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Academicians and researchers have been keen on climatic and maritime monitoring. Synthetic Aperture Radars (SAR) have been instrumental in capturing images of the ocean. Sea-ice classification using Sentinel-1, a type of SAR, has gained popularity over the years. Automatic image segmentation is a fundamental task in SAR image analysis. This paper proposes a novel, innovative approach to SAR image segmentation by integrating physics-based knowledge into a neural network framework. The implementation of a PDE for SAR image segmentation offers a promising avenue for advancing the field of remote sensing. PDEs provide the required physics for the model. This integration not only enhances the network’s capability to learn meaningful features but also enables it to generate more interpretable and physically meaningful segmentation maps. The Partial Differential Equations (PDE) based Physics-Guided Neural Network (PGNN) model can achieve an accuracy of about 96%, which is greater than all existing state-of-art techniques. By harnessing the synergy between deep learning and physics-based knowledge, this work not only improves segmentation accuracy but also facilitates a deeper understanding of SAR data, paving the way for more reliable and insightful remote sensing applications.

Due to the semi-enclosed nature of the Mediterranean Sea, natural disasters and anthropogenic activities impose stronger pressures on its coastal ecosystems than in any other sea of the world. With the aim of responding adequately to science priorities and societal challenges, littoral waters must be effectively monitored with high-frequency radar (HFR) systems. This land-based remote sensing technology can provide, in near-real time, fine-resolution maps of the surface circulation over broad coastal areas, along with reliable directional wave and wind information. The main goal of this work is to showcase the current status of the Mediterranean HFR network and the future roadmap for orchestrated actions. Ongoing collaborative efforts and recent progress of this regional alliance are not only described but also connected with other European initiatives and global frameworks, highlighting the advantages of this cost-effective instrument for the multi-parameter monitoring of the sea state. Coordinated endeavors between HFR operators from different multi-disciplinary institutions are mandatory to reach a mature stage at both national and regional levels, striving to do the following: (i) harmonize deployment and maintenance practices; (ii) standardize data, metadata, and quality control procedures; (iii) centralize data management, visualization, and access platforms; and (iv) develop practical applications of societal benefit that can be used for strategic planning and informed decision-making in the Mediterranean marine environment. Such fit-for-purpose applications can serve for search and rescue operations, safe vessel navigation, tracking of marine pollutants, the monitoring of extreme events, the investigation of transport processes, and the connectivity between offshore waters and coastal ecosystems. Finally, future prospects within the Mediterranean framework are discussed along with a wealth of socioeconomic, technical, and scientific challenges to be faced during the implementation of this integrated HFR regional network.

The Mediterranean Sea is a prominent climate-change hot spot, with many socioeconomically vital coastal areas being the most vulnerable targets for maritime safety, diverse met-ocean hazards and marine pollution. Providing an unprecedented spatial and temporal resolution at wide coastal areas, high-frequency radars (HFRs) have been steadily gaining recognition as an effective land-based remote sensing technology for continuous monitoring of the surface circulation, increasingly waves and occasionally winds. HFR measurements have boosted the thorough scientific knowledge of coastal processes, also fostering a broad range of applications, which has promoted their integration in coastal ocean observing systems worldwide, with more than half of the European sites located in the Mediterranean coastal areas. In this work, we present a review of existing HFR data multidisciplinary science-based applications in the Mediterranean Sea, primarily focused on meeting end-user and science-driven requirements, addressing regional challenges in three main topics: (i) maritime safety, (ii) extreme hazards and (iii) environmental transport process. Additionally, the HFR observing and monitoring regional capabilities in the Mediterranean coastal areas required to underpin the underlying science and the further development of applications are also analyzed. The outcome of this assessment has allowed us to provide a set of recommendations for future improvement prospects to maximize the contribution to extending science-based HFR products into societally relevant downstream services to support blue growth in the Mediterranean coastal areas, helping to meet the UN's Decade of Ocean Science for Sustainable Development and the EU's Green Deal goals.

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Phylogenetic inference and molecular taxonomy are becoming increasingly important approaches to classical morphological systematics and marine ecology. The number of molecular markers suitable for such goals is quite high, but general use restricts the list to a few of them, mainly mitochondrial (namely cytochrome c oxidase subunit I, COI and Cytochrome b), especially in copepods. The ribosomal cistronic regions have been widely used for broad phylogenetic analyses in different taxa. Among them, the internal transcribed spacers (ITS rDNA) are powerful tools for phylogenetic reconstructions at the different taxonomic levels, although not yet extensively used for copepods. In the present work, we tested the suitability of ITS2 rDNA marker to reconstruct the phylogenetic relationships of calanoid copepods using sequences retrieved from GenBank, complementing the phylogenetic positions of the species studied with their morphological and ecological traits. Through ITS2 rDNA we provided the first molecular evidence for the invasive calanoid Pseudodiaptomus marinus from the Mediterranean Sea (Lake Faro, Sicily, Italy), and compared it with the GenBank ITS2 sequences for P. marinus from Korea and other calanoid species. The divergence of the sequences of our P. marinus from those of Korean specimens was quite prominent (4.4%) and allowed us to hypothesise either a new forma living in the Mediterranean or a cryptic species. This study highlights the appropriateness of ITS2 for phylogenetic reconstructions and species identification, as well as for barcoding, meta-barcoding and phylogeographic approaches, and evidences the need for a more thorough knowledge of ribosomal regions in copepods from different sites.

Standards for data generation and collection are important for integration and for achieving data-driven actionable insights in dairy farming. Data integration and analysis are critical for advancing the dairy industry, enabling better decision-making, and improving operational efficiencies. This commentary paper discusses the challenges of and proposes pathways for standardizing data generation and collection based on insights from a multidisciplinary group of stakeholders. Drawing from a series of meetings of industry experts, academics, and farmers organized under the Dairy Brain Project's Coordinated Innovation Network (CIN), we explore the benefits of creating uniform data generation and collection protocols to ensure compatibility and reliability across different data sources. Key insights include the importance of defining standardization at both farm and industry levels, the role of education and incentives, and the potential for using existing frameworks such as the International Committee for Animal Recording. Additionally, we highlight industry-specific case studies, including successful examples from Brazil such as GERAR, which focuses on reproductive performance data, and Labor Rural, which integrates data from multiple farms to provide valuable insights to farmers and milk processors. The paper concludes with recommendations for implementing these protocols and highlights the need to foster collaboration among stakeholders for the successful implementation and adoption of standardized data generation and collection protocols in the dairy industry.

Endometriosis is a chronic inflammatory gynaecological disease characterized by the growth of endometrial tissue outside the uterine cavity. There are currently no definitive non-invasive diagnostic tools. Glycosylation is the most common posttranslational modification of proteins and altered glycosylation has been found in many diseases, including chronic inflammatory conditions and cancer. Sialylation and galactosylation on serum IgG have previously been found to be altered in endometriosis and serum sialylation changed after Zoladex (Goserelin Acetate) therapy. Using IgG and whole serum glycoproteins, we investigated N-glycosylation in two clinical cohorts of women with and without endometriosis. PNGase F-digested serum samples were fluorescently labelled and N-glycans were profiled by ultra-performance liquid chromatography. Clinical data was collected to link glycomic findings with metabolic and hormonal profiles. Total serum glycoprotein and IgG glycosylation differed in patients with endometriosis compared to control cases. The most significantly altered was glycan peak 3 from IgG, containing bisected biantennary glycans, which was decreased in the endometriosis cohorts (p = 0.0000005-0.018). In conclusion, this is the first pilot study to identify changes in N-glycans from whole serum glycoproteins associated with endometriosis. A larger validation study is now warranted and such studies should include the follow-up of surgically and pharmacologically treated patients., (© 2023. The Author(s).)

The aerial parts of Anthemis tinctoria L. and Angelica sylvestris L. and the roots of A. sylvestris have been used as traditional anticancer remedies in Estonian ethnomedicine. The aim of this study was to investigate content of essential oils (by gas chromatography) and polyphenolic compounds (using two different methods of high performance liquid chromatography-mass spectrometry (HPLC-MS)) of both plant species, as well as the in vitro anti-cancer effects of their essential oils and methanolic extracts. The average ( n = 5 samples) yield of essential oils was 0.15%, 0.13%, and 0.17%, respectively. The principal compounds of the essential oil from the aerial parts of A. tinctoria were palmitic acid (15.3%), p -cymene (12.6%), and α -muurolene (12.5%), and α -pinene (45.4%), p -cymene (15.5%), and β -myrcene (13.3%) in aerial parts of A. sylvestris , while isocaryophyllene oxide (31.9%), α -bisabolol (17.5%), and α -pinene (12.4%) were the main constituents in the roots. The most abundant phenolic compounds in aerial parts were the derivatives of caffeic acid, quinic acid, and quercetin; the main compounds in roots of A. sylvestris were chlorogenic acid, quinic acid, and naringenin. The strongest anticancer effects were observed in essential oils of A. sylvestris roots and aerial parts on human carcinoma in the mouth cells (KB, IC 50 19.73 μg/mL and 19.84 μg/mL, respectively). The essential oil of A. tinctoria showed a strong effect on KB and LNCaP cells (27.75-29.96 μg/mL). The methanolic extracts of both plants had no effect on the cancer cells studied.

The primary objective was to determine if Angus bull fertility varied by number of sperm inseminated. A secondary objective was to characterize the potential impact of random variation on fertility using two identical sperm per dose treatments, which differed only in straw color. Computer-assisted sperm analysis (CASA) and flow cytometry (FC) were used to identify post-thaw sperm characteristics associated with field fertility differences between bulls. Ejaculates from five Angus bulls were collected, extended, and cryopreserved at 10, 20, 20 or 40 × 10 6 sperm per dose in color-coded 0.5-mL French straws. Multiparous cows (n = 4866) from ten Brazilian farms were synchronized for first-service timed artificial insemination (TAI). Bull identification and straw color were recorded at TAI. Pregnancy per TAI (P/TAI) did not differ between sperm doses (43.8, 45.3, 43.8 and 47.1% for 10, 20, 20 or 40 × 10 6 sperm respectively; P = 0.31) nor was there an interaction between bull and dose (P = 0.53). The P/TAI differed between bulls and ranged from 40.7 to 48.1% (P < 0.01). The overall P/TAI between the two control groups were not different (45.3 vs 43.8%); however, the numerical variation within bull ranged from 0.5 to 4.9 percentage points. Numerous CASA and FC post-thaw sperm characteristics differed among bulls (P < 0.05), but these characteristics did not explain the fertility difference between bulls. Principal component analysis provided a multivariate description of the CASA and FC data, where three principal components (Prin1, Prin2, and Prin3) accounted for a combined total of 88.7% of the data variability. The primary components of each PCA axis were flow cytometric measures of sperm viability and DNA fragmentation (Prin1), and CASA-derived sperm movement patterns (Prin2) and motility (Prin3); however, the relative influence of these characteristics varied by bull. Although fertility differences between bulls were detected, neither sperm per dose nor post-thaw in vitro sperm analyses (CASA and FC) were able to explain the observed differences in field fertility between bulls, further illustrating the difficulties in predicting bull fertility., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Coronary artery disease contributes to noncommunicable disease deaths worldwide. In order to make preventive methods more accurate, we need to know more about the development and progress of this pathology, including the genetic aspects. Humanin is a small peptide known for its cytoprotective and anti-apoptotic properties. Our study looked for genomic associations between humanin-like nuclear isoform genes and coronary artery disease using CARDIoGRAMplusC4D Consortium data., Results: Lookup from meta-analysis datasets gave single nucleotide polymorphisms in all 13 humanin-like nuclear isoform genes with the lowest P value for rs6151662 from the MTRNR2L2 gene including the 50 kb flanking region in both directions (P-value = 0.0037). Within the gene region alone the top variant was rs78083998 from the MTRNR2L13 region (meta-analysis P-value = 0.042). None of the found associations were statistically significant after correction for multiple testing. Lookup for expression trait loci in these gene regions gave no statistically significant variants.

Appearance of autoantibodies represents the first detectable sign of autoimmune destruction of beta cells in type 1 diabetes (T1D). In addition, autoantibody levels represent an important predictive marker regarding the development of an autoimmune process. Recently, the zinc transporter (ZnT8) protein was identified as an autoimmune target in T1D; therefore, there is a need for reliable and simple methods for detection of ZnT8 autoantibodies. This report describes an assay designed to detect anti-ZnT8 autoantibodies in the serum of patients with T1D. This was carried out by generating a ZnT8 C-terminal dimer fused to the N-terminus of the Gaussia princeps luciferase that was overexpressed in insect cells using the baculovirus expression system. Recombinant protein was semi-purified and used as the target antigen in the liquid-phase luciferase immunoprecipitation system assay (LIPS), and results were compared to data obtained using a commercial ELISA designed to detect ZnT8 autoantibodies in T1D patient sera, particularly among adults. LIPS was less effective in detecting antibodies in children probably due to the relatively high prevalence of IgM anti-ZnT8 antibodies in children with T1D., (Copyright © 2014. Published by Elsevier B.V.)

The intestinal microbiota is essential to the maturation and homeostasis of the immune system. Immunoblot assays were used to establish the prevalence of serum IgG, IgM, and IgA antibodies specific for Bifidobacterium adolescentis, Bifidobacterium longum, and Lactobacillus rhamnosus GG proteins in young children presenting with or without type 1 diabetes (T1D). We demonstrated that children between the ages of 6 and 12 months had a substantial increase in the frequency of IgG antibodies specific for L. rhamnosus GG proteins. We measured IgG, IgM, and IgA class antibody reactivity against B. adolescentis DSM 20083, B. adolescentis DSM 20086, and B. longum DSM 20088 proteins demonstrating significantly higher IgA responses against B. adolescentis DSM 20083 strain proteins in children who developed islet autoimmunity and T1D later in life. B. adolescentis strains showed more IgM type antibodies in children who developed T1D later in life, but the difference was not statistically significant. B. longum proteins were recognized by IgG and IgA antibodies to a higher extent compared to other bacteria studied. These results confirm that differences in immune reactivity against some commensal strains in young children may represent a different risk factor for developing T1D.

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin-binding regions of human TG2 by studying binding kinetics of the predicted heparin-binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high-affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202-215) and P2 (261-274), were found to bind heparin. The amino acid sequences corresponding to the heparin-binding peptides were located close to each other on the surface of the TG2 molecule as part of the α-helical structures. The heparin-binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco-2 intestinal epithelial cell attachment to the FN and FN-TG2 coated surfaces. We propose that TG2 amino acid sequences 202-215 and 261-274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin-binding peptides of TG2 with CD patient's IgA supports the previously described role of anti-TG2 autoantibodies interfering with this interaction., (Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.)

Autoantibodies from patients with celiac disease (CD) can influence transglutaminase 2 (TG2) activity and its cellular functions, but the exact mechanisms have remained unknown. Our objective was to study whether autoantibodies could modulate TG2 binding to heparin/heparan sulfate (HS) and intestinal epithelial cell attachment to fibronectin-TG2 matrix. Anti-TG2 antibodies were purified by TG2 affinity chromatography from sera of patients with active CD. Serum and antibody effects on TG2 binding to heparin/HS, on transamidase activity of TG2, as well as on Caco-2 cell attachment to fibronectin-TG2 matrix were assessed using microplate assays. Both sera and purified anti-TG2 antibodies from CD patients with high anti-TG2 IgA levels reduced TG2 binding to heparin/HS as compared with those with low anti-TG2 IgA or controls. There was a negative correlation between anti-TG2 IgA levels and TG2 binding to heparin/HS. Treatment of fibronectin-TG2 coated wells with CD patients' sera or purified anti-TG2 antibodies reduced attachment of Caco-2 cells onto the plate as compared with the control samples. The effect of CD patients' antibodies on Caco-2 cell attachment to fibronectin-TG2 matrix occurred independently of the inhibition of cell adhesion by Arg-Gly-Asp sequence containing peptides. Anti-TG2 autoantibodies had no effect on transamidase activity of TG2 in vitro. We suggest that modulation of adhesion function of TG2 by autoantibodies from patients with CD could be related to the inhibition of TG2 binding to HS residues of cell surface proteoglycans and could have possible implications for CD pathogenesis.

Two common chronic childhood diseases-celiac disease (CD) and type 1 diabetes (T1D)-result from complex pathological mechanisms where genetic susceptibility, environmental exposure, alterations in intestinal permeability and immune responses play central roles. In this study, we investigated whether these characteristics were universal for CD independently of T1D association. For this purpose, we studied 36 children with normal small-bowel mucosa and 26 children with active CD, including 12 patients with T1D. In samples from the small-bowel mucosa, we detected the lowest expression of tight junction protein 1 (TJP1) mRNA in CD patients with T1D, indicating an increase in intestinal permeability. Furthermore, these samples displayed the highest expression of forkhead box P3 (FoxP3) mRNA, a marker for regulatory T cells, as compared with other patient groups. At the same time, serum levels of IgA antibodies specific for the CD-related antigens deamidated gliadin and tissue transglutaminase (tTG) were the highest in CD patients with T1D. In contrast, no significant differences were found in IgA or IgG antibodies specific for bovine beta-lactoglobulin or Bifidobacterium adolescentis DSM 20083-derived proteins. There were also no differences in the transamidating activity of serum autoantibodies between patients and control individuals. Our results show that patients with T1D and newly detected CD exhibit severely altered intestinal permeability, strong local immune activation and increased immunoregulatory mechanisms in the small bowel. Further study is required to determine whether these extreme changes in this CD subgroup are due to some specific environmental factors (virus infections), unknown genetic effects or autoimmune reactions to antigenic targets in intracellular tight junctions.

Immune responses to lactobacilli have been so far insufficiently investigated in patients with autoimmune diseases. We used whole-cell lysate of an indigenous Lactobacillus acidophilus strain isolated from an Estonian child to study serum IgG antibodies in children groups with type 1 diabetes [insulin dependent diabetes mellitus (IDDM)] (n = 21, age 4-18 yr) and with acute coeliac disease (CD) (n = 20, age 0.6-15 yr) and to compare the results with the controls (n = 24, age 2-17 yr). We found that our developed 1-D immunoblot assay readily enables to reveal antibodies against 28 L. acidophilus antigenic proteins in patients' and controls' sera. As verified by immunoproteomics analysis with 2-D and LC ESI-MS/MS the antigens of L. acidophilus were mainly common cytoplasmic proteins GroEL (HSP60), enolase, transcription factor EF-Ts and EF-Tu. However, in addition we identified formyl-CoA transferase being target for antibodies in every tested IDDM patients' serum. We have characterized for the first time the antigenic profile of L. acidophilus whole-cell lysate using sera from children with IDDM, CD, and controls. The different prevalence of reactions against tested antigens in patients and controls sera may indicate significant differences in immune system and commensal bacteria cross-talk in these groups.

Problem: Female infertility patients with diverse etiologies show increased production of autoantibodies., Method of Study: Immunoblot analysis of sera from patients with endometriosis and tubal factor infertility (TFI) and mass spectrometry identification of candidate antigens., Results: The immunoblot results demonstrated the presence of IgA and IgG anti-endometrial antibodies (AEA) to various antigens at molecular weights ranging from 10 to 200 kDa. Differences were detected in certain AEA reactions between the patients' groups and particular AEA were associated with in vitro fertilization (IVF) implantation failure. IgA AEA to a 47-kDa protein were more prevalent in TFI patients and were associated with unsuccessful IVF treatment. This antigen was subsequently identified as alpha-enolase., Conclusion: Determination of the presence and spectra of AEA in patients with endometriosis and TFI undergoing IVF may be a useful marker to predict their pregnancy outcome.

Background: Serum IgA antibodies against tissue transglutaminase (tTG) are reliable markers for celiac disease (CD), still the diagnostic performance of anti-tTG immunoassays can be improved. A novel ELISA, using fibronectin (FN) as tTG binding protein, was evaluated for the detection of anti-tTG antibodies in CD., Methods: Sera from 173 children with untreated CD and 97 controls were analyzed for IgA, IgG, and IgM anti-tTG antibodies with ELISA using human recombinant tTG with or without FN., Results: The areas under the ROC (receiver operating characteristic) curves were significantly higher for FN/tTG ELISA compared to tTG ELISA for IgG (0.930 versus 0.809; p < 0.001) and IgM (0.850 versus 0.811; p = 0.019), but not for IgA (0.977 versus 0.970; p = 0.356), respectively. At the fixed diagnostic specificity (100% for IgA and IgM, 99% for IgG), the sensitivity of all three FN/tTG ELISA (92.5% for IgA, 60.7% for IgG, 50.3% for IgM) exceeded those obtained in tTG ELISA (89.0% for IgA, 48.6% for IgG, 38.7% for IgM; p < 0.05). The combined use of IgA- and IgG-FN/tTG ELISA resulted in 95.4% sensitivity and 99.0% specificity for CD., Conclusions: Using FN to bind tTG improves the diagnostic performance of solid phase anti-tTG antibody assays for childhood CD.

Background: The prevalence of Helicobacter pylori infection is inversely associated with socioeconomic conditions in childhood. In Estonia, a high prevalence of H. pylori infection has been observed among children born in 1987 and earlier. Since 1991, after the dissolution of the USSR, profound social and economic changes have taken place in the country. The aim of the study was to evaluate changes in the seroprevalence of H. pylori infection among children in the period 1991-2002., Materials and Methods: The hospital-based study population consisted of two groups of children enrolled in 1991 (n = 425) and 2002 (n = 296) according to the same inclusion criteria. The immunoglobulin G antibodies to the cell surface proteins of H. pylori were determined by enzyme-linked immunosorbent assay, and the sera with the borderline results were analyzed by immunoblot analysis. Multiple regression analysis was used to determine the associations between H. pylori seropositivity and different variables such as demographic characteristics, diagnoses and year of enrollment., Results: The only two variables linked independently to H. pylori serostatus were age and year of enrollment: the adjusted odds of being H. pylori seropositive were 1.92 [95% confidence interval (CI) 1.33-2.76] times higher for the children enrolled in 1991 compared with the children enrolled in 2002. The age-standardized seroprevalence rate was 42.2% (95% CI 37.4-47.0%) for the group of 1991 and 28.1% (95% CI 23.1-33.6%) for the group of 2002., Conclusion: The prevalence of H. pylori infection among children has significantly decreased during the 11-year period of profound socioeconomic changes in Estonia.

Serological tests for Lyme disease are mostly not well standardized and cases of misinterpretation of test results by clinicians are rather common. The diagnostic value of serologic tests may also depend on the seroepidemiological situation of the population. The aim of the study was to compare the immunoblot pattern of Lyme borreliosis patients and control sera from endemic and non-endemic regions and to identify the most suitable interpretation criteria for our immunoblot test. Serum samples of 24 Estonian patients with Lyme disease, 12 sera from patients with tick-borne encephalitis, 40 Estonian control sera, and sera from 50 Laplanders from North Sweden where people usually never come into contact with ticks were tested for IgG antibodies to Borrelia. Sonicated lysate of Borrelia afzelii (strain ACA1) was used in immunoblot as source of antigens. In our test system the following interpretation criteria gave the specificity of 96% for Estonian population: > or = 1 band from p58, p21, p17 and p14 plus > or = 2 bands from p83/100, p39, p34, p30 and p25; or > or = 4 bands from p83/100, p39, p34, p30 and p25. The comparison of Estonian controls with Laplanders showed that subclinical infections with Borrelia are rather common in Estonia. Also the rate of other infections, giving rise to cross-reactive antibodies, may be more frequent in Estonians. The frequent reactions with Borrelia antigens in a healthy population complicate the serodiagnosis of Lyme disease.

The ecological niches occupied by various species of Helicobacter are not yet known and the full spectrum of diseases associated with Helicobacter infections are not yet defined. Since these fastidious microaerofilic bacteria require special growth conditions new and improved molecular and serologic diagnostic methods have been developed to increase our understanding of their pathogenesis and virulence characteristics. Immunogenic cell surface proteins of Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus were characterised by proteomic techniques using two-dimensional electrophoresis and immunoblotting with antisera from immunised rabbits. Cross-reactivity between the three Helicobacter species were analysed after a four-step cross-absorption experiment. For H. pullorum, H. bilis and H. hepaticus 21, 13 and 27 specific immunogenic proteins, respectively, were identified. These proteins could be of important sero-diagnostic value for analyses of sera from humans, laboratory animals and for the veterinarian field.

The analysis of Helicobacter pylori proteins is a demanding task for the elucidation of virulence factors, antigens and vaccines, all important for diagnosis, therapy and protection. In the "pre-genomic era" the purification of proteins was mostly performed by using various techniques such as detergent treatment of the bacterial cells, ultra-centrifugation, various chromatographic methods, antibody detection, N-terminal sequence determination and finally cloning and identification of the corresponding gene. In this review, the most representative methods used for purification, separation and identification of H. pylori proteins will be presented as well as some important developments in the "post-genomic era" that have improved the performance of these characterisation techniques.

Cell surface proteins of the human gastric pathogen Helicobacter pylori, reference strain CCUG 17874, were extracted with acid glycine and fractionated by heparin affinity chromatography. The extracts were subsequently analysed using high-resolution two-dimensional gel electrophoresis (2-DE) and immunoblotting. Four proteins of low molecular masses (25-30 kDa) stained by Coomassie R-350, were identified by peptide ESI-MS/MS sequencing after in-gel tryptic digestion. The identified proteins were recognised by sera from H. pylori-infected patients. Two of them are now described for the first time as immunogenic proteins of which one protein was determined to be distinct from all H. pylori proteins previously described. In addition, the specificity of the identified peptides was evaluated using both 1-D and 2-D immunoblotting against a panel of sera from patients with various bacterial infections. The present identification of highly specific antigens of H. pylori will encourage the improvement of serological diagnostic tests to diagnose and monitor H. pylori infection.

A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around 17 kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand factor and, weakly, fibrinogen. The N-terminal sequences of the 17-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins.

The Helicobacter pylori vacuolating cytotoxin or VacA toxin is a major virulence factor in H. pylori infection and type B gastritis. We predicted heparin/heparan sulfate (H/HS) binding properties of the 58-kDa subunit of VacA cytotoxin using bioinformatics tools and showed this by surface plasmon resonance (SPR)-based biosensor studies. Putative H/HS binding peptides were synthesized and binding to HS was shown by SPR in the absence or presence of trifluoroethanol. We found that a recombinant cytotoxin VacA polypeptide binds to surface-immobilized HS and propose that HS might be a receptor/co-receptor for H. pylori VacA cytotoxin.

Helicobacter pylori is a cause of chronic gastritis and leads to development of atrophy in some cases. There is evidence that the heat shock protein 60 (HSP60) of H. pylori is involved in induction of chronic inflammation. Seroprevalence of IgG antibodies to H. pylori HSP60 in an adult cohort from Saaremaa, Estonia (68 persons, median age 57 years), with a high prevalence of antibodies to cell surface proteins of H. pylori (92%) and a well characterized dynamics of chronic gastritis in an 18-year follow-up study, was tested using purified H. pylori HSP60 at a concentration of 1 microg ml(-1) with ELISA. The state of the gastric mucosa and the presence of H. pylori in histological sections in the samples of 1979 and 1997 were assessed in accordance with the Sydney system. Seropositivity for H. pylori HSP60 was 65%. Immunological response to H. pylori HSP60 is associated with the morphological presence of H. pylori in the antrum and corpus (P=0.01) and is strongly correlated with the grade of chronic inflammation, particularly in the antrum mucosa (r=0.34; P=0.003; OR=5.97 (95% CI 1.21-29.3)), but is not associated with development of atrophy during 18 years of follow-up, or with the activity of gastritis. This finding supports the evidence that immunological response to H. pylori HSP60 may play a role in triggering of the inflammatory process in the gastric mucosa.

Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one- and two-dimensional gelelectrophoresis (1-DE and 2-DE) and by 2-DE immunoblot. Broth-cultured H. pylori cells were stained with an acridine-orange dye to monitor the morphological status of the organism. In 2-day-cultures, 96% of the bacterial cells were spiral-shaped and four days later a morphological switch to coccoid forms occurred. In 10-day cultures spiral-shaped forms were not found. By 1-DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2-day cultures that disappeared in cells of 12-day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB-subunit, were identified in extracted proteins of 2-, 8-, and 12-day cultures. 2-DE revealed an increased number of silver-stained spots of 8-day cultures (in average 250 spots) compared with protein extracted from 2-day cells (in average 160 spots). 2-DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A- and B-subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2-, 8-, and 12-day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral-shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2-DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.

Biomaterial implants in various human body tissues are highly susceptible to bacterial colonization. We report here on the coating of heparinized biomaterials with heparin binding extracellular matrix proteins giving special regard to the efficient adsorption and slow release of antibiotics. Heparin was partially degraded and the resulting fragments were covalently end-point attached to 0.5 cm long silicone biomaterial surface. Collagen type I was immobilized on the heparinized biomaterials and then cross-linked with acyl-azide or carbodiimide. Finally, the resulting biosurfaces were exposed to antibiotics, i.e. rifampicin in combination with cefuroxime, fusidic acid, ofloxacin or vancomycin, respectively. The antibiotic bonded biomaterials were evaluated for their anti-staphylococcal activity after elution in NaCl, serum or blood by measuring the zones of inhibition for S. epidermidis strain RP12. Furthermore, we examined the in-vitro colonization resistance to S. epidermidis RP12 for these combinations of rifampicin-bonded biomaterials by an ATP bioluminescence assay. The ATP measurements showed that initially adherent bacteria were eradicated from the polymer surface, for at least 24 or 48 h (fusidic acid > cefuroxime > vancomycin > ofloxacin). The anti-staphylococcal activity of rifampicin-fusidic acid bonded heparinized biomaterials seems of sufficient duration and efficacy to merit testing in an animal model.

Heparan sulphate binding to cells of the gastric pathogen Helicobacter pylori at pH 4-6 is common. Binding was inhibited by various unlabelled sulphated polysaccharides and at high ionic strength and pH, but not by carboxylated or non-sulphated compounds. The inhibition by various sulphated compounds such as dextran sulphate and carrageenans was related to the sulphate content and not to the carbohydrate polymer backbone. The IC50 values for heparin and dextran sulphate for H. pylori strain 25 were calculated as 3.55 x 10(-7) M and 5.01 x 10(-6) M respectively. Heparin-binding proteins of H. pylori are exposed on the cell surface, as shown by biotinylation of cell-surface proteins before separation of outer membranes and by an indirect immunofluorescence assay. The strongest biotin-heparin binding by H. pylori was observed with a polypeptide in the 55-60 kDa region.

Helicobacter pylori adhere to Kato III and Hela S3 cells in monolayer cultures. To explore whether cell surface glycoconjugates on these two cell lines mediate binding of H. pylori, various carbohydrates, glycoproteins, and glycolipids were tested to inhibit H.pylori cell adhesion. The adhesion was measured (i) with a urease-based assay and (ii) by cells stained with fluorescein. Sodium periodate and sialidase treatment (but not alpha- or beta-galactosidase, heparitinase,lysozyme, or trypsin) inhibited H. pylori binding to both cell lines. Sulfatides and sulfated glycoconjugates (50 microg/ml) but not heparin or a number of simple carbohydrates inhibited binding (1 mg/ml). The two H.pylori strains studied (CCUG 17874 and strain 25) showed high binding of soluble 125I-labeled heparin and other sulfated carbohydrate compounds.

Heparan sulphate binding to Helicobacter pylori at pH 4 to 5 was inhibited with various sulphated polysaccharides (heparin and chondroitin sulphates, fucoidan, carrageenans and some others), but not by carboxylated or nonsulphated compounds. Heparin binding proteins are exposed on the cell surface.