Effect of ivermectin on the cellular and humoral immune responses of rabbits

The objective of this paper is to determine the effect of ivermectin administration on cell mediated (CMI) and humoral immunity (HI) of rabbits. CMI against dinitrochlorobenzene (DNCB) and sheep red blood cells (SRBC) in rabbits was determined by delayed-type hypersensitivity and macrophage engulfment assay (MEA), respectively; whereas, HI to Pasteurella multocida B2 vaccine and SRBC was determined by indirect haemagglutination assay (IHA) and Jerne hemolytic plaque formation assay (JHPFA), respectively. The rabbits were divided into four major groups (A through D) each subdivided into four sub-groups (1 through 4). Rabbits of group A served as vehicle control while those of groups B, C and D were treated with ivermectin at the dose rates of 200 microg/kg, 400 microg/kg and 600 microg/kg b.w., respectively. Cellular immunity was determined in sub-groups 1 and 2 through DNCB and MEA, respectively while HI was determined in sub-groups 3 and 4 through IHA and JHPFA, respectively. The skin sensitivity to DNCB at 24 and 48 h and macrophage engulfment of SRBC were highest (P>0.05) in rabbits administered with 600 microg/kg b.w. The highest geometric mean titers (14.00+/-0.31) and number of plaque forming units (1860+/-0.75) were found in rabbits that received ivermectin at a dose of 600 microg/kg b.w. followed, in order by the groups that received 400 microg/kg, 200 microg/kg b.w. and controls. Leukocyte counts were significantly higher in ivermectin-treated groups (C and D) than group A (vehicle control) and B (ivermectin at the rate of 200 microg/kg). A graded dose immune response suggested an immunopotentiating effect of ivermectin at higher doses.     

Role of the Akt/mTOR survival pathway in cisplatin resistance in ovarian cancer cells

The mechanism of cisplatin resistance in cancer cells is not fully understood. Here, we show that the Akt/mTOR survival pathway plays an important role in cisplatin resistance in human ovarian cancer cells. Specifically, we found that cisplatin treatment activates the Akt/mTOR survival pathway and that inhibition of this pathway by the PI3 K inhibitor LY294002 or knockdown of Akt sensitizes ovarian cancer cells to cisplatin. Furthermore, we generated cisplatin-resistant cells and found that resistant cells express a higher level of activated Akt as compared to their cisplatin sensitive counterparts. Importantly, inhibition of Akt or mTOR sensitized resistant cells to cisplatin-induced apoptosis. Taken together, our data indicate that activation of the Akt/mTOR pathway prevents cisplatin-induced apoptosis, leading to cisplatin resistance. Therefore, our study suggests that cisplatin resistance can be overcome by targeting the Akt/mTOR survival pathway in human ovarian cancer cells.     

Oridonin effectively reverses the drug resistance of cisplatin involving induction of cell apoptosis and inhibition of MMP expression in human acute myeloid leukemia cells

Cisplatin is the first generation platinum-based chemotherapy agent. However, the extensive application of cisplatin inevitably causes drug resistance, which is a major obstacle to cancer chemotherapy. Oridonin is a diterpenoid isolated from Rabdosia rubescens with potent anticancer activity. The aim of our study is to investigate the role of oridonin to reverse the cisplatin-resistance in human acute myeloid leukemia (AML) cells. The effect of oridonin on human AML cell proliferation was evaluated by MTT assay, cell migration and invasion were evaluated by transwell migration and invasion assays in cisplatin-resistant human AML cells. Furthermore, cell apoptosis was examined by flow cytometry. The inhibitive effect of oridonin in vivo was determined using xenografted nude mice. In addition, the expressions of MMP2 and MMP9 were detected by Western blot. There was a synergistic antitumor effect between cisplatin and oridonin on cisplatin-resistant human AML cells in vitro and in vivo. In addition, the combination of cisplatin and oridonin synergistically induced cell apoptosis. Furthermore, the combination treatment not only inhibited AML cell migration and invasion, but more significantly, decreased the expressions of MMP2 and MMP9 proteins. Our results suggest that the synergistic effect between both agents is likely to be driven by the inhibition of MMP expression and the resulting increased apoptosis.     

New extracellular resistance mechanism for cisplatin

The HSQC NMR spectrum of ¹⁵N-cisplatin in cell growth media shows resonances corresponding to the monocarbonato complex, cis-[Pt(NH₃)₂(CO₃)Cl]⁻, 4, and the dicarbonato complex, cis-[Pt(NH₃)₂(CO₃)₂]⁻², 5, in addition to cisplatin itself, cis-[Pt(NH₃)₂Cl₂], 1. The presence of Jurkat cells reduces the amount of detectable carbonato species by (2.8 ± 0.7) fmol per cell and has little effect on species 1. Jurkat cells made resistant to cisplatin reduce the amount of detectable carbonato species by (7.9 ± 5.6) fmol per cell and also reduce the amount of 1 by (3.4 ± 0.9) fmol per cell. The amount of detectable carbonato species is also reduced by addition of the drug to medium that has previously been in contact with normal Jurkat cells (cells removed); the reduction is greater when drug is added to medium previously in contact with resistant Jurkat cells (cells removed). This shows that the platinum species are modified by a cell-produced substance that is released to the medium. Since the modified species have been shown not to enter or bind to cells, and since resistant cells modify more than non-resistant cells, the modification constitutes a new extracellular mechanism for cisplatin resistance which merits further attention.     

In vivo imaging of tumor growth after electrochemotherapy with cisplatin

Imaging methods can give both temporal and spatial dimensions to characterize the processes in progression of and/or treatment of specific disease Subcutaneous tumors can be cured after electrochemotherapy (ECT). Growth and reduction of tumors as a result of cytotoxic therapy can be followed by fluorescence video imaging directly on the same animal after treatment. Imaging of tumors should bring more information on the cellular effects of ECT. Green fluorescent protein (eGFP) expressing B16F10 and LPB tumors implanted in C57Bl/6 mice were treated with ECT with cisplatin. The growth or regression of the tumors was monitored either classically by using a caliper or by a manual definition of the region of interest where critical fluorescence levels were detected on the animals. A very good correlation between the two methods was observed. The eGFP mean fluorescence emission was only slightly affected by ECT with intravenously injected cisplatin. Ex vivo observations under a fluorescence microscope showed that eGFP was only detected on the outer layer of the tumor. No fluorescence was detected in the central part of the tumors, which were necrotic.     

Insect immune resistance to parasitoids

Insect host-parasitoid interactions involve complex physiological, biochemical and genetic interactions. Against endoparasitoids, immune-competent hosts initiate a blood cell-mediated response that quickly destroys the intruders and envelops them in a multilayered melanotic capsule. During the past decade, considerable progress has been made in identifying some of the critical components of the host response, mainly because of the use of efficient molecular tools. This review examines some of the components of the innate immune response of Drosophila, an insect that has served as an exceptionally good experimental model for studying non-self recognition processes and immune cell signaling mechanisms. Topics considered in this review include hematopoiesis, proliferation and adhesion of hemocytes, melanogenesis and associated cytotoxic molecules, and the genetic aspects of the host-parasitoid interaction.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.     

Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats

An herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51+/-0.22 mg/dl (mean+/-SE) from 0.28+/-0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86+/-0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8+/-13.0% and 220.8+/-39.3% (mean+/-SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity.     

Regulatory T cells and tumor immunity

Central deletion of self-reactive T cells has been the textbook paradigm for inducing self-tolerance in the periphery and the concept of a role of T cell-mediated suppression in this process has long been controversial. A decisive shift in the opinion on suppressor T cells has lately occurred with the observations of Sakaguchis group that linked a class of CD4+CD25+ T cells to the prevention of autoimmunity from neonatal thymectomy in mice. These CD4+CD25+ T cells have been named T regulatory (Treg) cells. They are believed to be selected in the thymus as an anti-self repertoire. Hence they were referred to as natural T regulatory (nTreg) cells. Presently, in addition to their role in autoimmunity, they are believed to exert regulatory function in infection, in transplantation immunity as well as in tumor immunity. In contrast to these nTreg cells, another class of CD4+ Treg cells also exercises regulatory function in the periphery. These Treg cells are also CD4+ T cells and after activation they also become phenotypically CD4+CD25+. They are, however induced in the periphery as Treg cells. Hence, they are termed as induced Treg (iTreg) cells. There are major differences in the biology of these two types of Treg cells. They differ in their requirements for activation and in their mode of action. Nonetheless, evidence indicates that both nTreg cells and iTreg cells are involved in the control of tumor immunity. The question of how to circumvent their regulatory constraints, therefore, has become a major challenge for tumor immunologists.     

Active hexose correlated compound enhances tumor surveillance through regulating both innate and adaptive immune responses

Active hexose correlated compound (AHCC) is a mixture of polysaccharides, amino acids, lipids and minerals derived from cocultured mycelia of several species of Basidiomycete mushrooms. AHCC has been implicated to modulate immune functions and plays a protective role against infection. However, the potential role of AHCC in tumor immune surveillance is unknown. In this study, C57BL/6 mice were orally administered AHCC or water, followed by tumor cell inoculation. We showed that compared to pure water-treated mice, AHCC treatment significantly delayed tumor development after inoculation of either melanoma cell line B16F0 or lymphoma cell line EL4. Treatment with AHCC enhanced both Ag-specific activation and proliferation of CD4⁺ and CD8⁺ T cells, increased the number of tumor Ag-specific CD8⁺ T cells, and more importantly, increased the frequency of tumor Ag-specific IFN-γ producing CD8⁺ T cells. Interestingly, AHCC treatment also showed increased cell number of NK and γδ T cells, indicating the role of AHCC in activating these innate-like lymphocytes. In summary, our results demonstrate that AHCC can enhance tumor immune surveillance through regulating both innate and adaptive immune responses.     

NK cells rapidly remove B16F10 tumor cells in a perforin and interferon-gamma independent manner in vivo

Natural killer (NK) cells have been shown critical in reducing tumor lung metastasis in various murine cancer models. Effector molecules such as perforin and IFN-γ may play important roles in inhibition of metastasis. However, most of these conclusions were based on experiments that involved quantitation of metastatic colonies several weeks after tumor challenge. The roles of NK cells and their effector molecules (perforin and IFN-γ) in the initial immune responses against tumor metastasis in lungs are still unknown. By using the B16F10 melanoma tumor model combined with confocal microscopy, we observed an increase in numbers of B16F10 cells in NK-depleted mice at 60 min post tumor inoculation, but this effect was independent of perforin or IFN-γ. In addition, NK cell numbers in lungs after tumor injection rapidly increased suggesting a redistribution of NK cells in the lungs. However, NK cells were not found in contact with tumor cells until day 6 or later. Our data indicate that during early responses against B16F10 cells, NK cells use another mechanism(s) besides perforin and IFN-γ to prevent tumor metastasis.     

Loss of putative tumor suppressor EI24/PIG8 confers resistance to etoposide

Expression of p53-target gene EI24/PIG8 is lost in invasive breast cancers, suggesting that EI24/PIG8 is a tumor suppressor that prevents tumor spreading, and partially mediates p53-attributed tumor suppressor activity. EI24/PIG8 also has pro-apoptotic activity indicating that loss of EI24/PIG8 may modulate sensitivity to chemotherapy. Here it is demonstrated that suppression of EI24/PIG8 in fibroblasts and breast cancer cells significantly inhibits the apoptotic response to etoposide treatment. These findings suggest that loss of EI24/PIG8 contributes significantly to resistance of cells to chemotherapeutic agents that function through p53, and identify the EI24/PIG8 status as a potentially new prognostic marker of chemotherapy responsiveness.     

Epigenetic regulation of immune escape genes in cancer

According to the concept of immune surveillance, the appearance of a tumor indicates that it has earlier evaded host defenses and subsequently must have escaped immunity to evolve into a full-blown cancer. Tumor escape mechanisms have focused mainly on mutations of immune and apoptotic pathway genes. However, data obtained over the past few years suggest that epigenetic silencing in cancer may be as frequent a cause of gene inactivation as are mutations. Here, we discuss the evidence that tumor immune evasion is mediated by non-mutational epigenetic events involving chromatin and that epigenetics collaborates with mutations in determining tumor progression. Since epigenetic changes are potentially reversible, the relative contribution of mutations and epigenetics, to the gene defects in any given tumor, may be a factor in determining the efficacy of treatments. We review new developments in basic chromatin mechanisms and in this context describe the rationale for the current use of epigenetic agents in cancer therapy and for a novel epigenetically generated tumor vaccine model. We emphasize that epigenetic cancer treatments are currently a ‘blunt-sword’ and suggest future directions for designing chromatin-based programs of potential value in the diagnosis and treatment of cancer.     

凝结芽胞杆菌对免疫功能影响的实验研究

目的 研究凝结芽胞杆菌（TQ33）对实验动物免疫功能模型的影响。方法 以鸡红细胞混悬液对小鼠腹腔注射,观察TQ33对小鼠腹腔巨噬细胞吞噬功能的影响;制备绵羊红细胞（SRBC）,观察血球凝集程度,测定血清溶血素;以靶细胞（YAC-1细胞）与脾细胞（效应细胞）的反应检测TQ33。对小鼠NK细胞活性的影响;采用淋巴细胞转化法观察TQ33对细胞免疫的影响;计算小鼠胸腺／体重及脾脏／体重为脏器系数观察TQ33对小鼠脏器的影响。结果 与对照组比较,TQ33显著增加小鼠腹腔巨噬细胞吞噬功能;对小鼠体液免疫功能有一定的增强作用;可增强小鼠NK细胞活性;对ConA诱导下的小鼠淋巴细胞转化有增强作用;对脏器系数没有显著的影响。结论 凝结芽胞杆菌具有明显的免疫调节作用,预示有良好的应用前景。     

Liposome-aided metabolic engineering of tumor surface immunogenicity

Approaches to increase tumor immunogenicity are of therapeutic potentials. We herein reported the use of liposomes for covalent incorporation of neoantigen on tumor surfaces with DNP-conjugated sialic acid (^DNPSia). Relative to free ^DNPSia, sugar-encapsulated biotinylated liposomes (^DNPSia@LP@biotin) enables effective cell surface expression of ^DNPSia on biotin receptor (BR)-expressing cells over BR-free cells in vitro, and on tumor cell surfaces with high tumor-to-normal tissue contrast in a mice model. These findings suggest the potentials of targetable liposomes for modulating tumor surface immunity via metabolic oligosaccharide engineering.     

Annexin-I expression modulates drug resistance in tumor cells

The use of anti-cancer chemotherapy often leads to the rise of multidrug-resistant (MDR) tumors. We have previously reported the overexpression of a 40kDa protein (P-40) in several MDR tumor cell lines. In this report we describe the cloning of a 1.4kb cDNA with an open reading frame of 344 amino acids that encodes the P-40 protein. Analysis of the P-40 amino acid sequence showed it is identical to the human annexin I (Anx-I) protein. The identity of the isolated P-40 cDNA as Anx-I was confirmed by the specific binding of IPM96 mAb to a 40kDa protein following the in vitro expression of P-40 full-length cDNA. Northern blot analysis of total RNA from drug-sensitive and -resistant cells revealed an increase in P-40 (or Anx-I) mRNA in drug-resistant cells relative to drug-sensitive cells. Transfection of Anx-I cDNA into drug-sensitive MCF-7 cells was carried out without further drug selection and showed 2- to 5-fold increase in resistance of transfected cells to adriamycin, melphalan, and etoposide. Conversely, transfection of reverse Anx-I cDNA into SKOV-3 cells decreased the expression of Anx-I without affecting the expression of other members of the annexin family and showed a 3- to 8-fold increase in sensitivity to these drugs. Of interest was the correlation between the presence of Anx-I and MDR in MDA-MB-231 cells when compared to MCF-7 cells. MDA-MB-231 cells show 3- to 20-fold increase in resistance to adriamycin, melphalan, and etoposide in the absence of detectable levels of P-glycoprotein (P-gp1), the multidrug resistance protein (MRP1) or the breast cancer resistance protein (BCRP). Taken together, these results provide the first direct evidence for the role of Anx-I in MDR of tumor cells.     

Changes in biophysical parameters of plasma membranes influence cisplatin resistance of sensitive and resistant epidermal carcinoma cells

The mechanism of resistance of cancer cells to the anticancer drug cisplatin is not fully understood. Using cisplatin-sensitive KB-3-1 and -resistant KCP-20 cells, we found that the resistant cells have higher membrane potential, as determined by membrane potential sensing oxonol dye. Electron spin resonance and fluorescence polarization studies revealed that the resistant cells have more "fluid" plasma membranes than the sensitive cells. Because of this observed difference in membrane "fluidity," we attempted modification of the plasma membrane fluidity by the incorporation of heptadecanoic acid into KB-3-1 and KCP-20 cell membranes. We found that such treatment resulted in increased heptadecanoic acid content and increased fluidity in the plasma membranes of both cell types, and also resulted in increased cisplatin resistance in the KCP-20 cells. This finding is in accord with our results, which showed that the cisplatin-resistant KCP-20 cells have more fluid membranes than the cisplatin-sensitive KB-3-1 cells. It remains to be determined whether the observed differences in biophysical status and/or fatty acid composition alone, or the secondary effect of these differences on the structure or function of some transmembrane protein(s), is the reason for increased cisplatin resistance.     

Defining targets of modulation of human tumor cell response to cisplatin

We previously observed that in yeast cisplatin activates different pathways accounting for stress response. Here, we investigated whether genes involved in yeast drug response were modulated by cisplatin in human tumor cell lines (A2780, IGROV-1, A431, U2-OS) including cisplatin-resistant sublines (A2780/BBR, IGROV-1/Pt1, A431/Pt and U2-OS/Pt). Factors and pathways involved in stress response (glutathione-S-transferase, proteasome, checkpoint control and recombinational repair) were increased by cisplatin in human tumor sensitive and resistant cells. Moreover, sensitization to cisplatin by pharmacologically targeting glutathione or proteasome was observed in sensitive and resistant cells. Interestingly, only in IGROV-1/Pt1 cells, in which cisplatin up-regulated HSP70 and HSP90, targeting of HSP90 resulted in sensitization of resistant cells, suggesting a protective role of stress response. In conclusion, the present findings support the potential relevance of interfering with heat shock protein response to increase cisplatin cytotoxicity in resistant cells. Overall, pathways activated by cisplatin in human tumor cells appear cell-type specific, at least in part reflecting the stress response observed in yeast.     

A novel immunoregulatory axis of NKT cell subsets regulating tumor immunity

There are many mechanisms that regulate and dampen the immune response to cancers, including several types of regulatory T cells. Besides the T reg cell, we have identified another immunoregulatory circuit initiated by NKT cells that produce IL-13 in response to tumor growth and this IL-13 then induces myeloid cells to make TGF-beta that inhibits cytotoxic T cell-mediated tumor immunosurveillance in several mouse tumor models. This finding created a paradox in the role of NKT cells in tumor immunity, in that they can also contribute to protection. We resolve this paradox by the finding that the suppressive NKT cell is a type II NKT cell that lacks the canonical invariant T cell receptor, whereas the protective cell is a type I NKT cell that expresses the invariant receptor. Further, we see that these two subsets of NKT cells counter-regulate each other, defining a new immunoregulatory axis. The balance along this axis may determine the outcome of tumor immunosurveillance as well as influence the efficacy of anti-cancer vaccines and immunotherapy.