Vojtech Tambor

Intraamniotic inflammatory response to bacteria: analysis of multiple amniotic fluid proteins in women with preterm prelabor rupture of membranes.

Objective: To analyse whether intraamniotic inflammation in response to bacteria is different below and above gestational age 32 weeks in pregnancies complicated by preterm prelabor rupture of membranes (PPROM). Methods: A prospective study was performed, and 115 women with singleton pregnancies complicated by PPROM at gestational ages between 240/7 and 366/7 weeks were included in the study. Transabdominal amniocenteses were performed. Amniotic fluid was analysed using polymerase chain reactions for genital mycoplasmas and cultured for aerobic and anaerobic bacteria. The concentrations of 26 proteins in the amniotic fluid were determined simultaneously using multiplex technology. Results: Bacteria were found in the amniotic fluid of 43% (49/115) of the women. The women were stratified into two subgroups according to gestational age 32 weeks. The amniotic fluid levels of four (interleukin-6, interleukin-10, CC chemokine ligands 2, and 3) and one specific (CC chemokine ligands 2) proteins were higher in women with the presence of bacteria in the amniotic fluid below and above 32 gestational weeks, respectively. Conclusions: An intraamniotic inflammatory response to bacteria in pregnancies complicated by PPROM seems to be different below and above 32 weeks of gestation.

The microbial load with genital mycoplasmas correlates with the degree of histologic chorioamnionitis in preterm PROM

OBJECTIVE We sought to determine whether there is an association between bacterial load of genital mycoplasmas and histologic chorioamnionitis (HCA) in women with preterm premature rupture of membranes (PPROM). STUDY DESIGN A total of 103 women with PPROM between 24-36 weeks of gestation were included in the study. Amniocenteses were performed, and the amounts of target genital mycoplasma DNA in amniotic fluid samples were evaluated using real-time polymerase chain reaction. The bacterial load of the genital mycoplasmas was relatively assessed using the threshold cycle value. RESULTS The presence of genital mycoplasmas in amniotic fluid was found in 38% (39/103) of the women. The presence of HCA was associated with lower threshold cycle values (median 21.3, interquartile range, 16.5-28.5, vs median 29.4, interquartile range, 27.0-30.5; P = .005). CONCLUSION HCA in PPROM is associated with a higher bacterial load of genital mycoplasmas.

CysTRAQ — A combination of iTRAQ and enrichment of cysteinyl peptides for uncovering and quantifying hidden proteomes

Shotgun proteomics is capable of characterizing differences in both protein quality and quantity, and has been applied in various biomedical applications. Unfortunately, the high complexity and dynamic range of proteins in studied samples, clinical in particular, often hinders the identification of relevant proteins. Indeed, information-rich, low abundance proteins often remain undetected, whereas repeatedly reported altered concentrations in high abundance proteins are often ambiguous and insignificant. Several techniques have therefore been developed to overcome this obstacle and provide a deeper insight into the proteome. Here we report a novel approach, which enables iTRAQ reagent quantitation of peptides fractionated based on presence of a cysteine residue (thus CysTRAQ). For the first time, we prove that iTRAQ quantitation is fully compatible with cysteinyl peptide enrichment and is not influenced by the fractionation process. Moreover, the employment of the method combined with high-resolution TripleTOF 5600 mass spectrometer for very fast MS/MS acquisition in human amniotic fluid analysis significantly increased the number of identified proteins, which were simultaneously quantified owing to the introduction of iTRAQ labeling. We herein show that CysTRAQ is a robust and straightforward method with potential application in quantitative proteomics experiments, i.e. as an alternative to the ICAT reagent approach.

Amniotic Fluid Cathelicidin in PPROM Pregnancies: From Proteomic Discovery to Assessing Its Potential in Inflammatory Complications Diagnosis

Background Preterm prelabor rupture of membranes (PPROM) complicated by microbial invasion of the amniotic cavity (MIAC) leading to histological chorioamnionitis (HCA) significantly impacts perinatal morbidity. Unfortunately, no well-established tool for identifying PPROM patients threatened by these disorders is available. Methodology/Principal Findings We performed an unbiased exploratory analysis of amniotic fluid proteome changes due to MIAC and HCA. From among the top five proteins that showed the most profound and significant change, we sought to confirm results concerning cathelicidin (P49913, CAMP_HUMAN), since an ELISA kit was readily available for this protein. In our exploratory proteomic study, cathelicidin showed a ∼6-fold higher concentration in PPROM patients with confirmed MIAC and HCA. We verified significantly higher levels of cathelicidin in exploratory samples (women without both MIAC and HCA: median 1.4 ng/ml; women with both conditions confirmed: median 3.6 ng/ml; p = 0.0003). A prospective replication cohort was used for independent validation and for assessment of cathelicidin potential to stratify women with MIAC leading to HCA from women in whom at least one of these conditions was ruled out. We confirmed the association of higher amniotic fluid cathelicidin levels with MIAC leading to HCA (the presence of both MIAC and HCA: median 3.1 ng/ml; other women: median 1.4 ng/ml; p<0.0001). A cathelicidin concentration of 4.0 ng/ml was found to be the best cut-off point for identifying PPROM women with both MIAC and HCA. When tested on the validation cohort, a sensitivity of 48%, a specificity of 90%, a likelihood ratio of 5.0, and an area under receiver-operating characteristic curve of 71% were achieved for identification of women with MIAC leading to HCA. Conclusions Our multi-stage study suggests cathelicidin as a candidate marker that should be considered for a panel of amniotic fluid proteins permitting identification of PPROM women with MIAC leading to HCA.

iTRAQ quantitative analysis of Francisella tularensis ssp. holarctica live vaccine strain and Francisella tularensis ssp. tularensis SCHU S4 response to different temperatures and stationary phases of growth

Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.

Proteomic Biomarkers for Spontaneous Preterm Birth A Systematic Review of the Literature

This review aimed to identify, synthesize, and analyze the findings of studies on proteomic biomarkers for spontaneous preterm birth (PTB). Three electronic databases (Medline, Embase, and Scopus) were searched for studies in any language reporting the use of proteomic biomarkers for PTB published between January 1994 and December 2012. Retrieved citations were screened, and relevant studies were selected for full-text reading, in triplicate. The search yielded 529 citations, 51 were selected for full-text reading and 8 studies were included in the review. A total of 64 dysregulated proteins were reported. Only 14-3-3 protein sigma, annexin A5, protein S100-A8, protein S100-A12, and inter-α-trypsin inhibitor heavy chain H4 were reported in more than 1 study, but results could not be combined due to heterogeneity in type of sample and analytical platform. In conclusion, according to the existing literature, there are no specific proteomic biomarkers capable of accurately predicting PTB.

cGMP-phosphodiesterase 6, transducin and Wnt5a/Frizzled-2-signaling control cGMP and Ca(2+) homeostasis in melanoma cells.

Malignant melanoma is one of the most aggressive human neoplasms which develop from the malignant transformation of normal epithelial melanocytes and share the lineage with retinal cells. cGMP-phosphodiesterase 6 (PDE6) is one of the cancer-retina antigens newly identified in melanoma cells. Normally, PDE6 hydrolyzes the photoreceptor second messenger cGMP allowing the visual signal transduction in photoreceptor cells. cGMP also play an important signaling role in stimulating melanogenesis in human melanocytes. Here, we present evidence that PDE6 is a key enzyme regulating the cGMP metabolism in melanoma cells. Decrease in intracellular cGMP leads to calcium accumulation in melanoma cells. In these cells, cGMP-phosphodiesterase 6 can be activated by another cancer-retina antigen, transducin, through Wnt5a–Frizzled-2 cascade, which leads to a lowering of cGMP and an increase in intracellular calcium mobilization. Thus, the aberrant expression of PDE6 may control cGMP metabolism and calcium homeostasis in melanoma cells.

Amniotic fluid soluble Toll-like receptor 4 in pregnancies complicated by preterm prelabor rupture of the membranes.

Objective: To determine amniotic fluid soluble Toll-like receptor 4 (sTLR4) levels in women with preterm prelabor rupture of the membranes according to the presence of microbial invasion of the amniotic cavity and histological chorioamnionitis and its relation to neonatal outcome. Methods: One hundred two women with singleton pregnancies with a gestational age between 24 + 0 and 36 + 6 weeks were included in a prospective cohort study. Amniocenteses were performed, and the concentrations of sTLR4 in the amniotic fluid were determined using sandwich enzyme-linked immunosorbent assay technique. Results: Women with the presence of microbial invasion of the amniotic cavity had higher sTLR4 levels [median 54.2 ng/mL, interquartile range (IQR) 10.15–289.9] than those without this condition (median 18.1 ng/mL, IQR 8.1–29.9; p = 0.001). Women with the presence of histological chorioamnionitis had a higher sTLR4 level (median 28.0 ng/mL, IQR 11.15–178.1) compared with women without histological chorioamnionitis (median 13.0 ng/mL, IQR 7.8–28.7; p = 0.003). A mixed linear model was used to adjust for confounders. The difference was found only between women with and without microbial invasion of the amniotic cavity in this model. Conclusions: Microbial invasion of the amniotic cavity was associated with higher amniotic fluid sTLR4 levels independent of confounders.

Proteomics and bioinformatics analysis reveal underlying pathways of infection associated histologic chorioamnionitis in pPROM.

INTRODUCTION The presence of microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) is associated with adverse neonatal outcomes in pregnancies complicated by preterm prelabor rupture of membranes (pPROM). Therefore, there is an urgent need to identify new biomarkers revealing these conditions. The objective of this study is to identify possible biomarkers and their underlying biofunctions in pPROM pregnancies with and without MIAC and HCA. METHODS A total of 72 women with pPROM were recruited. Only women with both MIAC and HCA (n = 19) and all women without these complications (n = 19) having the same range of gestational ages at sampling were included in the study. Samples of amniotic fluid were obtained by transabdominal amniocentesis, processed and analyzed using quantitative shotgun proteomics. Ingenuity pathway analysis was used to identify molecular networks that involve altered proteins. RESULTS Network interaction identified by ingenuity pathway analysis revealed immunological disease and the inflammatory response as the top functions and disease associated with pPROM in the presence of MIAC and HCA. The proteins involved in these pathways were significantly altered between the groups with and without the presence of both MIAC and HCA. Proteins involved included histones H3, H4, H2B, cathelicidin antimicrobial peptide, myeloperoxidase, neutrophil gelatinase-associated lipocalin, matrix metalloproteinase-9, peptidoglycan recognition protein-1 and neutrophil defensin 1, all of which were found to be up-regulated in the presence of MIAC and HCA. CONCLUSION Bioinformatic analysis of proteomics data allowed us to project likely biomolecular pathology resulting in pPROM complicated by MIAC and HCA. As inflammation is not a homogeneous phenomenon, we provide evidence for oxidative-stress-associated DNA damage and biomarkers of reactive oxygen species generation as factors associated with inflammation and proteolysis.

Preterm prelabor rupture of membranes (PPROM) is not associated with presence of viral genomes in the amniotic fluid.

BACKGROUND The role of viral infections in preterm prelabor rupture of the membranes (PPROM) is not established. Studies on the presence of viral genomes in the amniotic fluid (AF) collected in pregnancies complicated by PPROM show contradictory outcomes. OBJECTIVES To investigate AF samples of PPROM pregnancies for the presence of viral genomes. STUDY DESIGN AF samples from patients with PPROM were collected during a 4-year (2008-2012) observational study. 174 women were included with selection criteria of singleton pregnancy, PPROM, and maternal age of 18 years and above. PCR was used for detection of human cytomegalovirus (HCMV), herpes simplex virus (HSV), parvovirus B19, human adenoviruses (HAdV), enteroviruses (EV) and human parechovirus (HPeV). The selection of these viral targets was based on literature regarding screening of AF for presence of viral genomes. RESULTS Only a single sample was positive out of the 174 tested AFs, HCMV DNA was detected. CONCLUSIONS PPROM is not associated with active viral infections.