CBD Inhibits In Vivo Development of Human Breast Cancer Tumors.

Inflammation is a critical component of cancer development. Previously, we showed in vitro that IL-1ß treatment of non-invasive human breast cancer MCF-7 cells promoted their transition to a malignant phenotype (6D cells). This epithelial-mesenchymal transition was reverted by exposure to cannabidiol (CBD). We show in a murine model that subcutaneous inoculation of 6D cells induced formation and development of tumors, the cells of which keep traits of malignancy. These processes were interrupted by administration of CBD under two schemes: therapeutic and prophylactic. In the therapeutic scheme, 6D cells inoculated mice developed tumors that reached a mean volume of 540 mm3 at 45 days, while 50% of CBD-treated mice showed gradual resorption of tumors. In the prophylactic scheme, mice were pre-treated for 15 days with CBD before cells inoculation. The tumors formed remained small and were eliminated under continuous CBD treatment in 66% of the animals. Histological and molecular characterization of tumors, from both schemes, revealed that CBD-treated cells decreased the expression of malignancy markers and show traits related with apoptosis. These results confirm that in vivo CBD blocks development of breast cancer tumors formed by cells induced to malignancy by IL-1ß, endorsing its therapeutic potential for cancer treatment.

CBD Reverts the Mesenchymal Invasive Phenotype of Breast Cancer Cells Induced by the Inflammatory Cytokine IL-1ß.

Cannabidiol (CBD) has been used to treat a variety of cancers and inflammatory conditions with controversial results. In previous work, we have shown that breast cancer MCF-7 cells, selected by their response to inflammatory IL-1ß cytokine, acquire a malignant phenotype (6D cells) through an epithelial-mesenchymal transition (EMT). We evaluated CBD as a potential inhibitor of this transition and inducer of reversion to a non-invasive phenotype. It decreased 6D cell viability, downregulating expression of receptor CB1. The CBD blocked migration and progression of the IL-1ß-induced signaling pathway IL-1ß/IL-1RI/ß-catenin, the driver of EMT. Cannabidiol reestablished the epithelial organization lost by dispersion of the cells and re-localized E-cadherin and ß-catenin at the adherens junctions. It also prevented ß-catenin nuclear translocation and decreased over-expression of genes for ∆Np63α, BIRC3, and ID1 proteins, induced by IL-1ß for acquisition of malignant features. Cannabidiol inhibited the protein kinase B (AKT) activation, a crucial effector in the IL-1ß/IL-1RI/ß-catenin pathway, indicating that at this point there is crosstalk between IL-1ß and CBD signaling which results in phenotype reversion. Our 6D cell system allowed step-by-step analysis of the phenotype transition and better understanding of mechanisms by which CBD blocks and reverts the effects of inflammatory IL-1ß in the EMT.

IL-1ß Inflammatory Cytokine-Induced TP63 Isoform ∆NP63α Signaling Cascade Contributes to Cisplatin Resistance in Human Breast Cancer Cells.

The mechanisms behind the induction of malignancy and chemoresistance in breast cancer cells are still not completely understood. Inflammation is associated with the induction of malignancy in different types of cancer and is highlighted as an important factor for chemoresistance. In previous work, we demonstrated that the inflammatory cytokine interleukin 1ß (IL-1ß)-induced upregulation of genes was associated with chemoresistance in breast cancer cells. Here, we evaluated the participation and the expression profile of TP63 in the induction of resistance to cisplatin. By loss-of-function assays, we identified that IL-1ß particularly upregulates the expression of the tumor protein 63 (TP63) isoform ΔNP63α, through the activation of the IL-1ß/IL-1RI/ß-catenin signaling pathway. Upregulation of ΔNP63α leads to an increase in the expression of the cell survival factors epidermal growth factor receptor (EGFR) and phosphatase 1D (Wip1), and a decrease in the DNA damage sensor, ataxia-telangiectasia mutated (ATM). The participation of these processes in the increase of resistance to cisplatin was confirmed by silencing TP63 expression or by inhibition of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) activity in the IL-1ß/IL-1RI/ß-catenin signaling pathway. These data reinforced the importance of an inflammatory environment in the induction of drug resistance in cancer cells and uncovered a molecular mechanism where the IL-1ß signaling pathway potentiates the acquisition of cisplatin resistance in breast cancer cells.

Primary cilia formation is diminished in schizophrenia and bipolar disorder: A possible marker for these psychiatric diseases.

Primary cilium (PC) is a microtubule-rich organelle that protrudes from the plasma membrane and acts as a cellular antenna sensing extracellular signals during brain development. DISC1 (Disrupted-in-Schizophrenia-1) is involved in PC formation and is considered a risk factor for neuropsychiatric disorders. We have previously described altered subcellular distribution of DISC1 and an aberrant microtubule organization in olfactory neuronal precursors (ONP) obtained from schizophrenia (SCZ) and bipolar disorder (BD) patients. Herein, we analyzed in vitro PC formation in healthy control subjects, SCZ and BD patients. The results indicated that 66.73±4.33% of ONP from control subjects showed immunostaining for the PC marker, acetylated α-tubulin. By contrast, only a small percentage of cells in culture from paranoid SCZ and BD patients showed PC staining (SCZ, 12.8±4.43%; BD, 12.32±5.86%). However, cells from an affected proband with disorganized SCZ and a subject with BD displayed a higher percentage of cells with cilia (SCZ, 42.20%; BD, 38.59%). Additionally, cilia elongation was observed in lithium-treated ONP derived from all groups, with a more evident response in cells from the BD group. The present study provides novel evidence that the molecular pathways involved in PC formation are defective in SCZ and BD, and impairment in these processes may be involved in the physiopathology of both diseases. Our observations also suggest that ONP is a patient-derived cell model with a potential use for diagnosis and high-throughput drug screening for brain diseases.

IL-1ß induced methylation of the estrogen receptor ERα gene correlates with EMT and chemoresistance in breast cancer cells.

Inflammation has been recently acknowledged as a key participant in the physiopathology of oncogenesis and tumor progression. The inflammatory cytokine IL-1ß has been reported to induce the expression of markers associated with malignancy in breast cancerous cells through Epithelial-Mesenchymal Transition (EMT). Aggressive breast cancer tumors classified as Triple Negative do not respond to hormonal treatment because they lack three crucial receptors, one of which is the estrogen receptor alpha (ERα). Expression of ERα is then considered a good prognostic marker for tamoxifen treatment of this type of cancer, as the binding of this drug to the receptor blocks the transcriptional activity of the latter. Although it has been suggested that inflammatory cytokines in the tumor microenvironment could regulate ERα expression, the mechanism(s) involved in this process have not yet been established. We show here that, in a cell model of breast cancer cells (6D cells), in which the inflammatory cytokine IL-1ß induces EMT by activation of the IL-1ß/IL-1RI/ß-catenin pathway, the up regulation of TWIST1 leads to methylation of the ESR1 gene promoter. This epigenetic modification produced significant decrease of the ERα receptor levels and increased resistance to tamoxifen. The direct participation of IL-1ß in these processes was validated by blockage of the cytokine-induced signaling pathway by wortmannin inactivation of the effectors PI3K/AKT. These results support our previous reports that have suggested direct participation of the inflammatory cytokine IL-1ß in the transition to malignancy of breast cancer cells.

IL-1ß induces up-regulation of BIRC3, a gene involved in chemoresistance to doxorubicin in breast cancer cells.

Epithelial to mesenchymal transition (EMT) of tumor cells facilitates their progress to metastasis. In the tumor microenvironment the inflammatory cytokine 1ß (IL-1ß) has been associated with tumor development and invasiveness. IL-1ß-induced EMT triggers the expression of markers associated with malignancy. We have recently reported that an IL-1ß-highly responsive clone (6D cells) from non-invasive MCF-7 breast cancer cells activates PI3K/Rac and IL-1RI/ß-catenin pathways that up-regulate the transcription of genes involved in an EMT-like process. However, a correlation between the EMT program induced by a pro-inflammatory environment, and the acquisition of chemoresistance has not been yet determined in these cells. In this work, we report the expression of cell survival genes after IL-1ß stimulation of 6D cells. The expression of CDKN1A, TP63, SFN and, particularly, BIRC3 was found to be up-regulated in a RNA-seq analysis and validated by qPCR. Cells stimulated with IL-1ß when challenged with doxorubicin showed resistance to the drug, whereas silencing of BIRC3 decreased viability of the cells treated with the drug. Our present results show that IL-1ß confers doxorubicin resistance to breast cancer cells, underlining the importance of an inflammatory environment in cancer malignancy.

Helicobacter pylori CagA and IL-1ß Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model.

Gastric cancer is the third cause of cancer death worldwide and infection by Helicobacter pylori (H. pylori) is considered the most important risk factor, mainly by the activity of its virulence factor CagA. H. pylori/CagA-induced chronic inflammation triggers a series of gastric lesions of increased severity, starting with gastritis and ending with cancer. IL-1ß has been associated with tumor development and invasiveness in different types of cancer, including gastric cancer. Currently, it is not clear if there is an association between CagA and IL-1ß at a cellular level. In this study, we analyzed the effects of IL-1ß and CagA on MCF-10A nontransformed cells. We found evidence that both CagA and IL-1ß trigger the initiation of the epithelial-to-mesenchymal transition characterized by ß-catenin nuclear translocation, increased expression of Snail1 and ZEB1, downregulation of CDH1, and morphological changes during MCF-10A acini formation. However, only CagA induced MMP9 activity and cell invasion. Our data support that IL-1ß and CagA target the ß-catenin pathway, with CagA leading to acquisition of a stage related to aggressive tumors.

Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.

Helicobacter pylori CagA Suppresses Apoptosis through Activation of AKT in a Nontransformed Epithelial Cell Model of Glandular Acini Formation.

H. pylori infection is the most important environmental risk to develop gastric cancer, mainly through its virulence factor CagA. In vitro models of CagA function have demonstrated a phosphoprotein activity targeting multiple cellular signaling pathways, while cagA transgenic mice develop carcinomas of the gastrointestinal tract, supporting oncogenic functions. However, it is still not completely clear how CagA alters cellular processes associated with carcinogenic events. In this study, we evaluated the capacity of H. pylori CagA positive and negative strains to alter nontransformed MCF-10A glandular acini formation. We found that CagA positive strains inhibited lumen formation arguing for an evasion of apoptosis activity of central acini cells. In agreement, CagA positive strains induced a cell survival activity that correlated with phosphorylation of AKT and of proapoptotic proteins BIM and BAD. Anoikis is a specific type of apoptosis characterized by AKT and BIM activation and it is the mechanism responsible for lumen formation of MCF-10A acini in vitro and mammary glands in vivo. Anoikis resistance is also a common mechanism of invading tumor cells. Our data support that CagA positive strains signaling function targets the AKT and BIM signaling pathway and this could contribute to its oncogenic activity through anoikis evasion.

A surface membrane protein of Entamoeba histolytica functions as a receptor for human chemokine IL-8: its role in the attraction of trophozoites to inflammation sites.

Entamoeba histolytica trophozoites respond to the presence of IL-8, moving by chemotaxis towards the source of the chemokine. IL-8 binds to the trophozoite membrane and triggers a response that activates signaling pathways that in turn regulate actin/myosin cytoskeleton organisation to initiate migration towards the chemokine, suggesting the presence of a receptor for IL-8 in the parasite. Antibodies directed to the human IL-8 receptor (CXCR1) specifically recognised a 29 kDa protein in trophozoite membrane fractions. The same protein was immunoprecipitated by this antibody from total amebic extracts. Peptide analysis of the immunoprecipitated protein revealed a sequence with high homology to a previously identified amebic outer membrane peroxiredoxin and a motif within the third loop of human CXCR1, which is an important site for IL-8 binding and activation of signaling processes. Immunodetection assays demonstrated that the anti-human CXCR1 antibody binds to the 29 kDa protein in a different but close site to where IL-8 binds to the trophozoite surface membrane, suggesting that human and amebic receptors for this chemokine share common epitopes. In the context of the human intestinal environment, a receptor for IL-8 could be a great advantage for E. histolytica trophozoite survival, as they could reach an inflammatory milieu containing abundant nutrients. In addition, it has been suggested that the high content of accessible thiol groups of the protein and its peroxidase activity could provide protection in the oxygen rich milieu of colonic lesions, allowing trophozoite invasion of other tissues and escape from the host immune response.

Altered subcellular distribution of the 75-kDa DISC1 isoform, cAMP accumulation, and decreased neuronal migration in schizophrenia and bipolar disorder: implications for neurodevelopment.

BACKGROUND: DISC1 (Disrupted-In-Schizophrenia-1) is considered a genetic risk factor for schizophrenia (SZ) and bipolar disorder (BD). DISC1 regulates microtubule stability, migration, and cAMP signaling in mammalian cell lines and mouse brain tissue. cAMP is a regulator of microtubule organization and migration in neurons. Aberrant microtubule organization has been observed in olfactory neuronal precursors (ONP) derived from patients with SZ and BD, which suggests involvement of DISC1 and cAMP. However, the biology of DISC1 in the physiopathology of psychiatric conditions remains elusive. AIMS: Herein, utilizing ONP obtained from SZ, BD patients and healthy subjects, we have studied DISC1 expression, protein levels, and subcellular distribution by qRT-PCR, immunoblotting, subcellular fractionation, and confocal microscopy. Cell migration and cAMP accumulation were assessed by Transwell and PKA competition assays. RESULTS: We found increased levels of the 75-kDa DISC1 isoform in total cell extracts of ONP from patients with SZ and BD compared with controls. Subcellular distribution showed a significant decrease of cytoplasmic DISC1 concomitant with its augmented levels in transcription sites. Moreover, significant cAMP accumulation and diminished migration were also observed in patients' cells. CONCLUSION: Alterations of DISC1 levels and its cellular distribution, which negatively modify cAMP homeostasis, microtubule organization, and cell migration, in ONP from patients with SZ and BD, suggest that their presence in early stages of brain development may impact brain maturation and function.

A novel ß-catenin signaling pathway activated by IL-1ß leads to the onset of epithelial-mesenchymal transition in breast cancer cells.

Interleukin 1ß has been associated with tumor development, invasiveness and metastasis in various types of cancer. However, the molecular mechanisms underlying this association have not been clearly elucidated. The present study is the first to show, in breast cancer cells, that an IL-1ß/IL-1RI/ß-catenin signaling pathway induces ß-catenin accumulation due to GSK3ß inactivation by Akt phosphorylation. Translocation to the nucleus of accumulated ß-catenin and formation of the TCF/Lef/ß-catenin complex induce sequential expression of c-MYC, CCDN1, SNAIL1 and MMP2, leading to up-regulation of proliferation, migration and invasion; all of the processes shown to be required, in cancerous cells, to initiate transition from a non-invading to an invasive phenotype.

Jak3 enables chemokine-dependent actin cytoskeleton reorganization by regulating cofilin and Rac/Rhoa GTPases activation.

We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3⁻/⁻ lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3⁻/⁻ lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines.

Lymphoid progenitor cells from childhood acute lymphoblastic leukemia are functionally deficient and express high levels of the transcriptional repressor Gfi-1.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34⁺ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

Toll-like receptor signaling activation by Entamoeba histolytica induces beta defensin 2 in human colonic epithelial cells: its possible role as an element of the innate immune response.

BACKGROUND: Entamoeba histolytica, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent in vitro studies have shown that Entamoeba histolytica trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria. METHODOLOGY/PRINCIPAL FINDINGS: We show here that E. histolytica activation of the classic TLR pathway in CaCo2 cells is required to induce ß defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic Escherichia coli. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody. CONCLUSIONS/SIGNIFICANCE: Entamoeba histolytica trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2's possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed.

Selection of a MCF-7 Breast Cancer Cell Subpopulation with High Sensitivity to IL-1ß: Characterization of and Correlation between Morphological and Molecular Changes Leading to Increased Invasiveness.

Cancer and inflammation are closely related in tumor malignancy prognosis. Breast cancer MCF-7 cells have a poor invasive phenotype, although, under IL-1ß stimulus, acquire invasive features. Cell response heterogeneity has precluded precise evaluation of the malignant transition. MCF-7A3 cells were selected for high sensitivity to IL-1ß stimulus, uniform expression of CXCR4, and stability of IL1-RI. Structural changes, colony formation ability, proliferation rate, chemotaxis, Matrigel invasion, E-cadherin mRNA expression and protein localization were determined in these cells and in MCF-7 parental cells under the stimulus of IL-1ß. Selected MCF-7A3 cells showed a uniform response to IL-1ß stimulation increasing features of invasive cells such as scattering, colony formation, proliferation, chemokinesis and invasion. Basal expression of E-cadherin mRNA was higher, and IL-1ß stimulus had no further effect at early times of cytokine exposure. Total E-cadherin levels remained unchanged in parental cells, whereas levels decreased, as MCF-7A3 cells became fibroblastoid or scattered. Triton X-100 soluble/insoluble E-cadherin ratios were highly increased in these cells, while, in MCF-7pl cells, ratios could not be correlated with morphology changes. MCF-7A3 cells uniform response to IL-1ß allowed characterization of changes induced by the cytokine that had not been assessed when using heterogeneous cell lines.

Early lymphoid development and microenvironmental cues in B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is a hematological disorder characterized by malignant and uncontrolled proliferation of B-lymphoid precursor cells in bone marrow. Over the last few years remarkable advances have been made in identifying genetic aberrations, patterns of abnormal transcriptional activity controlling early fate decisions and environmental cues that may influence leukemic development. In this review we focus on the structure of the early lymphoid system and the current knowledge about cell composition and function of the hematopoietic microenvironment that might control progenitor cell activity and lead to differentiation, proliferation and survival of developing B leukemic precursors. Learning the biology of special leukemic niches is central to understanding the pathogenesis of B-ALL and for the development of novel therapies.

Molecular and functional characterization of an Entamoeba histolytica protein (EhMLCI) with features of a myosin essential light chain.

Entamoeba histolytica, a protozoan parasite of humans, relays on its striking motility to survive and invade host tissues. Characterization of the molecular components involved in motile processes is crucial to understand its pathogenicity. Although protein components of myosin II hexamers have been predicted from E. histolytica genome data, only a heavy chain of myosin, EhmhcA, has been characterized so far. We have cloned an E. histolytica cDNA sequence that best matched Dictyostelium discoideum myosin essential light chain and found that the cloned sequence is transcribed as an mRNA of 0.445 kb which could encode a protein of 16.88 kDa, within the predicted range for a myosin light chain. In silico analyses revealed that the protein sequence, named EhMLCI, shows two consensus domains for binding MHC, but lacks the N-terminal sequence for actin binding, as in A2 type myosin essential light chains. A single EF-hand calcium-binding domain was identified in the C-terminus and several high score predictability sites for serine and tyrosine phosphorylation. Antibodies to recombinant EhMLCI identified two proteins of approximately 17 and 15 kDa in trophozoite extracts, the latter phophorylated in tyrosines. Serine phosphorylation was not detected. Immunomicroscopy revealed EhMLCI cortical and cytoplasmic distribution in trophozoites and true colocalization with EhmhcA determined by PCC. Co-immunoprecipitation corroborated EhMLCI interaction with EhmhcA. EhMLCI was also localized in actomyosin-containing complexes. Differential partition of phospho-tyrosinated EhMLCI into cell fractions containing the soluble form of EhmhcA and its lack of serine phosphorylation suggest its possible participation in a novel down regulatory mechanism of myosin II activity in E. histolytica.

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica.

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the "classic" signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram⁺) or Shigella dysenteriae (Gram⁻), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.