Pathogen interactions with cytokines and host defence: an overview

This review summarises some of the immune evasion tactics adopted by pathogens. They include the antagonism of immune function through the use of homologues of cytokine receptors, expression of viral proteins which interact with cytokine signal transduction and expression of cytokine mimics and host proteins that influence the Type I or II cytokine responses. Some of the viral defense molecules that interfere with the functions of cytokines include the EBV protein BCRF1 (viral IL-10) which blocks synthesis of cytokines such as IFN-γ, viral IL-17 and IL-8 receptor encoded by the herpesvirus saimiri genome and chemokine receptor homologues of Epstein–Barr virus, herpesvirus saimiri and cytomegalovirus. These immunomodulatory tactics function to protect the host from the lethal inflammatory effects as well as inhibit the local inflammatory response elicited to kill the foreign pathogen. Other strategies include the alterations in cytokine expression such as demonstrated with the hepatitis B virus (HBV) core protein and terminal protein which can inhibit interferon-β gene expression, the interactions of the hepatitis C virus core protein to lymphotoxin-β receptor and the effects of the interferon signal transduction pathway by adenovirus EIA oncogene and HBV by reducing levels or activity of the cytosolic latent transcriptional factors (STATS). Immune evasive strategies of helminth parasites related to cytokine activities will also be briefly discussed

Activation of phosphatidylinositol 3-kinase/Akt signaling by EGF downregulates membranous E-cadherin and â-catenin and enhances invasion in nasopharyngeal carcinoma cells.

Dysregulation of E-cadherin and β-catenin function in cell-cell adhesion is common in nasopharyngeal carcinoma (NPC) and correlates with metastatic disease. In this study, we examined the role of EGF-activated phosphatidylinositol 3-kinase (PI3K)-Akt signaling in E-cadherin and β-catenin regulation. We found that reduced membranous E-cadherin and β-catenin expression was positively correlated with Akt phosphorylation in NPC tissues. EGF treatment disrupted cell-cell adhesion and resulted in mesenchymal morphological features in NPC cell lines (TW01, TW04, and TW06). Western blot analysis showed that the E-cadherin protein level was partially reduced in TW04 cells only and the β-catenin levels were not considerably affected upon EGF treatment. In contrast, quantitative real-time RT-PCR showed that the E-cadherin, but not β-catenin, mRNA levels were markedly reduced by EGF in all cell lines. Immunofluorescent staining revealed that E-cadherin and β-catenin appeared to be markedly reduced on the cell surface and more localized in the cytoplasm. Inhibition of PI3K by LY294002 did not abolish the EGF-induced downregulation of E-cadherin protein or mRNA in TW04 cells but moderately increased the β-catenin protein level in TW01 cells and mRNA level in TW06 cells. However, LY294002 substantially restored or increased cell surface E-cadherin and β-catenin in all EGF-treated cell lines, in concordance with the inhibition of cell morphological changes. Moreover, LY294002 significantly blocked EGF-driven cell invasion, correlating with the elevation of membranous E-cadherin and β-catenin levels. In conclusion, EGF-induced epithelial-to-mesenchymal transition may not be only dependent on downregulation of E-cadherin protein/mRNA but also on mislocalization of E-cadherin and β-catenin. The mechanisms involved may be related, at least in part, to the PI3K-Akt pathway

Are there “Magic Bullets” for cancer theraphy

Despite continuing intensive research, cancer is still a devastating disease with high mortality rates. However, progress made in understanding the behavior of tumour cells has led to advances in the creation of ‘magic bullets’ for treating cancer as exemplified by imatinib, an inhibitor that targets an oncogenic kinase, namely, bcr-abl. This article will focus on the seminal discoveries made in oncogenes, growth factor receptors and tyrosine phosphorylation and contributions from my research on signal transduction, adoptive immunotherapy and drug discovery. Signal transduction is a process whereby a cascade of proteins is activated inside the cell causing the cancer cell to grow uncontrollably and spread to other parts of the body. By blocking the signals with molecular-targeted drugs, the cancer cell growth can be controlled. The success of molecularly-targeted drugs depends on the target and knowledge of the signal transduction network. Due to the limited information on tumours available in Malaysia, we set out to determine the incidence of expression of proteins such as the epidermal growth factor receptor (EGFR), phosphatidyl-inositol-3-kinase (PI3K), pTEN, Akt and downstream targets such as β-catenin, pBAD, pGSK, pmTOR and pFHKR, by immunohistochemistry. This can provide insights into the possible mechanism of survival of colorectal, breast and nasopharyngeal carcinoma cells. The contributions of activating mutations such as PI3K in colorectal, breast and nasopharyngeal carcinoma were also compared. This approach revealed the potential therapeutic targets relevant to our population. In the second phase of the research program, we attempted to isolate inhibitors of signal transduction from soil-derived Actinomycetes . The ability of natural compounds purified from extracts of soil-derived Actinomycetes (genus Streptomycetes) to induce apoptosis, cytotoxic and cell cycle effects were also measured. This led to the discovery of the ability to arrest cells in the G1 phase in a breast cancer cell line, MCF-7. Further, the synergistic effect with a PI3K inhibitor was also revealed. Another approach to cancer treatment is adoptive immunotherapy. Conditions for the generation of cytotoxic CD8+ T lymphocytes with recognition for HER2-peptide specific and Epstein Barr viral protein and latent membrane protein-2 (LMP-2) were established to obtain cells for treating breast and nasopharyngeal carcinoma patients. The existence of cells with T regulatory cell phenotype was detected in breast and nasopharyngeal carcinoma. An alternative state of the art approach using mesenchymal stem cells for cancer therapy was also attempted. Subsequently, the immunomodulatory effects of umbilical mesenchymal stem cells was demonstrated with K562, an undifferentiated erythromyeloblastoid leukemia cell line. Lastly, the development of molecular diagnostic tests for ten species of Candida species was achieved. Monoclonal antibodies against Candida glabrata and Aspergillus fumigatus were also produced. In summary, we have successfully demonstrated the aberrations of the signal transduction pathway in primary breast, colorectal and nasopharyngeal carcinoma. This study has given us an insight into the relevant biomolecules in the cascade. Results from the nasopharyngeal carcinoma tissues revealed that the PI3K/Akt/mTOR pathway is not a linear pathway. The potency of the natural compound for cancer treatment will need to be tested on animal models. The second approach of harnessing the immune system showed promising results. The effectiveness of the cytotoxic T cells and mesenchymal stem cells generated will need to be tested on animal models. Thus, further work is warranted to improve treatment outcomes for cancer patient

Cancer immunotherapy: current progress and applications

Cancer immunotherapy is a form of treatment protocol for cancer patients that have been studied intensively over the last two decades. The undesirable side effects during the course of conventional treatment have lead to the development of immunotherapy as an alternative treatment modality. This approach encompasses the use of three different strategies with various immunotherapeutic modalities including (i) cytokines and monoclonal antibodies; (ii) activation of antigen presentation cells (APC) by using antigen-specific peptides or sources of antigens such as tumour lysate; and finally (iii) adoptive transfer of ex vivo activated autologous cytotoxic T-cells. Due to specific-targeting by antigen-specific monoclonal antibodies, dendritic cells and activated CD8 + T-cells, immunotherapy can eliminate tumour cells efficiently but the normal tissues are unaffected. Despite years of investigation, the outcome of immunotherapy-based clinical trials are inconsistent with very low response rates from patients. Several mechanisms have been proposed to contribute to this failure including the presence of regulatory T-cells (Treg), immunomodulatory cytokines, and aberrant gene expression in tumour cells. This review summarises information from about 140 articles and review papers. In addition, it also provides an update on recent trends in combinational immunotherapy with conventional therapy and encouraging results have been obtained. Re- evaluation of previous studies is necessary to fine-tune the design and approach of immunotherapy to ensure better treatment outcomes

Immunomodulatory potential of mesenchymal stem cells on microglia

It is becoming increasingly evident that inflammatory reactions of microglia contribute to the pathology of neurodegenerative diseases. Although the focus for rescuing neurones previously lied on minimising direct insult (including limiting aggregation of misfolded proteins and antagonising the effects of glutamate), therapeutic approaches now include moderating the ensuing inflammatory responses of microglia. Microglia responses in the central nervous system (CNS) are diverse and their involvement in both neuroprotection and neurotoxicity may seem paradoxical. Accordingly, management of neuroinflammation must include an understanding of conditions that trigger neurotoxic responses by microglia and deciphering strategies to maintain their neuroprotective phenotype. Mesenchymal stem cells (MSC) are stem cells with great capacity for immunomodulation on a wide range of immune cells. Evidence presented here highlights the potential of using MSC to modulate the inflammatory responses of microglia. The mechanisms underlying the ability of MSC to moderate microglia responses are also explained in this review. Although many aspects of this approach will require defined characterisation, MSC serve as a potential cell-based therapy that may slow or halt the progression of inflammatory CNS diseases

Isolation of a potential anticancer agent with protein phosphatase inhibitory activity from soil-derived Penicillium sp. strain H9318

Purpose: To determine the effect of the secondary metabolites from Penicillium sp. H9318 on cytotoxicity and cell cycle progression. Methods: A yeast PP1 inhibitory screening system was carried out to confirm the presence of anti-PP1c activity in crude acetone extracts of strain H9318. The extracts were fractionated and identified as Fraction S1 and Citrinin 9318 (CTN9318). Various cancer cell lines were used to test for the toxicity of the crude acetone extracts, Fraction S1 and Citrinin 9318, using MTT viability assay. Results: It was found that a colorectal cancer cell line, HT-29, was susceptible to Fraction S1 and Citrinin 9318. A propidium iodide (PI)-incorporated DNA assay was used to show that there was G2/M arrest in HT-29 by Citrinin 9318. Conclusion: Citrinin 9318 inhibits the viability of HT-29 via mitotic block. The results suggest that Citrinin 9318 is capable of exerting cytotoxicity and mitotic arrest in a colon cancer cell line, HT29

Characterization of tumorspheres generated from nasopharyngeal carcinoma cell line, TW06 and chemoresistance to docetaxel and oxaliplatin

In this study, tumorspheres were generated from TW06 nasopharyngeal carcinoma cell line and examined their expression of putative cancer stem-like cell surface markers and drug sensitivity. The rate of tumorsphere expansion from dissociated late passage TW06 tumorspheres (≥ passage 15) was higher than that from parental cells and dissociated 10-day-old (passage 0) tumorspheres. The expression of CD24 surface marker was lost in the generation of tumorspheres and the loss was reversible after differentiating the tumorspheres in monolayer culture conditions. Drug sensitivity assay showed that late passage tumorspheres were resistant to docetaxel and oxaliplatin treatment. Our data suggest that serially passaged tumorspheres possess the characteristics of CSCs that render them a suitable preclinical in vitro model for evaluating anticancer drug efficacy and elucidating the underlying mechanisms of drug resistance

Selection of high affinity ligands to hepatitis B core antigen from a phage-displayed cyclic peptide library.

M13 phages that display random disulfide constrained heptapeptides on their gpIII proteins were used to select for high affinity ligands to hepatitis B core antigen (HBcAg). Phages bearing the amino acid sequences C-WSFFSNI-C and C-WPFWGPW-C were isolated, and a binding assay in solution showed that these phages bind tightly to full-length and truncated HBcAg with KDrel values less than 25 nM, which is at least 10 orders of magnitude higher than phage carrying the peptide sequence LLGRMK selected from a linear peptide library. Both the phages that display the constrained peptides were inhibited from binding to HBcAg particles by a monoclonal antibody that binds specifically to the immunodominant region of the particles. A synthetic heptapeptide with the amino acid sequence WSFFSNI derived from one of the fusion peptides inhibits the binding of large surface antigen (L-HBsAg) to core particles with an IC50 value of 12 ± 2 μM. This study has identified a smaller peptide with a greater inhibitory effect on L-HBsAg-HBcAg association

Transcriptome profiling of endothelial cells during infections with high and low densities of Candida albicans cells

Systemic infections of Candida albicans, the most prevalent fungal pathogen in humans, are on the rise in recent years. However, the exact mode of pathogenesis of this fungus is still not well elucidated. Previous studies using C. albicans mutants locked into the yeast form via gene deletion found that this form was avirulent and did not induce significant differential expression of host genes in vitro. In this study, a high density of C. albicans was used to infect human umbilical vein endothelial cells (HUVEC), resulting in yeast-form infections, whilst a low density of C. albicans resulted in hyphae infections. Transcriptional profiling of HUVEC response to these infections showed that high densities of C. albicans induced a stronger, broader transcriptional response from HUVEC than low densities of C. albicans infection. Many of the genes that were significantly differentially expressed were involved in apoptosis and cell death. In addition, conditioned media from the high-density infections caused a significant reduction in HUVEC viability, suggesting that certain molecules released during C. albicans and HUVEC interactions were capable of causing cell death. This study has shown that C. albicans yeast-forms, at high densities, cannot be dismissed as avirulent, but instead could possibly contribute to C. albicans pathogenesis