In vivo delivery of interferon-α gene enhances tumor immunity and suppresses immunotolerance in reconstituted lymphopenic hosts.

T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c(+) cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c(+) cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c(+) cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.

Application of innate immune molecules for a new class of drugs: infection, inflammation and beyond.

The innate immune system plays an important role systemically and locally in infectious and inflammatory diseases. Vaccines, vaccine adjuvants and anti-inflammatory drugs were developed by understanding mechanisms of the innate immune system and causative factors of infection and inflammatory diseases. Pattern-recognition receptors, such as Toll-like receptors, retinoic acid-inducible gene I (RIG-I)-like helicases and nucleotide-binding oligomerization domain(NOD)-like receptors, and their downstream signals have great potential as targets of therapeutics because they are involved in numerous diseases. Furthermore, proteolytic systems such as autophagy and immunoproteasomes play important roles in the innate immune system, making them potential therapeutic targets also. By taking advantage of the immune system, humankind has made a great effort to develop new therapeutic and preventive medicines. Accordingly, we have reported several studies on the development of vaccines and adjuvants based on novel mechanistic strategies. Additionally, we have elucidated the mechanism underlying an interaction between innate immunity and the endocrine system. This review introduces the possible use of innate immune molecules for the development of immunomodulatory drugs and the involvement of the immune system in endocrine metabolic diseases to discuss future applications of innate immune molecules to therapeutics of various inflammatory diseases.

Mutant mouse p53 transgene elevates the chemical induction of tumors that respond to gene silencing with siRNA.

To study the role of mutant p53 in the induction and cure of tumors, we generated transgenic mice carrying mutant p53 (mp53) containing a 9 bp deletion in exon 6 in addition to wild-type p53, expressing both p53 and mp53. The mp53 cDNA was cloned from a radiation-induced mouse tumor and ligated to the chicken beta-actin promoter/CMV-IE enhancer in the expression vector. The presence of mp53 suppressed p21 expression in primary fibroblasts after ionizing irradiation, indicating the dominant-negative activity of mp53 in the mice. These mice developed fibrosarcomas after the subcutaneous injection of 3-methylcholanthrene with an incidence 1.7-fold higher than that of wild-type mice (42% excess). The tumors were then treated via a potent atelocollagen delivery system with small interfering RNA (siRNA), that targeted the promoter/enhancer of the expression vector, resulting in the suppression of tumor growth in 30% of 44 autochthonous tumors, including four cures, and their transplants, the total fraction corresponding to the tumor excess. This suppressive effect involved the induction of apoptosis. These results indicate that mp53 activity causes tumors that can be suppressed by subsequent silencing of mp53 in the presence of wild-type p53 alleles.

Tryptophan aspartate-containing coat protein (CORO1A) suppresses Toll-like receptor signalling in Mycobacterium leprae infection.

Mycobacterium leprae is an intracellular pathogen that survives within the phagosome of host macrophages. Several host factors are involved in producing tolerance, while others are responsible for killing the mycobacterium. Tryptophan aspartate-containing coat protein (TACO; also known as CORO1A or coronin-1) inhibits the phagosome maturation that allows intracellular parasitization. In addition, the Toll-like receptor (TLR) activates the innate immune response. Both CORO1A and TLR-2 co-localize on the phagosomal membrane in the dermal lesions of patients with lepromatous leprosy. Therefore, we hypothesized that CORO1A and TLR-2 might interact functionally. This hypothesis was tested by investigating the effect of CORO1A in TLR-2-mediated signalling and, inversely, the effect of TLR-2-mediated signalling on CORO1A expression. We found that CORO1A suppresses TLR-mediated signal activation in human macrophages, and that TLR2-mediated activation of the innate immune response resulted in suppression of CORO1A expression. However, M. leprae infection inhibited the TLR-2-mediated CORO1A suppression and nuclear factor-kappaB activation. These results suggest that the balance between TLR-2-mediated signalling and CORO1A expression will be key in determining the fate of M. leprae following infection.

MicroRNAs as biomarkers and therapeutic drugs in human cancer.

MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, noncoding small RNAs that act as post-transcriptional gene regulators. Experimental evidence has shown that miRNAs can play roles as oncogenes or tumor suppressor genes, suggesting their contribution to cancer development and progression. Expression profiles of human miRNAs demonstrated that many miRNAs are deregulated in cancers and are differentially expressed in normal tissues and cancers. Therefore, miRNA profiling is used to create signatures for a variety of cancers, indicating that the profile will help further establish molecular diagnosis, prognosis and therapy using miRNAs. This paper introduces the aberrant expression of miRNAs in human cancer, and discusses the potential of these miRNAs as biomarkers and targets/molecules for molecular therapy.

Streptozotocin-induced partial beta cell depletion in nude mice without hyperglycaemia induces pancreatic morphogenesis in transplanted embryonic stem cells.

AIMS/HYPOTHESIS: It appears that the adult pancreas has limited regenerative ability following beta cell destruction by streptozotocin (STZ). However, it is not clear if this limitation is due to an inability to respond to, rather than an absence of, regenerative stimuli. In this study we aimed to uncouple the regenerative signal from the regenerative response by using an exogenous stem cell source to detect regenerative stimuli produced by the STZ-injured pancreas at physiological blood glucose levels. METHOD: Adult nude mice received 150 mg/kg STZ and 1x10(6) J1 mouse embryonic stem (ES) cells by i.p. injection. Permanent beta cell depletion of 50% was estimated from the ratio of beta:alpha cells in pancreata from STZ-treated mice compared with control animals after 24 days. RESULTS: Transplanted ES cells homed to the STZ-injured pancreas and formed tumours. Immunocytochemical analysis of pancreas-associated ES tumours revealed foci containing insulin/PDX-1 double-positive and glucagon-positive/PDX-1-negative cell clusters associated with PDX-1-positive columnar lumenal epithelium and extensive alpha-amylase-positive pancreatic acini comprising approximately 0.1% of ES tumour volume. CONCLUSIONS/INTERPRETATION: These data indicate that (1) the adult pancreas produces a milieu of regenerative stimuli following beta cell destruction, and (2) this is not dependent on hyperglycaemic conditions; (3) these regenerative stimuli appear to recapitulate the signalling pathways of embryonic development, since both exocrine and endocrine lineages are produced from PDX-1-positive precursor epithelium. This model will be useful for characterising the regenerative mechanisms in the adult pancreas.

Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV.

Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. This study examines the safety and immunogenicity of a replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) in BALB/c mice and rhesus monkeys. This novel Ad5/35 vector showed minimal hepatotoxicity after intramuscular administration with the novel Ad5/35 vector. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use.

Upregulation of T-cell-stimulating activity of mycobacteria-infected macrophages.

Macrophages are one of the most abundant host cells to come in contact with mycobacteria. However, the infected macrophages less efficiently stimulate autologous T cells in vitro. We investigated the effect of the induction of phenotypic change of macrophages on the host cell activities by using Mycobacterium leprae as a pathogen. The treatment of macrophages with interferon-gamma (IFN-gamma), GM-CSF and interleukin-4 deprived macrophages of CD14 antigen expression but instead provided them with CD1a, CD83 and enhanced CD86 antigen expression. These phenotypic features resembled those of monocyte-derived dendritic cells (DC). These macrophage-derived DC-like cells (MACDC) stimulated autologous CD4+ and CD8+ T cells when infected with M. leprae. Further enhancement of the antigen-presenting function and CD1a expression of macrophages was observed when treated with IFN-gamma. The M. leprae-infected and -treated macrophages expressed bacterial cell membrane-derived antigens on the surface and were efficiently cytolysed by the cell membrane antigen-specific CD8+ cytotoxic T lymphocytes (CTL). These results suggest that the induction of phenotypic changes in macrophages can lead to the upregulation of host defence activity against M. leprae.

Activation of microglia and astrocytes by CpG oligodeoxynucleotides.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate cells of the immune system to secrete a variety of cytokines and chemokines. This function can be carried out by microglia and astrocytes in the CNS. To evaluate the effect of CpG ODN on microglia and astrocytes, purified cells were isolated and cultured in vitro. CpG ODN rapidly up-regulated their production of IL-1beta, IL-6, IL-12, TNFalpha, MIP-1alpha and/or MIP-1beta. In vivo, systemically administered CpG ODN up-regulated the expression of mRNA encoding cytokines and chemokines in normal mouse brain. These findings suggest that CpG ODN can directly activate immune cells of the CNS.

Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells.

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.

Genomic DNA released by dying cells induces the maturation of APCs.

Mature APCs play a key role in the induction of Ag-specific immunity. This work examines whether genomic DNA released by dying cells provides a stimulus for APC maturation. Double-stranded but not single-stranded genomic DNA triggered APC to up-regulate expression of MHC class I/II and various costimulatory molecules. Functionally, dsDNA enhanced APC function in vitro and improved primary cellular and humoral immune responses in vivo. These effects were dependent on the length and concentration of the dsDNA but were independent of nucleotide sequence. The maturation of APC induced by dsDNA may promote host survival by improving immune surveillance at sites of tissue injury/infection.

Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor alpha 2 chain.

Interleukin (IL)-13 receptor alpha2 (IL-13Ralpha2) chain is an essential binding component for IL-13-mediated ligand binding. Recently, we have demonstrated that this receptor chain also plays an important role in the internalization of IL-13. To study the mechanism of IL-13 internalization, we generated mutated IL-13Ralpha2 chains that targeted trileucine residues (Leu(335), Leu(336), and Leu(337)) in the transmembrane domain and a tyrosine motif (Tyr(343)) in the intracellular domain and transfected these cDNAs in COS-7 cells. Cells that expressed a C-terminally truncated IL-13Ralpha2 chain (Delta335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13Ralpha2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Ralpha2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Ralpha2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Ralpha2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR) to cells that expressed IL-13Ralpha2 chain mutants. We further demonstrate that the IL-13Ralpha2 chain is not ubiquitinated and that internalization of IL-13Ralpha2 did not depend on ubiquitination. Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Ralpha2 internalization in distinct manners.

Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice.

For better understanding of cancer metastasis, we have established an in vivo model for induction of highly metastatic hepatocellular carcinomas (HCC) in male F344 rats. From 1 tumor, 4 cell lines with differing metastatic potential (C1, C2, C6, C5F) were established by subcloning using the limited-dilution cloning technique. Two other lines, N1 and L2, arose from another primary HCC and a lung metastatic lesion, respectively. Although cell adhesion of each cell line in culture medium was different, tumors developing in the subcutis of nude mice after transplantation were all moderately differentiated HCC with a trabecular pattern. On subcutaneous injection into nude mice, all 6 cell lines proved to be tumorigenic in the injection site and C5F was highly metastatic to the lung. With injection into the tail vein, N1 and L2 formed frequent metastases in the lung as well as in lymph nodes. Using intraperitoneal injection, C1, C6, N1 and L2 showed marked disseminated growth in the abdominal cavity with bloody ascitis. Northern blot analysis revealed expression of known metastasis-related genes, KAI1 and heparanase, to be decreased in C5F, but no differences in expression of nm23-H1 were evident. A point mutation in the GSK-3beta phosphorylation site of the beta-catenin gene was found in L2. These transplantable HCC cell lines that have different metastatic ability should be useful for elucidation of mechanisms of metastasis.

Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs.

Oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides trigger a strong innate immune response in vertebrates. CpG ODN show promise as vaccine adjuvants, anti-allergens, and immunoprotective agents in animal models. Their transition to clinical use requires the identification of motifs that are optimally stimulatory in humans. Analysis of hundreds of novel ODN resulted in the identification and characterization of two structurally distinct "clusters" of immunostimulatory CpG ODN. One cluster ("D") preferentially stimulates IFN-gamma production by NK cells, whereas the other ("K") stimulates cell proliferation and the production of IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory properties of K and D ODN can improve the design of CpG-based products to achieve specific therapeutic goals.

Mycobacterium leprae and leprosy: a compendium.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which was discovered by G.H.A. Hansen in 1873. M. leprae is an exceptional bacterium because of its long generation time and no growth in artificial media. Entire sequencing of the bacterial genome revealed numerous pseudogenes (inactive reading frames with functional counterparts in M. tuberculosis) which might be responsible for the very limited metabolic activity of M. leprae. The clinical demonstration of the disease is determined by the quality of host immune response. Th1-type immune response helps to kill the bacteria, but hosts are encroached upon when Th2-type response is predominant. The bacteria have affinity to the peripheral nerves and are likely to cause neuropathy. M. leprae/laminin-alpha2 complexes bind to alpha/beta dystroglycan complexes expressed on the Schwann cell surface. WHO recommends a chemotherapy protocol [multidrug therapy (MDT)] which effectively controls the disease and contributes to the global elimination program. Leprosy has been stigmatized throughout history, and recent topics regarding the disease in Japan are also discussed.

CpG ODN-mediated regulation of IL-12 p40 transcription.

Oligodeoxynucleotides (ODN) containing CpG motifs activate RAW 264.7 mouse macrophages and RPMI 8226 human myeloma cells to produce IL-12 p40. Using deletion and site-directed mutagenesis, the nuclear factor (NF)-kappaB half-site and the CCAAT/enhancer binding protein (C/EBP) recognition site were identified as potent cis-acting elements in CpG ODN-mediated IL-12 p40 promoter activation. Several NF-kappaB/Rel proteins competed for binding to the NF-kappaB half-site. The p65/c-Rel and p65/p50 heterodimer occupied this site shortly after CpG ODN administration (0.5-2 h), while the p50/c-Rel heterodimer dominated binding in the late stage (8-12 h). The induction of p50/c-Rel heterodimer was associated with a significant expression of IL-12 p40 mRNA. C/EBPbeta also contributed to CpG ODN-mediated IL-12 p40 promoter activation.

Positive and negative regulatory elements contribute to CpG oligonucleotide-mediated regulation of human IL-6 gene expression.

Oligonucleotides (ODN) expressing immunostimulatory "CpG motifs" activate human RPMI 8226 myeloma cells to secrete IL-6. Using deletion and site-directed mutagenesis of the human (h)IL-6 promoter region, two positive regulatory elements (the binding sites for the 5'-CCAAT / enhancer binding protein-beta and NF-kappaB) were identified. Two negative regulatory elements, the 3'-retinoblastoma control element (RCE) and the binding site for Epstein-Barr virus C-promoter binding factor 1 (CBF1), also contributed to CpG ODN induction of hIL-6 gene expression. Of interest, CpG ODN treatment induced the dissociation of a repressor protein from its 3'-RCE binding site. Thus, CpG ODN regulation of hIL-6 gene expression involves both enhancer and derepression mechanisms.

CpG oligodeoxynucleotides induce murine macrophages to up-regulate chemokine mRNA expression.

Intramuscular injection of synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs trigger the rapid development of a local inflammatory response. In vitro studies demonstrate that macrophages exposed to CpG ODN up-regulate expression of mRNA encoding the chemokines MIP-1alpha, MIP-1beta, MIP-2, RANTES, JE/MCP-1, and IP-10. Within 6 h of in vivo administration, CpG ODN induce a significant increase in chemokine mRNA levels at the site of injection and draining lymph nodes. These chemokines may contribute to the migration and stimulation of inflammatory cells that contribute to the development of CpG ODN-induced immune responses.

Immunotherapeutic applications of CpG-containing oligodeoxynucleotides.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs stimulate the mammalian immune system to mount a rapid innate immune response. This response is characterized by the production of polyreactive IgM, immunomodulatory cytokines and chemokines. CpG ODN directly stimulate lymphocytes, natural killer cells and professional antigen-presenting cells (such as macrophages and dendritic cells). Owing to the strength and nature of this stimulation, CpG ODN are being harnessed for a variety of therapeutic uses. They are being tested for their ability to act as immune adjuvants, boosting the immune response elicited by conventional and DNA vaccines. As a result of their ability to activate a strong interferon gamma-dominated Th1 response while blocking the development of Th2-dependent allergies, CpG ODN are being examined for their antiallergic properties. Finally, CpG ODN are being used as "immunoprotective agents", since the innate immune response they elicit can protect the host from a variety of pathogenic bacteria, viruses and parasites.