A possible way to prevent the progression of bone lesions in multiple myeloma via Src-homology-region-2-domain-containing-phosphatase-1 activation.

On the basis of our recent findings, in which multiple receptor-mediated mast cell functions are regulated via a common signaling cascade, we posit that the formation and functioning of osteoclasts are also controlled by a similar common mechanism. These cells are derived from the same granulocyte/monocyte progenitors and share multiple receptors except those that are cell-specific. In both types of cells, all known receptors reside in lipid rafts, form multiprotein complexes with recruited signaling molecules, and are internalized upon receptor engagement. Signal transduction proceeds in a chain of protein phosphorylations, where adaptor protein LAT (linker-for-activation-of-T-cells) plays a central role. The key kinase that associates LAT phosphorylation and lipid raft internalization is Syk (spleen-tyrosine-kinase) and/or an Src-family-kinase, most probably Lck (lymphocyte-specific-protein-tyrosine-kinase). Dephosphorylation of phosphorylated Syk and Lck by activated SHP-1 (Src-homology-region-2-domain-containing-phosphatase-1) terminates the signal transduction and endocytosis of receptors, resulting in inhibition of osteoclast differentiation and other functions. In malignant plasma cells (MM cells) too, SHP-1 plays a similar indispensable role in controlling signal transduction required for survival and proliferation, though BLNK (B-cell-linker-protein), a functional equivalent of LAT and SLP-76 (SH2-domain-containing-leukocyte-protein-of-76-kDa) in B cells, is used instead of LAT. In both osteoclasts and MM cells, therefore, activated SHP-1 acts negatively in receptor-mediated cellular functions. In osteoblasts, however, activated SHP-1 promotes differentiation, osteocalcin generation, and mineralization by preventing both downregulation of transcription factors, such as Ostrix and Runx2, and degradation of ß-catenin required for activation of the transcription factors. SHP-1 is activated by tyrosine phosphorylation and micromolar doses (M-dose) of CCRI-ligand-induced SHP-1 activation. Small molecular compounds, such as A770041, Sorafenib, Nitedanib, and Dovitinib, relieve the autoinhibitory conformation. Activation of SHP-1 by M-dose CCRI ligands or the compounds described may prevent the progression of bone lesions in MM.

Anti-Metastatic Benefits Produced by Hyperthermia and a CCL3 Derivative.

Significant numbers of malignant tumor cells that have spread to surrounding tissues and other distant organs are often too small to be picked up in a diagnostic test, and prevention of even such small metastases should improve patient outcomes. Using a mouse model, we show in this article that intravenous administration of a human CCL3 variant carrying a single amino acid substitution after mild local hyperthermia not only induces tumor growth inhibition at the treated site but also inhibits metastasis. Colon26 adenocarcinoma cells (1 × 105 cells/mouse) were grafted subcutaneously into the right hind leg of syngeneic BALB/c mice and after nine days, when tumor size reached ~11 mm in diameter, the local tumor mass was exposed to high-frequency waves, by which intratumoral temperature was maintained at 42 °C for 30 min. Mice received the CCL3 variant named eMIP (2 µg/mouse/day) intravenously for five consecutive days starting one day after heat treatment. We found that tumor growth in eMIP recipients after hyperthermia was inhibited markedly but no effect was seen in animals treated with either hyperthermia or eMIP alone. Furthermore, the number of lung metastases evaluated after 18 days was dramatically reduced in animals receiving the combination therapy compared with all other controls. These results encourage future clinical application of this combination therapy.

Reduced Number of Lymphocytes by X-ray Irradiation: A Problem in a Combination Therapy Trial that Elicits the Abscopal Effect in Preclinical Studies Using Electron Beam Irradiation.

In preclinical studies with model animals, intravenous administration of a derivative of chemokine CCL3, named eMIP, after local electron-beam irradiation, not only enhanced tumor growth inhibition at a target site but also induced tumor killing beyond the treated site (a phenomenon known as the abscopal effect). eMIP works with alarmins such as high mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70) released from overexpressed tumor cells by irradiation. These alarmins at the irradiated tumor bed trap injected eMIP and, by forming complexes with eMIP, play a key role to recruit and activate tumor inhibitory natural killer (NK) cells and CD4+ and CD8+ T cells. Tumor type-specific secretion of gamma interferon from splenocytes was also demonstrated, which may also activate NK cells. During Phase 1 clinical studies using X-rays, however, no apparent abscopal effect was observed. Instead, we saw frequent reduction in numbers of lymphocytes in the peripheral blood of irradiated patients. The reduced number of lymphocytes recovered poorly once depleted, in contrast to neutrophils, and persisted for months after the treatment. This might have affected outcome after combination treatment of irradiation and eMIP. To enhance host defense mechanisms during and after photon-beam (X-ray) radiotherapy of a deep-seated tumor, it seems essential to keep lymphocytes undamaged by eliminating reactive oxygen species that are formed in the peripheral blood during irradiation.

Identification of the active portion of the CCL3 derivative reported to induce antitumor abscopal effect.

BACKGROUND AND PURPOSE: Intravenous administration of a single amino acid-substituted chemokine CCL3 derivative named eMIP elicits the abscopal effect (an effect distal to the target), after local irradiation at a tumor-bearing site. To distinguish the active portion of eMIP, we tested the antitumor activity of chemically synthesized partial peptides of eMIP. Synthetic peptide has various advantages in its clinical application. MATERIAL AND METHODS: Colon26 adenocarcinoma cells were implanted subcutaneously in the right and left flanks of mice. eMIP, CCL3 or any of synthesized peptides was administered intravenously, either after irradiating the right flank. The effect was evaluated by tumor-growth inhibition. RESULTS: Q/C peptide, a synthetic peptide of amino acids 22-51 of eMIP has no chemotaxis-inducing ability but yet enhanced tumor growth inhibition at the non-irradiated sites, recapitulating the effect of eMIP with local irradiation. Co-administration of this peptide and HSP70 also inhibited tumor growth. CONCLUSIONS: Q/C peptide maps to the eMIP ß-sheet: 3 adjacent anti-parallel strands connected by the ß-hairpins, is the active portion of eMIP necessary for an immunomodulatory antitumor effect. This experimental reduction furthers our understanding of the underlying mechanism of the abscopal effect. The data will open the way for therapeutic application of like peptides.

Macrophage inflammatory protein derivative ECI301 enhances the alarmin-associated abscopal benefits of tumor radiotherapy.

Radiotherapy can produce antitumor benefits beyond the local site of irradiation, an immune-based phenomenon known as the abscopal effect, but the mechanisms underlying these benefits are poorly understood. Preclinical studies of ECI301, a mutant derivative of macrophage inhibitory protein-1α, have shown that its administration can improve the antitumor effects of radiotherapy in a manner associated with a tumor-independent abscopal effect. In this article, we report that i.v. administration of ECI301 after intratumoral injection of tumor cell lysates can inhibit tumor growth, not only at the site of injection but also at nontreated sites. Effects of the tumor lysate were further recapitulated by intratumoral injection [corrected] of the alarmins HSP70 or HMGB1, but not HSP60, and i.v. administration [corrected] of ECI301 + HSP70 were sufficient to inhibit tumor growth. Although i.v. administration [corrected] of ECI301 + HMGB1 did not inhibit tumor growth, we found that administration of a neutralizing HMGB1 antibody neutralized the cooperative effects of ECI301 on tumor irradiation. Moreover, mice genetically deficient in TLR4, an immune pattern receptor that binds alarmins, including HMGB1 and HSP70, did not exhibit antitumor responses to irradiation with ECI301 administration. Although ECI301 was cleared rapidly from peripheral blood, it was found to bind avidly to HSP70 and HMGB1 in vitro. Our results suggest a model in which sequential release of the alarmins HSP70 and HMGB1 from a tumor by irradiation may trap circulating ECI301, thereby licensing or restoring tumor immunosurveillance capabilities of natural killer cells or CD4(+) and CD8(+) T cells against tumor cells that may evade irradiation. Cancer Res; 74(18); 5070-8. ©2014 AACR.

Alarmins released during local antitumor treatments play an essential role in enhancing tumor growth inhibition at treated and non-treated sites via a derivative of CCL3.

ECI301 (eMIP), a single amino-acid substituted CCL3 (MIP-1α), enhanced tumor growth inhibition and the abscopal effect (an effect distal to the target) following local antitumor therapy such as radiation, radiofrequency ablation (RFA), or hyperthermia treatment. The recent elucidation of the underlying mechanism may lead to a better antitumor therapy.

A novel liposome surface modification agent that prolongs blood circulation and retains surface ligand reactivity.

Liposomes are recognized as potentially useful drug carriers but many problems preclude practical medical application. Liposomes bind with serum proteins (opsonization) and are captured by the reticuloendothelial system cells in the liver and spleen, which limits their ability to deliver drugs to other target sites. Modification of lipids with flexible, hydrophilic polymers such as poly(ethylene glycol) (PEG) to yield sterically stabilized liposomes is one approach to improve liposome blood circulation and tissue distribution properties. In this study, we examined liposomes prepared using lipids modified with a new branched oligoglycerol (BGL) moiety for steric stabilization. This novel BGL comprised 14 glycerol units (termed BGL014) connected with flexible ether linkages, resulting in a branched cascade-like structure that is highly expanded in aqueous solution. BGL014 was coupled to 1,2-distearoylphosphatidylethanolamine to yield BGL014-modified lipids. Incorporation of BGL014 into liposomes (BGL014L) resulted in long blood circulation times, despite a much thinner fixed aqueous layer thickness compared to PEG formulations. BGL014 produced a liposome surface coating that appears to function through steric inhibition of non-specific protein binding without strong interference of specific protein-binding reactions. Liposome structure and functionality was maintained following BGL014-modification, as the incorporation ratio of drug remained high. These results suggest that the BGL014 modification of liposomes is a promising approach to produce stable and long circulating drug carriers capable of selective binding to specific proteins.

Quantitative analysis of eosinophil chemotaxis tracked using a novel optical device -- TAXIScan.

We have reported previously the development of an optically accessible, horizontal chemotaxis apparatus, in which migration of cells in the channel from a start line can be traced with time-lapse intervals using a CCD camera (JIM 282, 1-11, 2003). To obtain statistical data of migrating cells, we have developed quantitative methods to calculate various parameters in the process of chemotaxis, employing human eosinophil and CXCL12 as a model cell and a model chemoattractant, respectively. Median values of velocity and directionality of each cell within an experimental period could be calculated from the migratory pathway data obtained from time-lapse images and the data were expressed as Velocity-Directionality (VD) plot. This plot is useful for quantitatively analyzing multiple migrating cells exposed to a certain chemoattractant, and can distinguish chemotaxis from random migration. Moreover precise observation of cell migration revealed that each cell had a different lag period before starting chemotaxis, indicating variation in cell sensitivity to the chemoattractant. Thus lag time of each cell before migration, and time course of increment of the migrating cell ratio at the early stages could be calculated. We also graphed decrement of still moving cell ratio at the later stages by calculating the duration time of cell migration of each cell. These graphs could distinguish different motion patterns of chemotaxis of eosinophils, in response to a range of chemoattractants; PGD(2), fMLP, CCL3, CCL5 and CXCL12. Finally, we compared parameters of eosinophils from normal volunteers, allergy patients and asthma patients and found significant difference in response to PGD(2). The quantitative methods described here could be applicable to image data obtained with any combination of cells and chemoattractants and useful not only for basic studies of chemotaxis but also for diagnosis and for drug screening.

SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) is caused by an immune response to self-thyroid antigens and has a significant genetic component. Antisense RNA transcripts have been implicated in gene regulation. Here we have identified a novel zinc-finger gene, designated ZFAT (zinc-finger gene in AITD susceptibility region), as one of the susceptibility genes in 8q23-q24 through an initial association analysis using the probands in the previous linkage analysis and a subsequent association analysis of the samples from a total of 515 affected individuals and 526 controls. The T allele of the single-nucleotide polymorphism (SNP), Ex9b-SNP10 located in the intron 9 of ZFAT, is associated with increased risk for AITD (dominant model: odds ratio = 1.7, P = 0.000091). The Ex9b-SNP10 falls into the 3'-UTR of truncated-ZFAT (TR-ZFAT) and the promoter region of the small antisense transcript of ZFAT (SAS-ZFAT). In peripheral blood lymphocytes, SAS-ZFAT is exclusively expressed in CD19+ B cells and expression levels of SAS-ZFAT and TR-ZFAT seemed to correlate with the Ex9b-SNP10-T-associated ZFAT-allele, inversely and positively, respectively. The Ex9b-SNP10 is critically involved in the regulation of SAS-ZFAT expression in vitro and this expression results in a decreased expression of TR-ZFAT. These results suggested that the SNP-associated ZFAT-allele plays a critical role in B cell function by affecting the expression level of TR-ZFAT through regulating SAS-ZFAT expression and that this novel regulatory mechanism of SNPs might be involved in controlling susceptibility or resistance to human disease.

Induction of Bach1 and ARA70 gene expression at an early stage of adipocyte differentiation of mouse 3T3-L1 cells.

Using a subtraction method, we have isolated genes that are induced early in the differentiation of mouse 3T3-L1 preadipocyte cells into adipocytes. These include the genes encoding transcription factors and signalling proteins, as well as unknown genes. Bach1, a transcription factor, and ARA70, a cofactor, were rapidly induced during differentiation. The induction of these two genes was observed only in growth-arrested 3T3-L1 cells, and not in proliferating cells. In NIH-3T3 cells, no induction was observed under either set of conditions. These results strongly indicate that Bach1 and ARA70 have valuable roles at the onset of adipocyte differentiation.