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The quest for new and enhanced vaccines requires improved means for identification of relevant antigens and the epitopes present within these. While CD8+ T cells recognize antigenic peptides presented in the context of MHC Class I, and play a key role in cellular immunity, CD4+ helper T-cells recognize antigens in the context of MHC Class II and assist other immune cells in orchestration of the defined immune response. Being a functional assay, ELISPOT can be used to define both MHC Class I and II epitopes as well as to validate them. Providing the quality of the cells used in the assay is sufficiently good, ELISPOT provides a solid platform for both screening and validating T-cell epitopes. While some ELISPOT assays rely upon loading of dendritic cells with antigen followed by incubation of HLA matched or autologous PBMC or enriched T cells, the assay presented here uses PBMC from diseased or vaccinated individuals to simultaneously identify and validate T-cell epitopes.

CD8+ T cells recognize antigenic peptides presented in the context of MHC class I. They play a key role in cellular immunity and are crucial for longterm protective immunity to many infectious diseases. The quest for new and enhanced vaccines requires improved means for identification of relevant antigens and the epitopes present within these. While there are several algorithms available for epitope prediction (all of which work to differing degrees of success), the definition of actual MHC class I-binding epitopes is very reliant on time-consuming and difficult to perform functional assays using often very limited biological material. The iTOPIA assay is quick and easy to perform and determines real-binding to MHC class I molecules. It provides an excellent platform for screening and elimination of potential epitopes and identification of novel epitopes prior to validation with a relevant functional assay.

In The Netherlands it has been shown that people in contact with pigs have a higher risk of meticillin-resistant Staphylococcus aureus (MRSA) carriage than the general population. Isolates of closely related spa types, corresponding to multilocus sequence type (MLST) ST398, were found in pig farmers, pig veterinarians and pigs. The objective of this study was to investigate whether contact with pigs and veal calves or other livestock is a risk factor for MRSA carriage in Dutch healthcare workers (HCWs). HCWs at four general hospitals and one university hospital were asked to fill in questionnaires covering contact with animals and to take MRSA cultures of their throat and nares. Cultures of HCWs in contact with livestock were processed with samples from HCWs with no contact with livestock as controls. Seventy-seven of 1721 HCWs (4.4%) reported direct or indirect contact with pigs and/or veal calves and 145 reported contact with other livestock animals. The MRSA carriage rate in the group in contact with pigs and veal calves was 1.7% and in the control group was 0.15%. No carriers were found among HCWs in contact with other livestock. An estimated 3% of hospital staff working in Dutch hospitals serving rural populations belong to a high risk group for MRSA carriage according to the Dutch guidelines. Although MRSA carriage in HCWs in contact with livestock is 10-fold higher than in other HCWs, the difference is not statistically significant.

The physico-chemical properties of three fully hydrated monoacyl maltoside glycolipids were investigated with Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS). The different synthesized maltoside glycoconjugates vary in the length and saturation of the fatty acid moiety, whereas the constant head group region contains a beta-linked maltose with a OC(2)-NH spacer. The compounds with saturated acyl chains showed a complex pattern of temperature-dependent behaviour, regarding the adopted three-dimensional aggregate structure of the molecules and the main phase transition from the gel to liquid crystalline phase of the acyl chains. A substitution of the saturated acyl chain with an unsaturated acyl chain led to a complete change of the structural preferences, from a high ordered stacking of the bilayers to an unilamellar arrangement of completely disordered and fluid membranes. The presence of the NH group in the spacer, compared to the compounds lacking the NH group allows the formation of a hydrogen bonding network, which influences the observed phase properties. The results of these studies of the hydrated monoacylated maltose glycolipids are discussed in relation to the thermotropic phase properties of the pure compounds in the absence of water.

Pig farmers and veterinarians in contact with livestock in The Netherlands have a higher risk of methicillin-resistant Staphylococcus aureus (MRSA) carriage than the general population. The objective of this study was to investigate whether this is also true for other professionals in contact with pigs in an international setting. A convenience sample of 272 participants at an international conference on pig health in Denmark was screened for MRSA carriage using combined nose/throat swabs and were asked to complete a questionnaire concerning animal contacts, exposure to known MRSA risk-factors, and the protective measures taken when entering pig farms. In total, 34 (12.5%) participants from nine countries carried MRSA. Thirty-one of these isolates were non-typeable by pulsed-field gel electrophoresis following SmaI digestion of chromosomal DNA. All of the non-typeable isolates belonged to spa types (t011, t034, t108, t571, t567 and t899) that correspond to multilocus sequence type 398. All of the above-mentioned spa types, with the exception of t899, have been isolated previously from either Dutch pigs, pig farmers and/or veterinarians. Protective measures, e.g., masks, gowns and gloves, did not protect against MRSA acquisition. Transmission of MRSA from pigs to staff tending to these animals appears to be an international problem, creating a new reservoir for community-acquired MRSA (CA-MRSA) in humans in Europe, and possibly worldwide. The rise of a new zoonotic source of MRSA could have a severe impact on the epidemiology of CA-MRSA, and may have consequences for the control of MRSA, especially in those countries that maintain a low prevalence by means of search-and-destroy policies.

The structural preferences of 1,2-dioleoyl-sn-glycerol glycolipids with glucose, galactose, maltose, and cellobiose as sugar head group were investigated under near physiological conditions with Fourier-transform infrared spectroscopy (FT-IR) and synchrotron radiation small-angle X-ray scattering (SAXS). Whereas all glycolipids have a very high fluidity at temperatures above 0 degrees C, the mono- and disaccharide compounds differ considerably in their aggregate structures. The monosaccharide compounds adopt only inverted hexagonal (H(II)) structures in the temperature range 5-70 degrees C, while the disaccharide compounds adopt only multilamellar structures. Since these and similar glycolipids are frequently found in nature, these data should be of relevance for the function of their host cell membranes.

Challenged by scattered understanding of protective immunity to Mycobacterium tuberculosis (MTB), we have mapped peptide epitopes to human leukocyte antigen (HLA)-A*0101, A*0201, A*1101, A*2402, B*0702, B*0801 and B*1501 of the secreted mycobacterial antigen Ag85B, a vaccine candidate that may be associated with immune protection. Affinity (ED(50)) and half-life (t(1/2), off-rate) analysis for individual peptide species on HLA-A and HLA-B molecules revealed binding ranges between 10(-3) and 10(-7) M. After selection of the best matches, major histocompatibility complex class I/peptide tetramer complexes were constructed to measure the CD8+ T-cell responses directly ex vivo in peripheral blood mononuclear cells (PBMC) derived from 57 patients with acute pulmonary tuberculosis. Three patterns of (allele-) specific CD8+ recognition were identified: (a). Focus on one dominant epitope with additional recognition of several subdominant T-cell epitopes (HLA-A*0301, A*2402, B*0801 and B*1501); (b). Co-dominant recognition of two distinct groups of peptides presented by HLA-B*0702; and (c). Diverse and broad recognition of peptides presented by HLA-A*0201. Peptides that bound with slow off-rates to class I alleles, that is HLA-A*0201, were associated with low frequency of CD8+ T cells in PBMCs from patients with tuberculosis. HLA-B alleles showed fast off-rates in peptide binding and restricted high numbers (up to 6%) of antigen-specific CD8+ T cells in patients with pulmonary tuberculosis.

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the Netherlands, at 1.0%, is among the lowest in Europe. In 2004, a relationship between pig farming and a high risk for MRSA carriage was found. To investigate if those in professional contact with livestock are at higher risk for MRSA carriage, we screened 80 veterinary students and 99 veterinarians and questioned them about animal contacts and known MRSA risk factors. Of these, 27 students who did not have livestock contact were excluded from further analysis. We found 7 carriers of MRSA, a prevalence of 4.6%, which is similar to that found in patients who had previously been treated at foreign hospitals. A correlation of MRSA carriage with a specific animal group could not be established. To preserve the low prevalence of MRSA in the Netherlands, persons involved in the care of livestock should be isolated and screened on admission to the hospital.

We conducted a study among a group of 26 regional pig farmers to determine the methicillin-resistant Staphylococcus aureus prevalence rate and found it was >760 times greater than the rate of patients admitted to Dutch hospitals. While spa-type t108 is apparently a more widespread clone among pig farmers and their environment, we did find other spa-types.

A 76-year-old female, presented with a recurrent cough and haemoptysis after an aspiration of a 'grain de Valls'. Chest radiography and computed tomography demonstrated an atelectasis of the medial segment of the right middle pulmonary lobe. Bronchoscopy was negative for any malignancy but the onco-PET-scan showed a pathologic hypermetabolic lesion in the right middle lobe. A lobectomy was performed and pathology revealed a 'destroyed lung' with images of foreign body reactions and microabscesses without any sign of malignancy. Postoperatively, clinical improvement with complete resolution of the symptoms was observed. With this case report we demonstrate the irritative and clastogenic characteristics of phenolphtalein, the main active ingredient of the 'grain de Valls'. This case also illustrates an example of a false positive PET-scan.

The concept of sustainability in forestry has a long tradition in Europe. However, the perception of 'sustainable management of forests' has changed in accordance with the changes in society, which has made growing demands on ecological, economical, and social functions on forests in recent years. Following the so-called 'Helsinki-process', the EU member states have decided on general guidelines for sustainable management and for the conservation of forest biodiversity. Six criteria and 27 descriptive and quantitative indicators have been defined for the assessment and monitoring of sustainable forest management. Criterion no. 4, relating to semi-natural ancient woodland as a potential vector and source of high biodiversity and respective forest policies, provides the central theme for this paper. The paper is focused on the diversity of vascular plants and explores the possibility that isolated recent woodland could contribute to plant species diversity. Measures are suggested for forest management at forest stand and landscape levels which could enhance the diversity of typical forest plants.

Purpose: Evaluation of a new surface treatment method to obtain a good bond strength between a luting composite and (1) a light-cured, (2) a heat-cured and (3) a thermoplastic resin., Materials and Methods: Specimens were prepared and tests conducted according to ISO 10477, Amend. 1. The surfaces of Targis (light cured), SR Isosit (heat cured), and Dental D (thermoplastic) were ground under water cooling with 400-grit grinding paper, polished with 800-grit paper and air dried. Each resin material was divided into 3 groups of 10 specimens each. Group 1 was flame treated with a PyrosilPen for 5 s/2 cm2, group 2 for 10 s/2 cm2, and group 3 for 20 s/2 cm2. Subsequently, a methacryl silane was applied, followed by a luting composite. Prior to measuring shear bond strength, the specimens were thermocycled 5000 times in a water bath between 5 degrees C and 55 degrees C. SEM, FTIR investigations, and fracture analysis were also done. Etched and silanized Empress II - the gold standard - was used as a control., Results: The following shear bond strengths were found: treatment time 5 s/2 cm2, Targis 25 (+/- 12) MPa, SR Isosit 17 (+/- 11) MPa; treatment time 10 s/2 cm2, Targis 23 (+/- 12) MPa, SR Isosit 26 (+/- 8) MPa; treatment time 20 s/2 cm2, Targis 29 (+/- 5) MPa, SR Isosit 26 (+/- 9) MPa. All Dental D specimens failed completely so that shear bond strength could not be measured. The control achieved 27 (+/- 6) MPa. No significant differences were found between the materials or the flaming times. On all flamed surfaces, Si was detected by FTIR. SEM showed that no heat destruction occurred at a flaming time of 5 s/2 cm2, a slight change at 10 s/2 cm2, and a significant change at 20 s/2 cm2., Conclusion: This new bonding technology is an effective method for surface-treating polymerized composite resin materials to obtain good bonding to luting composites. The method fails on thermoplastic resins.

Despite the wide use of extracts from the rhizome of black cohosh (Cimicifuga racemosa) for the treatment of menopausal complaints, surprisingly little is known on their potential estrogenic properties, e.g. on estrogen dependent gene transcription. In addition, available informations on the effects on cell proliferation are contradictory. We therefore, tested for estrogenic and antiestrogenic effects of Cimicifuga racemosa extracts on proliferation of MCF-7 cells and on gene expression using ethanolic and iso-propanolic extracts of this medical plant. Estrogenic properties of plant extracts could neither be detected in proliferation assays, nor on gene expression using an estradiol-inducible yeast assay or the estrogen-inducible MVLN cells. In contrast, in all three experimental systems Cimicifuga racemosa antagonized estradiol induced activities. Estradiol induced stimulation of proliferation was inhibited by a dosage >1 microg/ml of extract concentration, gene expression was suppressed by doses of 100-1000 microg/ml of Cimicifuga racemosa extracts. From these results we conclude, that extracts from the rhizome of Cimicifuga racemosa contain compounds with antiestrogenic properties.

High-energy collisions of Cp*P(6)(+) cations yield neutral P(6) molecules (see reaction), thus providing the first experimental evidence for the existence of this new allotrope of phosphorus.

Tumor growth and metastasis are angiogenesis-dependent processes initiated and regulated by a number of cytokines. Vascular endothelial growth factor (VEGF) is a potent angiogenic protein with a selective mitogenic effect on vascular endothelial cells, known to be involved in physiological (embryogenesis) and pathophysiological (rheumatoid arthritis, tumor) angiogenesis. An increased expression of matrix metalloproteinase type IV collagenase has been reported in invading endothelial cells in vitro and in malignant cells, degrading structures of the basement membranes in various human malignancies. In the present study we investigated the expression of the genes for type IV collagenase and vascular endothelial growth factor (VEGF) in 40 cases of primary non-small-cell lung cancer (NSCLC). Specimens were immunostained by an antibody directed against VEGF and mRNA transcripts of VEGF and type IV collagenase were localized by non-radioactive in situ hybridization. VEGF mRNA was detected in 33 neoplasms, while in 23 cases transcripts of the type IV collagenase gene were visualized by digoxigenin-labeled cDNA probes. Transcripts of both mRNAs were detected in malignant cells. Furthermore, anti-VEGF immunostaining was present in newly formed microvessels close to the atypical cells, and mRNA of type IV collagenase was present in stromal cells adjacent to the tumor. A statistically significant correlation was found between the expression of type IV collagenase and VEGF (P = 0.0061). These data suggest a double role for type IV collagenase in the metastatic process of NSCLC: (1) facilitating the invasion of tumor cells by the proteolytic cleavage of the basement membrane and (2) similarly supporting the endothelial cell invasion essential for tumor angiogenesis. Furthermore, our findings sustain the hypothesis that metastatic spread and angiogenesis are associated with a clonal expansion of highly angiogenic and invasive tumor cell clones.

Heterotopic Ossification (HO) occurs as a consequence of several diseases and of various forms of trauma. HO is particularly frequent in paraplegic patients with spinal cord lesions. It is obvious that extraskeletal cells are able to differentiate into an osteogenic direction. However, the mechanisms of the induction process of HO and the stimulating agents are not precisely known. A novel tool for studying the ossification process at the level of transcription is the technique of non-radioactive in situ hybridisation. Using digoxigenin labeled cDNA probes we investigated the distribution patterns of types I, II and III collagen mRNAs and the mRNA of Transforming Growth Factor beta 1 (TGF-beta 1) in heterotopic ossification of pressure sores of paraplegic patients. The three collagen mRNAs as well as the TGF-beta 1 mRNA exhibited substantially divergent distribution patterns. Type I (alpha 1) collagen mRNA was predominantly detectable in preosteoblasts, chondroblasts and chondrocytes of the ossification zone. Type II (alpha 1) collagen mRNA was nearly exclusively found in cells of the chondrogenic lineage. Type III (alpha 1) collagen mRNA was detectable at low levels in soft tissue, but was strongly expressed by chondroblasts and chondrocytes of heterotopic cartilage. In contrast expression of TGF-beta 1 mRNA was found in a spatial different distribution pattern in areas of proliferation of mesenchymal tissue and in different stages of ectopic bone formation. As in the case of collagen Type I (alpha 1) and III (alpha 1) mRNAs the maximum of localization of TGF-beta 1 was detected in chondroblastic areas of heterotopic ossification. Taken together our in situ hybridization experiments provide evidence that chondrogenic cells play a central role in the process of HO with a phenotypic alteration in collagen type expression and a strong expression of TGF-beta 1 mRNA. These findings support individual in vivo function for TGF-beta 1 in local cellular regulation of ectopic bone formation.

Heterotopic ossification is a metabolically active process which shares several properties of orthotopic bone formation and, therefore, represents an excellent model for the investigation of matrix components. A novel tool for studying the ossifying process at the level of transcription is the technique of non-radioactive in situ hybridization. Using digoxigenin labeled cDNA probes we investigated the distribution patterns of types I, II and III collagen mRNAs in heterotopic ossification of pressure sores of paraplegic patients. The three collagen mRNAs exhibited substantially divergent distribution patterns. Type I (alpha 1) collagen mRNA was predominantly detectable in preosteoblasts, prechondroblasts and chondrocytes of the ossification zone. Type II (alpha 1) collagen mRNA was nearly exclusively found in cells of the chondrogenic lineage. Type III (alpha 1) collagen mRNA was detectable at low levels in soft tissue, but was strongly expressed by prechondroblasts and chondrocytes of heterotopic cartilage. Our in situ hybridization experiments provide evidence that chondrogenic cells in heterotopic ossification show a phenotypic alteration in collagen type expression. These results indicate that chondrocytes of heterotopic cartilage show a co-expression of types I (alpha 1), II (alpha 1) and III (alpha 1) collagen mRNAs.

A retrospective statistical analysis of the acute lymphoblastic leukemia study BFM 70/76 revealed the prognosis being strongly dependent on the initial leukemia cell count/mm3 in the peripheral blood, and the enlargement of liver and spleen below the costal margin. Based on postmortem examinations reported in the literature a method was developed to estimate the tumor mass in these three compartments in an order of magnitude. The estimated blast cell mass related to the patient's body weight proved to be highly significant (p much less than 0.001). Simplification of the method led to the definition of a "risk factor" allowing the separation of patients into 3 groups with a distinctly different risk for relapse. In the current study BFM 81 the risk factor is used to stratify patients with respect to the individually needed therapeutic intensity.