-I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA-I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune-related genes to characterize PDAC tumor microenvironments. Lower expression of HLA-I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA-E and HLA-G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA-I antigens was significantly correlated with shorter survival. Higher HLA-I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD-L1 and PD-L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA-I in three PDAC cell lines. It also upregulated surface expression of HLA-E, HLA-G and immune checkpoint molecules, including PD-L1 and PD-L2. These results suggest that the higher expression of HLA-I, HLA-E and HLA-G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA-I expression on PDAC cells.

The association between consuming bivalves, and acute gastroenteritis and norovirus in Tokyo, Japan.

A prospective matched case-control study was conducted to evaluate associations between dietary histories, including consumption of bivalves, diarrhea, and norovirus positive diarrhea in adult ambulatory patients at an outpatient clinic of a hospital in Tokyo, Japan. Ambulatory cases with diarrhea were matched with nondiarrheal control patients, who visited the same clinic. A standardized questionnaire was used to obtain patients' information, including histories of food consumption and clinical information. Norovirus infection was confirmed using real-time reverse transcription polymerase chain reaction. A total of 207 patients, including 69 diarrheal cases and 138 nondiarrheal cases were included in the analysis. Among them, 60 (29.0%) participants reported consuming bivalves. Norovirus was detected in 35% (24/69) of diarrheal cases. Of those, 10 (41.7%) reported consumption of bivalves and of those, 6 (60.0%) consumed raw bivalves. The proportion of those who consumed raw bivalves was significantly higher in norovirus-positive diarrheal cases than in norovirus-negative diarrheal cases (25.0% vs 6.7%; odds ratio [OR], 4.67; 95% confidence interval [CI], 1.1-20.7) and matched nondiarrheal controls (25.0% vs 6.3%, OR: 5.00; 95% CI, 1.1-22.2). The attributable fraction of consuming raw bivalves for norovirus-associated diarrhea to matched nondiarrheal controls was 20.0%. Consuming raw bivalves was substantially attributed to norovirus-associated diarrhea in adult ambulatory patients and preventive measures for reducing the risk associated with consumption of raw bivalves could decrease the incidence of norovirus-associated diarrhea.

Phase 1/2 study of the WT1 peptide cancer vaccine WT4869 in patients with myelodysplastic syndrome.

WT4869 is a synthetic peptide vaccine derived from the Wilms' tumor gene 1 (WT1) protein. This phase 1/2 open-label study evaluated the safety and efficacy of WT4869, and biomarkers for response, in patients with myelodysplastic syndrome. WT4869 (5-1200 µg/dose) was administered intradermally every 2 weeks, according to a 3 + 3 dose-escalation method in higher-risk (International Prognostic Scoring System score ≥1.5) or lower-risk (score <1.5) red blood cell transfusion-dependent patients with myelodysplastic syndrome. Twenty-six patients were enrolled and treated (median age, 75 years; range, 32 to 89). The most common adverse event was injection site reaction (61.5%). Main grade 3 or 4 adverse events were neutropenia (30.8%), febrile neutropenia, pneumonia, elevated blood creatine phosphokinase levels and hypoalbuminemia (all 7.7%). Dose-limiting toxicities occurred in 1 patient in the 50 µg/dose cohort (pyrexia, muscle hemorrhage and hypoalbuminemia) and 1 patient in the 400 µg/dose cohort (pneumonitis); however, the maximum tolerated dose could not be determined from this trial. The overall response rate was 18.2%, the disease control rate was 59.1% and median overall survival was 64.71 weeks (95% confidence interval: 50.29, 142.86) as assessed by the Kaplan-Meier method. Subgroup analysis of azacitidine-refractory patients with higher-risk myelodysplastic syndrome (11 patients) showed median overall survival of 55.71 weeks (approximately 13 months). WT1-specific cytotoxic T lymphocyte induction was observed in 11 of 25 evaluable patients. WT4869 was well tolerated in patients with myelodysplastic syndrome and preliminary data suggest that WT4869 is efficacious. This trial was registered at www.clinicaltrials.jp as JapicCTI-101374.

A new peptide vaccine OCV-501: in vitro pharmacology and phase 1 study in patients with acute myeloid leukemia.

Wilms' tumor 1 (WT1) is a promising target of new immunotherapies for acute myeloid leukemia (AML) as well as for other cancers. OCV-501 is a helper peptide derived from the WT1 protein. OCV-501 induced OCV-501-specific Type 1 T-helper (Th1) responses dose-dependently and stimulated helper activity of the specific Th1 cells in peripheral blood mononuclear cells from healthy donors. OCV-501 also enhanced the increase in WT1-killer peptide-specific cytotoxic T lymphocytes. OCV-501 stimulated the OCV-501-specific Th1 clones in an HLA class-II restricted manner and formed a complex with HLA class-II protein. OCV-501-specific Th1 clones demonstrated significant OCV-501-specific cytolytic activity against OCV-501-pulsed B-lymphoblastoid cell line cells. Based on the pre-clinical results, phase 1 clinical trial was conducted. The result of this trial suggested that the subcutaneous administration of OCV-501 once weekly for 4 weeks at doses of 0.3, 1, and 3 mg in older patients with AML during complete remission was safe and well tolerated. The maximum tolerated dose was considered to be ≥3 mg. Of the nine subjects enrolled, neither relapse nor blast cells were observed during the study. Immunological responses were observed in OCV-501-specific delayed-type hypersensitivity test. This trial was registered at http://www.clinicaltrials.gov as NCT 01440920.

CTIA-4 blockade therapy; biology and clinical activity.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a key inhibitory regulator for priming phase in T cell adaptive immunity against cancer. CTLA-4 blockade monotherapy or combination therapy with PD-1 blockade induces T cell activation and shows clinical activity in melanoma patients. Anti-CTLA-4 antibody is the first drug for immune checkpoint blockade and has been clinically used over the past decades. Several clinical characteristics and biomarkers in cancer immunotherapy were identified during developing CTLA-4 blockade therapy for mela- noma patients. Further roles of CTLA-4 blockade in other cancers are still to be determined. Well-designed clinical trial considering immune biology and findings from translational re- search is warranted to emerge potential efficacy of CTLA-4 blockade in cancer treatment.

Phase I study of a new cancer vaccine of ten mixed peptides for advanced cancer patients.

A phase I study of a new cancer vaccine (KRM-10), consisting of a mixture of 10 different short peptides, was conducted for patients with advanced gastrointestinal cancers. Primary or secondary endpoints included the dose-limiting toxicity (DLT), or safety and immune responses, respectively. Peptide-specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG), together with soluble inflammatory factors, were measured before and after vaccination. Twenty-one patients were vaccinated with KRM-10 at dose levels of 10 (n = 6), 20 (n = 8) or 30 mg (n = 7) of peptides every week for 6 weeks. No DLT were observed in the dose range evaluated. Common treatment-related adverse events were a grade 1 injection site reaction in 15 patients, and fever in three patients (grade 1 in two patients and grade 2 in one patient). CTL activity to at least one peptide at the time of the third and sixth vaccination increased in 2 and 3 of 6 (10 mg), 2 of 8 and 4 of 6 (20 mg), or 2 and 1 of 6 (30 mg) patients, respectively. IgG levels, at the third and sixth vaccination, were also increased in 1 and 1 of 6 (10 mg), 2 of 8 and 4 of 6 (20 mg), or 1 and 3 of 6 (30 mg) patients, respectively. The KRM-10 vaccine consisting of 20 mg of peptides was determined as the optimal dose for a coming phase II trial because of its safety, and also for demonstrating the most potent activity for augmenting the immune response of the three doses tested. This trial was registered at the UMIN Clinical Trials Registry as UMIN000008820.

Prognostic significance of HLA class I and II expression in patients with diffuse large B cell lymphoma treated with standard chemoimmunotherapy.

Loss of tumor cell human leukocyte antigen (HLA) is an immune escape mechanism for malignancies. However, the effect of low HLA class I or class II expression in diffuse large B cell lymphoma (DLBCL) treated with chemoimmunotherapy with the monoclonal antibody rituximab is largely unknown. We retrospectively analyzed samples and other data from 144 patients with DLBCL who were newly diagnosed in our institution and treated with standard R-CHOP therapy. We used antibodies against pan-HLA class I and pan-HLA class II molecules to assess HLA expression and its effect on prognosis. In a multivariate analysis, loss of HLA class II expression was a significantly independent adverse factor for progression-free survival (PFS; hazard ratio 2.3; 95 % confidence interval 1.2-4.6; P = 0.01). Although HLA class I loss of expression did not correlate with prognosis, the combination of HLA class I(+) with either low peripheral lymphocyte count or CD3(+) lymphocyte count was an adverse prognostic factor for PFS. Loss of HLA class II is an International Prognostic Index (IPI)-independent adverse factor for PFS in patients with DLBCL treated with standard therapy. However, in contrast to other solid cancers, HLA class I loss was not solely a prognostic factor in DLBCL.

Donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene: detailed immunological function following add-back after haplo-identical transplantation.

BACKGROUND AIMS: Haplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. METHODS: We investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vß repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. RESULTS: A comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8(+) non-TK cells were also depleted in response to ganciclovir administration. The TCR Vß-chain repertoire of both TK cells and non-TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non-TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. CONCLUSIONS: We demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells.

Positive impact of chronic graft-versus-host disease on the outcome of patients with de novo myelodysplastic syndrome after allogeneic hematopoietic cell transplantation: a single-center analysis of 115 patients.

To evaluate the impact of graft-versus-host disease (GVHD) and prognostic factors for patients with myelodysplastic syndrome (MDS) after allogeneic hematopoietic cell transplantation (allo-HCT), we retrospectively reviewed 115 patients with MDS or acute myeloid leukemia with multilineage dysplasia (AML-MLD) after allo-HCT at our center. Eighty one patients received reduced-intensity conditioning (RIC) regimens, whereas 34 received myeloablative conditioning regimens. Although the RIC group was significantly older and included more patients with poor cytogenetic risk, no difference in 4-yr overall survival (OS) was seen between the two groups. In a multivariate analysis, covariates associated with a worse OS were the French-American-British stage of refractory anemia excess blasts in transformation/AML-MLD at peak, poor cytogenetic risk, bone marrow blasts of 20% or higher at HCT and the absence of chronic GVHD (cGVHD). By using semi-landmark analyses, we found that the presence of cGVHD significantly improved OS in high-risk patients or the RIC group. However, there was no difference in OS between those with and without cGVHD among low-risk MDS patients. These findings suggest that the graft-versus-leukemia effect may be more beneficial in high-risk patients who do not receive intensive preparative regimens.

Comparison of outcomes after allogeneic hematopoietic stem cell transplantation in patients with follicular lymphoma, diffuse large B-cell lymphoma associated with follicular lymphoma, or de novo diffuse large B-cell lymphoma.

The outcome after allogeneic hematopoietic stem cell transplantation (allo-HCT) for diffuse large B-cell lymphoma (DLBCL) associated with follicular lymphoma (FL), which includes DLBCL with pre- or co-existing FL, remains controversial, and few previous reports have compared the outcomes after allo-HCT for FL, DLBCL associated with FL, and de novo DLBCL. We retrospectively analyzed 97 consecutive patients with FL (n = 46), DLBCL associated with FL (n = 22), or de novo DLBCL (n = 29) who received allo-HCT at our institute between 2000 and 2010. With a median follow-up of 53 months, the 5-year overall survival (OS) and progression-free survival (PFS) were, respectively, 77% and 70% for FL, 62% and 57% for DLBCL associated with FL, and 26% and 23% for de novo DLBCL. The 5-year cumulative incidences of non-relapse mortality and disease progression/relapse were, respectively, 16% and 15% for FL, 19% and 24% for DLBCL associated with FL, and 36% and 41% for de novo DLBCL. By a multivariate analysis, the OS and PFS for DLBCL associated with FL were significantly better than those for de novo DLBCL, whereas they were not significantly different from those for FL. These results suggest that allo-HCT may be a promising option for patients with not only advanced FL but also DLBCL associated with FL.

Natural adjuvants (PC and G2) induce activated natural killer cells with NKG2D expression and cytotoxic properties in colorectal cancer.

Aim: This study aimed to investigate the effects of natural adjuvants (G2 and PC) to activate natural killer cells in colorectal cancer. Background: Natural killer (NK) cells are an element of the innate immune system that can recognize and kill cancer cells and provide hope for cancer therapy. One of the current methods in cancer immunotherapy is NK cell therapy. Immunotherapy with NK cells has been limited because of the low number and cytotoxicity level of NK cells. Natural adjuvants such as PC and G2 may stimulate the immune system. It seems that these adjuvants could increase cytotoxic NK cells. Methods: Twelve patients with colorectal cancer and six healthy individuals qualified for inclusion in this study. Peripheral blood mononuclear cells (PBMCs) from each patient with two distinctive concentrations (105and 5×104 cells/well) were treated with Interleukin2 (IL2), PC, and G2 adjuvant separately. The NK cell's surface markers, including CD16, CD56, and NKG2D, were evaluated by flow cytometry. The cytotoxicity effect of treated PBMCs as effector cells against NK sensitive cell line (K562) was assessed using the LDH assay method. Results: The results revealed a significant increase in the level of CD16+NKG2D+ NK cells in PBMCs treated with the G2 group compared with the control group in CRC PBMC (p<0.001) as well as the normal PBMC group (p < 0.01). In addition, the results indicated a significant increase in the level of CD56+NKG2D+ cells in the PBMC treated with PC (p < 0.05) and G2 (p < 0.001) groups compared with the PBMC group. The cytotoxicity result of PBMC from CRC patients in 10:1 ratio of the effector: target showed that the cells' cytotoxicity in the PBMCs treated with PC (p<0.01) and G2 (p<0.05) was significantly higher than the untreated PBMC. Conclusion: According to the result of this study, it can be stated that the PC and G2 adjuvants could be candidates for inducing cytotoxic natural killer cells.

Autologous Natural Killer Cell-enrichment for Immune Cell Therapy: Preclinical Setting Phase, Shiraz Experience.

Natural killer (NK) cell therapy has proven to be a promising approach for the treatment of malignancies. Osaki method for ex-vivo autologous NK cell expansion has been recently introduced in Japan. To start clinical trial phase I at Shiraz University of Medical Sciences in collaboration with the Japanese group, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells, and the likely unwanted contamination of the final cell product. Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy individuals' peripheral blood and transferred directly to the specified initial culture bag containing anti-CD52 and anti-CD3 and Interleukin (IL)-2. The cells were cultured for 14-17 days in an incubator, during which the cells received condition media, and underwent several passages into bigger culture bags. All the procedures were carried out in a cleanroom and associated facilities. Before and after activation PBMCs were analyzed for their phenotype and cytotoxic activity; using flow cytometry and cytokine release assay. Our results indicated that NK (CD3-CD16+/-CD56+) cells were expanded 510-fold on average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay, with a strong expression of Natural killer group 2 member D (NKG2D) and CD16. The prepared final cell products were negative for HCV, HBV, HIV, mycoplasma, and endotoxin. In the preclinical phase, large numbers of activated and un-contaminated NK cells from healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination and has the potential to be used for NK cell therapy in future clinical trials, suitable to be infused back to the donors in phase I clinical trial.

Long-term administration of Wilms tumor-1 peptide vaccine in combination with gemcitabine causes severe local skin inflammation at injection sites.

The skin toxicity of vaccine therapy at injection sites is generally limited to Grades 1-2 due to the nature of their function. We experienced two cases of severe and prolonged local adverse effects in 25 patients following a Phase I study of gemcitabine and Wilms tumor-1 peptide vaccine mixed with incomplete Freund's adjuvant for inoperable pancreatic or biliary tract cancer. These patients requested to continue the treatment after the study period; however, in the course of compassionate use, they developed unacceptable local skin reactions and terminated their vaccine treatment. One patient (human leukocyte antigen, A0201, 3 mg) developed Grade 3 ulceration at the 10th vaccination and another (human leukocyte antigen, A2402, 1 mg) developed Grade 2 indulation and fibrosis at the 16th vaccination. Skin toxicity occurred at 6.4-8.4 months and continued for several months after the final vaccination during gemcitabine treatment. In these cases, activation or induction of Wilms tumor-1-specific T lymphocytes was not apparent in the peripheral blood despite their severe local reactions. Therefore, we need to monitor patients for late-onset, severe and long-lasting skin reactions at injection sites in Wilms tumor-1 cancer vaccine therapy, particularly for combination treatment with gemcitabine.

Facile Fabrication of Thin-Bottom Round-Well Plates Using the Deformation of PDMS Molds and Their Application for Single-Cell PCR.

Recently, microdevices made of resins have been strongly supporting cell analysis in a range of fields, from fundamental life science research to medical applications. Many microdevices are fabricated by molding resin to a mold made precisely from rigid materials. However, because dimensional errors in the mold are also accurately printed to the products, the accuracy of the product is limited to less than the accuracy of the rigid mold. Therefore, we hypothesized that if dimensional errors could be self-corrected by elastic molds, microdevices could be facilely fabricated with precision beyond that of molds. In this paper, we report a novel processing strategy in which an elastic mold made of polymethylsiloxane (PDMS) deforms to compensate for the dimensional error on the products. By heat-press molding a polycarbonate plate using a mold that has 384 PDMS convexes with a large dimensional error of height of ± 15.6 µm in standard deviation, a 384-round-well plate with a bottom thickness 13.3 ± 2.3 µm (n = 384) was easily fabricated. Finally, single-cell observation and polymerase chain reactions (PCRs) demonstrated the application of the products made by elastic PDMS molds. Therefore, this processing method is a promising strategy for facile, low-cost, and higher precision microfabrication.

Camouflage makeup improves quality of life in cancer patients with treatment-related skin changes.

BACKGROUND: Cancer treatment causes various skin appearance changes, which affect quality of life (QoL) in patients with cancer. We examined whether camouflage makeup improves QoL in these patients. METHODS: Skindex-16 and visual analogue scale scores of 39 female patients with cancer treatment-related skin changes were compared before and 2-3 months after self-administration of camouflage makeup. RESULTS: Camouflage makeup was able to conceal almost all skin changes, improving QoL scores regardless of age, diagnosis, and site of skin changes. Use frequency was significantly higher in patients with skin changes on exposed sites compared with patients with unexposed sites. CONCLUSIONS: Even though the patients applied the makeup only when required, they were satisfied with its effect, which improved their QoL. Moreover, the makeup had a positive effect even in patients with changes in unexposed sites, suggesting that clinicians can recommend camouflage makeup to all patients to improve QoL.

Outcome of 93 patients with relapse or progression following allogeneic hematopoietic cell transplantation.

Relapse/progression after allogeneic hematopoietic cell transplantation (allo-HCT) remains the major cause of treatment failure. In this study, the subsequent clinical outcome was overviewed in 292 patients with leukemia/myelodysplastic syndrome who received allo-HCT. Among them, 93 (32%) showed relapse/progression. Cohort 1 was chosen to receive no interventions with curative intent (n = 25). Cohort 2 received reinduction chemotherapy and/or donor lymphocyte infusion (n = 48), and Cohort 3 underwent a second allo-HCT (n = 20). Sixty-three patients received reinduction chemotherapy, and 27 (43%) achieved subsequent complete remission (CR). The incidence of nonrelapse mortality (NRM) was similar among the three cohorts (4, 15, and 5%). The 1-year overall survival (OS) after relapse was significantly better in patients with a second HCT (58%) than in others (14%, Cohorts 1 and 2; P <.001). However, the 2-year OS did not differ between the two groups, which suggests that it is difficult to maintain CR after the second HCT. Multivariate analysis showed that reinduction chemotherapy, CR after intervention, second HCT, and longer time to post-transplant relapse were associated with improved survival. In conclusion, for patients with relapse after allo-HCT, successful reinduction chemotherapy and a second HCT may be effective for prolonging survival without excessive NRM. However, effective measures to prevent disease progression after a second HCT clearly need to be developed.

Regular dose of gemcitabine induces an increase in CD14+ monocytes and CD11c+ dendritic cells in patients with advanced pancreatic cancer.

OBJECTIVE: Chemotherapy and immunotherapy often seem to contradict each other. However, recent reports suggested that the anticancer effects in some chemotherapeutic agents were concerned with immune response. This study was designed to evaluate the immunological reaction by gemcitabine for future clinical trial of combination therapy with gemcitabine and cancer vaccines. METHODS: We evaluated several immunological parameters in patients with advanced pancreatic cancer who received a conventional dose of gemcitabine for 2 months. Twenty-eight patients with metastasis or locally advanced tumor, including 18 gemcitabine-naïve and 10 with a history of preceding gemcitabine treatment, were enrolled in this study. The patients received gemcitabine 1000 mg/m(2) for 3 weeks, followed by 1 week of rest. We monitored the kinetics of lymphocytes, natural killer cells, monocytes, dendritic cells (DC), human leukocyte antigen (HLA)-multimer conjugated with CMV or WT1 peptide, and intracellular cytokine production of interferon-gamma and interleukin-4 by flow cytometry. The T cell receptor (TCR) repertoire was also analyzed. RESULTS: The absolute number and percentage of CD14(+) monocytes and CD11c(+) (myeloid) DC increased with gemcitabine treatment (P = 0.033 and P = 0.021). The percentage of CD123(+) (plasmacytoid) DC also increased (P = 0.034), whereas no significant change was observed in other immune parameters, including multimer, intracellular cytokine production and TCR repertoire. CONCLUSIONS: Our finding that gemcitabine treatment induced the proliferation of CD14(+) monocytes and CD11c(+) DC could support combination therapy with gemcitabine and specific immunotherapy such as peptide vaccination against pancreatic cancers.

[Gene therapy for myeloid leukemia].

Among several immune-gene therapies against myeloid leukemia, "T cell depleted haplo-identical transplantation with HSV-TK gene modified T cell add-back" and "donor lymphocyte infusion using HSV-TK gene modified T cell in patients experiencing recurrence after allogeneic stem cell transplantation" were in advanced stages of clinical development. In this chapter, I will summarize the strategies of gene therapy using HSV-TK gene modified T lymphocyte in allogeneic stem cell transplantation. I will also point out the current situation of other gene therapies such as cytokine gene modified CML-DC and TCR gene modified T lymphocyte against myeloid leukemia.

Cell Energy: A New Hypothesis in Decoding Cancer Evolution.

The present study deviates from previous approaches as it focuses on the concept of energy to illuminate cancer-related issues. Energy is a prerequisite for any function; cellular function is no exception, and thus, reduced energy in human cells can impair their performance. This hypothesis provides a novel view of cancer formation. It shows that a normal cell transforms into its cancerous counterpart in response to cellular adenosine triphosphate (ATP) depletion. Moreover, it presents a new definition for the origin of cancer stem cells and how they can regenerate cancer. This article regards a distinct aspect of cancer that helps to differentiate various phases of its progression and shed light on some of the uncharted zones of its pathway for the first time that needs further confirmation by empirical studies.