Cancer-ID: Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood.

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 µm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1 µm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic.

Sonoprinting liposomes on tumor spheroids by microbubbles and ultrasound.

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed "sonoprinting". However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues.

A medium throughput device to study the effects of combinations of surface strains and fluid-flow shear stresses on cells.

We report a medium throughput device to study the effects of combinations of two mechanical stimuli - surface strains and fluid flow shear stresses, on cells. The first generation prototype can screen combinations of five strain and five shear stress levels. Computational modeling and empirical measurements were used to determine the generated strains and flows. Uniform equibiaxial strains up to 20% and shear stresses up to 0.3 Pa can be generated. Compatibility of the device with cell culture and end point fixation, staining and imaging is shown using C2C12 mouse myoblast cells.

Ruthenium oligonucleotides, targeting HPV16 E6 oncogene, inhibit the growth of cervical cancer cells under illumination by a mechanism involving p53.

High-risk Human Papillomaviruses (HPV) has been found to be associated with carcinomas of the cervix, penis, vulva/vagina, anus, mouth and oro-pharynx. As the main tumorigenic effects of the HPV have been attributed to the expression of E6 and E7 genes, different gene therapy approaches have been directed to block their expression such as antisense oligonucleotides (ASO), ribozymes and small interfering RNAs. In order to develop a gene-specific therapy for HPV-related cancers, we investigated a potential therapeutic strategy of gene silencing activated under illumination. Our aim according to this antisense therapy consisted in regulating the HPV16 E6 oncogene by using an E6-ASO derivatized with a polyazaaromatic ruthenium (Ru(II)) complex (E6-Ru-ASO) able, under visible illumination, to crosslink irreversibly the targeted sequence. We examined the effects of E6-Ru-ASO on the expression of E6 and on the cell growth of cervical cancer cells. We demonstrated using HPV16(+) SiHa cervical cancer cells that E6-Ru-ASO induces after illumination, a reactivation of p53, the most important target of E6, as well as the inhibition of cell proliferation with a selective repression of E6 at the protein level. These results suggest that E6-Ru ASOs, activated under illumination and specifically targeting E6, are capable of inhibiting HPV16(+) cervical cancer cell proliferation.

Bax-mediated mitochondrial membrane permeabilization after heat treatment is caspase-2 dependent.

Heat-induced apoptosis proceeds via mitochondria by permeabilization of the outer mitochondrial membrane (MOMP), resulting in the release of cytochrome c. This essential step is mediated by Bcl-2 family proteins, such as Bax. Recently, caspase-2 was assigned a prominent role in regulating Bax. Therefore, we studied the initiation of heat-induced apoptosis by monitoring Bcl-2 family members and the release of cytochrome c with or without caspase-2 inhibition. Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1) were exposed to heat treatment and/or X-radiation. Expression and localization of Bax and Bcl-2 proteins was investigated by flow cytometry (FCM) and confocal microscopy respectively. Cytochrome c release was measured with FCM as evidence for MOMP. In addition, the role of caspase-2 in heat- and radiation-induced apoptosis was assessed using the specific caspase-2 inhibitor zVDVAD-fmk. Here we present evidence that heat treatment, and not irradiation, increases intracellular Bax protein expression and subsequently stimulates MOMP, resulting in the release of cytochrome c. Furthermore, by selective blocking of caspase-2 using zVDVAD-fmk less Bax was expressed and subsequently a significant decrease in cytochrome c release was observed. In conclusion, heat treatment of hematopoietic cells does require caspase-2 activation for the initiation of Bax-mediated MOMP.

High degree of correlation between molecular polymorphism and geographic origin of wine yeast strains.

AIMS: To guarantee the endemic genetic background of the isolates obtained in yeast isolation programs, it is necessary to differentiate between endemic and commercial strains because the progressive use of commercial yeast in wine areas around the world would affect the autochthonous yeast populations. METHODS AND RESULTS: Mitochondrial DNA restriction analysis, electrophoretic karyotyping and random amplification of polymorphic DNA (RAPD) were evaluated as experimental approaches to correlate genomic polymorphism and geographic origin of native wine yeast strains. The three molecular methods were capable of detecting a European commercial strain among native Chilean strains; however, RAPD proved to have the best performance. CONCLUSIONS: The molecular polymorphism analysis is useful to evaluate the geographical origin of native yeast isolates and confirms or refutes the genetic background of currently marketed strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits a genetic characterization of native yeast populations and confirms its utility as a tool for evaluating if a native isolate derives from the region where it was collected, permitting, furthermore, to develop studies on the evolution of native yeast populations and to evaluate the effect of introduced yeasts on these populations.

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America.

AIMS: The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. METHODS AND RESULTS: Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. CONCLUSIONS: Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.

Synthesis, characterisation and in vivo behaviour of a norfloxacin-poly(L-lysine citramide imide) conjugate bearing mannosyl residues.

With the aim of promoting the targeting of macrophage mannose receptors and the internalisation of the norfloxacin antibiotic, which is active against some intracellular bacteria, a macromolecular prodrug was synthesised where the antibiotic and mannosyl moieties were coupled to a polymeric carrier, namely poly(L-lysine citramide imide). This carrier, which derived from two metabolites, citric acid and L-lysine, is known to be biocompatible and slowly degradable under slight acidic conditions. Norfloxacin was coupled onto the acid groups present along the polymer chains, and conjugates were characterised by UV, TLC and SEC. The mannosyl groups selected to promote the targeting of the mannose-specific lectin present on the outer membrane of macrophages were incorporated through a biodegradable glycolic spacer arm. Two different strategies were considered to synthesise the full conjugates, namely coupling norfloxacin onto mannosylated conjugates, and coupling mannose onto PLCAI/Nflx conjugates. The second pathway led to better results regarding mannosylation. The presence of norfloxacin and mannose caused chain aggregation, especially for conjugates with a high content of mannosyl residues. The targeting ability of the prodrug was investigated using a method based on the competition between the mannosylated macromolecules and glucose oxidase, a mannosyl-bearing non-human protein. This method showed that prodrug macromolecules competed effectively with glucose oxidase and thus should be able to bring the drug up to the mannosyl receptor-bearing membranes of macrophages infected by intracellular bacteria.

Binding analysis of 95 HIV gp120 peptides to HLA-DR1101 and -DR0401 evidenced many HLA-class II binding regions on gp120 and suggested several promiscuous regions.

To identify HLA-DR-binding peptides within the human immunodeficiency virus (HIV)-1 proteins. 95 overlapping synthetic peptides representing the entire sequence of gp120-LAI were screened for their capacity to bind to two HLA-DR molecules with distant sequences (DR0401 and DR1101). By using a cell surface competitive binding assay, 56 DR-binding peptides were identified, of which 35 bound to both DR1101 and DR0401. A highly significant concordance was evidenced by statistical analysis between binding of peptides to one and to the other DR molecule, suggesting a high proportion of promiscuity among gp120 peptides, even though no clear sequence pattern accounting for such promiscuity was found. DR-binding peptides were located along the entire gp120 sequence. Yet, the majority of them (42 among 56) were concentrated in seven multiagretopic regions that were arbitrarily defined as regions containing four or more overlapping continuous peptides binding to DR1011 and/or DR0401. A good correlation was found between DR-binding regions or DR-binding peptides defined in this study and promiscuous T helper gp120 epitopes previously described in seropositive individuals. All these results suggest that the identification of multiagretopic DR-binding regions may be a great help for the predicition of protein determinants that have the likelihood of being promiscuous T helper epitopes in humans.

Defective cytokine expression but adult-type T-cell receptor, CD8, and p56lck modulation in CD3- or CD2-activated T cells from neonates.

Expression of IL-2, interferon-gamma, and IL-3 mRNA and proteins was investigated in peripheral blood mononuclear cells from cord blood after activation with phytohemagglutinin, CD2, or CD3 MAb. The results showed that interferon-gamma and IL-3 expression was decreased in cord peripheral blood mononuclear cells when compared with expression observed in adult peripheral blood mononuclear cells, irrespective of the stimulation used. In addition, in newborn cells a defect in IL-2 secretion and mRNA expression was observed in response to CD2 or CD3 MAb but not in response to phytohemagglutinin-mediated activation. We further analyzed the modulation of nonlymphokine genes under the same protocol of stimulations. The results indicate that in newborn cells, despite a reduced lymphokine expression observed after CD2 or CD3 MAb activation, the up-regulation of the T-cell receptor, CD8, and p56lck was similar to that found in adult cells, as was also found after phytohemagglutinin activation of both types of cells. These data are in favor of a deficient T-cell responsiveness to CD2 or CD3 MAb in newborn cells. This impairment of the T-cell response appears to selectively affect lymphokine gene expression because the modulation of other genes also implicated in T cell activation is not altered.

Requirements for lysis of activated T cells by class-II-restricted cytolytic T-lymphocytes.

In the present study, we explored the specific requirements for lysis of human activated T cells by CD4+ CTLs. This was achieved by using human CD4+ T cell lines or clones specific for a peptidic fragment of influenza virus as both CTL effectors and target T cells (TTCs). Our results further establish that human activated T cells expressing HLA-DR molecules can present Ag to and be lysed by CD4+ HLA-DR restricted CTLs. This killing is Ag specific and HLA-DR restricted. It can be observed whether TTCs are heterologous or autologous, CD4+ or CD8+. However, we find that in our model: (a) TTCs are able to present artificially processed peptidic fragments of Ag, but not the corresponding natural Ag in the context of class II determinants, even if they can process whole virus in the context of class I determinants; (b) TTCs must express high density of HLA-DR molecules on their membrane; (c) preincubation of TTCs with high concentrations of peptide is required; and (d) interestingly enough, addition of free peptide at similar concentration during the cytolytic assay to replace TTC preincubation inhibits TTC lysis by at least two different mechanisms, i.e., cold-target inhibition in which CTLs serve as their own cold targets and inhibition at the effector cell level. From these results, one can conclude that stringent conditions are required for lysis of activated T cells by class-II-restricted CTLs.

Comparison of T cell functional changes during childhood with the ontogeny of CDw29 and CD45RA expression on CD4+ T cells.

The ontogeny of the peripheral blood mononuclear cells' responsiveness to various activators during childhood was studied and compared to the expression of CDw29 and CD45RA molecules at the surface of CD4+ T cells. The results show that newborn peripheral blood mononuclear cells are characterized by a responsiveness to mitogens that is higher than that observed in adults, at least shortly after stimulation. This contrasts with a clear decreased response to CD2 and CD3 MAb at any time after stimulation. These functional characteristics correlate with a low density of CDw29 antigen on virtually all CD4+ T cells and a high density of CD45RA antigen on most CD4+ T cells at birth. These patterns of reactivity and phenotype are similar to those found among naive adult T cells. When ageing, the response to mitogens becomes rapidly similar to the adult's values, whereas the responses to CD2 or CD3 MAb are more gradually acquired. This slow rate of functional changes grossly parallels the increase of CDw29+ CD4+ and the decrease of CD45RA+ CD4+ T cell subsets. These changes finally lead to the immunophenotypic and functional characteristics that are typical of adult memory T cells. These results suggest that iterative antigenic stimulations both induce memory T cells and create the conditions to improve the overall immune competence.

Survey of multiply resistant Providencia stuartii in a chronic care unit.

Providencia stuartii was cultured over a 4-year period from the urinary tract, throat, perineum, axilla and stools of patients in a long-term chronic care unit, the most common site of colonization being the urinary tract. A total of 17 patients had Prov. stuartii bacteraemia and manipulation of the urinary tract preceded bacteraemia in 10 cases. Eighty-two per cent of the patients had long-term urinary tract colonization from 1 month to 4 years. Although most isolates were resistant to all aminoglycosides except amikacin, the organisms were all susceptible to thienamycin, ceftazidime, cefotaxime, ceftizoxime and moxalactam.