Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg

BACKGROUND: Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA) and cysteine protease (SpeB) as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells. RESULTS: Streptococcal mutants were readily labelled with CFDA-SE and their binding to epithelial cells could be effectively studied by flow cytometry. A strain deficient in Rgg expression showed increased binding to the analyzed epithelial cell lines of various origin. Inactivation of SpeB had no effect on the adhesion, while PulA knock-out strains displayed decreased binding to the cell lines. CONCLUSION: These results suggest that the flow cytometric assay is a valuable tool in the analysis of S. pyogenes adherence to host cells. It appears to be an efficient and sensitive tool for the characterization of interactions between the bacteria and the host at the molecular level. The results also suggest a role for Rgg regulated surface molecules, like PulA, in the adhesion of S. pyogenes to host cells

Internalization of a polysialic acid-binding Escherichia coli bacteriophage into eukaryotic neuroblastoma cells

Eukaryotic organisms are continuously exposed to bacteriophages, which are efficient gene transfer agents in bacteria. However, bacteriophages are considered not to pass the eukaryotic cell membrane and enter nonphagocytic cells. Here we report the binding and penetration of Escherichia coli PK1A2 bacteriophage into live eukaryotic neuroblastoma cells in vitro. The phage interacts with cell surface polysialic acid, which shares structural similarity with the bacterial phage receptor. Using fluorescence and electron microscopy, we show that phages are internalized via the endolysosomal route and persist inside the human cells up to one day without affecting cell viability. Phage capsid integrity is lost in lysosomes, and the phage DNA is eventually degraded. We did not detect the entry of phage DNA into the nucleus; however, we speculate that this might occur as a rare event, and propose that this potential mechanism could explain prokaryote-eukaryote gene flow.Peer reviewe

Design of a cytotoxic neuroblastoma-targeting agent using an enzyme acting on polysialic acid fused to a toxin

Polysialic acid, an abundant cell surface component of the developing nervous system, which declines rapidly postnatally to virtual absence in the majority of adult tissues, is highly expressed in some malignant tumors including neuroblastoma. We found that the binding of a noncatalytic endosialidase to polysialic acid causes internalization of the complex from the surface of neuroblastoma kSK-N-SH cells, a subline of SK-N-SH, and leads to a complete relocalization of polysialic acid to the intracellular compartment. The binding and uptake of the endosialidase is polysialic acid-dependent as it is inhibited by free excess ligand or removal of polysialic acid by active endosialidase, and does not happen if catalytic endosialidase is used in place of inactive endosialidase. Afusion protein composed of the noncatalytic endosialidase and the cytotoxic portion of diphtheria toxin was prepared to investigate whether the cellular uptake observed could be used for the specific elimination of polysialic acid-containing cells. The conjugate toxin was found to be toxic to polysialic acid-positive kSKN-SH with an IC50 of 1.0 nmol/L. Replacing the noncatalytic endosialidase with active endosialidase decreased the activity to the level of nonconjugated toxin. Normal nonmalignant cells were selectively resistant to the toxin conjugate. The results demonstrate that noncatalytic endosialidase induces a quantitative removal and cellular uptake of polysialic acid from the cell surface which, by conjugation with diphtheria toxin fragment, can be exploited for the selective elimination of polysialic acid-containing tumor cells.Peer reviewe

RAI-arviointijärjestelmä - mitä hyötyä lääkärille?

Vertaisarvioitu.• RAI-arviointijärjestelmä on tarkoitettu hoito- ja palvelusuunnitelmansekäkuntoutussuunnitelman laatimiseen, palvelutarpeen arviointiin sekä toiminnan laadun seurantaan. • Se mahdollistaa asiakkaiden toimintakyvyn osa-alueiden systemaattisen ja samansisältöisen arvioinnin. • RAI-arvio antaa perinteisiä sairauskertomusmerkintöjä laajemman kuvan iäkkään potilaan terveydentilaan ja toimintakykyyn vaikuttavista tekijöistäsekä puuttumista edellyttävistä ongelma-alueista.Peer reviewe

The role of geriatric syndromes in predicting unplanned hospitalizations : a population-based study using Minimum Data Set for Home Care

Background: The predictive accuracies of screening instruments for identifying home-dwelling old people at risk of hospitalization have ranged from poor to moderate, particularly among the oldest persons. This study aimed to identify variables that could improve the accuracy of a Minimum Data Set for Home Care (MDS-HC) based algorithm, the Detection of Indicators and Vulnerabilities for Emergency Room Trips (DIVERT) Scale, in classifying home care clients’ risk for unplanned hospitalization. Methods: In this register-based retrospective study, factors associated with hospitalization among home care clients aged ≥ 80 years in the City of Tampere, Finland, were analyzed by linking MDS-HC assessments with hospital discharge records. MDS-HC determinants associated with hospitalization within 180 days after the assessment were analyzed for clients at low (DIVERT 1), moderate (DIVERT 2–3) and high (DIVERT 4–6) risk of hospitalization. Then, two new variables were selected to supplement the DIVERT algorithm. Finally, area under curve (AUC) values of the original and modified DIVERT scales were determined using the data of MDS-HC assessments of all home care clients in the City of Tampere to examine if addition of the variables related to the oldest age groups improved the accuracy of DIVERT. Results: Of home care clients aged ≥ 80 years, 1,291 (65.4%) were hospitalized at least once during the two-year study period. Unplanned hospitalization occurred following 15.9%, 22.8%, and 33.9% MDS-HC assessments with DIVERT group 1, 2–3 and 4–6, respectively. Infectious diseases were the most common diagnosis within each DIVERT groups. Many MDS-HC variables not included in the DIVERT algorithm were associated with hospitalization, including e.g. poor self-rated health and old fracture (other than hip fracture) (p 0.001) in DIVERT 1; impaired cognition and decision-making, urinary incontinence, unstable walking and fear of falling (p < 0.001) in DIVERT 2–3; and urinary incontinence, poor self-rated health (p < 0.001), and decreased social interaction (p 0.001) in DIVERT 4–6. Adding impaired cognition and urinary incontinence to the DIVERT algorithm improved sensitivity but not accuracy (AUC 0.64 (95% CI 0.62–0.65) vs. 0.62 (0.60–0.64) of the original DIVERT). More admissions occurred among the clients with higher scores in the modified than in the original DIVERT scale. Conclusions: Certain geriatric syndromes and diagnosis groups were associated with unplanned hospitalization among home care clients at low or moderate risk level of hospitalization. However, the predictive accuracy of the DIVERT could not be improved. In a complex clinical context of home care clients, more important than existence of a set of risk factors related to an algorithm may be the various individual combinations of risk factors.Peer reviewe

Bacterial adhesion of Streptococcus suis to host cells and its inhibition by carbohydrate ligands

Peer reviewe

Polysialic acid is associated with better prognosis and IDH1-mutation in diffusely infiltrating astrocytomas

Peer reviewe

Expression of neural cell adhesion molecule and polysialic acid in human bone marrow-derived mesenchymal stromal cells

BACKGROUND: In order to develop novel clinical applications and to gain insights into possible therapeutic mechanisms, detailed molecular characterization of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) is needed. Neural cell adhesion molecule (NCAM, CD56) is a transmembrane glycoprotein modulating cell–cell and cell–matrix interactions. An additional post-translational modification of NCAM is the α2,8-linked polysialic acid (polySia). Because of its background, NCAM is often considered a marker of neural lineage commitment. Generally, hBM-MSCs are considered to be devoid of NCAM expression, but more rigorous characterization is needed. METHODS: We have studied NCAM and polySia expression in five hBM-MSC lines at mRNA and protein levels. Cell surface localization was confirmed by immunofluorescence staining and expression frequency in the donor-specific lines by flow cytometry. For the detection of poorly immunogenic polySia, a fluorochrome-tagged catalytically defective enzyme was employed. RESULTS: All five known NCAM isoforms are expressed in these cells at mRNA level and the three main isoforms are present at protein level. Both polysialyltransferases, generally responsible for NCAM polysialylation, are expressed at mRNA level, but only very few cells express polySia at the cell surface. CONCLUSIONS: Our results underline the need for a careful control of methods and conditions in the characterization of MSCs. This study shows that, against the generally held view, clinical-grade hBM-MSCs do express NCAM. In contrast, although both polysialyltransferase genes are transcribed in these cells, very few express polySia at the cell surface. NCAM and polySia represent new candidate molecules for influencing MSC interactions.Peer reviewe

Costs and Risk Assessment of the Health Effects of the Food System

The project Costs and Risk Assessment of the Health Effects of the Food System employed biological, chemical and nutritional examples to investigate the health and economic risks of food and nutrition in Finland and the cost effectiveness of controls. The project explored the causes and consequences of the health risks associated with food in Finland and sought appropriate risk-management measures. The onset of foodborne health hazards can be slow, and therefore even strong efforts to control them at the population level may take years or decades. Based on the project results, several proposals for action were presented, the most important of which are to: 1. deepen and broaden the risk assessment of the public health impact of the food system to compare different health risks, 2. undertake cost–benefit analyses of food control and health care and 3. to study policy instruments for change. Food contributes significantly to the health of the population, which can be influenced by nutrition and food safety. According to the project results, the main burden of disease (BoD) was caused by the inadequate consumption of fruit and vegetables and the excessive intake of salt and saturated fats, resulting in disease burdens of 36,000, 29,000, 32,000 and 9,200 disability-adjusted life years (DALYs)/year, respectively. Reducing the intake of salt and saturated fats in particular would be a cost-effective way to reduce the BoD. Despite the relatively high level of food hygiene achieved in Finland, up to thousands of foodborne infections occur every year, and food contaminants increase the BoD on consumers. Of the biological food hazards, Listeria caused the largest BoD, about 670 DALYs annually. Of the chemical hazards, lead caused the largest BoD, about 570 DALYs annually. Most of the control costs are borne by food businesses. Therefore, the scenarios that most affect businesses also have the greatest financial impact.This publication is part of the implementation of the Government Plan for Analysis, Assessment and Research. (tietokayttoon.fi) The content is the responsibility of the producers of the information and does not necessarily represent the view of the Government