Animal models are effective for assessing tumor localization of nanosystems but difficult to use for studying penetration beyond the vasculature. Here, we have used well-characterized HCT116 ...colorectal cancer spheroids to study the effect of nanoparticle (NP) physicochemical properties on penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient, which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry. This approach was used to compare doxorubicin and liposomal doxorubicin (Caelyx) and a range of model poly(styrene) nanoparticles of different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistries (50 nm uniform plain, carboxylated, aminated and a range of NPs and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified poly(styrene) nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT116 spheroids more efficiently than larger poly(styrene) nanoparticles (100 nm). Surprisingly, penetration of 30 and 50 nm particles was as good as clinically relevant doxorubicin concentrations. However, penetration was reduced with higher surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm poly(styrene) nanoparticles, which may be related to polymer flexibility. PEG surface modification of polymeric particles significantly improved penetration into the spheroid core. The new model combining the use of spheroids Hoechst staining and flow cytometry was a useful model for assessing NP penetration and gives useful insights into the effects of NPs’ physical properties when designing nanomedicines.
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The aim of this work was to prepare and characterize solid dispersions of abietic acid (AB) and chitosan (CS) to investigate how formulation of the mixture may help in the battle ...against microbial colonization in different areas, such as the biomedical field or the food industry. Solid dispersions were characterized by differential scanning calorimetry, infrared spectroscopy, Raman spectroscopy, polarized optical microscopy, zeta potential and size analysis. The data showed that the dispersion/solvent evaporation method formed solid dispersions in which abietic acid was molecularly dispersed in the carrier. A synergistic effect between the two components in terms of antioxidant and antimicrobial properties was found, especially in the formulations obtained with 1/1 AB/CS molar ratio. Interestingly, the aggregation state (amorphous/crystalline) of AB seemed to affect the antimicrobial activity of the formulation, suggesting increased bioactivity when the drug was in the amorphous state. These findings, together with the demonstrated biocompatibility of the formulations, seem to open promising perspectives for a successful application of the developed AB/CS formulations in the biomedical field or in the food industry.
A previous study suggested that Helicobacter pylori strains possessing dupA are positively associated with duodenal ulceration and negatively associated with gastric adenocarcinoma. We determined the ...prevalence of dupA in H. pylori strains recovered from 4 independent populations and found a significant association with gastric cancer but not with duodenal ulceration.
Helicobacter pylori causes peptic ulceration and gastric adenocarcinoma; the latter is common in Iran but not in Iraq. We hypothesized that more virulent H. pylori strains may be found in Iran than ...in Iraq and so compared established and newly described virulence factors in strains from these countries. We studied 59 unselected dyspeptic patients from Iran and 49 from Iraq. cagA was found in similar proportions of strains from both countries (76% in Iran versus 71% in Iraq) and was significantly associated with peptic ulcer disease in Iraq (P <= 0.01) but not in Iran. cagA alleles encoding four or more tyrosine phosphorylation motifs were found in 12% of the Iranian strains but none of the Iraqi strains (P = 0.02). There were no significant differences in the vacA signal-, middle-, or intermediate-region types between Iranian and Iraqi strains. Among the strains from Iran, vacA genotypes showed no specific peptic ulcer associations, but among the strains from Iraq, vacA i1 strains were associated with gastric ulcer (P <= 0.02), mimicking their previously demonstrated association with gastric cancer in Iran. dupA was found in similar proportions of Iranian and Iraqi strains (38% and 32%, respectively) and was associated with peptic ulceration in Iraqi patients (P <= 0.01) but not Iranian patients. H. pylori strains from Iraq and Iran possess virulence factors similar to those in Western countries. The presence of cagA with more phosphorylation motifs in Iranian strains may contribute to the higher incidence of gastric cancer. However, the association between strain virulence markers and disease in Iraq but not Iran suggests that other host and environmental factors may be more important in the disease-prone Iranian population.
Lifelong Helicobacter pylori infection and its associated gastric inflammation underlie peptic ulceration and gastric carcinogenesis. The immune and inflammatory responses to H. pylori are doubly ...responsible: gastric inflammation is the main mediator of pathology, and the immune and inflammatory response is ineffective, allowing lifelong bacterial persistence. However, despite inducing gastric inflammation, most infections do not cause disease, and bacterial, host and environmental factors determine individual disease risk. Although H. pylori avoids many innate immune receptors, specific virulence factors (including those encoded on the cag pathogenicity island) stimulate innate immunity to increase gastric inflammation and increase disease risk. An acquired T helper 1 response upregulates local immune effectors. The extent to which environmental factors (including parasite infection), host factors and H. pylori itself influence T-helper differentiation and regulatory T-cell responses remains controversial. Finally, effective vaccines have still not been developed: a better understanding of the immune response to H. pylori may help.
Background. Infection with Helicobacter pylori possessing a newly described virulence factor—duodenal ulcer-promoting gene A (dupA)—has been associated with duodenal ulceration and increased gastric ...inflammation. Methods. The dupA locus of 34 strains was sequenced. A panel of dupA mutants was generated and cocultured with human gastric epithelial cells and peripheral blood mononuclear cells; proinflammatory cytokine release was measured. IL8 expression was measured in human gastric biopsy specimens and related to the dupA and cagA status of infecting strains. Results. Most H. pylori strains had a dupA allele that was longer (1884 bp; dupA1) than previously described dupA alleles, although some had truncated versions (dupA2). Unlike the best-characterized H. pylori virulence determinant, the cag pathogenicity island (cag PaI), neither dupA type induced release of interleukin (IL)–8 from gastric epithelial cells. However, infections due to dupA-positive strains were associated with higher-level mucosal IL-8 messenger RNA expression in the human stomach than were infections due to dupA-negative strains. To explain this paradox, we found that dupA1 (but not dupA2 or the cag PaI) substantially increased H. pylori–induced IL-12p40 and IL-12p70 production from CD14 + mononuclear cells. Other T helper 1–associated cytokines were also modestly induced. Conclusion. We suggest that virulent H. pylori strains cause inflammation by stimulating epithelial cells through cag-encoded proteins and mononuclear inflammatory cells through dupA1 products.
Helicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of ...epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines.
Three gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT.
The pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins.
The upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric cancer, potentially through EMT, by indirectly increasing levels of soluble HB-EGF.
Pancreatic ductal adenocarcinoma (PDAC) is characterised by desmoplasia, thought to support progression and chemotherapeutic resistance. The Hedgehog pathway is known to play an important role in ...this cancer. While the upregulation of Sonic hedgehog (Shh) in the epithelium of PDAC is known, we investigated its expression in the tumour microenvironment in order to find new targets for new chemotherapeutical approaches. Immunohistochemistry was used for the investigation of Shh and Vimentin in primary human pancreatic tissues. Gene (qRT-PCR) and protein (immunofluorescence) expression of Shh, αSMA (a marker of the mesenchymal phenotype) and periostin (a marker of mesenchymal cells within a mixed population) were investigated in in vitro cell models. Shh expression was significantly upregulated in the stromal and epithelial compartments of poorly-differentiated PDAC samples, with a strong correlation with the amount of stroma present. Characterisation of stromal cells showed that there was expression of Shh ligand in a mixed population comprising αSMA
myofibroblasts and αSMA
mesenchymal stem cells. Moreover, we demonstrated the interaction between these cell lines by showing a higher rate of mesenchymal cell proliferation and the upregulation of periostin. Therefore, targeting stromal Shh could affect the equilibrium of the tumour microenvironment and its contribution to tumour growth.
Helicobacter pylori strains from East Asia have an 'East Asian' type of CagA that is more active and predominantly comprises a single type. Strains from other countries have a 'western' type of CagA, ...which is less active and comprises many different types generated by intragenomic recombination. Co-culture of AGS gastric epithelial cells with isolates of western strains that displayed microevolution in CagA showed that isolates with additional copies of the C motif induced significantly more interleukin (IL)-8 secretion. Co-culture of AGS cells with western and East Asian strains, each expressing CagA with a single copy of the C or D motif, showed that East Asian strains induced significantly more IL-8 secretion. Analysis of the different CagA types from data deposited in GenBank and from the literature showed that western CagA is significantly more likely to undergo duplication of tyrosine phosphorylation motif C than East Asian CagA is of the corresponding D motif. Taken together, the data suggest that the already highly active East Asian CagA with one D motif has no requirement to increase its virulence, whereas the less active western CagA displays flexibility in its capacity to increase its number of tyrosine phosphorylation motifs to become more virulent.
The Helicobacter pylori virulence factors CagA and VacA are implicated in the development of gastroduodenal diseases. Most strains possessing CagA also possess the more virulent vacuolating form of ...VacA. This study assessed the significance of possession of both virulence factors in terms of their effect on gastric epithelial cells, using a set of minimally passaged, isogenic VacA, CagA and CagE mutants in H. pylori strains 60190 and 84-183. The cagA and cagE mutants were found to significantly increase VacA-induced vacuolation of epithelial cells, and the vacA mutants significantly increased CagA-induced cellular elongations, compared with wild-type strains, indicating that CagA reduces vacuolation and VacA reduces hummingbird formation. Although epithelial cells incubated with the wild-type H. pylori strains may display both vacuolation and hummingbird formation, it was found that (i) hummingbird length was significantly reduced in vacuolated cells compared with those without vacuolation; (ii) the number of vacuoles was significantly reduced in vacuolated cells with hummingbird formation compared with those without hummingbirds; and (iii) cells displaying extensive vacuolation did not subsequently form hummingbirds and vice versa. VacA did not affect the phosphorylation of CagA. These data show that VacA and CagA downregulate each other's effects on epithelial cells, potentially allowing H. pylori interaction with cells whilst avoiding excessive cellular damage.
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