Search

Generalized nonsegmental vitiligo is often associated with the activation of melanocyte-specific autoimmunity. Because chemokines play an important role in the maintenance of immune responses, we examined chemotactic signatures in cultured vitiligo melanocytes and skin samples of early (≤2 months) and advanced (≥6 months) vitiligo. Analysis showed that melanocytes in early lesions have altered expression of several chemotaxis-associated molecules, including elevated secretion of CXCL12 and CCL5. Higher levels of these chemokines coincided with prominent infiltration of the skin with antigen presenting cells (APCs) and T cells. Most of the intralesional APCs expressed the CD86 maturation marker and co-localized with T cells, particularly in early vitiligo lesions. These observations were confirmed by in vivo animal studies showing preferential recruitment of APCs and T cells to CXCL12- and CCL5-expressing transplanted melanocytes, immunotargeting of the chemokine-positive cells, continuous loss of the pigment-producing cells from the epidermis, and development of vitiligo-like lesions. Taken together, our studies show that melanocyte-derived CXCL12 and CCL5 support APC and T-cell recruitment, antigen acquisition, and T-cell activation in early vitiligo and reinforce the role of melanocyte-derived CXCL12 and CCL5 in activation of melanocyte-specific immunity and suggest inhibition of these chemotactic axes as a strategy for vitiligo stabilization., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

CXCR1 and CXCR2 chemokine receptors and their ligands (CXCL1/2/3/7/8) play an important role in tumor progression. Tested to date CXCR1/2 antagonists and chemokine-targeted antibodies were reported to affect malignant cells in vitro and in animal models. Yet, redundancy of chemotactic signals and toxicity hinder further clinical development of these approaches. In this pre-clinical study we investigated the capacity of a novel small molecule dual CXCR1/2 inhibitor, Ladarixin (LDX), to attenuate progression of experimental human melanomas. Our data showed that LDX-mediated inhibition of CXCR1/2 abrogated motility and induced apoptosis in cultured cutaneous and uveal melanoma cells and xenografts independently of the molecular defects associated with the malignant phenotype. These effects were mediated by the inhibition of AKT and NF-kB signaling pathways. Moreover, systemic treatment of melanoma-bearing mice with LDX also polarized intratumoral macrophages to M1 phenotype, abrogated intratumoral de novo angiogenesis and inhibited melanoma self-renewal. Collectively, these studies outlined the pre-requisites of the successful CXCR1/2 inhibition on malignant cells and demonstrated multifactorial effects of Ladarixin on cutaneous and uveal melanomas, suggesting therapeutic utility of LDX in treatment of various melanoma types.

Background: Dystrophic epidermolysis bullosa (DEB), a rare genodermatosis, is characterized by the formation of intra-epidermal blistering and the development of chronic nonhealing skin wounds. Recently, attempts have been made to develop cell-based therapies for this currently intractable disorder. The molecular mechanisms that govern directional migration of the adult stem cells, allowing their efficient and controlled homing to the skin affected with DEB, are poorly understood. The key mechanism that regulates recruitment of leukocytes and progenitor stem cells to distal anatomical tissues affected with disease is chemotaxis, which depends on the signaling molecules, chemokines, and acts primarily as part of the host defense and repair mechanism., Methods: Comprehensive proteomic screening of chemokines in the blister fluids of DEB-affected mice was conducted to define the inflammatory and immune activities, thus providing potential to examine local biological mechanisms and define the protein signature within lesional skin as a potential marker of disease activity. Also, the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations., Results: Assessment of blister fluid-derived chemokines showed a persistent presence of several chemotactic molecules, including CXCL1 + 2 and CXCL5. The majority of blister-originated chemotactic signals were associated with preferential recruitment of CD45(+)CXCR2(+) and CD11b(+)CXCR2(+) leukocytes. Systemic transplantation of an enriched CXCR2 population of mouse adipose-derived stem cells (mADSC) into DEB-affected mice demonstrated effective recruitment of cells to the blistering skin under the influence of blister-derived ligands and deposition of therapeutic type VII collagen., Conclusions: Collectively, these studies demonstrate that recruitment of mADSC into DEB skin is tightly controlled by disease-site chemotactic activities and suggest a potential mechanism for effective application of therapeutic stem cells for DEB.

Melanoma is one of the most aggressive metastatic cancers with resistance to radiation and most chemotherapy agents. This study highlights an alternative treatment for melanoma based on nanosecond pulsed dielectric barrier discharge (nsP DBD). We show that a single nsP DBD treatment, directly applied to a 5 mm orthotopic mouse melanoma tumor, completely eradicates it 66% ( n = 6; p ≤ 0.05) of the time. It was determined that reactive oxygen and nitrogen species produced by nsP DBD are the main cause of tumor eradication, while nsP electric field and heat generated by the discharge are not sufficient to kill the tumor. However, we do not discount that potential synergy between each plasma generated component (temperature, electric field and reactive species) can enhance the killing efficacy.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR's role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bone marrow suppression due to exposure to ionizing radiation is a significant clinical problem associated with radiation therapy as well as with nonmedical radiation exposure. Currently, there are no small molecule agents available that can enhance hematopoietic regeneration after radiation exposure. Here, we report on the effective mitigation of acute hematopoietic radiation syndrome in mice by the synthetic triterpenoid, RTA 408. The administration of a brief course of RTA 408 treatment, beginning 24 h after lethal doses of radiation to bone marrow, significantly increased overall survival. Importantly, treatment with RTA 408 led to the full recovery of steady state hematopoiesis with normalization of the frequency of hematopoietic stem and progenitor cells. Moreover, hematopoietic stem cells from RTA 408-mitigated mice showed lineage-balanced, long-term, multilineage potential in serial transplantation assays, indicative of their normal self-renewal activity. The potency of RTA 408 in mitigating radiation-induced bone marrow suppression makes it an attractive candidate for potential clinical use in treating both therapy-related and unanticipated radiation exposure.

The data of literature on possibility of the use of perfusion technologies in combined treatment for malignant tumors of different sites are presented. Possible complications during hyperthermic chemoperfusion are discussed and the effectiveness of this method is analyzed.

A comparative study of safety and efficacy of normothermic and hyperthermic intraperitoneal chemoperfusion (IPEC and HIPEC) with cisplatin and dioxadet was carried out in 143 female Wistar rats. Ovarian cancer was inoculated intraperitoneally (i.p.). In 48 hours after ovarian cancer inoculation the drugs were administered i.p. or IPEC and HIPEC with the drugs were performed using maximum tolerated doses (MTD). Content of cisplatin was determined in the perfusate and blood plasma during HIPEC with the drug. The leukocyte count was measured using veterinary hematologic analyzer in peripheral blood of rats at different time points after HIPEC with dioxadet. Efficacy of the treatment was estimated in increase in median survival time (MST). During HIPEC cisplatin was accumulated in the abdominal cavity in a considerable amount with minimal systemic absorption. HIPEC with dioxadet didn't significantly affect the leukocyte count in peripheral blood while i.p. administration of dioxadet suppressed leukopoiesis. MST of rats after IPEC with cisplatin was 37.5 days which was significantly higher compared to MST after i.p. administration of cisplatin (19.5 days, p = 0.037). HIPEC with dioxadet was the most effective regimen of treatment with MST of rats reaching 49 days which was significantly higher compared to MST after HIPEC with cisplatin (25.5 days, p = 0.002).

Normal tissue toxicity markedly reduces the therapeutic index of genotoxic anticancer agents, including ionizing radiation. Countermeasures against tissue damage caused by radiation are limited by their potential to also protect malignant cells and tissues. Here, we tested a panel of signal transduction modifiers for selective radioprotection of normal but not tumor tissues. These included three inhibitors of GSK3 (LiCl, SB216763, and SB415286) and two inhibitors of NF-κB (ethyl pyruvate and RTA 408). Among these, the thiol-reactive triterpenoid RTA 408 emerged as a robust and effective protector of multiple organ systems (gastrointestinal, skin, and hemopoietic) against lethal doses of radiation. RTA 408 preserved survival and proliferation of intestinal crypt cells in lethally irradiated mice while reducing apoptosis incidence in crypts and villi. In contrast, RTA 408 uniformly inhibited growth of established CWR22Rv1, LNCaP/C4-2B, PC3, and DU145 xenografts either alone or combined with radiation. Antitumor effects in vivo were associated with reduced proliferation and intratumoral apoptosis and with inhibition of NF-κB-dependent transcription in PC3 cells. Selective protection of normal tissue compartments by RTA 408 critically depended on tissue context and could not be replicated in vitro. Collectively, these data highlight the potential of RTA 408 as a cytoprotective agent that may be safely used in chemoradiation approaches., (©2014 American Association for Cancer Research.)

Introduction: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness within the first two years of life. Collagen VI-related muscle disorders have recently emerged as one of the most common types of CMD. COL6 CMD is caused by deficiency and/or dysfunction of extracellular matrix (ECM) protein collagen VI. Currently, there is no specific treatment for this disabling and life-threatening disease. The primary cellular targets for collagen VI CMD therapy are fibroblasts in muscle, tendon and skin, as opposed to muscle cells for other types of muscular dystrophies. However, recent advances in stem cell research have raised the possibility that use of adult stem cells may provide dramatic new therapies for treatment of COL6 CMD., Methods: Here, we developed a procedure for isolation of human stem cells from the adipose layer of neonatal skin. The adipose-derived stem cells (ADSC) were examined for expression of ECM and related genes using gene expression array analysis. The therapeutic potential of ADSC was assessed after a single intramuscular transplantation in collagen VI-deficient mice., Results: Analysis of primary cultures confirmed that established ADSC represent a morphologically homogenous population with phenotypic and functional features of adult mesenchymal stem cells. A comprehensive gene expression analysis showed that ADSC express a vast array of ECM genes. Importantly, it was observed that ADSC synthesize and secrete all three collagen VI chains, suggesting suitability of ADSC for COL6 CMD treatment. Furthermore, we have found that a single intramuscular transplantation of ADSC into Col6a1-/-Rag1-/- mice under physiological and cardiotoxin-induced injury/regeneration conditions results in efficient engraftment and migration of stem cells within the skeletal muscle. Importantly, we showed that ADSC can survive long-term and continuously secrete the therapeutic collagen VI protein missing in the mutant mice., Conclusions: Overall, our findings suggest that stem cell therapy can potentially provide a new avenue for the treatment of COL6 CMD and other muscular disorders and injuries.

We have demonstrated that priming of intratumoral and intradermal vaccination sites with chemokines enhances cytotoxic immune response against established neoplasms. Additional insights into the molecular mechanisms that underlie these findings and the optimization of such an approach may lead to the development of cost-effective and generic immunotherapeutic regimens against cancer.

Background Aims: Adult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin., Methods: We examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin., Results: Analysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue., Conclusions: Collectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

IL-10 is an immunomodulatory cytokine that is frequently upregulated in various types of cancer. The biological role of IL-10 in cancer is quite complex; however, the presence of IL-10 in advanced metastases and the positive correlation between serum IL-10 levels and progression of disease indicates a critical role of IL-10 in the tumor microenvironment. IL-10 has been shown to directly affect the function of antigen-presenting cells by inhibiting the expression of MHC and costimulatory molecules, which in turn induces immune suppression or tolerance. Additionally, IL-10 downregulates the expression of Th1 cytokines and induces T-regulatory responses. Taken together, a combination of IL-10 antagonism and immunostimulatory treatments such as cancer vaccines, Toll-like receptor agonists, Th1 cytokines, and chemokines would be a logical approach to enhance an antitumor immune response.

Background Aims: Multiple studies have demonstrated that mesenchymal stromal cells (MSC) can be utilized therapeutically for various congenital and acquired disorders. The involvement of MSC in the maintenance of skin homeostasis and their curative application for the treatment of skin wounds have also been documented. However, it is not known whether MSC can commit to cutaneous lineages, produce structural proteins essential for the skin integrity or be used for hereditary skin disorders., Methods: To address these questions, we conducted a comparative expression analysis between MSC and potentially adjacent cutaneous cells, fibroblasts and keratinocytes, with specific emphasis on extracellular matrix encoding and related genes., Results: Our data demonstrated that MSC share many features with cutaneous fibroblasts. We also observed that under direct influence of cutaneous fibroblasts in vitro and fibroblast-derived matrix in vivo, MSC acquired a fibroblastic phenotype, suggesting that specific cell-cell interactions play a key regulatory role in the differentiation of MSC. Additionally, the observed fibroblastic transition of MSC was underlined by a significant up-regulation of several cutaneous-specific genes encoding lumican, decorin, type VII collagen, laminin and other structural proteins. As many of the identified genes have considerable therapeutic value for dermatologic afflictions, particularly type VII collagen, we evaluated further the therapeutic potential of congenic MSC in the skin of Col7a1-null mice recapitulating human recessive dystrophic epidermolysis bullosa (RDEB). Remarkably, MSC-derived type VII collagen was sufficient for restoration of the damaged dermal-epidermal junction and partial reversal of the RDEB phenotype., Conclusions: Collectively, our results suggest that MSC may offer promising therapeutics for the treatment of RDEB and potentially other genodermatoses.

Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.

Interleukin 10 (IL-10) is produced by various types of human cancer, including malignant melanoma, and plays an important role in negative regulation of cell-mediated immune responses against tumors. We have developed chimeric molecules (immunoadhesins), combining the extracellular domain of human interleukin 10 receptor 1 (IL-10R1) with the Fc regions of human IgG1 heavy chain and investigated their capability of blocking the biological activities of human IL-10. Monomeric and dimeric immunoadhesins (IL-10R1/IgG1) constructs were tested for capturing human IL-10 and blocking its biological activities. Plasmid vectors that contained the IL-10 immunoadhesin constructs were directly transfected into human melanoma cell lines. Transfection of plasmid vectors into melanoma cell lines resulted in capturing of exogenously added as well as endogeneously produced IL-10. The supernatants obtained from an IL-10 non-producing melanoma cell line transfected with monomeric IL-10 immunoadhesin plasmids most efficiently captured exogenously added IL-10, compared to those obtained with the dimeric IL-10R1/IgG1 plasmid vector. Transfection of IL-10-producing melanoma cells with the monomeric IL-10 immunoadhesin plasmids totally captured endogenously produced IL-10 and enhanced T cell responses against allogeneic melanoma cells. Furthermore, purified monomeric IL-10 immunoadhesin protein showed IL-10 capturing efficacy compatible with that of IL-10-specific monoclonal antibodies. Collectively, these studies indicate that IL-10 immunoadhesins, especially in monomeric form, are potent inhibitors of biological activities of IL-10 and suggest that these molecules, alone or in conjunctions with other immunotherapeutic approaches, can be utilized for the immuno-targeting of IL-10 producing tumors.

The use of recombinant DNA has become a powerful tool in the analysis of functional and structural properties of the extracellular matrix proteins. During last decade, various procedures of plasmid DNA delivery using liposome-based or electroporation-based transfection have been developed. However, in many instances, these procedures were shown to be not effective in DNA transfer or toxic for the mammalian cells. On contrary, retrovirus-mediated infection represents a superior mode of gene delivery with a success rate and viability of the cells approaching 100% in in vitro conditions. The use of the retroviral system also allows permanent insertion of the gene of interest into the chromosome of the infected cell, resulting in efficient gene transfer in which most recipient cells will incorporate and express the transduced gene. In this chapter, we will describe several retrovirus-based systems and provide step-by-step protocols applicable for the production of the recombinant virus and efficient delivery of the ECM genes.

Synthetic oligodeoxynucleotides (ODNs) had been employed in gene modification and represent an alternative approach to 'cure' genetic disorders caused by mutations. To test the ability of ODN-mediated gene repair in bone marrow-derived mesenchymal stem cells (MSCs), we established MSCs cell lines with stably integrated mutant neomycin resistance and enhanced green fluorescent protein reporter genes. The established cultures showed morphologically homogenous population with phenotypic and functional features of mesenchymal progenitors. Transfection with gene-specific ODNs successfully repaired targeted cells resulting in the expression of functional proteins at relatively high frequency approaching 0.2%. Direct DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. The position of the mismatch-forming nucleotide was shown to be important structural feature for ODN repair activity. The genetically corrected MSCs were healthy and maintained an undifferentiated state. Furthermore, the genetically modified MSCs were able to engraft into many tissues of unconditioned transgenic mice making them an attractive therapeutic tool in a wide range of clinical applications.

Junctional epidermolysis bullosa (JEB) is an inherited mechanobullous disease characterized by reduced adherence of the epidermal keratinocytes to the underlying dermis, and is often caused by the absence of functional laminin 332 due to the lack or dysfunction of its beta3 chain. As there are no specific therapies for JEB, we tested whether a protein replacement strategy could be applicable for the restoration of the laminin 332 assembly and reversion of the JEB phenotype in human keratinocytes that lack beta3 subunit. Here, we developed the protocol for production and purification of the biologically active recombinant beta3 chain. Next, we demonstrated that delivery of recombinant beta3 polypeptide into the endoplasmic reticulum of the immortalized beta3-null keratinocytes led to the restoration of the laminin 332 assembly, secretion, and deposition into the basement membrane zone, as confirmed by Western blot analysis, confocal immunofluorescent microscopy in vitro, and on cultured organotypic human JEB skin reconstructs. Although the amount of laminin 332 produced by protein-treated beta3-null keratinocytes is lower than that in normal human keratinocytes, our results demonstrate the applicability of the recombinant proteins for JEB treatment and open new perspectives for the development of novel therapeutics for this inherited, currently intractable, skin disorder.

Gene therapy using viral vectors for liver diseases, particularly congenital disorders, is besought with difficulties, particularly immunologic reactions to viral antigens. As a result, nonviral methods for gene transfer in hepatocytes have also been explored. Gene repair by small synthetic single-stranded oligodeoxynucleotides (ODNs) produces targeted alterations in the genome of mammalian cells and represents a great potential for nonviral gene therapy. To test the feasibility of ODN-mediated gene repair within chromosomal DNA in human hepatocytes, two new cell lines with stably integrated mutant reporter genes, namely neomycin and enhanced green fluorescent protein were established. Targeting theses cells with ODNs specifically designed for repair resulted in site-directed and permanent gene conversion of the single-point mutation of the reporter genes. Moreover, the frequency of gene alteration was highly dependent on the mitotic activity of the cells, indicating that the proliferative status is an important factor for successful targeting in human hepatocytes. cDNA array expression profiling of DNA repair genes under different cell culture conditions combined with RNA interference assay showed that mismatch repair (MMR) in actively growing hepatocytes imposes a strong barrier to efficient gene repair mediated by ODNs. Suppression of MSH2 activity in hepatocytes transduced with short hairpin RNAs (shRNAs) targeted to MSH2 mRNA resulted in 25- to 30-fold increase in gene repair rate, suggesting a negative effect of MMR on ODN-mediated gene repair. Taken together, these data suggest that under appropriate conditions nonviral chromosomal targeting may represent a feasible approach to gene therapy in liver disease.

The incidence of cutaneous malignant melanoma is increasing at a faster rate than any other cancer worldwide. Despite new advances in surgical management of melanoma, this malignancy remains one of the most aggressive and intractable to treat among other solid tumors. Continuous search for better therapeutics led to the development of various immunological approaches applicable to the treatment of this melanocytic malignancy. Multiple peptide, dendritic cell, adjuvant, lymphocyte, and virus-based strategies were established and tested in preclinical and clinical studies with varying degrees of clinical success. However, the most recent investigations in melanoma immunotherapy have clearly demonstrated that complex vaccines and the combination of different approaches, such as the use of dendritic cell vaccines in conjunction with costimulatory molecules, are superior to conventional immunization protocols in induction of tumor-specific immune responses. These recent studies open new perspectives for the development of efficient melanoma immunotherapeutics suitable for the treatment of primary and metastatic disease.

Previous studies have shown that secondary lymphoid chemokine, CCL21, can be used for modulation of tumor-specific immune responses. Here, using B16F0 melanoma cells stably expressing CCL21 under the control of cytomegalovirus and ubiquitin promoters, we showed that CCL21-activated immune responses depend on the amount of melanoma-derived chemokine, which, in turn, depends on the strength of the promoter. We showed that ubiquitin promoter-driven expression of CCL21 enabled massive infiltration of tumors with CD4(+)CD25(-), CD8(+) T lymphocytes, and CD11c(+) dendritic cells, and consequent activation of cellular and humoral immune responses sufficient for complete rejection of CCL21-positive melanomas within 3 weeks in all tumor-inoculated mice. Mice that rejected CCL21-positive tumors acquired protective immunity against melanoma, which was transferable to naive mice via splenocytes and central memory T cells. Moreover, melanoma-derived CCL21 facilitated immune-mediated remission of preestablished, distant wild-type melanomas. Overall, these results suggest that elevated levels of tumor-derived CCL21 are required for the activation of strong melanoma-specific immune responses and generation of protective immunologic memory. They also open new perspectives for the development of novel vaccination strategies against melanoma, which use intratumoral delivery of the optimized CCL21-encoding vectors in conjunction with DNA-based vaccines.

In the paper, dynamics of risk factors of cardiovascular diseases and their contribution to mortality rate in the male population of Yakutsk are analyzed according to the results of standardized epidemiological researches carried out in the period of 1986-2005. It was revealed that the unfavourable cardiovascular epidemiologic situation in Yakutia is caused by the prevalence of such risk factors as smoking, excess body weight and arterial hypertension.

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy., (Copyright 2006 Wiley-Liss, Inc.)

The cKit receptor plays a critical role in melanocyte physiology, influencing melanogenesis, proliferation, migration, and survival of the pigment-producing cells. However, pathways of cKit-mediated intracellular signaling and molecular mechanisms, which regulate specific cellular responses to the activation of the receptor in melanocytes, remain incompletely understood. Here, by using the genetically altered mouse melanocytes expressing an endogenous, constitutively active mutant (D814Y) cKit receptor, we investigated physiological cellular responses to the ligand-independent activation of the receptor tyrosine kinase. It was anticipated that such activation would either trigger uncontrolled proliferation of the melanocytes or stimulate melanin biosynthesis. In contrast to the expectation, we found that constitutive signaling from the cKit receptor did not stimulate melanogenesis and proliferation, but significantly promoted migration of the melanocytes both in vitro and in vivo. We also showed that such signaling is not associated with tumorigenic transformation of the pigment-producing cells. Taken together, our observations suggest that, in mammalian melanocytes, activation of the cKit receptor tyrosine kinase is primarily responsible for transmission of pro-migration signals, which may antagonize proliferation and melanogenesis. Our data also provide an additional explanation as to why malignant melanocytes lose cKit expression during melanoma progression.

Oligodeoxynucleotide (ODN)-mediated gene alteration was postulated to occur in two steps, DNA strand pairing and DNA repair. Once alignment has occurred through homologous strand pairing, a single mismatch is formed between an oligonucleotide and one of the target strands. Because of this mismatch, it has been suggested that proteins involved in a mismatch repair pathway (MMR) participate in the process. We proposed an alternative model, in which a transient assimilation of ODN to the target DNA can interrupt the trafficking of RNA polymerase, and the stalled RNA polymerase may signal for recruitment of DNA repair proteins, including transcription-coupled (TCR) DNA repair and nucleotide excision repair (NER) pathways. Recently, we found that transcription of many genes participating in NER and MMR was induced by the presence of plasmid DNA, and the extent of induction correlated with episomal gene repair rates. To investigate whether an increased level of induction of genes involved in specific DNA repair pathways has a functional role in ODN-directed gene repair, we performed episomal targeting in several cell lines with a specific defective gene in NER and MMR pathways. Comparison among several genetically related cell lines harboring a specific defective gene and complementation of missing activities showed that a primary pathway for gene correction involves some of the proteins participating in NER, primarily two endonucleases processing a DNA lesion, but not MMR.

During the last decade, chimeric RNA-DNA oligonucleotides (RDOs) and single-stranded oligodeoxynucleotides have been used to make permanent and specific sequence changes in the genome, with the ultimate goal of curing human genetic disorders caused by mutations. There have been large variations observed in the rate of gene repair in these studies. This has been due, at least in part, to the lack of standardized assay conditions and the paucity of mechanistic studies in the early developmental stages. Previously, it was proposed that strand pairing is the rate-limiting step and mismatch DNA repair is involved in the gene repair process. We propose an alternative model, in which an oligonucleotide is assimilated to the target DNA during active transcription, leading to formation of a transient D-loop. The trafficking of RNA polymerase is interrupted by the D-loop, and the stalled RNA polymerase complex may signal for recruitment of DNA repair proteins, including transcription-coupled DNA repair and nucleotide-excision repair. Thus, oligonucleotides can be considered as a class of DNA-damaging agents that cause a transient but major structural change in DNA. Understanding of the recognition and repair pathways to process this unusual DNA structure may have relevance in physiologic processes, transcription, and DNA replication.

Within the last decade, a number of nucleic acid-based gene targeting strategies have been developed with the ultimate goal to cure human genetic disorders caused by mutations. Thus far, site-directed gene targeting is the only procedure that can make predefined alterations in the genome. The advantage of this approach is that expression of the corrected gene is regulated in the same way as a normal gene. In addition, targeted specific mutations can be made in the genome for functional analysis of proteins. Several approaches, including chimeric RNA-DNA oligonucleotides, short single-stranded oligonucleotides, small fragment homologous replacements, and triple-helix-forming oligonucleotides have been used for targeted modification of the genome. Due to the absence of standardized assays and mechanistic studies in the early developmental stages of oligonucleotide-directed gene alteration, it has been difficult to explain the large variations and discrepancies reported. Here, we evaluate the progress in the field, summarize the achievements in understanding the molecular mechanism, and outline the perspective for the future development. This review will emphasize the importance of reliable, sensitive and standardized assays to measure frequencies of gene repair and the use of these assays in mechanistic studies. Such studies have become critical for understanding the gene repair process and setting realistic expectations on the capability of this technology. The conventionally accepted but unproven dogmas of the mechanism of gene repair are challenged and alternative points of view are presented. Another important focus of this review is the development of general selection procedures that are required for practical application of this technology.

The role of transcription in oligonucleotide (ODN)-directed gene modification has been investigated in mammalian cells. The importance of transcription is demonstrated using mammalian cell lines with varying degrees of transcription of the mutant LacZ reporter gene, residing in both episome and chromosome. Gene correction occurs more efficiently when the target gene is actively transcribed and antisense ODN is more active than sense ODN. Using an approach that combines biochemical studies with a cell-based assay to measure the functional activity of intermediates it is shown that a joint molecule, consisting of supercoiled DNA and homologous ODN targeted to correct the mutated base, is a functional intermediate in the gene repair process. Furthermore, this approach showed that a resected joint molecule is a downstream intermediate of the D-loop. These results indicate that the primary reason for efficient gene repair exhibited by the antisense ODN is its increased accessibility to the non-transcribed strand, and as a consequence an increased formation of intermediate during active transcription. Moreover, the processing of intermediates was also affected by transcription, suggesting that ODN-directed gene repair may be linked to transcription-coupled repair. Thus, transcription plays an important role in ODN-directed gene repair by affecting the formation and processing of key intermediates.

We compared strand pairing and gene correction activities between different constructs of oligonucleotides, using homologous supercoiled DNA and eukaryotic nuclear extracts. The RNA-DNA chimeric oligonucleotide was more efficient in strand pairing and gene correction than its DNA-DNA homolog. Single-stranded deoxyoligonucleotides showed similar strand pairing and correction activity to the modified RNA-DNA chimeric oligonucleotides, whereas single-stranded ribooligonucleotides did not show either activity. However, the correlations were not always linear, suggesting that only a fraction of the joint molecules may be processed to cause the final gene correction. Several mammalian extracts with markedly different in vitro activity showed the similar amounts of the joint molecules. These results led us to conclude that strand pairing is a necessary event in gene correction but may not be the rate-limiting step. Furthermore, depletion of HsRad51 protein caused large decreases in both strand-pairing and functional activities, whereas supplementation of HsRad51 produced only a slight increase in the repair activity, indicating that HsRad51 participates in the strand pairing, but subsequent steps define the frequency of gene correction. In addition, we found that the structure and stability of intermediates formed by single-stranded deoxyoligonucleotides and RNA-DNA chimeric oligonucleotides were different, suggesting that they differ in their mechanisms of gene repair.

An oligonucleotide composed of a contiguous stretch of RNA and DNA residues has been developed to facilitate the correction of single-base mutations of episomal and chromosomal targets in mammalian cells. The design of the oligonucleotide exploited the highly recombinogenic RNA-DNA hybrids and featured hairpin capped ends avoiding destruction by cellular helicases or exonucleases. The RNA-DNA oligonucleotide (RDO) was designed to correct a point mutation in the tyrosinase gene and caused a permanent gene correction in mouse albino melanocytes, determined by clonal analysis at the level of genomic sequence, protein and phenotypic change. Recently, we demonstrated correction of the tyrosinase gene using the same RDO in vivo, as detected by dark pigmentation of several hairs and DOPA staining of hair follicles in the treated skin of albino mice. Such RDOs might hold a promise as a therapeutic method for the treatment of skin diseases. However, the frequency of gene correction varies among different cells, indicating that cellular activities, such as recombination and repair, may be important for gene conversion by RDOs. As this technology becomes more widely utilized in the scientific community, it will be important to understand the mechanism and to optimize the design of RDOs to improve their efficiency and general applicability.

We recently demonstrated that an RNA-DNA oligonucleotide corrected a point mutation in the mouse tyrosinase gene, resulting in permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in cultured melanocytes. In this study, we extended gene correction of melanocytes from tissue culture to live animals, using a chimeric oligonucleotide designed to correct a point mutation in the tyrosinase gene. Both topical application and intradermal injection of this oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area. The restored tyrosinase enzymatic activity was detected by dihydroxyphenylacetic acid (DOPA) staining of hair follicles in the treated skin. Tyrosinase gene correction was also confirmed by restriction fragment length polymorphism analysis and DNA sequencing from skin that was positive for DOPA staining and melanin synthesis. Localized gene correction was maintained three months after the last application of the chimeric oligonucleotides. These results demonstrated correction of the tyrosinase gene point mutation by chimeric oligonucleotides in vivo.

Experimental strategies have been developed to correct point mutations using chimeric oligonucleotides composed of RNA and DNA. We used these RNA-DNA oligonucleotides to correct a point mutation in mouse tyrosinase, a key enzyme for melanin synthesis and pigmentation. Melanocytes derived from albino mice contain a homozygous point mutation (TGT-->TCT) in the tyrosinase gene, resulting in an amino acid change from Cys-->Ser. Correction of this point mutation results in the restoration of tyrosinase activity and melanin synthesis, thus changing the pigmentation of the cells. Upon transfection of the RNA-DNA oligonucleotide to albino melanocytes, we detected black-pigmented cells and isolated multiple single clones. All black-pigmented clones exhibited a correction of the point mutation in a single allele of the tyrosinase gene. A full-length tyrosinase was detected by an antityrosinase antibody, and the enzymatic activity was restored in all converted black-pigmented clones. Only degraded fragments were detected in albino cells due to proteolytic cleavage of mutant tyrosinase. The phenotype and genotype of converted black-pigmented clones was stable. These results demonstrate a permanent and stable gene correction by the RNA-DNA oligonucleotide at the level of genomic sequence, protein, and phenotypic change by clonal analysis.

In seven families from a Siberian founder population with autosomal dominant spinocerebellar ataxia (SCA) genetic analysis of the polymorphisms flanking the SCA1 locus on chromosome 6p showed allelic association with disease inheritance. While the association was absolute in the case of microsatellite D6S274, an allele switch was observed for D6S89 in two families, suggesting a historic recombinant. Further genetic and physical study of this recombinant event could be instrumental for the precise localization and identification of the SCA1 gene.