Quantification of D-dimer levels in human saliva

Background: Plasma D-dimer tests are currently used to exclude deep vein thrombosis and pulmonary embolism. Human saliva has numerous advantages over blood as a diagnostic sample. The aims of our study were to develop a reliable immunoassay to detect D-dimer levels in saliva, and to determine the correlation between salivary and blood D-dimer levels. Results/methodology: Saliva and blood samples were collected from 40 healthy volunteers. We developed a AlphaLISA((R)) immunoassay with acceptable analytical performances to quantify D-dimer levels in the samples. The median salivary D-dimer levels were 138.1 ng/ml (morning) and 140.7 ng/ml (afternoon), and the plasma levels were 75.0 ng/ml. Salivary D-dimer levels did not correlate with plasma levels (p = 0.61). Conclusion: For the first time, we have quantified D-dimer levels and found twofold increase in saliva (p < 0.05) than in plasma. Further studies are required to demonstrate the clinical relevance/utility of salivary D-dimer in patients with confirmed deep vein thrombosis and/or pulmonary embolism.

Validation of an immunoassay to measure plasminogen-activator inhibitor-1 concentrations in human saliva

Introduction We have previously shown that the concentrations of D-dimer are significantly elevated in saliva compared with plasma. Saliva offers several advantages compared with blood analysis. We hypothesised that human saliva contains plasminogen activator inhibitor-1 (PAI-1) and that the concentrations are not affected by the time of saliva collection. The aim was to adopt and validate an immunoassay to quantify PAI-1 concentrations in saliva and to determine whether saliva collection time has an influence in the measurement. Materials and methods Two saliva samples (morning and afternoon) from the same day were collected from healthy subjects (N = 40) who have had no underlying heart conditions. A customized AlphaLISA® immunoassay (PerkinElmer®, MA, USA) was adopted and used to quantify PAI-1 concentrations. We validated the analytical performance of the customized immunoassay by calculating recovery of known amount of analyte spiked in saliva. Results: The recovery (95.03%), intra- (8.59%) and inter-assay (7.52%) variations were within the acceptable ranges. The median salivary PAI-1 concentrations were 394 pg/mL (interquartile ranges (IQR) 243.4-833.1 pg/mL) in the morning and 376 (129.1-615.4) pg/mL in the afternoon and the plasma concentration was 59,000 (24,000-110,000) pg/mL. Salivary PAI-1 did not correlate with plasma (P = 0.812). Conclusions The adopted immunoassay produced acceptable assay sensitivity and specificity. The data demonstrated that saliva contains PAI-1 and that its concentration is not affected by the time of saliva collection. There is no correlation between salivary and plasma PAI-1 concentrations. Further studies are required to demonstrate the utility of salivary PAI-1 in CVD risk factor studies.

Antidepressants, but not antipsychotics, modulate GR function in human whole blood: An insight into molecular mechanisms

Clinical studies have demonstrated an impairment of glucocorticoid receptor (GR)-mediated negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis in patients with major depression (GR resistance), and its resolution by antidepressant treatment. Recently, we showed that this impairment is indeed due to a dysfunction of GR in depressed patients (Carvalho et al., 2009), and that the ability of the antidepressant clomipramine to decrease GR function in peripheral blood cells is impaired in patients with major depression who are clinically resistant to treatment (Carvalho et al. 2008). To further investigate the effect of antidepressants on GR function in humans, we have compared the effect of the antidepressants clomipramine, amytriptiline, sertraline, paroxetine and venlafaxine, and of the antipsychotics, haloperidol and risperidone, on GR function in peripheral blood cells from healthy volunteers (n=33). GR function was measured by glucocorticoid inhibition of lypopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) levels. Compared to vehicle-treated cells, all antidepressants inhibited dexamethasone (DEX, 10-100nM) inhibition of LPS-stimulated IL-6 levels (p values ranging from 0.007 to 0.1). This effect was specific to antidepressants, as antipsychotics had no effect on DEX-inhibition of LPS-stimulated IL-6 levels. The phosphodiesterase (PDE) type 4 inhibitor, rolipram, potentiated the effect of antidepressants on GR function, while the GR antagonist, RU-486, inhibited the effect of antidepressants on GR function. These findings indicate that the effect of antidepressants on GR function are specific for this class of psychotropic drugs, and involve second messenger pathways relevant to GR function and inflammation. Furthermore, it also points towards a possible mechanism by which one maybe able to overcome treatment-resistant depression. Research in this field will lead to new insights into the pathophysiology and treatment of affective disorders.

Assessment of dynamic susceptibility contrast cerebral blood flow response to amphetamine challenge: A human pharmacological magnetic resonance imaging study at 1.5 and 4 T

Pharmacological MRI (phMRI) techniques can be used to monitor the neurophysiological effects of central nervous system (CNS) active drugs. In this study, we investigated whether dynamic susceptibility contrast (DSC) perfusion imaging employing the use of superparamagnetic iron oxide nanoparticles (Resovist) could be used to measure hemodynamic response to d-amphetamine challenge in human subjects at both 1.5 and 4 T. Significant changes in cerebral blood flow (CBF) were found in focal regions associated with the nigrostriatal circuit and mesolimbic and mesocortical dopaminergic pathways. More significant CBF responses were found at higher field strength, mainly within striatal structures. The results from this study indicate that DSC perfusion imaging using Resovist can be used to assess the efficacy of CNS-active drugs and may play a role in the development of novel psychiatric therapies at the preclinical level. © 2005 Wiley-Liss, Inc.

The effect of ongoing blood loss on human serum concentrations of perfluorinated acids

Perfluorinated alkyl acids (PFAAs) have been detected in serum at low concentrations in background populations. Higher concentrations haven been observed in adult males compared to females, with a possible explanation that menstruation offers females an additional elimination route. In this study, we examined the significance of blood loss as an elimination route of PFAAs. Pooled serum samples were collected from individuals undergoing a medical procedure involving ongoing blood withdrawal called venesection. Concentrations from male venesection patients were approximately 40% lower than males in the general population for perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). A simple pharmacokinetic model was used to test the hypothesis that blood loss could explain why adult males have higher concentrations of PFAAs than females, and why males undergoing venesections had lower concentrations compared to males in the general population. The model application generally supported these hypotheses showing that venesection might reduce blood serum concentrations by 37% (PFOA) and 53% (PFOS) compared to the observed difference of 44% and 37%. Menstruation was modeled to show a 22% reduction in PFOA serum concentrations compared to a 24% difference in concentrations between males and females in the background population. Uncertainties in the modeling and the data are identified and discussed.

The high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Genistein and 17β-estradiol fatty acid esters in blood and adipose tissue and the structure-related antioxidant activity of estrogens on lipoproteins

Soy-derived phytoestrogen genistein and 17β-estradiol (E2), the principal endogenous estrogen in women, are also potent antioxidants protecting LDL and HDL lipoproteins against oxidation. This protection is enhanced by esterification with fatty acids, resulting in lipophilic molecules that accumulate in lipoproteins or fatty tissues. The aims were to investigate, whether genistein becomes esterified with fatty acids in human plasma accumulating in lipoproteins, and to develop a method for their quantitation; to study the antioxidant activity of different natural and synthetic estrogens in LDL and HDL; and to determine the E2 esters in visceral and subcutaneous fat in late pregnancy and in pre- and postmenopause. Human plasma was incubated with [3H]genistein and its esters were analyzed from lipoprotein fractions. Time-resolved fluoroimmunoassay (TR-FIA) was used to quantitate genistein esters in monkey plasma after subcutaneous and oral administration. The E2 esters in women s serum and adipose tissue were also quantitated using TR-FIA. The antioxidant activity of estrogen derivatives (n=43) on LDL and HDL was assessed by monitoring the copper induced formation of conjugated dienes. Human plasma was shown to produce lipoprotein-bound genistein fatty acid esters, providing a possible explanation for the previously reported increased oxidation resistance of LDL particles during intake of soybean phytoestrogens. Genistein esters were introduced into blood by subcutaneous administration. The antioxidant effect of estrogens on lipoproteins is highly structure-dependent. LDL and HDL were protected against oxidation by many unesterified, yet lipophilic derivatives. The strongest antioxidants had an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups. E2 ester levels were high during late pregnancy. The median concentration of E2 esters in pregnancy serum was 0.42 nmol/l (n=13) and in pre- (n=8) and postmenopause (n=6) 0.07 and 0.06 nmol/l, respectively. In pregnancy visceral fat the concentration of E2 esters was 4.24 nmol/l and in pre- and postmenopause 0.82 and 0.74 nmol/l. The results from subcutaneous fat were similar. In serum and fat during pregnancy, E2 esters constituted about 0.5 and 10% of the free E2. In non-pregnant women most of the E2 in fat was esterified (the ester/free ratio 150 - 490%). In postmenopause, E2 levels in fat highly exceeded those in serum, the majority being esterified. The pathways for fatty acid esterification of steroid hormones are found in organisms ranging from invertebrates to vertebrates. The evolutionary preservation and relative abundance of E2 esters, especially in fat tissue, suggest a biological function, most likely in providing a readily available source of E2. The body s own estrogen reservoir could be used as a source of E2 by pharmacologically regulating the E2 esterification or hydrolysis.

Ischemia-reperfusion injury in human liver transplantation : Mechanisms and effects on graft function

Liver transplantation is an established therapy for both acute and chronic liver failure. Despite excellent long-term outcome, graft dysfunction remains a problem affecting up to 15-30% of the recipients. The etiology of dysfunction is multifactorial, with ischemia-reperfusion injury regarded as one of the most important contributors. This thesis focuses on the inflammatory response during graft procurement and reperfusion in liver transplantation in adults. Activation of protein C was examined as a potential endogenous anti-inflammatory mechanism. The effects of inflammatory responses on graft function and outcome were investigated. Seventy adult patients undergoing liver transplantation in Helsinki University Central Hospital, and 50 multiorgan donors, were studied. Blood samples from the portal and the hepatic veins were drawn before graft procurement and at several time points during graft reperfusion to assess changes within the liver. Liver biopsies were taken before graft preservation and after reperfusion. Neutrophil and monocyte CD11b and L-selectin expression were analysed by flow cytometry. Plasma TNF-α, IL-6, IL-8, sICAM-1, and HMGB1 were determined by ELISA and Western-blotting. HMGB1 immunohistochemistry was performed on liver tissue specimens. Plasma protein C and activated protein C were determined by an enzyme-capture assay. Hepatic IL-8 release during graft procurement was associated with subsequent graft dysfunction, biliary in particular, in the recipient. Biliary marker levels increased only 5 7 days after transplantation. Thus, donor inflammatory response appears to influence recipient liver function with relatively long-lasting effects. Hepatic phagocyte activation and sequestration, with concomitant HMGB1 release, occurred during reperfusion. Neither phagocyte activation nor plasma cytokines correlated with postoperative graft function. Thus, activation of the inflammatory responses within the liver during reperfusion may be of minor clinical significance. However, HMGB1 was released from hepatocytes and were also correlated with postoperative transaminase levels. Accordingly, HMGB1 appears to be a marker of hepatocellular injury.

Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.

Flow cytometry analysis of human fetal osteoblast fate processes on spark plasma sintered hydroxyapatite-titanium biocomposites

Hydroxyapatite (HA)-based biocomposites have been widely investigated for a multitude of applications and these studies have been largely driven to improve mechanical properties (toughness and strength) without compromising cytocompatibility properties. Apart from routine cell viability/proliferation analysis, limited efforts have been made to quantify the fate processes (cell proliferation, cell cycle, and cell apoptosis) of human fetal osteoblast (hFOB) cells on HA-based composites, in vitro. In this work, the osteoblast cell fate process has been studied on a model hydroxyapatite-titanium (HA-Ti) system using the flow cytometry. In order to retain both HA and Ti, the novel processing technique, that is, spark plasma sintering, was suitably adopted. The cell fate processes of hFOBs, as evaluated using a flow cytometry, revealed statistically insignificant differences among HA-10 wt % Ti and HA and control (tissue culture polystyrene surface) in terms of osteoblast apoptosis, proliferation index as well as division index. For the first time, we provide quantified flow cytometry results to demonstrate that 10 wt % Ti additions to HA do not have any significant influence on the fate processes of human osteoblast-like cells, in vitro.

Study of the effect of alcohol on single human red blood cells using near-infrared laser tweezers Raman spectroscopy

The effect of alcohol solution on single human red blood Cells (RBCs) was investigated using near-infrared laser tweezers Raman spectroscopy (LTRS). In our system, a low-power diode laser at 785 nm was applied for the trapping of a living cell and the excitation of its Raman spectrum. Such a design could simultaneously reduce the photo-damage to the cell and suppress the interference from the fluorescence on the Raman signal. The denaturation process of single RBCs in 20% alcohol solution was investigated by detecting the time evolution of the Raman spectra at the single-cell level. The vitality of RBCs was characterized by the Raman band at 752 cm(-1), which corresponds to the porphyrin breathing mode. We found that the intensity of this band decreased by 34.1% over a period of 25 min after the administration of alcohol. In a further study of the dependence of denaturation on alcohol concentration, we discovered that the decrease in the intensity of the 752 cm(-1) band became more rapid and more prominent as the alcohol concentration increased. The present LTRS technique may have several potential applications in cell biology and medicine, including probing dynamic cellular processes at the single cell level and diagnosing cell disorders in real time. Copyright (c) 2005 John Wiley T Sons, Ltd.

Efficacy and safety of autologous platelet rich plasma for the treatment of vascular ulcers in primary care: Phase III study

Background: Vascular ulcers are commonly seen in daily practice at all levels of care and have great impact at personal, professional and social levels with a high cost in terms of human and material resources. Given that the application of autologous platelet rich plasma has been shown to decrease healing times in various different studies in the hospital setting, we considered that it would be interesting to assess the efficacy and feasibility of this treatment in primary care. The objectives of this study are to assess the potential efficacy and safety of autologous platelet rich plasma for the treatment of venous ulcers compared to the conventional treatment (moist wound care) in primary care patients with chronic venous insufficiency (C, clinical class, E, aetiology, A, anatomy and P, pathophysiology classification C6). Design: We will conduct a phase III, open-label, parallel-group, multicentre, randomized study. The subjects will be 150 patients aged between 40 and 100 years of age with an at least 2-month history of a vascular venous ulcer assigned to ten primary care centres. For the treatment with autologous platelet rich plasma, all the following tasks will be performed in the primary care setting: blood collection, centrifugation, separation of platelet rich plasma, activation of coagulation adding calcium chloride and application of the PRP topically after gelification. The control group will receive standard moist wound care. The outcome variables to be measured at baseline, and at weeks 5 and 9 later include: reduction in the ulcer area, Chronic Venous Insufficiency Quality of Life Questionnaire score, and percentage of patients who require wound care only once a week. Discussion: The results of this study will be useful to improve the protocol for using platelet rich plasma in chronic vascular ulcers and to favour wider use of this treatment in primary care.