Mice homozygous for a single tyrosine mutation in LAT (linker for activation of T cells) exhibited an early block in T cell maturation but later developed a polyclonal lymphoproliferative disorder and signs of autoimmune disease. T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) and of nuclear factor of activated T cells, calcium influx, interleukin-2 production, and cell death were reduced or abrogated in T cells from LAT mutant mice. In contrast, TCR-induced Erk activation was intact. These results identify a critical role for integrated PLC-gamma1 and Ras-Erk signaling through LAT in T cell development and homeostasis.

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5−/− mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5−/−) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5−/− phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.

BackgroundThe screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers.ResultsWe present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O)) induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O) enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O) without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells.ConclusionsOur overall results show that Lipoplex(O) is a potent adjuvant and that complexes of peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) is a naturally occurring hydroxylated compound commonly found in species of the subfamily Nepetoideae of the Lamiaceae and Boraginaceae, such as Rosmarinus officinalis, Salvia officinalis, and Perilla frutescens. RA is biosynthesized from the amino acids L-phenylalanine and L-tyrosine by eight enzymes that include phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase. RA can also be chemically produced by the esterification of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA and its numerous derivatives containing one or two RA with other aromatic moieties are well known and include lithospermic acid, yunnaneic acid, salvianolic acid, and melitric acid. Recently, RA and its derivatives have attracted interest for their biological activities, which include anti-inflammatory, anti-oxidant, anti-angiogenic, anti-tumor, and anti-microbial functions. Clinically, RA attenuates T cell receptor-mediated signaling, attenuates allergic diseases like allergic rhinitis and asthma, and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like symptoms, protects from neurotoxicity, and slows the development of Alzheimer's disease. These attributes have increased the demand for the biotechnological production and application of RA and its derivatives. The present review discusses the function and application of RA and its derivatives including the molecular mechanisms underlying clinical efficacy.

Allergy is characterized by an overreaction of the immune system. Perilla frutescens leaf extract has been reported to exhibit antiallergic inflammatory activity. To investigate precisely the effect and mechanism of 30% ethanol extract powder of P. frutescens var. acuta Kudo (EPPF) and rosmarinic acid (RA), a component of EPPF in allergic rhinitis and rhinoconjunctivitis, the antiallergic effects of EPPF and RA were analyzed using in vivo and in vitro models. Cytokine production was analyzed by means of an enzyme-linked immunosorbent assay. Cytokine expression was analyzed via reverse transcription-polymerase chain reaction and Western blotting. Transcription factor and caspase-1 activity were analyzed by a luciferase assay and caspase-1 assay, respectively. The number of nasal, ear and eye rubs after an ovalbumin (OVA) challenge in OVA-sensitized mice was significantly higher than that in OVA-unsensitized mice. Increased number of rubs was inhibited by administration of EPPF or RA. Increased levels of IgE in the serum, spleen and nasal mucosa of OVA-sensitized mice were reduced by EPPF or RA administration. The histamine level was also reduced by EPPF or RA administration in the serum of OVA-sensitized mice. Protein levels and mRNA expressions of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α were inhibited by EPPF or RA administration in the nasal mucosa tissue or spleen of OVA-sensitized mice. In EPPF or RA-administered mice, the mast cell and eosinophil infiltration increase as caused by OVA-sensitization was decreased. In addition, EPPF or RA inhibited both cyclooxygenase-2 protein expression and caspase-1 activity in the same nasal mucosa tissue. In activated human mast cells, nuclear factor-kappa B (NF-κB)/Rel A and caspase-1 activation increased, whereas NF-κB/Rel A and caspase-1 activation was inhibited after a treatment of EPPF or RA. These results indicate that EPPF and RA ameliorate allergic inflammatory reactions such as allergic rhinitis and allergic rhinoconjunctivitis.

MicroRNAs (miRNAs) are small, noncoding RNAs that play a crucial role in regulation of gene expression. Recent studies have shown that miRNAs implicated in initiation and progression of various human cancers, including breast cancer and also analysis of miRNA expression profiles in cancer provide new insights into potential mechanisms of carcinogenesis. Melatonin, N-acetyl-5-methoxytryptamine, is synthesized by the pineal gland in response to the dark/light cycle and has been known to act as a synchronizer of the biological clock. Melatonin has a variety of therapeutic effects, such as immunomodulatory actions, anti-inflammatory effects, and antioxidant actions. Furthermore, melatonin is reported to have an anticancer function including suppression of the metabolism of tumor cells and induction of tumor suppressor genes in cancer cells, including breast cancer cells. In this study, we determined whether miRNAs play a role in regulation of various gene expression responses to melatonin in MCF-7 human breast cancer cells. We examined whole-genome miRNA and mRNA expression and found that 22 miRNAs were differentially expressed in melatonin-treated MCF-7 cells. We further identified a number of mRNAs whose expression level shows a high inverse correlation with miRNA expression. The Gene Ontology (GO) enrichment analysis and pathways analysis were performed for identification of the signaling pathways and biological processes affected by differential expression of miRNA and miRNA-related genes. Our findings suggested that melatonin may modulate miRNA and gene expression as an anticancer mechanism in human breast cancer cells.

Although peptide vaccines have been actively studied in various animal models, their efficacy in treatment is limited. To improve the efficacy of peptide vaccines, we previously formulated an efficacious peptide vaccine without carriers using the natural phosphodiester bond CpG-DNA and a special liposome complex (Lipoplex(O)). Here, we show that immunization of mice with a complex consisting of peptide and Lipoplex(O) without carriers significantly induces peptide-specific IgG2a production in a CD4+ cells- and Th1 differentiation-dependent manner. The transmembrane 4 superfamily member 5 protein (TM4SF5) has gained attention as a target for hepatocellular carcinoma (HCC) therapy because it induces uncontrolled growth of human HCC cells via the loss of contact inhibition. Monoclonal antibodies specific to an epitope of human TM4SF5 (hTM4SF5R2-3) can recognize native mouse TM4SF5 and induce functional effects on mouse cancer cells. Pre-immunization with a complex of the hTM4SF5R2-3 epitope and Lipoplex(O) had prophylactic effects against tumor formation by HCC cells implanted in an mouse tumor model. Furthermore, therapeutic effects were revealed regarding the growth of HCC when the vaccine was injected into mice after tumor formation. These results suggest that our improved peptide vaccine technology provides a novel prophylaxis measure as well as therapy for HCC patients with TM4SF5-positive tumors.