Overlapping Atypical Hemolytic Uremic Syndrome and C3 Glomerulopathy with Mutation in CFI in a Japanese Patient: A Case Report.

A 34-year-old Japanese man presented with blurred vision, headache, nausea, anemia, thrombocytopenia, and severe renal dysfunction. Thrombotic microangiopathy was initially suspected to have been caused by malignant hypertension. Antihypertensive medications did not improve his thrombocytopenia or renal dysfunction, and other diseases causing thrombotic microangiopathy were ruled out. Therefore, the patient was diagnosed with atypical hemolytic uremic syndrome. A renal biopsy revealed an overlap of thrombotic microangiopathy and C3 glomerulopathy. Genetic testing revealed c.848A>G (p.Asp283Gly), a missense heterozygous variant in the gene encoding complement factor I. Overlapping atypical hemolytic uremic syndrome and C3 glomerulopathy with complement factor I mutation is very rare, especially in Japan.

Increased infiltration of CD4+ T cell in the complement deficient lymphedema model.

BACKGROUND: Lymphedema is an intractable disease that can be caused by injury to lymphatic vessels, such as by surgical treatments for cancer. It can lead to impaired joint mobility in the extremities and reduced quality of life. Chronic inflammation due to infiltration of various immune cells in an area of lymphedema is thought to lead to local fibrosis, but the molecular pathogenesis of lymphedema remains unclear. Development of effective therapies requires elucidation of the immunological mechanisms involved in the progression of lymphedema. The complement system is part of the innate immune system which has a central role in the elimination of invading microbes and acts as a scavenger of altered host cells, such as apoptotic and necrotic cells and cellular debris. Complement-targeted therapies have recently been clinically applied to various diseases caused by complement overactivation. In this context, we aimed to determine whether complement activation is involved in the development of lymphedema. RESULTS: Our mouse tail lymphedema models showed increased expression of C3, and that the classical or lectin pathway was locally activated. Complement activation was suggested to be involved in the progression of lymphedema. In comparison of the C3 knockout (KO) mouse lymphedema model and wild-type mice, there was no difference in the degree of edema at three weeks postoperatively, but the C3 KO mice had a significant increase of TUNEL+ necrotic cells and CD4+ T cells. Infiltration of macrophages and granulocytes was not significantly elevated in C3 KO or C5 KO mice compared with in wild-type mice. Impaired opsonization and decreased migration of macrophages and granulocytes due to C3 deficiency should therefore induce the accumulation of dead cells and may lead to increased infiltration of CD4+ T cells. CONCLUSIONS: Vigilance for exacerbation of lymphedema is necessary when surgical treatments have the potential to injure lymphatic vessels in patients undergoing complement-targeted therapies or with complement deficiency. Future studies should aim to elucidate the molecular mechanism of CD4+ T cell infiltration by accumulated dead cells.

Metabolic profiling analysis of head and neck squamous cell carcinoma.

OBJECTIVE: Tumor cells can acquire a large amount of energy and structural components by reprogramming energy metabolism; moreover, metabolic profiles slightly differ according to cancer type. This study compared and assessed the metabolic profile of head and neck squamous cell carcinoma (HNSCC) and normal tissues, which were collected from patients without cancer. SUBJECTS AND METHODS: Overall, 23 patients with HNSCC and 6 patients without cancer were included in the analysis. Metabolomic profiles were analyzed using capillary electrophoresis-mass spectrometry. Gene expression was evaluated using real-time reverse transcription-polymerase chain reaction. RESULTS: Glycolysis, the pentose phosphate pathway, tricarboxylic acid cycle, and glutamine metabolism were upregulated in HNSCC tissues based on gene expression analysis. HNSCC could then have enhanced energy production and structural component. The levels of lactate, succinate, glutathione, 2-hydroxyglutarate, and S-adenosylmethionine, considered as oncometabolites, increased and these had accumulated in HNSCC tissues. CONCLUSIONS: The level of metabolites and the expression of enzymes differ between HNSCC and normal tissues. Reprogramming metabolism in HNSCC provides an energy source as well as structural components, creating a system that offers rapid proliferation, progression, and is less likely to be eliminated.

Potent adjuvant effect elicited for tumor immunotherapy by a liposome conjugated pH-sensitive polymer and dendritic cell-targeting Toll-like-receptor ligand.

The generation of DCs with augmented functions is a strategy for obtaining satisfactory clinical outcomes in tumor immunotherapy. We developed a novel synthetic adjuvant comprising a liposome conjugated with a DC-targeting Toll-like-receptor ligand and a pH-sensitive polymer for augmenting cross-presentation. In an in vitro study using mouse DCs, these liposomes were selectively incorporated into DCs, significantly enhanced DC function and activated immune responses to present an epitope of the incorporated antigen on the major histocompatibility complex class I molecules. Immunization of mice with liposomes encapsulating a tumor antigen significantly enhanced antigen-specific cytotoxicity. In tumor-bearing mice, vaccination with liposomes encapsulating a tumor antigen elicited complete tumor remission. Furthermore, vaccination significantly enhanced cytotoxicity, targeting not only the vaccinated antigen but also the other antigens of the tumor cell. These results indicate that liposomes are an ideal adjuvant to develop DCs with considerably high potential to elicit antigen-specific immune responses; they are a promising tool for cancer therapy with neoantigen vaccination.

Early Elevation of Complement Factor Ba Is a Predictive Biomarker for Transplant-Associated Thrombotic Microangiopathy.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a fatal complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous reports suggest that TA-TMA is caused by complement activation by complement-related genetic variants; however, this needs to be verified, especially in adults. Here, we performed a nested case-control study of allo-HSCT-treated adults at a single center. Fifteen TA-TMA patients and 15 non-TA-TMA patients, matched according to the propensity score, were enrolled. Based on a previous report showing an association between complement-related genes and development of TA-TMA, we first sequenced these 17 genes. Both cohorts harbored several genetic variants with rare allele frequencies; however, there was no difference in the percentage of patients in the TA-TMA and non-TA-TMA groups with the rare variants, or in the average number of rare variants per patient. Second, we measured plasma concentrations of complement proteins. Notably, levels of Ba protein on Day 7 following allo-HSCT were abnormally and significantly higher in TA-TMA than in non-TA-TMA cases, suggesting that complement activation via the alternative pathway contributes to TA-TMA. All other parameters, including soluble C5b-9, on Day 7 were similar between the groups. The levels of C3, C4, CH50, and complement factors H and I in the TA-TMA group after Day 28 were significantly lower than those in the non-TA-TMA group. Complement-related genetic variants did not predict TA-TMA development. By contrast, abnormally high levels of Ba on Day 7 did predict development of TA-TMA and non-relapse mortality. Thus, Ba levels on Day 7 after allo-HSCT are a sensitive and prognostic biomarker of TA-TMA.

Paroxysmal nocturnal hemoglobinuria caused by CN-LOH of constitutional PIGB mutation and 70-kbp microdeletion on 15q.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number-neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.

[The Warburg Effect and M2 Macrophage Polarization in Head and Neck Cancer].

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect. The Warburg effect is caused by tumor cells not only to adapt their metabolism to the demand for and limited supply of oxygen but also to obtain large amounts of nucleotides, amino acids and lipids for excessive proliferation of tumor cells. The Warburg effect results in increased production of lactic acid, as the final product of glycolysis, in the tumor microenvironment. Lactic acid secreted by tumor cells functions as an immunosuppressive mediator and converts macrophages into M2 macrophages. M2 macrophages reduce inflammatory responses and adaptive Th1 immunity, and promote angiogenesis and tissue remodeling. Tumor-associated macrophages(TAMs)polarize into the M2 phenotype and suppress the host anti-cancer immune response, leading to tumor progression. We have demonstrated that tumor-secreted lactic acid is linked to the induction of M2-macrophage polarization in human head and neck squamous cell carcinoma(HNSCC). FDG, which is a glucose analog, uptake measured by positron emission tomography/computed tomography(PET/CT)indicates the Warburg effect in tumor tissue. M2-macrophage polarization is promoted in HNSCC with increased glucose uptake, maximum standardized uptake value(SUVmax), mean SUV(SUVmean). Tumor cells mediate an immunosuppressive microenvironment via inducing M2-macrophage polarization by reprogramming of glucose metabolism, called Warburg effect.

The importance of FDG-PET/CT parameters for the assessment of the immune status in advanced HNSCC.

OBJECTIVE: Cancer cells secrete large amounts of lactic acid via aerobic glycolysis. We have shown that lactic acid plays an important role as a proinflammatory and immunosuppressive mediator and promotes tumor progression. Fluorine-18 fluorodeoxyglucose (FDG) uptake detected by positron emission tomography/computed tomography (PET/CT) is considered as a good indicator of aerobic glycolysis in cancer. In this study, we examined the relationships between systemic inflammatory parameters and FDG-PET/CT parameters in advanced head and neck squamous cell carcinoma (HNSCC). Furthermore, we investigated the relationships between FDG-PET/CT parameters and M2-macrophage polarization in HNSCC by assessing the ratio of CD163, a M2-macrophage marker, to CD68, a pan-macrophage marker. METHODS: This study included 73 advanced HNSCC patients. We assessed the C-reactive protein (CRP) level, white blood cell (WBC) count, neutrophil count, lymphocyte count, and monocyte count as systemic inflammatory markers. Additionally, we assessed the maximum standardized uptake value (SUVmax), mean SUV (SUVmean), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) as FDG-PET/CT parameters. RESULTS: The CRP level, WBC count, and neutrophil count were correlated with whole-body FDG-PET/CT parameters. The CD163/CD68 ratio was correlated with SUVmax and SUVmean. Our results suggest that systemic inflammation, which is associated with neutrophils, develops in patients with HNSCC having tumors with a larger volume and increased glucose uptake and that M2-macrophage polarization is promoted in HNSCC with increased glucose uptake, SUVmax, and SUVmean. FDG-PET/CT has the potential to reflect cancer-related chronic inflammation and immunosuppressive conditions in cancer patients. CONCLUSIONS: FDG-PET/CT parameters appear to be useful in assessing the immune status in HNSCC.

Eculizumab for Severe Thrombotic Microangiopathy Secondary to Surgical Invasive Stress and Bleeding.

Atypical hemolytic uremic syndrome (aHUS) is an extremely rare condition caused by an excessive activation of the complement pathway based on genetic or acquired dysfunctions in complement regulation, leading to thrombotic microangiopathy (TMA). A complement-amplifying condition (CAC) can trigger aHUS occurrence along with complement abnormality. We herein report a case of severe TMA after laparoscopic myomectomy in a healthy woman. This case was eventually diagnosed as complement-mediated TMA secondary to surgical invasive stress as a CAC, with no definitive diagnosis of aHUS despite a genetic test. The patient fully recovered after several eculizumab administrations.

An Immune-Stimulatory Helix-Loop-Helix Peptide: Selective Inhibition of CTLA-4-B7 Interaction.

Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix-loop-helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4-B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a KD of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4-B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4-B7-1 interaction with an IC50 of 1.1 ± 0.03 µM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.

Complement and inflammasome overactivation mediates paroxysmal nocturnal hemoglobinuria with autoinflammation.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent autoinflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients' leukocytes and of PIGT-KO THP-1 cells we show that, through increased IL-1ß secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of autoinflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also autoinflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.

Effects of laughter therapy on quality of life in patients with cancer: An open-label, randomized controlled trial.

BACKGROUND: Few randomized controlled trials have assessed the effects of laughter therapy on health-related quality of life (QOL) in cancer patients. This study aimed to evaluate these effects as an exploratory endpoint in cancer patients as part of a randomized controlled trial conducted at a single institution in Japan. METHODS: The Initiative On Smile And CAncer (iOSACA) study was an open-label randomized controlled trial conducted in 2017 in which participants aged 40-64 years with cancer were randomly assigned to either an intervention group (laughter therapy) or control group (no laughter therapy). Each participant in the intervention group underwent a laughter therapy session once every two weeks for seven weeks (total of four sessions). Each session involved a laughter yoga routine followed by Rakugo or Manzai traditional Japanese verbal comedy performances. We assessed QOL as a secondary endpoint in this intention-to-treat population using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30). The questionnaire was completed at baseline (Week 0) and at Weeks 3 and 7. Mixed-effects models for repeated measures were developed to compare time-dependent changes in each QOL domain from baseline between the intervention and control groups. RESULTS: Four participants retracted consent and one participant was retrospectively excluded from analysis due to unmet inclusion criteria. The analysis was conducted using 56 participants, with 26 in the intervention group and 30 in the control group. Questionnaire completion rates were high (>90%), with similar QOL scores reported at baseline in both groups. The mixed-effects models showed that the intervention group had significantly better cognitive function and less pain than the control group for a short period. CONCLUSION: Laughter therapy may represent a beneficial, noninvasive complementary intervention in the clinical setting. Further studies are needed to verify the hypotheses generated from this exploratory study.

Development of a vaccine based on bacteria-mimicking tumor cells coated with novel engineered toll-like receptor 2 ligands.

For a successful tumor vaccine, it is necessary to develop effective immuno-adjuvants and identify specific tumor antigens. Tumor cells obtained from surgical or biopsy tissues are a good source of tumor antigens but, unlike bacteria, they do not induce strong immune responses. Here, we designed 2 novel lipopeptides that coat tumor cell surfaces and mimic bacterial components. Tumor cells coated with these lipopeptides (called bacteria-mimicking tumor cells [BMTC]) were prepared and their efficacy as a tumor vaccine examined. Natural bacterial lipopeptides act as ligands for toll-like receptor 2 (TLR2) and activate dendritic cells (DC). To increase the affinity of the developed lipopeptides for the negatively charged plasma membrane, a cationic polypeptide was connected to Pam2Cys (P2C), which is the basic structure of the TLR2 ligand. This increased the non-specific binding affinity of the peptides for the cell surface. Two such lipopeptides, P2CSK11 (containing 1 serine and 11 lysine residues) and P2CSR11 (containing 1 serine and 11 arginine residues) bound to irradiated tumor cells via the long cationic polypeptides more efficiently than the natural lipopeptide MALP2 (P2C-GNNDESNISFKEK) or a synthetic lipopeptide P2CSK4 (a short cationic polypeptide containing 1 serine and 4 lysines). BMTC coated with P2CSR11 or P2CSK11 were efficiently phagocytosed by DC and induced antigen cross-presentation in vitro. They also induced effective tumor-specific cytotoxic T cell responses and inhibited tumor growth in in vivo mouse models. P2CSR11 activated DC but induced less inflammation-inducing cytokines/interferons than other lipopeptides. Thus, P2CSR11 is a strong candidate antigen-specific immuno-adjuvant, with few adverse effects.

Development of effective tumor immunotherapy using a novel dendritic cell-targeting Toll-like receptor ligand.

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.

M2-like macrophage polarization in high lactic acid-producing head and neck cancer.

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect and results in increased lactic acid secretion into the tumor microenvironment. We have previously shown that lactic acid has important roles as a pro-inflammatory and immunosuppressive mediator and promotes tumor progression. In this study, we examined the relationship between the lactic acid concentration and expression of LDHA and GLUT1, which are related to the Warburg effect, in human head and neck squamous cell carcinoma (HNSCC). Tumors expressing lower levels of LDHA and GLUT1 had a higher concentration of lactic acid than those with higher LDHA and GLUT1 expression. Lactic acid also suppressed the expression of LDHA and GLUT1 in vitro. We previously reported that lactic acid enhances expression of an M2 macrophage marker, ARG1, in murine macrophages. Therefore, we investigated the relationship between the lactic acid concentration and polarization of M2 macrophages in HNSCC by measuring the expression of M2 macrophage markers, CSF1R and CD163, normalized using a pan-macrophage marker, CD68. Tumors with lower levels of CD68 showed a higher concentration of lactic acid, whereas those with higher levels of CSF1R showed a significantly higher concentration of lactic acid. A similar tendency was observed for CD163. These results suggest that tumor-secreted lactic acid is linked to the reduction of macrophages in tumors and promotes induction of M2-like macrophage polarization in human HNSCC.

Characterization of the T-cell receptor beta chain repertoire in tumor-infiltrating lymphocytes.

Tumor-infiltrating lymphocytes (TILs) are direct effectors of tumor immunity, and their characterization is important for further development of immunotherapy. Recent advances in high-throughput sequencing technologies have enabled a comprehensive analysis of T-cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, which may provide information of therapeutic importance. We developed a high-fidelity target sequencing method with the ability for absolute quantitation, and performed large-scale sequencing of TCR beta chain (TCRB) CDR3 regions in TILs and peripheral blood lymphocytes (PBLs). The estimated TCRB repertoire sizes of PBLs from four healthy individuals and TILs from four colorectal cancer tissue samples were 608,664-1,003,098 and 90,228-223,757, respectively. The usage of J- and V-regions was similar in PBLs and TILs. Proportions of CDR3 amino acid (aa) sequences occupying more than 0.01% of the total molecular population were 0.33-0.43% in PBLs and 1.3-3.6% in TILs. Additional low coverage sequencing of 15 samples identified five CDR3 aa sequences that were shared by nine patients, one sequence shared by 10 patients, and one sequence shared by 12 patients. The estimated size of the TCRB repertoire in TILs was significantly smaller than that in PBLs. The proportion of abundant species (>0.01%) in TILs was larger than that in PBLs. Shared CDR3 aa sequences represent a response to common antigens, and the identification of such CDR3 sequences may be beneficial in developing clinical biomarkers.

Development of a dendritic cell-targeting lipopeptide as an immunoadjuvant that inhibits tumor growth without inducing local inflammation.

Materials used for the past 30 years as immunoadjuvants induce suboptimal antitumor immune responses and often cause undesirable local inflammation. Some bacterial lipopeptides that act as Toll-like receptor (TLR) 2 ligands activate immune cells as immunoadjuvants and induce antitumor effects. Here, we developed a new dendritic cell (DC)-targeting lipopeptide, h11c (P2C-ATPEDNGRSFS), which uses the CD11c-binding sequence of intracellular adhesion molecule-1 to selectively and efficiently activate DCs but not other immune cells. Although the h11c lipopeptide activated DCs similarly to an artificial lipopeptide, P2C-SKKKK (P2CSK4), via TLR2 in vitro, h11c induced more effective tumor inhibition than P2CSK4 at low doses in vivo with tumor antigens. Even without tumor antigens, h11c lipopeptide significantly inhibited tumor growth and induced tumor-specific cytotoxic T cells. P2CSK4 was retained subcutaneously at the vaccination site and induced severe local inflammation in in vivo experiments. In contrast, h11c was not retained at the vaccination site and was transported into the tumor within 24 hr. The recruitment of DCs into the tumor was induced by h11c more effectively, while P2CSK4 induced the accumulation of neutrophils leading to severe inflammation at the vaccination site. Because CD11b+ cells, but not CD11c+ cells, produced neutrophil chemotactic factors such as macrophage inflammatory protein (MIP)-2 in response to stimulation with TLR2 ligands, the DC-targeting lipopeptide h11c induced less MIP-2 production by splenocytes than P2CSK4. In this study, we succeeded in developing a novel immunoadjuvant, h11c, which effectively induces antitumor activity without adverse effects such as local inflammation via the selective activation of DCs.

Production of canine soluble CD40 ligand to induce maturation of monocyte derived dendritic cells for cancer immunotherapy.

CD40 ligand (CD40L) expressed by activated T cells is shown to induce maturation of immature dendritic cells (DCs) and this maturation is a vital part in DC based tumor immunotherapy. We constructed an expression vector by cloning the extracellular domain of canine CD40L fused to the signal sequence of canine IL-12p40. When PBMCs were incubated with canine granulocyte-macrophage (GM) -CSF and IL-4, expression of CD86 was significantly elevated, but the majority of cells displayed the morphology of immature DCs. Following addition of the expressed canine soluble CD40L (csCD40L) to the DC-inducing culture, the cell morphology shifted to that of mature DCs, and expression of CD80, CD86, MHC class II and CD1a was significantly enhanced. This morphological change and enhancement of expression was observed even when the csCD40L was present only in the second half period of the culture. Furthermore, the csCD40L caused a significant increase in IL-12 production from DCs. These results show that the csCD40L significantly promotes the maturation and activation of canine monocyte derived DCs.

Dichloroacetate improves immune dysfunction caused by tumor-secreted lactic acid and increases antitumor immunoreactivity.

The activation of oncogenic signaling pathways induces the reprogramming of glucose metabolism in tumor cells and increases lactic acid secretion into the tumor microenvironment. This is a well-known characteristic of tumor cells, termed the Warburg effect, and is a candidate target for antitumor therapy. Previous reports show that lactic acid secreted by tumor cells is a proinflammatory mediator that activates the IL-23/IL-17 pathway, thereby inducing inflammation, angiogenesis and tissue remodeling. Here, we show that lactic acid, or more specifically the acidification it causes, increases arginase I (ARG1) expression in macrophages to inhibit T-cell proliferation and activation. Accordingly, we hypothesized that counteraction of the immune effects by lactic acid might suppress tumor development. We show that dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinases, targets macrophages to suppress activation of the IL-23/IL-17 pathway and the expression of ARG1 by lactic acid. Furthermore, lactic acid-pretreated macrophages inhibited CD8+ T-cell proliferation, but CD8+ T-cell proliferation was restored when macrophages were pretreated with lactic acid and DCA. DCA treatment decreased ARG1 expression in tumor-infiltrating immune cells and increased the number of IFN-γ-producing CD8+ T cells and NK cells in tumor-bearing mouse spleen. Although DCA treatment alone did not suppress tumor growth, it increased antitumor immunotherapeutic activity of Poly(IC) in both CD8+ T cell- and NK cell-sensitive tumor models. Therefore, DCA acts not only on tumor cells to suppress glycolysis but also on immune cells to improve the immune status modulated by lactic acid and to increase the effectiveness of antitumor immunotherapy.