Increased tube formation and up-regulation of FGFR3 mRNA expression in microvascular endothelial cell by exosomes derived from SW480-7.

INTRODUCTION: Extracellular vesicles (exosome-like vesicles) are small membrane vesicles ranging from 20-200nm in size that are released by various cells into the extracellular space. These extracellular vesicles play a major role in cell-to-cell communication and contain materials, such as proteins, mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). The effect of exosomes derived from an invasive colon cancer cell line on angiogenesis is unclear. Hence, the aim of this study is to investigate the effect of exosomes derived from an invasive colon cancer cell line on angiogenesis of endothelial cells. MATERIALS AND METHODS: In the present study, the exosomes from the cell culture supernatants of an invasive colon cancer cell line SW480-7 were characterised. The effect on tube formation and expression of angiogenic genes in a microvascular endothelial cell, telomerase-immortalised microvascular endothelial cell (TIME) was examined after co-cultured with exosomes secreted from SW480-7. RESULTS: Zetasizer result showed average diameter of exosomes derived from SW480-7 was 246.2 nm and morphological analysis showed the size of majority of exosomes were less than 200 nm. Results showed that exosomes derived from SW480-7 increased tube formation and up-regulated FGFR3 mRNA expression in TIME. CONCLUSION: Our findings suggest that exosomes derived from SW480-7 increased tube formation and up-regulated expression of FGFR3 mRNA in TIME.

An insight of dysregulation of microRNAs in the pathogenesis of diabetic kidney disease.

BACKGROUND: Worldwide, diabetic kidney disease (DKD) remains the leading cause of chronic kidney disease (CKD) and its successor, the end stage renal disease, both of which constitute major morbidity and mortality concerns. CONTENT: The residual risk of disease progression remains despite the advert of newer therapeutic modalities and current biomarkers. Meanwhile, microRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression post-translationally by binding to specific mRNAs. Circulating miRNAs are increasingly recognised as novel biomarker or therapeutic targets, owing to their unique characteristics, such as their resilience to degradation by endogenous RNases, multiple downstream targets, involvement in biological processes, some degree of tissue specificity, relatively easy access and quantification. Unlike proteins, there are far less miRNAs and mature miRNAs are highly stable, structurally less complex without post-translational modification with high degree of conservation across species. Aberrant expression of miRNAs has been established in both in vitro and in vivo models of DKD. An up-to-date compilation of previous studies involving selected circulating miRNAs in blood and urine samples of DKD patients is discussed herein. SUMMARY: This review highlights the unmet clinical challenges and dysregulation of miRNAs in the pathogenesis of DKD.

HER3 overexpression and hypomethylation in colorectal adenocarcinoma.

INTRODUCTION: Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cancer in Malaysia. Despite advanced therapies, many cases of recurrence and resistance have been reported. Aberrant DNA methylation of HER3 has been implicated in carcinogenesis of CRC mainly through the regulation of gene expression. Hence, the objective of this study was to determine the status of HER3 DNA methylation and its effects on gene expression in CRC. MATERIALS AND METHODS: Fifty-nine of archival formalin-fixed, paraffin-embedded CRC cases with the adjacent normal colon tissues were retrieved. Manual micro-dissection was performed prior to RNA and DNA extraction. HER3 gene expression and DNA methylation status was evaluated by qPCR and methylation-specific PCR (MSP) techniques respectively. RESULTS: Upregulation of HER3 mRNA was found in CRC tissue compared to its adjacent normal colon tissue (8.04-fold). Of 59 CRC samples, 8.5% were methylated and 91.5% were unmethylated (hypomethylation). In the adjacent normal colon tissues, methylated and unmethylated tissue were observed in 6.8%and 93.2% respectively. DNA methylation of HER3 showed a significant association with tumour differentiation and tumour location. CONCLUSION: This study showed upregulation and hypomethylation of the HER3 gene in CRC cases. Epigenetic alterations were also found in the adjacent normal colon tissues. Thus, upregulation and hypomethylation of HER3 may play a key role in carcinogenesis of CRC. Hypomethylation of CpG islands might be associated with early steps during carcinogenesis. The findings of this biomarker serve a powerful approach to improve the current diagnostic and therapeutic measures.

Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 exhibit strong antifungal effects against vulvovaginal candidiasis-causing Candida glabrata isolates.

AIMS: This study investigates the antagonistic effects of the probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 against vulvovaginal candidiasis (VVC)-causing Candida glabrata. METHODS AND RESULTS: Growth inhibitory activities of Lact. rhamnosus GR-1 and Lact. reuteri RC-14 strains against C. glabrata were demonstrated using a spot overlay assay and a plate-based microtitre assay. In addition, these probiotic lactobacilli strains also exhibited potent candidacidal activity against C. glabrata, as demonstrated by a LIVE/DEAD yeast viability assay performed using confocal laser scanning microscopy. The metabolic activities of all C. glabrata strains were completely shut down in response to the challenges by the probiotic lactobacilli strains. In addition, both probiotic lactobacilli strains exhibited strong autoaggregation and coaggregation phenotypes in the presence of C. glabrata, which indicate that these lactobacilli strains may exert their probiotic effects through the formation of aggregates and, thus the consequent prevention of colonization by C. glabrata. CONCLUSIONS: Probiotic Lact. rhamnosus GR-1 and Lact. reuteri RC-14 strains exhibited potent antagonistic activities against all of the tested C. glabrata strains. These lactobacilli exhibited antifungal effects, including those attributed to their aggregation abilities, and their presence caused the cessation of growth and eventual cell death of C. glabrata. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report on the antagonistic effects of these probiotic lactobacilli strains against the non-Candida albicans Candida (NCAC) species C. glabrata.

Candida and invasive candidiasis: back to basics.

The ubiquitous Candida spp. is an opportunistic fungal pathogen which, despite treatment with antifungal drugs, can cause fatal bloodstream infections (BSIs) in immunocompromised and immunodeficient persons. Thus far, several major C. albicans virulence factors have been relatively well studied, including morphology switching and secreted degradative enzymes. However, the exact mechanism of Candida pathogenesis and the host response to invasion are still not well elucidated. The relatively recent discovery of the quorum-sensing molecule farnesol and the existence of quorum sensing as a basic regulatory phenomenon of the C. albicans population behavior has revolutionized Candida research. Through population density regulation, the quorum-sensing mechanism also controls the cellular morphology of a C. albicans population in response to environmental factors, thereby, effectively placing morphology switching downstream of quorum sensing. Thus, the quorum-sensing phenomenon has been hailed as the 'missing piece' of the pathogenicity puzzle. Here, we review what is known about Candida spp. as the etiological agents of invasive candidiasis and address our current understanding of the quorum-sensing phenomenon in relation to virulence in the host.

Mesenchymal stem cells inhibit proliferation of lymphoid origin haematopoietic tumour cells by inducing cell cycle arrest.

We have previously shown that mesenchymal stem cells (MSC) inhibit tumour cell proliferation, thus promising a novel therapy for treating cancers. In this study, MSC were generated from human bone marrow samples and characterised based on standard immunophenotyping. When MSC were co-cultured with BV173 and Jurkat tumour cells, the proliferation of tumour cells were profoundly inhibited in a dose dependent manner mainly via cell to cell contact interaction. Further cell cycle analysis reveals that MSC arrest tumour cell proliferation in G0/G1 phase of cell cycle thus preventing the entry of tumour cells into S phase of cell cycle.

Increase in tumour-infiltrating lymphocytes with regulatory T cell immunophenotypes and reduced zeta-chain expression in nasopharyngeal carcinoma patients.

The pathological significance of the mechanisms of tumour immune-evasion and/or immunosuppression, such as loss of T cell signalling and increase in regulatory T cells (T(regs)), has not been well established in the nasopharyngeal carcinoma (NPC) microenvironment. To evaluate the T(reg) immunophenotypes in tumour-infiltrating lymphocytes (TILs), we performed a double-enzymatic immunostaining for detection of forkhead box P3 (FoxP3) and other markers including CD4, CD8, and CD25 on 64 NPC and 36 non-malignant nasopharyngeal (NP) paraffin-embedded tissues. Expression of CD3 zeta and CD3 epsilon was also determined. The prevalence of CD4(+)FoxP3(+) cells in CD4(+) T cells and the ratio of FoxP3(+)/CD8(+) were increased significantly in NPC compared with those in NP tissues (P < 0.001 and P = 0.025 respectively). Moreover, the ratio of FoxP3(+)/CD25(+)FoxP3(-) in NPC was significantly lower than that in NP tissues (P = 0.005), suggesting an imbalance favouring activated phenotype of T cells in NPC. A significant negative correlation between the abundance of FoxP3(+) and CD25(+)FoxP3(-) cells (P < 0.001) was also identified. When histological types of NPC were considered, a lower ratio of FoxP3(+)/CD25(+)FoxP3(-) was found in non-keratinizing and undifferentiated carcinomas. Increased CD4(+)FoxP3(+)/CD4(+) proportion and FoxP3(+)/CD8(+) ratio were associated with keratinizing squamous cell carcinoma. A reduced expression of CD3 zeta in TILs was found in 20.6% of the NPC tissues but none of the NP tissues. These data provide evidence for the imbalances of T(reg) and effector T cell phenotypes and down-regulation of signal-transducing molecules in TILs, supporting their role in suppression of immune response and immune evasion of NPC.

Cord blood-derived mesenchymal stem cell does not stimulate nor inhibits T lymphocytes activation.

The immune modulatory properties of mesenchymal stem cell (MSC) had brought a new insight in cell-based neotherapy. However, recent works of MSC are focused exclusively on bone marrow-derived MSC. We evaluated the immunogenicity of cord blood-derived MSC (CB-MSC) on T lymphocytes. Human peripheral blood mononuclear cells (PBMC) were prepared by density gradient separation and culture with the presence or absence of CB-MSC. PBMC were collected for activation analysis by flow cytometry at 24-, 48-, and 72- hours. The results showed that, CB-MSC does not stimulate nor inhibit T lymphocyte activation.

Investigations into the nature of growth-related proteolysis in human fibroblasts.

Previous experiments have shown that cultured human fibroblasts possess a cell-surface proteinase (the growth-related proteinase; GRP) which is essential to cell proliferation. In the present work, proteinase inhibition in defined and complex serum-free media and in pre-conditioned normal medium, still resulted in a corresponding inhibition of cell proliferation. Proteinase inhibition also blocked the action of a range of peptide growth factors and of a phorbol ester. Elevated extracellular calcium concentrations were still mitogenic in the presence of proteinase inhibitors. Proteinase inhibition did not affect the mobilisation of intracellular calcium, nor the metabolism of inositol phosphate derivatives in response to a mitogenic stimulus.

The nucleotide sequence of the bovine interleukin-5-encoding cDNA.

The nucleotide (nt) sequence encoding the bovine homologue of interleukin-5 (boIL-5) was determined. Total cDNA generated from stimulated bovine peripheral blood lymphocytes was used to amplify the boIL-5 gene using primers based on the 5' and 3' untranslated regions of the ovine IL-5 cDNA sequence. The boIL-5 coding sequence is 405 bp long, coding for a 15.2 kDa precursor protein of 134 amino acids (aa). Cleavage of a putative signal peptide of 19 aa yields a mature form of 13.1 kDa. Comparisons at the nt level revealed 96%, 81%, 74% and 73% identity with the ovine, human, mouse and rat IL-5 sequences, respectively, and 97%, 66%, 59% and 58% aa identity, respectively.

Cloning and sequencing an ovine interleukin-4-encoding cDNA.

We have cloned a cDNA containing the complete coding sequence of ovine(ov) interleukin 4 (IL4) by the polymerase chain reaction using primers based on the 5' and 3' untranslated regions of the human IL4 gene. RNA was isolated from phorbol myristate acetate- and calcium ionphore A23187-stimulated mesenteric lymph node cells. The ovIL4 cDNA is 535 bp in length and contains an open reading frame of 408 nucleotides (nt) coding for a 15.1-kDa IL4 precursor of 135 amino acids (aa). Cleavage of the putative signal peptide of 22 aa yields the mature form of 13.2 kDa. Analysis of the mature aa sequence shows two potential N-linked glycosylation sites and six Cys residues. Ovine and bovine IL4 are shorter than human, mouse and rat IL4, because of a 51-nt deletion in the coding region. Comparison of the predicted aa sequence shows that ovIL4 shares 92, 57, 37 and 42% identity with the bovine, human, mouse and rat IL4s, respectively.

ELISA for IgG-class antibody to hepatitis E virus based on a highly conserved, conformational epitope expressed in Escherichia coli.

In assays based on most recombinant hepatitis E virus (HEV) antigens, the IgG antibody responses to HEV are observed commonly to wane or disappear after the acute phase of infection. Such IgG assays have therefore been used for the diagnosis of acute HEV infection, but they have limited usefulness in seroepidemiological studies. Using western immunoblotting, it was shown previously that the open reading frame (ORF) 2.1 antigen, representing the carboxy-terminal 267 amino acids (aa) of the capsid protein, exposes a conformational epitope which allows optimal detection of convalescent antibody compared to other proteins expressed in Escherichia coli. This conformational epitope is shown to be highly conserved between divergent human HEV isolates, and the development of a sensitive and highly specific enzyme immunoassay (ELISA) based on this recombinant antigen is described. The ORF2.1 ELISA allows the detection and quantitation of both acute- and convalescent phase HEV-specific IgG, and will help to define better the antibody responses to the virus and the prevalence of HEV infection worldwide.

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-gamma, 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-beta gene expression, the interactions of the hepatitis C virus core protein to lymphotoxin-beta 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.

T cell cytokines and disease prevention.

Until recently, work on cytokines has been dominated by the use of murine or human molecules. In the last 5 years we have seen a rapid expansion in the production of bovine, ovine and porcine cytokine reagents. cDNA clones, recombinant proteins and monoclonal antibody probes are not available for a wide variety of cytokines from veterinary species. One of the most interesting recent proposals in immunology has been the division of T helper cells into two classes. Th1 cells have been characterised by the production of gamma-interferon, interleukin (IL)-2, tumour necrosis factor-beta (lymphotoxin-alpha) and the ability to mediate delayed-type hypersensitivity responses, and Th2 cells by their production of IL-4, IL-5, IL-6 and IL-10 and the ability to stimulate production of mast cells, eosinophils and IgE. An important issue for us is to determine whether polarisation of T helper cells to Th1 or Th2 occurs in veterinary species. This paper will attempt to review the status of the Th1 and Th2 debate for sheep, cattle and pigs. It will also discuss the potential for the use of cytokines in modulating the type of immune response following vaccination. By incorporation of particular cytokines into vaccine formulations or the inhibition of production of specific cytokines it may be possible to redirect the nature of the immune response to a particular antigen.

Expression and purification of recombinant ovine interleukin-1 beta from Escherichia coli.

An expression vector bearing the gene segment encoding the mature form of ovine interleukin-1 beta (OvIL-1 beta) was constructed. This vector provided a rapid method for obtaining Escherichia coli derived recombinant OvIL-1 beta (rOvIL-1 beta) using the expression plasmid pGEX-2T. The level of expression of fusion protein in the soluble fraction was approximately 20% of the total accumulated proteins. Affinity purification by glutathione-Sepharose yielded a fusion protein and subsequent thrombin cleavage of this material yielded rOvIL-1 beta. The specific activity of the purified recombinant protein was 10(3)-10(4) times higher than the fusion protein. The rOvIL-1 beta was 10-100 times more potent than human interleukin-1 beta (HuIL-1 beta) in an ovine thymocyte proliferation assay, although they were of equal potency in the NOB-1/CTLL assay. This simple purification method, which produces purified rOvIL-1 beta with a high specific activity (approximately 10(8) U mg-1), will now make it possible to evaluate the in vivo effects of IL-1 beta in sheep.

Production and characterisation of ovine GM-CSF expressed in mammalian and bacterial cells.

A cDNA encoding ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) was isolated and two forms of recombinant ovine GM-CSF were produced. A glycosylated form was produced in mammalian cells infected with a recombinant vaccinia virus encoding ovine GM-CSF. Recombinant ovine GM-CSF was also produced in Escherichia coli and purified by affinity chromatography. Both forms of the protein were detected by ovine GM-CSF-specific monoclonal antibodies, and exhibited activity on ovine bone marrow haemopoetic progenitor cells.

Effects of purified bovine whey factors on cellular immune functions in ruminants.

The immunomodulatory properties of bovine milk and whey have long been documented. The recent advance of whey protein fractionation technology has now allowed us to study the immunobiological properties of some highly purified components of whey, with a view to exploiting their possible industrial and biomedical applications. The effects of fractionated bovine whey proteins on cellular immune responses were therefore examined using a panel of in vitro assays. Both lactoferrin (LF) and lactoperoxidase (LP) were found to inhibit proliferation and interferon-gamma (IFN-gamma) production of ovine blood lymphocytes in response to mitogenic stimulation. However, their effects in a combined fraction or in whey protein concentrate (WPC) were either diminished or eliminated. LF and LP had no effect on lipopolysaccharide (LPS)-induced ovine blood lymphocyte proliferation, production of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF alpha) by ovine bronchoalveolar lavage (BAL) macrophages, major histocompatibility complex (MHC) Class II antigen expression by ovine BAL macrophages and bovine natural killer (NK) cell activity. However, alpha-lactalbumin (alpha LA) exhibited an enhancing effect on IL-1 beta production. It is noteworthy that as bovine whey fractions become progressively more purified, their modulatory effects on the immune response also become more clear-cut. The effects of LF, LP and alpha LA may be eliminated by their combination in whey or by other minor components of whey. Further investigation of industrial applications for whey proteins of high purity is warranted.

Expression of ovine interleukin-2 cDNA in Escherichia coli.

The expression plasmids pGEX-2T and pT7-7 were used to express ovine (Ov) IL-2 cDNA in Escherichia coli. The pGEX-2T vector contained glutathione-S-transferase (GST) as the affinity handle and resulted in high level expression of the GST-IL-2 fusion protein. However, only a small proportion of this fusion protein was present in the soluble fraction. The insoluble fraction was extracted with a detergent, sarkosyl, and even though a large amount of fusion product was obtained, it would not bind to glutathione beads efficiently. Thus, only low yields of biologically active rOvIL-2 were obtained. The yields were not significantly improved when other detergents were used for extraction except for a non-ionic detergent, Zwittergent 3-14, where there was a two- to three-fold increase compared with extraction with sarkosyl. An alternative vector, pT7-7 was used with a 6 x histidine tag followed by a thrombin cleavage site at the amino terminus of the mature ovine IL-2 protein to allow affinity purification by Ni-NTA resin. A large proportion of the rOvIL-2 was partitioned to the insoluble fraction. This expression system was more useful than the pGEX-2T as large quantities of biologically active rOvIL-2 of at least 10 mg l-1 were obtained. The presence of the six histidine residues at the amino end of rOvIL-2 did not reduce its biological activity. Both systems yielded rOvIL-2 with a high specific activity of about 1 x 10(7) U mg-1 as measured by the ability to maintain proliferation of ovine ConA lymphoblasts. Recombinant OvIL-2 was active on bovine but not porcine ConA lymphoblasts.

Expression, biological activity and kinetics of production of recombinant ovine TNF-alpha.

Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNF alpha (rOvTNF-alpha) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30 degrees C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-alpha from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione-Sepharose. Yields of pure rOvTNF-alpha achieved in E. coli JM109 and AM207 were approximately 1 mg L-1. Both rOvTNF-alpha and recombinant human TNF-alpha (rhTNF-alpha) exerted cytotoxicity on L929 cells. However, rOvTNF-alpha but not rhTNF-alpha stimulated proliferation of ovine thymocytes. Maximum levels of TNF-alpha mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.

Cytokine-induced inflammation in the ovine teat and udder.

The inflammatory response, as measured by the accumulation of leukocytes and ovine serum albumin, induced by interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), interleukin-beta (IL-8), and granulocyte-macrophage colony stimulating factor (GM-CSF) was studied in lactating ovine udders, and in test cisterns of dry ewes after surgical closure of the passage between the teat and udder cisterns. In the lactating udders, IL-1 beta and TNF-alpha, but not IL-8 and GM-CSF, induced significant accumulation of cells. In the teat cisterns, all four cytokines IL-1 beta, TNF-alpha, IL-8, and GM-CSF induced significant cell accumulation. IL-1 beta was the most potent cytokine. A slight increase in serum albumin, paralleling the changes in leukocyte numbers, was observed after infusion of IL-1 beta and, to some extent, TNF-alpha. The cell accumulation induced by IL-1 beta or TNF-alpha was dose and time dependent in lactating udders, and time-dependent in teat cisterns. The cell numbers were considerably higher in lactating udders than in teat cisterns after infusion with IL-1 beta. The first influx of cells was observed earlier, and the cell numbers peaked earlier in the teat cisterns than in the lactating udders. IL-8 and GM-CSF induced dose and time dependent cell accumulation in teat cisterns only. The differences between lactating udders and teat cisterns may be attributable to the differences in tissue area involved and the number of receptors available, or to dilution of cytokines in milk, or to presence of inhibitory factors. Differences between cytokines in their inflammatory effects may be explained by their modes of action. IL-1 beta and TNF-alpha have a wide range of cellular functions enabling them to induce a more prominent response than IL-8 and GM-CSF. Furthermore, receptors for IL-1 beta and TNF-alpha are present on a larger number of cell types. Finally, the results indicated that the teat cisterns, being the port of entry for udder infections, as well as the lactating udders are capable of a diversified inflammatory response which is important in the defence against udder infections.