Isobutyric acid enhances the anti-tumour effect of anti-PD-1 antibody.

The low response rate of immune checkpoint inhibitors (ICIs) is a challenge. The efficacy of ICIs is influenced by the tumour microenvironment, which is controlled by the gut microbiota. In particular, intestinal bacteria and their metabolites, such as short chain fatty acids (SCFAs), are important regulators of cancer immunity; however, our knowledge on the effects of individual SCFAs remains limited. Here, we show that isobutyric acid has the strongest effect among SCFAs on both immune activity and tumour growth. In vitro, cancer cell numbers were suppressed by approximately 75% in humans and mice compared with those in controls. Oral administration of isobutyric acid to carcinoma-bearing mice enhanced the effect of anti-PD-1 immunotherapy, reducing tumour volume by approximately 80% and 60% compared with those in the control group and anti-PD-1 antibody alone group, respectively. Taken together, these findings may support the development of novel cancer therapies that can improve the response rate to ICIs.

Butyricimonas is a key gut microbiome component for predicting postoperative recurrence of esophageal cancer.

BACKGROUND: Recently, intestinal bacteria have attracted attention as factors affecting the prognosis of patients with cancer. However, the intestinal microbiome is composed of several hundred types of bacteria, necessitating the development of an analytical method that can allow the use of this information as a highly accurate biomarker. In this study, we investigated whether the preoperative intestinal bacterial profile in patients with esophageal cancer who underwent surgery after preoperative chemotherapy could be used as a biomarker of postoperative recurrence of esophageal cancer. METHODS: We determined the gut microbiome of the patients using 16S rRNA metagenome sequencing, followed by statistical analysis. Simultaneously, we performed a machine learning analysis using a random forest model with hyperparameter tuning and compared the data obtained. RESULTS: Statistical and machine learning analyses revealed two common bacterial genera, Butyricimonas and Actinomyces, which were abundant in cases with recurrent esophageal cancer. Butyricimonas primarily produces butyrate, whereas Actinomyces are oral bacteria whose function in the gut is unknown. CONCLUSION: Our results indicate that Butyricimonas spp. may be a biomarker of postoperative recurrence of esophageal cancer. Although the extent of the involvement of these bacteria in immune regulation remains unknown, future research should investigate their presence in other pathological conditions. Such research could potentially lead to a better understanding of the immunological impact of these bacteria on patients with cancer and their application as biomarkers.

Nivolumab receptor occupancy on effector regulatory T cells predicts clinical benefit.

Immune checkpoint inhibitor discovery represents a turning point in cancer treatment. However, the response rates of solid tumors remain ~10%-30%; consequently, prognostic and immune-related adverse event (irAE) predictors are being explored. The programmed cell death protein 1 (PD-1) receptor occupancy (RO) of PD-1 inhibitors depends on the number of peripheral blood lymphocytes and their PD-1 expression levels, suggesting that the RO may be related to efficacy and adverse events. As PD-1 inhibition affects each T-cell subset differently, the RO of each cell population must be characterized. However, relevant data have not been reported, and the prognostic relevance of this parameter is not known. In this study, we aimed to clarify the association between the nivolumab RO in each T-cell population and patient prognosis and reveal the development of irAEs in nivolumab-treated patients. Thirty-two patients were included in the study, and the mean follow-up period was 364 days. The nivolumab RO on effector regulatory T cells (eTregs) was significantly lower in the group that presented clinical benefits, and a significant negative association was observed between PD-1 occupancy on eTregs and all-cause mortality. The results suggest that the nivolumab RO on eTregs may be a prognostic factor in PD-1 inhibitor therapy, implying that the inhibition of PD-1/PD-ligand 1 (PD-L1) signaling on eTregs may attenuate antitumor effects.

Turicibacter and Acidaminococcus predict immune-related adverse events and efficacy of immune checkpoint inhibitor.

Introduction: Immune checkpoint inhibitors have had a major impact on cancer treatment. Gut microbiota plays a major role in the cancer microenvironment, affecting treatment response. The gut microbiota is highly individual, and varies with factors, such as age and race. Gut microbiota composition in Japanese cancer patients and the efficacy of immunotherapy remain unknown. Methods: We investigated the gut microbiota of 26 patients with solid tumors prior to immune checkpoint inhibitor monotherapy to identify bacteria involved in the efficacy of these drugs and immune-related adverse events (irAEs). Results: The genera Prevotella and Parabacteroides were relatively common in the group showing efficacy towards the anti-PD-1 antibody treatment (effective group). The proportions of Catenibacterium (P = 0.022) and Turicibacter (P = 0.049) were significantly higher in the effective group than in the ineffective group. In addition, the proportion of Desulfovibrion (P = 0.033) was significantly higher in the ineffective group. Next, they were divided into irAE and non-irAE groups. The proportions of Turicibacter (P = 0.001) and Acidaminococcus (P = 0.001) were significantly higher in the group with irAEs than in those without, while the proportions of Blautia (P = 0.013) and the unclassified Clostridiales (P = 0.027) were significantly higher in the group without irAEs than those with. Furthermore, within the Effective group, Acidaminococcus and Turicibacter (both P = 0.001) were more abundant in the subgroup with irAEs than in those without them. In contrast, Blautia (P = 0.021) and Bilophila (P= 0.033) were statistically significantly more common in those without irAEs. Discussion: Our Study suggests that the analysis of the gut microbiota may provide future predictive markers for the efficacy of cancer immunotherapy or the selection of candidates for fecal transplantation for cancer immunotherapy.

Antibiotic Usage Reduced Overall Survival by over 70% in Non-small Cell Lung Cancer Patients on Anti-PD-1 Immunotherapy.

BACKGROUND/AIM: There is an increasing use of immunotherapy for non-small cell lung cancer (NSCLC) patients. The present study analysed the effect of antibiotic use on the outcome of NSCLC patients undergoing treatment with anti-programmed cell death-1 (anti-PD-1) immunotherapy. PATIENTS AND METHODS: This was a retrospective study of 69 NSCLC patients. Eighteen out of 69 patients received antibiotics within 21 days before or within 21 days after start of anti-PD-1 therapy. RESULTS: Patients treated with anti-PD-1 antibodies receiving antibiotics had greatly decreased objective response rate (ORR), overall survival (OS) and progression-free survival (PFS) compared to those who did not use antibiotics. Multivariate analysis showed that antibiotic treatment of patients on anti-PD-1 antibody therapy was an independent negative predictive factor of PFS; however, it was not a significant independent predictive factor of OS. CONCLUSION: Use of antibiotics within 21 days before and after anti-PD-1 treatment initiation in patients with NSCLC strongly reduced OS and PFS, suggesting the two treatments should not be combined.

Calcium/calmodulin-dependent regulation of Rac GTPases and Akt in histamine-induced chemotaxis of mast cells.

Histamine induces chemotaxis of mast cells through the histamine H4 receptor. This involves the activation of small GTPases, Rac1 and Rac2, downstream of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Activation of the H4 receptor also results in phospholipase C (PLC)-mediated calcium mobilization; however, it is unclear whether the PLC­calcium pathway interacts with the PI3K-Rac pathway. Here, we demonstrated that calcium mobilization regulates the PI3K-dependent activation of Rac GTPases through calmodulin. A PLC inhibitor (U73122) and an intracellular calcium chelator (BAPTA-AM) suppressed the histamine-induced activation of Rac, whereas the calcium ionophore ionomycin increased the active Rac GTPases, suggesting that intracellular calcium regulates the activation of Rac. The calmodulin antagonist (W-7) inhibited the histamine-induced activation of Rac and migration of mast cells, indicating that calmodulin mediates the effect of calcium. Inhibition of calcium/calmodulin signaling suppressed histamine-induced phosphorylation of Akt. The Akt inhibitor MK-2206 attenuated histamine-induced migration of mast cells. However, it did not suppress the activation of Rac GTPases. These results suggest that Rac GTPases and Akt play independent roles in the histamine-induced chemotaxis of mast cells. Our findings enable further elucidation of the molecular mechanism of histamine-induced chemotaxis of mast cells and help identify therapeutic targets for allergic and inflammatory conditions involving mast cell accumulation.

Distinct Roles of Small GTPases Rac1 and Rac2 in Histamine H4 Receptor-Mediated Chemotaxis of Mast Cells.

Histamine induces chemotaxis of mast cells through the H4 receptor. However, little is known about the precise intracellular signaling pathway that mediates this process. In this study, we identified small GTPases Rac1 and Rac2 as intracellular binding partners of the H4 receptor and characterized their roles in H4 receptor signaling. We showed that histamine induced Rac GTPase activation via the H4 receptor. A Rac inhibitor NSC23766 attenuated chemotaxis of mast cells toward histamine, as well as histamine-induced calcium mobilization and extracellular signal-regulated kinase (ERK) activation. Histamine-induced migration of mast cells was also sensitive to PD98059, an inhibitor of the mitogen-activated protein kinase kinase, indicating that the Rac-ERK pathway was involved in chemotaxis through the H4 receptor. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) by LY294002 suppressed the histamine-induced chemotaxis and activation of Rac GTPases, suggesting that PI3K regulates chemotaxis upstream of Rac activation. Specific knockdown of Rac1 and Rac2 by short-hairpin RNA revealed that both Rac GTPases are necessary for histamine-induced migration. Downregulation of Rac1 and Rac2 led to attenuated response in calcium mobilization and ERK activation, respectively. These observations suggested that Rac1 and Rac2 have distinct and essential roles in intracellular signaling downstream of H4 receptor-PI3K in histamine-induced chemotaxis of mast cells.

Expression levels of UL16 binding protein 1 and natural killer group 2 member D affect overall survival in patients with gastric cancer following gastrectomy.

UL16 binding protein 1 (ULBP1) expressed on the tumor cell surface binds to the natural killer group 2 member D (NKG2D) receptor presenting on natural killer (NK), cluster of differentiation (CD)8+ T, and γ δ T cells. However, the roles of ULBP1 and NKG2D expression and associated immune responses in gastric cancer are unclear. The present study investigated the associations between ULBP1 and NKG2D expression and clinical outcomes in patients with gastric cancer. The levels of ULBP1 and NKG2D expression were examined in human gastric cancer cell lines and gastric cancer tissues from 98 patients who underwent surgery from 2004 to 2008. MKN-74 cells expressed ULBP1 with ULBP2, -5, or -6. NKG2D was expressed at a higher level following activation of T cells and NK cells. Among the tissue sections positive for NKG2D expression, 6 patients were positive for CD8 and CD56. In all tissues, NKG2D-expressing cells were typically aCD8+ T cells. Patients with NKG2D expression in tumors exhibited significantly longer overall survival (OS) compared with patients without NKG2D expression in tumors (P=0.0217). The longest OS was observed in patients positive for ULBP1 and NKG2D, whereas the shortest OS was observed in patients negative for ULBP1 and NKG2D. The interaction between ULBP1 and NKG2D may improve OS in patients with gastric cancer, and may have applications in immunotherapy for the induction of adaptive immunity in patients with cancer. Additionally, ULBP1 and NKG2D may be useful as prognostic biomarkers in gastric cancer.

Correlation Between NKG2DL Expression and Antitumor Effect of Protein-bound Polysaccharide-K in Tumor-bearing Mouse Models.

BACKGROUND/AIM: We investigated the relationship between the expression of natural killer group 2, member D ligands (NKG2DLs) and the antitumor effects of protein-bound polysaccharide-K (PSK). MATERIALS AND METHODS: PSK was administered to evaluate its effectiveness against tumor growth. The expression of Rae-1 and H60 were analyzed in multiple cell lines. RESULTS: PSK showed the highest antitumor effects in mice implanted with cells expressing neither Rae-1 nor H60. PSK had little antitumor effect in mice implanted with cells expressing both Rae-1 and H60. A correlation between the expression of NKG2DLs and the antitumor effect of PSK was observed. After PSK administration, INF-γ production in CD8+ T cells increased in mice with cells expressing neither Rae-1 nor H60, but did not change in mice implanted with cells expressing both Rae-1 and H60. CONCLUSION: We demonstrated that the expression of NKG2DLs affects tumor immunity and the efficacy of immuno therapy in tumor-bearing mouse model.

Calreticulin is highly expressed in pancreatic cancer stem-like cells.

Cancer stem-like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P-CSLCs). A P-CSLC-enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by 2-D electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P-CSLCs compared to parental cells. Flow cytometry analysis indicated that CRT was mostly localized to the surface of P-CSLCs and did not correlate with the levels of CD44v9, another P-CSLC biomarker. Furthermore, the side population in the CRThigh /CD44v9low population was much higher than that in the CRTlow /CD44v9high population. Calreticulin expression was also assessed by immunohistochemistry in pancreatic cancer tissues (n = 80) obtained after radical resection and was found to be associated with patients' clinicopathological features and disease outcomes in the Cox proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and postoperative therapy. Our results suggest that CRT can serve as a biomarker of P-CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be considered as a therapeutic target for cancer immunotherapy.

Combination immunotherapy with 4-1BB activation and PD-1 blockade enhances antitumor efficacy in a mouse model of subcutaneous tumor.

BACKGROUND/AIM: The purpose of the present study was to establish an effective immunotherapy by skewing the cosignal balance to be on the positive side by using the combination of monoclonal antibody (mAb) against 4-1BB also known as Cluster of Differentiation (CD) 137 as a co-stimulatory effector and to programmed death-1 (PD-1) to blockade the immune checkpoint. MATERIALS AND METHODS: Mice implanted with 1×10(5) CT26 cells were treated with anti 4-1BB mAb alone, anti PD-1 mAb alone, or both anti 4-1BB mAb and anti PD-1 mAb. Immune cell populations were analyzed by flow cytometry. Tumor-infiltrating T-cells were evaluated by immunohistochemistry. RESULTS: Mice treated with the combination therapy had the best antitumor response that resulted in complete tumor rejection. The numbers of CD4(+) interferon (IFN)-γ(+) and CD8(+) IFN-γ(+) T-cells were significantly higher in the combination group. The number of tumor-infiltrating T-cells was significantly increased in the combination therapy. CONCLUSION: The therapeutic strategy of targeting co-signal molecules has promising clinical applications in the future.

A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population.

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLC-enriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.

Expression of anti-HVEM single-chain antibody on tumor cells induces tumor-specific immunity with long-term memory.

Genetic engineering of tumor cells to express immune-stimulatory molecules, including cytokines and co-stimulatory ligands, is a promising approach to generate highly efficient cancer vaccines. The co-signaling molecule, LIGHT, is particularly well suited for use in vaccine development as it delivers a potent co-stimulatory signal through the Herpes virus entry mediator (HVEM) receptor on T cells and facilitates tumor-specific T cell immunity. However, because LIGHT binds two additional receptors, lymphotoxin ß receptor and Decoy receptor 3, there are significant concerns that tumor-associated LIGHT results in both unexpected adverse events and interference with the ability of the vaccine to enhance antitumor immunity. In order to overcome these problems, we generated tumor cells expressing the single-chain variable fragment (scFv) of anti-HVEM agonistic mAb on the cell surface. Tumor cells expressing anti-HVEM scFv induce a potent proliferation and cytokine production of co-cultured T cells. Inoculation of anti-HVEM scFv-expressing tumor results in a spontaneous tumor regression in CD4+ and CD8+ T cell-dependent fashion, associated with the induction of tumor-specific long-term memory. Stimulation of HVEM and 4-1BB co-stimulatory signals by anti-HVEM scFv-expressing tumor vaccine combined with anti-4-1BB mAb shows synergistic effects which achieve regression of pre-established tumor and T cell memory specific to parental tumor. Taken in concert, our data suggest that genetic engineering of tumor cells to selectively potentiate the HVEM signaling pathway is a promising antitumor vaccine therapy.

Herpesvirus entry mediator regulates hypoxia-inducible factor-1α and erythropoiesis in mice.

Erythropoiesis, the production of red blood cells, must be tightly controlled to ensure adequate oxygen delivery to tissues without causing thrombosis or stroke. Control of physiologic and pathologic erythropoiesis is dependent predominantly on erythropoietin (EPO), the expression of which is regulated by hypoxia-inducible factor (HIF) activity in response to low oxygen tension. Accumulating evidence indicates that oxygen-independent mediators, including inflammatory stimuli, cytokines, and growth factors, also upregulate HIF activity, but it is unclear whether these signals also result in EPO production and erythropoiesis in vivo. Here, we found that signaling through herpesvirus entry mediator (HVEM), a molecule of the TNF receptor superfamily, promoted HIF-1α activity in the kidney and subsequently facilitated renal Epo production and erythropoiesis in vivo under normoxic conditions. This Epo upregulation was mediated by increased production of NO by renal macrophages. Hvem-deficient mice displayed impaired Epo expression and aggravated anemia in response to erythropoietic stress. These data reveal that HVEM signaling functions to promote HIF-1α activity and Epo production, and thus to regulate erythropoiesis. Furthermore, our findings suggest that this molecular mechanism could represent a therapeutic target for Epo-responsive diseases, including anemia.

The hydrophobic amino acids in putative helix 8 in carboxy-terminus of histamine H(3) receptor are involved in receptor-G-protein coupling.

Functional roles of putative helix 8 in the carboxy-terminal tail of the human histamine H(3) receptor were investigated using deleted and alanine-substituted mutant receptors. While the deletion of the carboxy-terminal tail did not decrease the total expression level, surface expression, or ligand binding affinity, the agonist-stimulated cAMP response, [((35))S] GTPγS binding, and MAPK activation were totally abolished. The receptor lacking the carboxy-terminal tail also failed to respond to an inverse agonist, thioperamide, suggesting that the carboxy-terminal tail is involved in the regulation of receptor activity by changing G-protein coupling with the receptor. Site-directed mutagenesis revealed that hydrophobic amino acids in the putative helix 8 such as phenylalanines at position 419 (F7.60) and 423 (F7.64) or leucines at 426 (L7.67) and 427 (L7.68) were important for the agonist-induced activation of H(3) receptor. Substitution of F7.60 also resulted in a receptor that was less responsive to inactivation by the inverse agonist, implying the existence of an intermediate conformation that can be either activated or inactivated. Our results suggest that hydrophobic interface of putative helix 8 is important for the regulation of H(3) receptor activity, presumably by stabilizing the helix to the plasma membrane.

Dichotomous regulation of GVHD through bidirectional functions of the BTLA-HVEM pathway.

B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor that interacts with herpesvirus entry mediator (HVEM), and this interaction regulates pathogenesis in various immunologic diseases. In graft-versus-host disease (GVHD), BTLA unexpectedly mediates positive effects on donor T-cell survival, whereas immunologic mechanisms of this function have yet to be explored. In this study, we elucidated a role of BTLA in GVHD by applying the newly established agonistic anti-BTLA monoclonal antibody that stimulates BTLA signal without antagonizing BTLA-HVEM interaction. Our results revealed that provision of BTLA signal inhibited donor antihost T-cell responses and ameliorated GVHD with a successful engraftment of donor hematopoietic cells. These effects were dependent on BTLA signal into donor T cells but neither donor non-T cells nor recipient cells. On the other hand, expression of BTLA mutant lacking an intracellular signaling domain restored impaired survival of BTLA-deficient T cells, suggesting that BTLA also serves as a ligand that delivers HVEM prosurvival signal in donor T cells. Collectively, current study elucidated dichotomous functions of BTLA in GVHD to serve as a costimulatory ligand of HVEM and to transmit inhibitory signal as a receptor.

Roles of hypothalamic subgroup histamine and orexin neurons on behavioral responses to sleep deprivation induced by the treadmill method in adolescent rats.

Sleep deprivation induces several negative effects on behavior, emotion, attention, and learning ability. Sleep appears to be particularly important during adolescent brain development. In the present study, we examined the effects of sleep deprivation on behavior and hypothalamic neurotransmission including histamine and orexin neurons in adolescent rats using the treadmill method. Adolescent male rats were divided into three groups: treadmill sleep-deprived, treadmill control, and cage control groups. Energy expenditure, anxiety-like behavior, and locomotor activity were examined among the three groups. Histamine concentration in the cortex and diencephalon and the number of c-Fos-positive neurons in the hypothalamus were also examined. In addition, histamine and orexin neurons in the hypothalamus were simultaneously identified using rat histidine decarboxylase and orexin-A immunohistochemistry, respectively. Both energy expenditure and anxiety-related behavior significantly increased by the experimental 3-day sleep deprivation, while exploratory locomotor activity significantly decreased. Histamine contents did not change in the cortex, but significantly decreased in the diencephalon of sleep-deprived rats. Increased expression of c-Fos-positive neurons, including subgroup histamine and orexin neurons, was observed in the hypothalamus. These findings indicate that sleep deprivation increases energy expenditure and anxiety in adolescent rats and provide evidence for the pivotal role of hypothalamus subgroup histamine and orexin neurons in the behavioral response to sleep deprivation.

B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance.

T-cell tolerance is the central program that prevents harmful immune responses against self-antigens, in which inhibitory PD-1 signal given by B7-H1 interaction plays an important role. Recent studies demonstrated that B7-H1 binds CD80 besides PD-1, and B7-H1/CD80 interaction also delivers inhibitory signals in T cells. However, a role of B7-H1/CD80 signals in regulation of T-cell tolerance has yet to be explored. We report here that attenuation of B7-H1/CD80 signals by treatment with anti-B7-H1 monoclonal antibody, which specifically blocks B7-H1/CD80 but not B7-H1/PD-1, enhanced T-cell expansion and prevented T-cell anergy induction. In addition, B7-H1/CD80 blockade restored Ag responsiveness in the previously anergized T cells. Experiments using B7-H1 or CD80-deficient T cells indicated that an inhibitory signal through CD80, but not B7-H1, on T cells is responsible in part for these effects. Consistently, CD80 expression was detected on anergic T cells and further up-regulated when they were re-exposed to the antigen (Ag). Finally, blockade of B7-H1/CD80 interaction prevented oral tolerance induction and restored T-cell responsiveness to Ag previously tolerized by oral administration. Taken together, our findings demonstrate that the B7-H1/CD80 pathway is a crucial regulator in the induction and maintenance of T-cell tolerance.

Leucocyte-associated immunoglobulin-like receptor-1 is an inhibitory regulator of contact hypersensitivity.

Leucocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a membrane receptor of the immunoglobulin (Ig) superfamily that is expressed on most types of haematopoietic cells, and delivers inhibitory signals through interacting with collagens. In order to elucidate the immunological functions of LAIR-1 in vivo, we established transgenic mice expressing a chimeric protein composed of the extracellular domain of LAIR-1 fused with an Ig tag (LAIR-1-Ig), which acts as a decoy by competing with endogenous LAIR-1. The transgenic mice showed an increased susceptibility for development of contact hypersensitivity (CHS), an experimental model of allergic contact dermatitis, in association with enhanced hapten-specific T-cell responses. When T cells from the hapten-sensitized donor mice were transferred into non-sensitized recipients, treatment of either donor mice or recipient mice with LAIR-1-Ig protein accelerated CHS, suggesting a potentially negative role of LAIR-1 in both the sensitization and the elicitation of hapten-reactive T cells. In vitro assays revealed that LAIR-1 decreased the production of interleukin-6 and interleukin-12 in dendritic cells, and inhibited the proliferation and cytokine production of naïve and memory T cells along with G(0)/G(1) cell cycle arrest. Collectively, our findings suggest that LAIR-1 plays a crucial inhibitory role in CHS by regulating antigen-presenting cell and T-cell functions.

Roles of the histaminergic neurotransmission on methamphetamine-induced locomotor sensitization and reward: a study of receptors gene knockout mice.

Methamphetamine (METH) is often abused as a psychostimulant, and its administration induces several abnormal behaviors. We propose that neuronal histamine has an inhibitory role on the METH-induced locomotor hyperactivity and development of behavioral sensitization. We examined the roles of the histaminergic neuron system on behavioral sensitization and conditioned place preference (CPP) induced by METH using single and multiple histamine receptors deficient mice. Mice were injected intraperitoneally seven times with METH (1mg/kg) once in every 3 days. After drug-free intervals of 7 days, METH was rechallenged. The locomotor activities were gradually increased in histamine H1, H3 receptor gene double knockout (H1/H3-DKO), H1, H2, and H3 receptor gene triple knockout (TKO), and their wild-type (WT) mice when METH was repeatedly administrated, suggesting that these mice developed behavioral sensitization. The ratios of the locomotor activity in METH-administrated group to saline-treated group were not significantly changed among the different genotypes. The order of ratios were H1/H3-DKO > WT mice > TKO mice. We also examined METH-induced CPP in histamine H1 receptor gene knockout mice (H1-KO), H3 receptor gene knockout mice (H3-KO), and their WT mice. The CPP scores were increased by repeated METH administration. Especially, H1-KO mice showed higher METH-induced CPP scores than those of the WT and H3-KO mice. Our results suggest that the neuronal histamine could inhibit the METH-induced abnormal behaviors through the interactions of H1, H2, and H3 receptors.