Preterm delivery and selected biomarkers : phosphorylated insulin-like growth factor-binding protein-1 and matrix metalloproteinase-8 – in cervical fluid

Premature delivery is a major cause of neonatal morbidity and mortality. The incidence of premature deliveries has increased around the world. In Finland 5.3%, or about 3,000 children per year are born prematurely, before 37 weeks of gestation. The corresponding figure in the United States is about 13%. The morbidity and mortality are highest among infants delivered before 32 weeks of gestation - about 600 children each year in Finland. Approximately 70% of premature deliveries are unexplained. Preterm delivery can be caused by an asympto-matic infection between uterus and the fetal membranes, such can begin already in early pregnancy. It is difficult to predict preterm delivery, and many patients are therefore unnecessarily admitted to hospital for observation and exposed to medical treatments. On the other hand, the high risk women should be identified early for the best treatment of the mother and preterm infant. --- In the prospective study conducted at the Department of Obstetric and Gynecology, Helsinki University Central Hospital two biochemical inflammation related markers were measured in the lower genital tract fluids of asymp-tomatic women in early and mid pregnancy in an order to see whether these markers could identify women with an increased risk of preterm delivery. These biomarkers were phosphorylated insulin-like growth factor binding protein-1 (phIGFBP-1) and matrix metalloproteinase-8 (MMP-8). The study involved 5180 asymptomatic pregnant women, examined during the first and second ultrasound screening visits. The study samples were taken from the vagina and cervicix. In addition, 246 symptomatic women were studied (pregnancy weeks 22 – 34). The study showed that increased phIGFBP-1 concentration in cervical canal fluid in early pregnancy increased the risk for preterm delivery. The risk for very premature birth (before 32 weeks of gestation) was nearly four-fold. Low MMP-8 concentration in mid pregnancy increased the risk of subsequent premature preterm rupture of fetal membranes (PPROM). Significantly high MMP-8 concentrations in the cervical fluid increased the risk for prema-ture delivery initiated by preterm labour with intact membranes. Among women with preterm contractions the shortened cervical length measured by ultrasound and elevated cervical fluid phIGFBP-1 both predicted premature delivery. In summary, because of the relatively low sensitivity of cervical fluid phIGFBP-1 this biomarker is not suitable for routine screening, but provides an additional tool in assessing the risk of preterm delivery. Cervical fluid MMP-8 is not useful in early or mid pregnancy in predicting premature delivery because of its dual role. Further studies on the role of MMP-8 are therefore needed. Our study confirms that phIGFBP-1 testing is useful in predicting pre-term delivery.

Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis

Osteoporosis is a disease of low bone mass most often caused by an increase in bone resorption that is not sufficiently compensated for by a corresponding increase in bone formation(1). As gut-derived serotonin (GDS) inhibits bone formation(2), we asked whether hampering its biosynthesis could treat osteoporosis through an anabolic mechanism (that is, by increasing bone formation). We synthesized and used LP533401, a small molecule inhibitor of tryptophan hydroxylase-1 (Tph-1), the initial enzyme in GDS biosynthesis. Oral administration of this small molecule once daily for up to six weeks acts prophylactically or therapeutically, in a dose-dependent manner, to treat osteoporosis in ovariectomized rodents because of an isolated increase in bone formation. These results provide a proof of principle that inhibiting GDS biosynthesis could become a new anabolic treatment for osteoporosis.

Circulating Glucocorticoid Bioactivity and Serum Glucocorticoid-Responsive Biomarkers during Steroid Therapy in Children and Adolescents with Inflammatory Bowel Disease

Objective: Glucocorticoid therapy is used worldwide to treat various inflammatory and immune conditions, including inflammatory bowel disease (IBD). In IBD, 80% of the patients obtain a positive response to the therapy; however the development of glucocorticoid-related side-effects is common. Our aim was therefore to study the possibility of optimizing glucocorticoid therapy in children and adolescents with IBD by measuring circulating glucocorticoid bioactivity (GBA) and serum glucocorticoid-responsive biomarkers in patients receiving steroid treatment for active disease. Methods: A total of sixty-nine paediatric IBD patients from the Paediatric Outpatient Clinics of the University Hospitals of Helsinki and Tampere participated in the studies. Control patients included 101 non-IBD patients and 41 disease controls in remission. In patients with active disease, blood samples were withdrawn before the glucocorticoid therapy was started, at 2-4 weeks after the initiation of the steroid and at 1-month intervals thereafter. Clinical response to glucocorticoid treatment and the development of steroid adverse events was carefully registered. GBA was analyzed with a COS-1 cell bioassay. The measured glucocorticoid therapy-responsive biomarkers included adipocyte-derived adiponectin and leptin, bone turnover-related collagen markers amino-terminal type I procollagen propeptide (PINP) and carboxyterminal telopeptide of type I collagen (ICTP) as well as insulin-like growth factor 1 (IGF-1) and sex hormone-binding globulin (SHBG), and inflammatory marker high-sensitivity C-reactive protein (hs-CRP). Results: The most promising marker for glucocorticoid sensitivity was serum adiponectin that associated with steroid therapy–related adverse events. Serum leptin indicated a similar trend. In contrast, circulating GBA rose in all subjects receiving glucocorticoid treatment but did not associate with the clinical response to steroids or with glucocorticoid therapy-related side-effects. Of notice, young patients (<10 years) showed similar GBA levels than older patients, despite receiving higher weight-adjusted doses of glucocorticoid. Markers of bone formation were lower in children with active IBD than in the control patients, probably reflecting the suppressive effect of the active inflammation. The onset of the glucocorticoid therapy further suppressed bone turnover. Inflammatory marker hs-CRP decreased readily after the initiation of the steroid, however the decrease did not associate with the clinical response to glucocorticoids. Conclusions: This is the first study to show that adipocyte-derived adiponectin associates with steroid therapy-induced side-effects. Further studies are needed, but it is possible that the adiponectin measurement could aid the recognition of glucocorticoid-sensitive patients in the future. GBA and the other markers reflecting glucocorticoid activity in different tissues changed during the treatment, however their change did not correlate with the therapeutic response to steroids or with the development of glucocorticoid-related side effects and therefore cannot guide the therapy in these patients. Studies such as as the present one that combine clinical data with newly developed biomolecular technology are needed to step-by-step build a general picture of the glucocorticoid actions in different tissues.

Metabolomic and proteomic analysis of breast cancer patient samples suggests that glutamate and 12-HETE in combination with CA15-3 may be useful biomarkers reflecting tumour burden

Evaluation of protein and metabolite expression patterns in blood using mass spectrometry and high-throughput antibody-based screening platforms has potential for the discovery of new biomarkers for managing breast cancer patient treatment. Previously identified blood-based breast cancer biomarkers, including cancer antigen 15.3 (CA15-3) are useful in combination with imaging (computed tomography scans, magnetic resonance imaging, X-rays) and physical examination for monitoring tumour burden in advanced breast cancer patients. However, these biomarkers suffer from insufficient levels of accuracy and with new therapies available for the treatment of breast cancer, there is an urgent need for reliable, non-invasive biomarkers that measure tumour burden with high sensitivity and specificity so as to provide early warning of the need to switch to an alternative treatment. The aim of this study was to identify a biomarker signature of tumour burden using cancer and non-cancer (healthy controls/non-malignant breast disease) patient samples. Results demonstrate that combinations of three candidate biomarkers from Glutamate, 12-Hydroxyeicosatetraenoic acid, Beta-hydroxybutyrate, Factor V and Matrix metalloproteinase-1 with CA15-3, an established biomarker for breast cancer, were found to mirror tumour burden, with AUC values ranging from 0.71 to 0.98 when comparing non-malignant breast disease to the different stages of breast cancer. Further validation of these biomarker panels could potentially facilitate the management of breast cancer patients, especially to assess changes in tumour burden in combination with imaging and physical examination.

Noninvasive biomarkers for early smoking related lung disease

Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airway inflammation and largely irreversible airflow limitation. One major risk factor for COPD is cigarette smoking. Since the inflammatory process starts many years prior to the onset of clinical symptoms and still continues after smoking cessation, there is an urgent need to find simple non-invasive biomarkers that can be used in the early diagnosis of COPD and which could help in predicting the disease progression. The first aim of the present study was to evaluate the involvement of different oxidative/nitrosative stress markers, matrix metalloproteinases (MMPs) and their tissue inhibitor-1 (TIMP-1) in smokers and in COPD. Elevated numbers of inducible nitric oxide synthase (iNOS), nitrotyrosine, myeloperoxidase (MPO) and 4-hydroxy-2-nonenal (4-HNE) positive cells and increased levels of 8-isoprostane and lactoferrin were found in sputum of non-symptomatic smokers compared to non-smokers, and especially in subjects with stable mild to moderate COPD, and they correlated with the severity of airway obstruction. This suggests that an increased oxidant burden exists already in the airways of smokers with normal lung function values. However, none of these markers could differentiate healthy smokers from symptomatic smokers with normal lung function values i.e. those individuals who are at risk of developing COPD. In contrast what is known about asthma exhaled nitric oxide (FENO) was lower in smokers than in non-smokers, the reduced FENO value was significantly associated with neutrophilic inflammation and the elevated oxidant burden (positive cells for iNOS, nitrotyrosine and MPO). The levels of sputum MMP-8 and plasma MMP-12 appeared to differentiate subjects who have a risk for COPD development but these finding require further investigations. The levels of all studied MMPs correlated with the numbers of neutrophils, and MMP-8 and MMP-9 with markers of neutrophil activation (MPO, lactoferrin) suggesting that especially neutrophil derived oxidants may stimulate the tissue destructive MMPs already in lungs of smokers who are not yet experiencing any airflow limitation. When investigating the role of neutrophil proteases (neutrophil elastase, MMP-8, MMP-9) during COPD exacerbation and its recovery period, we found that levels of all these proteases were increased in sputum of patients with COPD exacerbation as compared to stable COPD and controls, and decreased during the one-month recovery period, giving evidence for a role of these enzymes in COPD exacerbations. In the last study, the effects of subject`s age and smoking habits were evaluated on the plasma levels of surfactant protein A (SP-A), SP-D, MMP-9 and TIMP-1. Long-term smoking increased the levels of all of these proteins. SP-A most clearly correlated with age, pack years and lung function decline (FEV1/FVC), and based on the receiver operating characteristic curve analysis, SP-A was the best marker for discriminating subjects with COPD from controls. In conclusion, these findings support the hypothesis that especially neutrophil derived oxidants may activate MMPs and induce an active remodeling process already in the lungs of smokers with normal lung function values. The marked increase of sputum levels of neutrophil proteases in smokers, stable COPD and/or during its exacerbations suggest that these enzymes play a role in the development and progression of COPD. Based on the comparison of various biomarkers, SP-A can be proposed to serve as sensitive biomarker in COPD development.

Identification of collagenolytic MMP networks as potential biomarkers in progressive chronic periodontitis subjects and MMP-8 null allele model

Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.

Biomarkers in acute respiratory failure

Acute respiratory failure (ARF) is the most common type of organ failure leading to the need for intensive care. It is often secondary to acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). ARF, and especially ALI and ARDS, cause increased morbidity, and mortality rates remain high (up to 40%). These disorders are characterised by inflammatory reaction and tissue damage. In some cases, inflammation continues and leads to an overwhelming repair process with ongoing fibrosis, accompanied by organ dysfunction and eventually a loss of function. Measuring the magnitude of the inflammation, and the repair process, would theoretically offer information concerning outcome. Early identification of patients whose disease process is likely to proceed unfavourably, would help clinicians to optimise their treatment. The aim of this study was to evaluate the epidemiology of ARF, its treatment, and outcome in Finland, with special interest in biomarkers, and their value in the prediction of mortality. Altogether, 958 adult patients treated with ventilatory support were prospectively included in this study during an eight week period in 2007 in 25 intensive care units. Plasma aminoterminal pro-brain natriuretic peptide (NT-pro-BNP) was assessed in 602 patients, and plasma cell-free DNA in 580 patients, to evaluate their prognostic value in ARF. Markers of collagen metabolism were studied in longitudinal serum samples in 68 patients in order to evaluate their evolution in ARF and the association to multiple organ dysfunction (MOD). Ventilatory support was used in 39% of all ICU patients. The estimated incidence of ARF was 149.5/100 000 per year. Median tidal volumes used were higher than recommended. Overall mortality at 90 days was 31%. Plasma NT-pro-BNP and cell-free DNA were highly increased in the majority of patients. Both markers were independent predictors of 90-day mortality, but their discriminative power was at most moderate when used separately. The mortality was highest in those patients, in whom both biomarkers were over their separate cut-off values. Thus, combined use of these biomarkers may increase their clinical value in the mortality prediction. The markers of collagen metabolism changed significantly over time in surviving patients. None of these markers did associate with MOD in these patients.

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/ mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2(-/-) mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnf alpha and Ifn gamma in sera are not significantly affected, Nos2(-/-) mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.

Synthesis, characterization and pharmacological study of 4,5-dihydropyrazolines carrying pyrimidine moiety

A series of 5-bromo-2-(3,5-diaryl-4,5-dihydro-1H-Pyrazol-1-yl)pyrimidine were prepared under conventional heating as well as microwave reaction condition. The newly synthesized compounds were characterized on the basis of elemental, spectral and single crystal X-ray studies. These new compounds were screened for their antioxidant, anti-inflammatory and analgesic activities. Some of these compounds exhibited potent biological activities compared to the standard drug. (C) 2012 Elsevier Masson SAS. All rights reserved.

Comparative Evaluation of Zoledronic Acid, Alfacalcidol and Propranolol in Pharmacological Correction of Experimental Osteoporosis

Propranolol, a beta-adrenergic receptor blocker, is presently considered to be a potential therapeutic intervention under investigation for its role in prevention and treatment of osteoporosis. However, no studies have compared the osteoprotective properties of propranolol with well accepted therapeu-tic interventions for the treatment of osteoporosis. To address this question, this study was designed to evaluate the bone protective effects of zoledronic acid, alfacalcidol and propranolol in an animal model of postmenopausal osteoporosis. Five days after ovariectomy, 36 ovariectomized (OVX) rats were divided in- to 6 equal groups, randomized to treatments zoledronic acid (100 μg/kg, intravenous single dose); alfacal-cidol (0.5 μg/kg, oral gauge daily); propranolol (0.1mg/kg, subcutaneously 5 days per week) for 12 weeks. Untreated OVX and sham OVX were used as controls. At the end of the study, rats were killed under anesthesia. For bone porosity evaluation, whole fourth lumbar vertebrae (LV4) were removed. LV4 were also used to measure bone mechanical propeties. Left femurs were used for bone histology. Propranolol showed a significant decrease in bone porosity in comparison to OVX control. Moreover, propranolol sig- nificantly improved bone mechanical properties and bone quality when compared with OVX control. The osteoprotective effect of propranolol was comparable with zoledronic acid and alfacalcidol. Based on this comparative study, the results strongly suggest that propranolol might be new therapeutic intervention for the management of postmenopausal osteoporosis in humans.

High-Throughput Sequencing of microRNAs in Peripheral Blood Mononuclear Cells: Identification of Potential Weight Loss Biomarkers

INTRODUCTION: MicroRNAs (miRNAs) are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. OBJECTIVE: The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. METHODS: Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when <5% of initial body weight (non-responders) and successful when >5% (responders). At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC) was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. RESULTS: Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772) and three others were down-regulated (mir-223, mir-224 and mir-376b). Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b) also correlated with weight loss. CONCLUSIONS: This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet.

Molecular biomarkers and toxic consequences of impact by organic pollution in aquatic organisms

Organic contaminants are readily bioaccumulated by aquatic organisms. Exposure to and toxic effects of contaminants can be measured in terms of the biochemical responses of the organisms (i.e. molecular biomarkers). The hepatic biotransformation enzyme cytochrome P4501A (CYP1A) in vertebrates is specifically induced by organic contaminants such as aromatic hydrocarbons, PCBs and dioxins, and is involved in chemical carcinogenesis via catalysis of the covalent binding of organic contaminants to DNA (DNA-adducts). Hepatic CYP1A induction has been used extensively and successfully as a biomarker of organic contaminant exposure in fish. Fewer but equally encouraging studies in fish have used hepatic bulky, hydrophobic DNA-adducts as biomarkers of organic contaminant damage. Much less is known of the situation in marine invertebrates, but a CYPlA-like enzyme with limited inducibility and some potential for biomarker application is indicated. Stimulation of reactive oxygen species (ROS) production is another potential mechanism of organic contaminant-mediated DNA and other damage in aquatic organisms. A combination of antioxidant (enzymes, scavengers) and pro-oxidant (oxidised DNA bases, lipid peroxidation) measurements may have potential as a biomarker of organic contaminant exposure (particularly those chemicals which do not induce CYP1A) and/or oxidative stress, but more studies are required. Both CYP1A- and ROS-mediated toxicity are indicated to result in higher order deleterious effects, including cancer and other aspects of animal fitness.