Biomedical subjects

T Soukka

Publications and source records attributed to T Soukka.

17 recordsLinked to original sources

PubMed2004

Estrogen receptor-beta is the predominant estrogen receptor subtype in human oral epithelium and salivary glands.

Many studies have shown that the oral mucosa and salivary glands are sensitive to estrogen action. However, the expression of estrogen receptors (ERs) within these tissues is an area of controversy. ERs exist as two subtypes (ERalpha and ERbeta), and we hypothesized that the incongruity between ER expression and estrogen sensitivity may result from differential expression of ER subtypes in oral tissues. To test this hypothesis, we analyzed oral mucosal and salivary gland samples for ERalpha and ERbeta protein expression by immunohistochemistry from a cross-section of patients attending hospital for surgical problems of the head and neck. ERalpha was not detected in oral buccal and gingival epithelium or in salivary glands. In contrast, ERbeta was widely expressed at high levels in all oral tissues studied. Within these tissues, ERbeta was observed primarily in keratinocytes and salivary gland acinar and ductal cells. Our results demonstrating the expression of only the ERbeta subtype within oral tissues may explain the contradictory results from previous studies investigating ER expression in these tissues. Importantly, these results suggest that estrogens may act via ERbeta in oral tissues and explain the effect of hormonal changes on the oral mucosa as well as on saliva secretion and composition.

Adult↗

PubMed2003

Different mechanisms of syndecan-1 activation through a fibroblast-growth-factor-inducible response element (FiRE) in mucosal and cutaneous wounds.

Syndecan-1 expression is enhanced in cutaneous and mucosal wounds. We have previously demonstrated that wounding-induced syndecan-1 expression in the skin occurs transcriptionally, through a fibroblast-growth-factor-inducible element (FiRE). Here, we show that FiRE is also activated in mucosal wounds. However, both the expression patterns and the activation mechanisms of FiRE are different from those in the skin. In the mucosa in vivo, the activation starts and ends earlier than in cutaneous wounds. FiRE is first detected at around 12 hours in keratinocytes, and the activation declines by the third day after wounding occurs. The activation is seen on the migrating sheet of epithelial mucosa, as in the case of cutaneous wounding. In contrast to the situation in vivo, organ-cultured mucosal wounds exhibit no FiRE activity, while organ-cultured cutaneous wounds show robust activity. Activation in mucosal wounds is enhanced, however, by the application of epidermal growth factor. This suggests that exogenous growth factor activity is required for activation of syndecan-1 in mucosal wounds but not in cutaneous wounds.

Animals↗

PubMed2001

Utilization of kinetically enhanced monovalent binding affinity by immunoassays based on multivalent nanoparticle-antibody bioconjugates.

The monovalent binding affinity of high binding site density nanoparticle-antibody bioconjugates is shown to exceed the intrinsic affinity of the original, monoclonal antibody. The nanoparticle-antibody bioconjugates were prepared by covalent coupling of antibodies to long-lifetime fluorescent, europium(III) chelate nanoparticles, 107 nm in diameter. Experiments were carried out in standard microtitration wells to determine solid-phase association and dissociation rate constants, nonspecific binding, and affinity constants of the various binding site density nanoparticle-antibody bioconjugates and the conventionally labeled monoclonal antibody. The affinity constant for monovalent binding of a high binding site density bioconjugate (5.4 x 10(10) M(-1)) was 8-fold higher than the intrinsic affinity of the antibody (6.6 x 10(9) M(-1)). The separately measured association (2.5 x 10(6) M(-1) s(-1)) and dissociation (3.7 x 10(-5) s(-1)) rate constants of the bioconjugate were 2-fold higher and 4-fold lower, respectively, compared to the antibody. The dependence of the association rate constant of the density of the binding sites enhanced the kinetics and the affinity of the high binding site density bioconjugates. The nanoparticle labels with high specific activity, low nonspecific binding, and enhanced binding affinity of the nanoparticle-antibody bioconjugates contribute to the design of the next generation immunoassays with extreme sensitivity.

Antibodies↗

PubMed2001

Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen.

BACKGROUND: Nanoparticle-based detection technologies have the potential to improve detection sensitivity in miniature as well as in conventional biochemical assays. We introduce a detection technology that relies on the use of europium(III) nanoparticles and time-resolved fluorometry to improve the detection limit of biochemical assays and to visualize individual molecules in a microtiter plate format. METHODS: Streptavidin was covalently coated on 107-nm nanoparticles containing >30 000 europium molecules entrapped with beta-diketones. In a model assay system, these nanoparticles were used to trace biotinylated prostate-specific antigen (PSA) in a microtiter plate format. RESULTS: The detection limit (mean + 3 SD of the zero calibrator) of biotinylated PSA was 0.38 ng/L, corresponding to 10 fmol/L or 60 zeptomoles (60 x 10(-21) moles) of PSA. Moreover, single nanoparticles, representing individual PSA molecules, were visualized in the same microtiter wells with a time-resolved fluorescence microscope using a x10 objective. Single nanoparticles, possessing high specific activity, were also detected in solution by a standard time-resolved plate fluorometer. CONCLUSIONS: The universal streptavidin-coated europium(III) nanoparticle label is suitable for detection of any biotinylated molecule either in solution or on a solid phase. The europium(III) nanoparticle labeling technology is applicable to many areas of modern biochemical analysis, such as immunochemical and multianalyte DNA-chip assays as well as histo- and cytochemistry to improve detection sensitivities.

Biotinylation↗

PubMed2001

Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology.

BACKGROUND: The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and the enhanced monovalent binding affinity of multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay. METHODS: We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (107 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrinsic affinity, 6 x 10(9) L/mol). The nanoparticle-antibody bioconjugates had an average of 214 active binding sites per particle and a monovalent binding affinity of 7 x 10(10) L/mol. The assay was performed in a low-fluorescence microtitration well passively coated with an another monoclonal anti-PSA antibody (affinity, 2 x 10(10) L/mol), and the europium(III) fluorescence was measured directly from the bottom of the well by a standard time-resolved microtitration plate fluorometer. RESULTS: The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 x 10(5) molecules/mL), and the dynamic detection range covered four orders of magnitude in a 3-h total assay time. The imprecision (CV) over the whole assay range was 2-10%. The detection limit of the assay was limited by the fractional nonspecific binding of the bioconjugate to the solid phase (0.05%), which was higher than the nonspecific binding of the original antibody (<0.01%). CONCLUSIONS: The sensitivity of the new assay is equal to that of the ambient-analyte, microspot immunoassay and will be improved by use of optimized, high binding-site density nanoparticle-antibody bioconjugates with reduced nonspecific binding and improved monovalent binding affinity.

Acetates↗

PubMed2000

A new label technology for the detection of specific polymerase chain reaction products in a closed tube.

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5'-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5'-->3' exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10(7)initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5'-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

Chelating Agents↗

PubMed2000

Reduction of syndecan-1 expression is associated with dysplastic oral epithelium.

Syndecans are a family of integral membrane proteoglycans that participate in cell-matrix interactions and growth factor binding. Syndecan-1 expression is induced during keratinocyte differentiation and reduced in squamous cell carcinomas. The purpose of this study was to examine the alteration in syndecan-1 expression in dysplastic oral epithelium. Sixty-six oral biopsy specimens (43 epithelial dysplasias, 3 carcinoma in situ and 20 squamous cell carcinomas) were studied using immunohistochemical methods. The normal epithelium specimens were highly positive for syndecan-1. Fifteen of 46 dysplasias or carcinoma in situ specimens showed negative or weak staining for syndecan-1, two of which were totally negative. Intermediate and strong staining were observed in 17 and 14 dysplasias or carcinoma in situ specimens, respectively. Thirteen (65%) squamous cell carcinomas showed negative or weak staining for syndecan-1, seven of which were totally negative. Only three carcinomas had a strong syndecan-1 expression. Four of the 34 patients with dysplasia who were followed up developed squamous cell carcinoma. All these dysplasias had weak or totally negative syndecan-1 expression. The results suggest that the loss of syndecan-1 is associated with dysplastic changes in oral epithelium.

Adult↗

PubMed2000

Miniature single-particle immunoassay for prostate-specific antigen in serum using recombinant Fab fragments.

BACKGROUND: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay. METHODS: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-microm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode. RESULTS: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 microg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06+/-0.03 and 1.03+/-0.02, respectively (S(y|x) = 0.084 and 0.057 microg/L), with intercepts of 0.013+/-0.018 and 0.013+/-0.017 microg/L (R>0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed. CONCLUSIONS: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.

Antibodies, Monoclonal↗

PubMed1999

Population-based genetic screening for the estimation of Type 1 diabetes mellitus risk in Finland: selective genotyping of markers in the HLA-DQB1, HLA-DQA1 and HLA-DRB1 loci.

AIMS: To improve sensitivity and specificity of the diabetes risk assessment of the population-based genetic screening used in the Finnish Diabetes Prediction and Prevention (DIPP) trial. METHODS: One thousand consecutive newborns enrolled in the DIPP were compared with 316 samples from children with Type 1 diabetes mellitus. A modification of the previously described technique based on hybridization of relevant PCR products with five lanthanide-labelled probes detected by time-resolved fluorometry (TRF) was used. A new probe was designed and allowed discrimination between DQB1*0602 and 0603 alleles, in addition to DQB1*02, *0301 or *0302, each of which required specific probes. A new, added screening strategy was developed for individuals carrying low-risk genotypes through specific typing of DQA1 *05 and *0201 alleles in DQB1*02 positive, and DRB1 typing for DR4 subtypes in DQB1*0302 positive subjects, with a new specifically designed high-resolution TRF-based DR4 subtyping technique. RESULTS: This two-step screening approach enhanced the sensitivity of the detection of genetic risk for Type 1 diabetes mellitus in this cohort up to 85.4%. In the general population cohort, 24.4% were identified for prospective follow-up, 2.6% of these are expected to develop Type 1 diabetes mellitus before the age of 15 years. Exclusive typing for HLA-DQB1 locus as an alternative screening strategy had sensitivities of 26.3-77.2% with general population cohorts of 2.3-23.1% identified for follow-up. CONCLUSIONS: The described strategy for genetic prediction of Type 1 diabetes mellitus relies on the convenient genotyping procedure and could be applied in large scale screening projects such as DIPP.

Biomarkers↗

PubMed1997

Regulation of CD44v6-containing isoforms during proliferation of normal and malignant epithelial cells.

CD44 is a family of molecules involved in cell-cell and cell-matrix interactions. Various isoforms of CD44 arise by insertion of one or more of the variant exons into the common backbone shared by all forms of CD44. In this work, we studied the expression of CD44 and exon v6-containing CD44 isoforms (CD44v6) in several nonmalignant and malignant conditions and the possibilities for regulating the expression of CD44v6. In primary squamocellular carcinomas of the head and neck, CD44 and CD44v6 were down-regulated in poorly differentiated tumors, whereas these molecules were uniformly expressed in the normal squamocellular epithelium, in proliferating skin diseases, and in nonmalignant tumors. When CD44v6 expression of original tumors and that of squamocellular carcinoma cell lines derived from them were compared, no CD44v6 up-regulation could be observed on in vitro growing cells. Moreover, several regulators were unable to up-regulate CD44v6 expression on cultured cell lines in vitro. When the same cell lines formed tumors after s.c. injection into severe combined immunodeficient mice, some of them up-regulated their CD44v6 expression. These data suggest that cell lines at certain differentiation stages can be induced to express CD44v6. Our results further indicate that CD44v6 positivity cannot be used as a universal indicator of tumor metastasis. Instead, the down-regulation of CD44v6 in squamocellular tumors is a sign of malignant transformation of the epithelium.

Adult↗

PubMed1993

Agglutination of Streptococcus mutans serotype C cells but inhibition of Porphyromonas gingivalis autoaggregation by human lactoferrin.

The ability of various forms of human lactoferrin (LF) to agglutinate oral Streptococcus mutans, Strep. sobrinus, Strep. rattus, Strep. sanguis, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans cells was studied spectrophotometrically. Fe3+ saturated LF was unable to agglutinate these bacteria, whereas iron-free LF (apo LF) effectively agglutinated Strep. mutans cells but not the other bacteria. The efficiency and rate of agglutination of Strep. mutans were somewhat lower with apo LF than with human whole saliva. However, secretory IgA, phosphate and whole saliva almost totally abolished the apo LF-mediated agglutination of Strep. mutans, suggesting binding to the same target sites on bacterial cell surfaces, or to each other. The presence of exogenous iron (Fe2+, Fe3+), lactoperoxidase or serum albumin did not affect the agglutination by apo LF. Low Ca2+ (50-100 microns) slightly enhanced the agglutination by apo LF but higher concentrations (0.5-1.0 mM) totally blocked the apo LF-mediated agglutination of Strep. mutans. Both saliva and apo LF significantly delayed the rapid autoaggregation of P. gingivalis cells. Aggregation of P. gingivalis is considered a potential virulence factor and a protective mechanism against the host's cellular defences in the gingival crevice. These findings show a novel, strain-specific antibacterial mechanism for LF against Strep. mutans and P. gingivalis and adds a new compound to the group of agglutinating proteins in human saliva.

Agglutination↗

PubMed1992

Fungicidal effect of human lactoferrin against Candida albicans.

Human lactoferrin (LF) in its iron-free state (apo LF), killed Candida albicans in a time- and dose-dependent way. The lethal effect was stronger at pH 7.0 than at pH 5.5 and maximum inhibition at neutral pH was achieved in 25 min when the fungal cells were exposed to LF in 0.05 mM KCl at 37 degrees C. Fe(3+)-saturated LF had no fungicidal activity. Apo LF-mediated killing was also temperature-dependent with enhanced inhibition at higher temperatures (37 degrees, 42 degrees C). The presence of 1 mM D-glucose did not affect the candidacidal activity of apo LF but both phosphate and bicarbonate ions at physiological salivary concentrations completely blocked the anti-fungal effect. Therefore it seems unlikely that LF belongs to the major host defence factors against oral candidosis.

Antifungal Agents↗

PubMed1992

Antimicrobial factors of saliva in relation to dental caries and salivary levels of mutans streptococci.

The possible association between salivary non-immunoglobulin (lysozyme, lactoferrin, hypothiocyanite, agglutinins) or immunoglobulin (total IgA, anti-Streptococcus mutans IgA) antimicrobial factors, and the prevalence of dental caries was studied in 59 young adults. These antimicrobial factors were also analysed in relation to the salivary levels of mutans streptococci (MS). The amount of MS correlated significantly (+0.31, p less than 0.05) with the number of initial caries lesions (Di) but not with other caries indices (DMFT, DMFS, DS). The group with no Di (N = 17) had significantly (p less than 0.05) more hypothiocyanite (HOSCN/OSCN-) and anti-S. mutans IgA antibodies in whole saliva than those with initial caries lesions (N = 42). None of the antimicrobial factors alone showed any significant association with salivary MS counts. Our results suggest that HOSCN/OSCN- and anti-S. mutans IgA may be involved in the prevention of the early phases of dental caries.

Adult↗

PubMed1991

Inhibition of the growth of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei by oral peroxidase systems in human saliva.

Streptococcus mutans, Strep. sobrinus and Lactobacillus casei were grown in glucose-supplemented, sterilized, human whole saliva, adjusted to pH 5, 6 or 7. Components of the antibacterial peroxidase system--hypothiocyanous acid (HOSCN) and hypothiocyanite ions (OSCN-)--were generated by adding exogenous H2O2 to sterilized saliva containing endogenous peroxidases and thiocyanate (SCN-) ions. HOSCN/OSCN- generation was proportional to the amount of H2O2 added, and more HOSCN/OSCN- was detected in saliva at pH 7 than at pH 5. However, the growth of mutans streptococci and L. casei was inhibited at pH 5 by HOSCN/OSCN-, whereas no inhibition was found at pH 7. The findings show that (a) sufficient amount of HOSCN/OSCN- will inhibit the growth of cariogenic bacteria in human saliva at pH 5; (b) this amount of HOSCN/OSCN- can be generated in saliva by exogenously added H2O2; and (c) peroxidase systems have stronger antistreptococcal effects in human whole saliva than in phosphate buffer.

Anti-Bacterial Agents↗

PubMed1991

Combined inhibitory effect of lactoferrin and lactoperoxidase system on the viability of Streptococcus mutans, serotype c.

We have studied the effects of iron-free lactoferrin (apo LF) and lactoperoxidase system (lactoperoxidase, LP/SCN-/H2O2), separately and together, on the viability of Streptococcus mutans (serotype c) in vitro. The bacteria were incubated in buffered KCl (pH 5.5) with and without the above components which were used at concentrations normally present in human saliva. Both apo LF and LP-system had a bactericidal effect against S. mutans at low pH. Together they showed an additive, but not a synergistic, antibacterial effect against S. mutans. Apo LF enhanced the LP enzyme activity but decreased the yield of the antimicrobial component, hypothiocyanite (HOSCN/OSCN-), when incorporated into the reaction mixtures. This decrease, which was most pronounced at low pH, was due to an LP-independent reaction between apo LF and HOSCN/OSCN-. Our study indicates that the LP-system and apo LF can be combined to combat oral S. mutans.

Anti-Bacterial Agents↗

PubMed1991

Salivary lysozyme, lactoferrin and peroxidases: antibacterial effects on cariogenic bacteria and clinical applications in preventive dentistry.

Many antimicrobial agents in human saliva are known to have bacteriostatic or bactericidal effects on cariogenic bacteria, in particular against Streptococcus mutans. Studies have usually been conducted with purified agents (proteins) in vitro. Very little proof exists to show that they also affect oral cariogenic flora in vivo. Recent studies have shown that some salivary systems can act synergistically against Streptococcus mutans. Such synergistic antibacterial activity is likely to exist in the human mouth. Attempts to enhance the anticariogenic properties of saliva have been made by adding antimicrobial proteins such as peroxidase, lactoferrin and lysozyme to oral health products. Although clinical evidence is still limited, the idea of using such antimicrobial agents--"natural antibiotics"--rather than synthetic agents against cariogenic bacteria seems promising.

Dental Caries↗

PubMedDate not supplied

Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence.

Prostate-specific antigen (PSA) was detected in microtitre wells coated with a PSA-specific antibody using biotinylated antibody and streptavidin-coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium ions entrapped by beta-diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensitivity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomoles (250 x 10(-21) mol/L) of PSA. The high specific activity and low non-specific binding of the streptavidin-coated nanoparticles improved the sensitivity of the PSA assay 100-fold compared to the conventional europium-labelled streptavidin tracer in the same assay format. Additionally, the streptavidin-coated nanoparticle label made very rapid assays possible, due to the high affinity of the streptavidin-biotin complex and a high number of binding sites available for tracing the biotinylated antibody on the surface. Due to the inherent problems of tracing analyte with a complex of biotinylated antibody and streptavidin-coated nanoparticles, the streptavidin-coated nanoparticles reacting with the surface-captured analyte and biotinylated antibody was favoured and factors influencing this are discussed. This universal labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sensitivities in many areas of biochemical analysis, such as cyto- and histochemistry, multianalyte DNA-chip assays and single-particle assays.

Antibodies, Monoclonal↗