Transfer of efficient anti-melanocyte T cells from vitiligo donors to melanoma patients as a novel immunotherapeutical strategy

Background

Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune-mediated loss of epidermal melanocytes. High frequencies of self-reactive T lymphocytes directed toward melanocyte differentiation antigens are found in vitiligo patients and might be directly responsible for the pathogenesis of the disease. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self antigens shared by normal melanocytes and melanoma cells are found in both conditions, but the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research.

Presentation of the hypothesis

Our working hypothesis is that vitiligo patients might represent a unique source of therapeutic cells to be used in allo-transfer for HLA-matched melanoma patients. The adoptive transfer of ex-vivo generated autologous tumor-specific T cells is a therapy that has met with only limited success, essentially because of inability to isolate therapeutically valuable T cells from the majority of tumor patients. Ideally, model systems where strong and efficient responses against the same (tumor) antigens are achieved would represent a better source of therapeutic cells. We believe it is possible to identify one such model in the melanoma-vitiligo dichotomy: T lymphocytes specific for different melanocyte differentiation antigens are found in vitiligo and represent the effective anti-melanocyte reactivity that is often ineffective in melanoma.

Testing the hypothesis

Melanocyte-specific T cell clones can be isolated from the peripheral blood of vitiligo patients and tested for their capacity to efficiently expand in vitro without loosing their cytotoxic activity and to migrate to the skin. Cytotoxicity against melanoma patients' non-tumor cells can also be tested. In addition, it would be interesting to attempt an in vivo animal model. If the results obtained from these validation steps will be satisfactory, it might be possible to plan the clinical grade preparation of relevant clones for transfer.

Implications of the hypothesis

When translated into a clinical trial, the possibility of in vitro selecting few effective tumor-specific T cell clones for infusion, inherent with this approach, could enhance the therapeutic graft-versus-tumor effect while possibly decreasing the risk of graft-versus-host disease.

     

APS8, a Polymeric Alkylpyridinium Salt Blocks α7 nAChR and Induces Apoptosis in Non-Small Cell Lung Carcinoma

Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer.     

Differential cytotoxicity of the isolated protein fraction of Escumite bean (Phaseolus acutifolius)

The Escumite bean (Phaseolus acutifolius) is an edible legume which in raw form is highly toxic to rats. Proteins were separated by DEAE cellulose and affinity chromatography. Hemagglutinating activity, trypsin inhibitory effect, cytotoxicity on human peripheral lymphocytes and on intestinal epithelial cells of rats, and mitogenic activity were assayed with each protein fraction. Hemagglutinins and trypsin inhibitory fractions showed differential toxicity with lymphocytes as compared to intestinal epithelial cells. A protein fraction without the previous activities was cytotoxic mainly to intestinal epithelial cells.     

Enhanced Control of Bladder-Associated Tumors Using Shrimp Anti-Lipopolysaccharide Factor (SALF) Antimicrobial Peptide as a Cancer Vaccine Adjuvant in Mice

Shrimp anti-lipopolysaccharide factor (SALF) is an antimicrobial peptide with reported anticancer activities, such as suppression of tumor progression. In this study, we prepared a potential cancer vaccine comprised of SALF in conjunction with the cell lysate of inactivated murine bladder carcinoma cells (MBT-2), and evaluated its efficacy in a mouse tumor model. Our study shows that SALF added to cell culture media inhibits growth progression of MBT-2, and that SALF together with inactivated MBT-2 lysate elevates the level of inflammasome activity, and modulates the levels of IL-1β, MCP-1, IL-6, IL-12, and TNF-α in mouse macrophages. Immunization of 7, 14, and 21 day-old mice with the vaccine prevented growth of MBT-2 cell-mediated tumors. The vaccine was found to enhance expression of T-cell, cytotoxic T cells, and NK cells in the immunized mice groups. Recruitment of macrophages, T-helper cells, and NK cells was enhanced, but levels of VEGF were decreased in immunized mice. This report provides empirical evidence that our SALF as vaccine adjuvant enhances antitumor immunity in mice.     

A ω-secalin contained decamer shows a celiac disease prevention activity

Celiac disease (CD) is an autoimmune permanent enteropathy that is triggered in susceptible individuals after the ingestion of gluten, a storage protein fraction presents in wheat, rye and barley endosperm. Specific gluten peptides can bind to HLA-DQ2/8 and induce lamina propria CD4⁺ T cell responses causing damage of the small intestine mucosa. Recent studies suggested that beside immunodominant and toxic epitopes, wheat gluten also contains epitopes capable of preventing the mucosal response in vitro. Among them, a decapeptide (QQPQDAVQPF) from wheat was reported to have an antagonist effect on the agglutination of K562(S) cells and celiac T-cell activation, although the corresponding nucleotidic sequence remained unknown. This study was therefore designed to clone the sequence encoding the protein carrying the decapetide with CD protective properties. A ω-secalin gene encoding containing the decapeptide QQPQRPQQPF was isolated. Although the decapeptide was not identical to the one previously described, QQPQRPQQPF showed the same capability to prevent K562(S) cell agglutination and celiac mucosa immune activation induced by toxic gliadins. The ω-secalin gene was found in wheat carrying the wheat–rye chromosomal translocations 1BL.1RS. The identification of this immunomodulatory gliadin sequence, naturally occurring in cultivars of wheat toxic for celiac patients, might offer new therapeutic strategies for CD.     

Immunogenetic mechanisms for the coexistence of organ-specific and systemic autoimmune diseases

Background

Organ-specific autoimmune diseases affect particular targets in the body, whereas systemic diseases engage multiple organs. Both types of autoimmune diseases may coexist in the same patient, either sequentially or concurrently, sustained by the presence of autoantibodies directed against the corresponding autoantigens. Multiple factors, including those of immunological, genetic, endocrine and environmental origin, contribute to the above condition. Due to association of certain autoimmune disorders with HLA alleles, it has been intriguing to examine the immunogenetic basis for autoantigen presentation leading to the production of two or more autoantibodies, each distinctive of an organ-specific or systemic disease. This communication offers the explanation for shared autoimmunity as illustrated by organ-specific blistering diseases and the connective tissue disorders of systemic nature.

Presentation of the hypothesis

Several hypothetical mechanisms implicating HLA determinants, autoantigenic peptides, T cells, and B cells have been proposed to elucidate the process by which two autoimmune diseases are induced in the same individual. One of these scenarios, based on the assumption that the patient carries two disease-susceptible HLA genes, arises when a single T cell epitope of each autoantigen recognizes its HLA protein, leading to the generation of two types of autoreactive B cells, which produce autoantibodies. Another mechanism functioning whilst an epitope derived from either autoantigen binds each of the HLA determinants, resulting in the induction of both diseases by cross-presentation. Finally, two discrete epitopes originating from the same autoantigen may interact with each of the HLA specificities, eliciting the production of both types of autoantibodies.

Testing the hypothesis

Despite the lack of immediate or unequivocal experimental evidence supporting the present hypothesis, several approaches may secure a better understanding of shared autoimmunity. Among these are animal models expressing the transgenes of human disease-associated HLA determinants and T or B cell receptors, as well as in vitro binding studies employing purified HLA proteins, synthetic peptides, and cellular assays with antigen-presenting cells and patient's lymphocytes. Indisputably, a bioinformatics-based search for peptide motifs and the modeling of the conformation of bound autoantigenic peptides associated with their respective HLA alleles will reveal some of these important processes.

Implications of the hypothesis

The elucidation of HLA-restricted immune recognition mechanisms prompting the production of two or more disease-specific autoantibodies holds significant clinical ramifications and implications for the development of more effective treatment protocols.

     

Lack of correlation between the levels of soluble cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60 genotypes

Background

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulation of antigen-activated immune response and polymorphisms at the CTLA-4 gene have been shown to be associated with several autoimmune diseases including type-1 diabetes (T1D). The etiological mutation was mapped to the CT60-A/G single nucleotide polymorphism (SNP) that is believed to control the processing and production of soluble CTLA-4 (sCTLA-4).

Methods

We therefore determined sCTLA-4 protein levels in the sera from 82 T1D patients and 19 autoantibody positive (AbP) subjects and 117 autoantibody negative (AbN) controls using ELISA. The CT-60 SNP was genotyped for these samples by using PCR and restriction enzyme digestion of a 268 bp DNA segment containing the SNP. Genotyping of CT-60 SNP was confirmed by dye terminating sequencing reaction.

Results

Higher levels of sCTLA-4 were observed in T1D (2.24 ng/ml) and AbP (mean = 2.17 ng/ml) subjects compared to AbN controls (mean = 1.69 ng/ml) with the differences between these subjects becoming significant with age (p = 0.02). However, we found no correlation between sCTLA-4 levels and the CTLA-4 CT-60 SNP genotypes.

Conclusion

Consistent with the higher serum sCTLA-4 levels observed in other autoimmune diseases, our results suggest that sCTLA-4 may be a risk factor for T1D. However, our results do not support the conclusion that the CT-60 SNP controls the expression of sCTLA-4.

     

Cytotoxic effect of two legumes in epithelial cells of the small intestine

The previously known high toxicity and hemagglutinin concentration found in Escumite bean (Phaseolous acutifolius), widely consumed in the south-east of Mexico, induced us to study the cytotoxic effect of these beans on the epithelial cells of the small intestine of rats. Another, less toxic leguminosae, chick-pea (Cicer arietinum), was used for comparison. In vitro tests showed that the viability of the cells was much more affected by the Escumite bean extract than by the chick-pea extract (P<0.001). The cellularity was much diminished when the epithelial cells were treated with Escumite extract (P<0.001), but not with chick-pea extract. In vivo experiments confirmed these findings. The present results suggest that the deleterious effect of some toxicants present in the raw legume is due to an interaction with intestinal epithelium, which, in turn, may cause a reduced absorption of nutrients.     

海南粗榧内生真菌F127发酵液的次级代谢产物研究

对海南粗榧(Cephalotaxus hainanensis Li.)内生真菌F127发酵液化学组分进行研究。用多种色谱技术对F127代谢产物进行分离纯化,以LC-MS、ESI-MS和超导核磁共振分析鉴定化合物结构,最后采用MTT法测定化合物的抗肿瘤活性。从海南粗榧内生真菌F127的发酵液中分离得到5个单体化合物,分别鉴定为oblongolide T(1)、sorbicillin(2)、邻苯二甲酸二丁酯(3)、phomopsolide B(4)、6,8-二羟基-3-甲基-3,4-二氢异香豆素(5),且化合物1、4、5对肿瘤细胞K562、NB4、HL-60、Hep G-2和Lovo表现出不同抑制活性。化合物4对5株肿瘤细胞均表现出抑制活性,对K562、NB4、HL-60抑制效果显著,IC50值分别为3.35、0.014和0.16μg/m L,对Hep G-2和Lo Vo只有温和抑制作用;化合物1对K562、NB4、HL-60抑制作用良好,IC50值分别为51.82、54.25和29.31μg/m L;化合物5对NB4和Hep G-2则具有一定抑制活性;化合物2和3对测试细胞株未表现出抑制活性。5个化合物均是首次从海南粗榧内生真菌中分离得到,并首次报道了化合物1、4对K562、NB4、HL-60的优良细胞毒活性,为进一步研究海南粗榧内生真菌中的活性天然产物奠定了基础。     

A lactose-binding lectin from the marine sponge Cinachyrella apion (Cal) induces cell death in human cervical adenocarcinoma cells

Cancer represents a set of more than 100 diseases, including malignant tumors from different locations. Strategies inducing differentiation have had limited success in the treatment of established cancers. Marine sponges are a biological reservoir of bioactive molecules, especially lectins. Several animal and plant lectins were purified with antitumor activity, mitogenic, anti-inflammatory and antiviral, but there are few reports in the literature describing the mechanism of action of lectins purified from marine sponges to induce apoptosis in human tumor cells. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated with respect to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death in tumor cells. The antiproliferative activity of CaL was tested against HeLa, PC3 and 3T3 cell lines, with highest growth inhibition for HeLa, reducing cell growth at a dose dependent manner (0.5-10 μg/mL). Hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC(50) (10 μg/mL) for both trials and twice the IC(50) for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. Results showed that lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase and acting as both dependent and/or independent of caspases pathway. These results indicate the potential of CaL in studies of medicine for treating cancer.     

Salternamide A Suppresses Hypoxia-Induced Accumulation of HIF-1α and Induces Apoptosis in Human Colorectal Cancer Cells

Hypoxia inducible factor-1α (HIF-1α) is an essential regulator of the cellular response to low oxygen concentrations, activating a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1α is overexpressed in various cancers and therefore represents a considerable chemotherapeutic target. Salternamide A (SA), a novel small molecule that is isolated from a halophilic Streptomyces sp., is a potent cytotoxic agent against a variety of human cancer cell lines. However, the mechanisms by which SA inhibits tumor growth remain to be elucidated. In the present study, we demonstrate that SA efficiently inhibits the hypoxia-induced accumulation of HIF-1α in a time- and concentration-dependent manner in various human cancer cells. In addition, SA suppresses the upstream signaling of HIF-1α, such as PI3K/Akt/mTOR, p42/p44 MAPK, and STAT3 signaling under hypoxic conditions. Furthermore, we found that SA induces cell death by stimulating G2/M cell cycle arrest and apoptosis in human colorectal cancer cells. Taken together, SA was identified as a novel small molecule HIF-1α inhibitor from marine natural products and is potentially a leading candidate in the development of anticancer agents.     

Virus-mediated autoimmunity in Multiple Sclerosis

Epidemiological data suggest the notion that in Multiple Sclerosis (MS) is an acquired autoimmune disease and the cause may be an environmental factor(s), probably infectious, in genetically susceptible individuals. Several cases of viral induced demyelinatimg encephalomyelitis in human beings and in experimental models as well as the presence of IgG oligoclonal bands in the cerebrospinal fluid indicate that the infectious factor may be viral. However, the absence of a specific virus identification in MS central nervous system may hardly support this notion. On the other hand, the partial response of patients with MS to immunosuppressive and immunomodulatory therapy support the evidence of an autoimmune etiology for MS. However, the autoimmune hypothesis shares the same criticism with the infectious one in that no autoantigen(s) specific to and causative for MS has ever been identified. Nevertheless, the absence of identifiable infectious agent, especially viral does not rule out its presence at a certain time – point and the concomitant long term triggering of an autoimmune cascade of events thereafter. Several concepts have emerged in an attempt to explain the autoimmune mechanisms and ongoing neurodegeneration in MS on the basis of the infectious – viral hypothesis.     

Cytotoxic effect of organotin(IV) benzylisopropyldithiocarbamate compounds on Chang liver cell and hepatocarcinoma HepG2 cell

Cancer is one of the main causes of mortality and morbidity in world. New compounds are currently being synthesized to combat this disease. The organotins are gaining more attention as anti-cancer agents due to their potent cytotoxicity properties. In this study, a series of newly synthesized organotins namely dimethyltin (IV) (compound 1), dibutyltin (IV) (compound 2) and triphenyltin (IV) benzylisopropyldithiocarbamate (compound 3) were assessed for their cytotoxic activities against human Chang liver cells and hepatocarcinoma HepG2 cells. The cytotoxicity of these organotins in both cells upon 24 h treatment was assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Compound 2 and 3 exhibited potent cytotoxic activities towards both cells where the IC50 values were less then 10 microM. The IC50 value for compound 2 was 2.5 microM in Chang liver cells and 7.0 microM in HepG2 cells whereas compound 3 exhibited an IC50 value of 1.5 microM in Chang liver cells and 2.5 microM in HepG2 cells. Therefore, compound 2 and 3 were more toxic against human Chang liver cells as compared to hepatocarcinoma HepG2 cells. Interestingly, compound 1 did not have any IC50 value in both cells and hence can be classified as non-toxic. In conclusion, organotin (IV) benzylisopropyldithiocarbamate with insertion of dibutyl and triphenyl functional group possess potent cytotoxicity properties. Structural modification of these compounds can be carried out in further studies to produce less or non toxic effects towards normal human cell.     

Mitochondrial genome-knockout cells demonstrate a dual mechanism of action for the electron transport complex I inhibitor mycothiazole

Mycothiazole, a polyketide metabolite isolated from the marine sponge Cacospongia mycofijiensis, is a potent inhibitor of metabolic activity and mitochondrial electron transport chain complex I in sensitive cells, but other cells are relatively insensitive to the drug. Sensitive cell lines (IC(50) 0.36-13.8 nM) include HeLa, P815, RAW 264.7, MDCK, HeLa S3, 143B, 4T1, B16, and CD4/CD8 T cells. Insensitive cell lines (IC(50) 12.2-26.5 μM) include HL-60, LN18, and Jurkat. Thus, there is a 34,000-fold difference in sensitivity between HeLa and HL-60 cells. Some sensitive cell lines show a biphasic response, suggesting more than one mechanism of action. Mitochondrial genome-knockout ρ(0) cell lines are insensitive to mycothiazole, supporting a conditional mitochondrial site of action. Mycothiazole is cytostatic rather than cytotoxic in sensitive cells, has a long lag period of about 12 h, and unlike the complex I inhibitor, rotenone, does not cause G(2)/M cell cycle arrest. Mycothiazole decreases, rather than increases the levels of reactive oxygen species after 24 h. It is concluded that the cytostatic inhibitory effects of mycothiazole on mitochondrial electron transport function in sensitive cell lines may depend on a pre-activation step that is absent in insensitive cell lines with intact mitochondria, and that a second lower-affinity cytotoxic target may also be involved in the metabolic and growth inhibition of cells.     

Anti-Hepatocellular Carcinoma (HepG2) Activities of Monoterpene Hydroxy Lactones Isolated from the Marine Microalga Tisochrysis Lutea

Tisochrysis lutea is a marine haptophyte rich in omega-3 polyunsaturated fatty acids (e.g., docosahexaenoic acid (DHA)) and carotenoids (e.g., fucoxanthin). Because of the nutraceutical applications of these compounds, this microalga is being used in aquaculture to feed oyster and shrimp larvae. In our earlier report, T. lutea organic crude extracts exhibited in vitro cytotoxic activity against human hepatocarcinoma (HepG2) cells. However, so far, the compound(s) accountable for the observed bioactivity have not been identified. Therefore, the aim of this study was to isolate and identify the chemical component(s) responsible for the bioactivity observed. Bioassay-guided fractionation through a combination of silica-gel column chromatography, followed by preparative thin layer chromatography (PTLC), led to the isolation of two diastereomers of a monoterpenoid lactone, namely, loliolide (1) and epi-loliolide (2), isolated for the first time in this species. The structural elucidation of both compounds was carried out by GC-MS and 1D (¹H and ¹³C APT) and 2D (COSY, HMBC, HSQC-ed, and NOESY) NMR analysis. Both compounds significantly reduced the viability of HepG2 cells and were considerably less toxic towards a non-tumoral murine stromal (S17) cell line, although epi-loliolide was found to be more active than loliolide.     

Gas Cell Stabilisation and Gas Retention in Wheat Bread Dough

Gas cell stabilisation and gas retention are of considerable interest because of their technological significance in bread making. We review recent studies in relation to the stabilisation of gas cells and the mechanisms of gas retention, and discuss how these may be affected by the liquid phase of dough. The possibility is discussed of the involvement of surface active materials, such as proteins and pentosans dissolved in the dough aqueous phase, and, perhaps more importantly, non-starch polar lipids in the formation and stabilisation of gas cells. There is accumulating evidence for the hypothesis that liquid films play a critical role in the mechanisms of gas retention in dough. The hypothesis proposes that two closely related, consecutive stages are involved in dough expansion. During the first stage, the expanding gas cells remain discrete until discontinuities develop in the starch–protein matrix, leaving areas containing only a liquid film. The timing and the degree to which such discontinuities occur is largely dependent on gluten proteins. The second stage involves an increase in the surface area of the liquid film as discontinuities become increasingly frequent during expansion. Failure of the lamellar film to maintain the rate at which new surface area is generated leads to the rupture of this film and, consequently, the loss of gas retention. Consideration is also given to the role of bakery fat in gas retention and to additional factors that affect gas retention in wholemeal doughs, in particular the physical disruption of the foam structure of such doughs by components of the outer layers of the grain.     

Marine Bromophenol Bis (2,3-Dibromo-4,5-dihydroxy-phenyl)-methane Inhibits the Proliferation,Migration, and Invasion of Hepatocellular Carcinoma Cells via Modulating β1-Integrin/FAK Signaling

Bis (2,3-dibromo-4,5-dihydroxy-phenyl)-methane (BDDPM) is a natural bromophenol compound derived from marine algae. Previous reports have shown that BDDPM possesses antimicrobial activity. In the present study, we found that BDDPM has cytotoxic activity on a wide range of tumor cells, including BEL-7402 cells (IC₅₀ = 8.7 μg/mL). Further studies have shown that prior to the onset of apoptosis, the BDDPM induces BEL-7402 cell detachment by decreasing the adherence of cells to the extracellular matrix (ECM). Detachment experiments have shown that the treatment of BEL-7402 cells with low concentrations of BDDPM (5.0 μg/mL) significantly inhibits cell adhesion to fibronectin and collagen IV as well as cell migration and invasion. High doses of BDDPM (10.0 μg/mL) completely inhibit the migration of BEL-7402 cells, and the expression level of MMPs (MMP-2 and MMP-9) is significantly decreased. Moreover, the expression of β1-integrin and focal adhesion kinase (FAK) is found to be down-regulated by BDDPM. This study suggests that BDDPM has a potential to be developed as a novel anticancer therapeutic agent due to its anti-metastatic activity and also indicates that BDDPM, which has a unique chemical structure, could serve as a lead compound for rational drug design and for future development of anticancer agents.