Changes in anti-Müllerian hormone serum concentrations over time suggest delayed ovarian ageing in normogonadotrophic anovulatory infertility

BACKGROUND: Anti-Müllerian hormone (AMH), produced by growing pre-antral and early antral ovarian follicles, has been shown to be a useful marker for ovarian ageing. Serum AMH concentrations are elevated during reproductive life in anovulatory women, especially in those patients exhibiting polycystic ovaries (PCO). The current study was designed to investigate whether the decrease in AMH serum concentrations over time is different comparing women with normogonadotrophic anovulation [World Health Organization (WHO) group 2 (including polycystic ovary syndrome (PCOS)] and normo-ovulatory controls. METHODS AND RESULTS: AMH serum levels were assessed on two occasions in 98 patients suffering from WHO 2 anovulatory infertility as well as in 41 normo-ovulatory premenopausal women. Median time interval between both visits was 2.6 years (range 0.3-9.0) for WHO 2 patients compared with 1.6 years (range 1.0-7.3) in controls. Serum AMH concentrations were significantly (P < 0.0001) elevated on both occasions in WHO 2 patients (AMH1, median = 7.5 microg/l, range 0.1-35.8; and AMH2, median = 6.7 microg/l, range 0.0-30.6) compared with controls (AMH1, median = 2.1 microg/l, range 0.1-7.4; and AMH2, median = 1.3 microg/l, range 0.0-5.0). Regression analysis, corrected for age, indicated a significant relative decrease in serum AMH concentrations over time for both groups (P < 0.001). However, the decline in serum AMH in WHO 2 patients was significantly less compared with controls (P = 0.03). CONCLUSION: The present longitudinal study shows that serum AMH concentrations decrease over time both in women presenting with WHO 2 anovulatory infertility and in normo-ovulatory controls. The decrease in WHO 2 patients is less pronounced despite distinctly elevated concentrations. This observation may suggest retarded ovarian ageing and hence a sustained reproductive life span in these patients.     

White Matter Fiber Tracking Computation Based on Diffusion Tensor Imaging for Clinical Applications

Fiber tracking allows the in vivo reconstruction of human brain white matter fiber trajectories based on magnetic resonance diffusion tensor imaging (MR-DTI), but its application in the clinical routine is still in its infancy. In this study, we present a new software for fiber tracking, developed on top of a general-purpose DICOM (digital imaging and communications in medicine) framework, which can be easily integrated into existing picture archiving and communication system (PACS) of radiological institutions. Images combining anatomical information and the localization of different fiber tract trajectories can be encoded and exported in DICOM and Analyze formats, which are valuable resources in the clinical applications of this method. Fiber tracking was implemented based on existing line propagation algorithms, but it includes a heuristic for fiber crossings in the case of disk-shaped diffusion tensors. We successfully performed fiber tracking on MR-DTI data sets from 26 patients with different types of brain lesions affecting the corticospinal tracts. In all cases, the trajectories of the central spinal tract (pyramidal tract) were reconstructed and could be applied at the planning phase of the surgery as well as in intraoperative neuronavigation.     

Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling

Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin B1, DNA content, and caspase 3 expression were measured by flow cytometry to assess cell cycle kinetics and apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting, and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression, contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression of interleukin-2 (IL-2), tumor necrosis factor alpha, and gamma interferon but increased IL-10 production in PBT. Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT proliferation by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious antigens in order to maintain immunological homeostasis.     

Histopathological grading of soft tissue tumours. Prognostic significance in a prospective study of 278 consecutive cases

A consecutive 10-year series of 278 soft tissue sarcomas was prospectively graded, using a system based on the number of mitoses and taking into account parameters such as cellularity, anaplasia, necrosis, and histogenetic type and subtype of tumour. Prognostic factors in relation to metastasis-free survival were studied by uni- and multivariate analysis. Fifty-seven (20.5 per cent) were low-grade tumours, 43 (15.5 per cent) were intermediate, and 178 (64 per cent) were high grade. High-grade tumours were divided into two groups; 80 (29 per cent) grade 3A (= 5-20 mitoses per 10 high power fields (HPF)) and 78 grade 3B (28 per cent) (= more than 20 mitoses/10 HPF); 10 HPF corresponds to 2.5 mm2. Twenty (7.2 per cent) high-grade tumours could not be further subdivided. Grading was found to be the prognostic factor associated with the strongest predictive value. Five-year survival in low-grade and intermediate tumours (95 and 86 percent, respectively) differed significantly (P less than 0.0001) from high grade (50 per cent) and (p = 0.0018) between grade 3A (64 per cent) and grade 3B (41 per cent). Other prognostic indicators of importance in high-grade tumours were age, local recurrence at presentation (primary operation outside the Centre), and localization (superficial vs. deep).     

Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies.

Sera from Brucella-infected bovines were analyzed by immunoblotting by using sonicated cell extracts of B. melitensis or B. abortus and a competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against outer membrane proteins (OMPs) with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa. Antibody responses against OMPs were compared with antibody responses against smooth lipopolysaccharide. Immunoblot analysis indicated that the antibody response in infected animals was largely different from one animal to another. The antigens of concern were OMPs with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa and other proteins with molecular masses of between 40 and 80 kDa. According to the specificity of the competitive ELISA, OMPs useful for the detection of infected animals are the OMPs of 10, 16.5, 19, 25 to 27, and 36 to 38 kDa. A competitive ELISA with the anti-89 kDa monoclonal antibody was not specific. Results of the competitive ELISA confirmed the individual variability of the humoral immune response against OMPs. It therefore seems that a combination of several protein antigens is necessary for the development of an immunoassay with a sensitivity comparable to that of the smooth lipopolysaccharide ELISA.     

Brucella melitensis cell envelope protein and lipopolysaccharide epitopes involved in humoral immune responses of naturally and experimentally infected sheep.

Cell envelope fraction (CEF) of Brucella melitensis B115 was used to investigate antibody responses of B. melitensis naturally and strain H38 experimentally infected sheep by immunoblotting, indirect enzyme-linked immunosorbent assay (ELISA) (I-ELISA), and competitive ELISA (C-ELISA) with monoclonal antibodies (MAbs). MAbs used were directed to outer membrane proteins with molecular masses of 10, 16.5, 19, 25 to 27, 31 to 34, 36 to 38, and 89 kDa; to the heat shock protein DnaK, to O-polysaccharide (O-PS) common (C) and M epitopes; and to rough lipopolysaccharide (R-LPS) epitopes. In immunoblotting, all infected sheep sera tested recognized a large number of protein bands, including the above-cited proteins and other proteins for which MAbs have not been defined. The antibody response pattern was different from one animal to another, even within the experimentally infected sheep which were infected under the same experimental conditions. A number of protein bands were recognized by the sheep sera prior to experimental infection and by other uninfected sheep sera. The antibody reactivity to these antigens and others might explain the nonspecific antibody reactivity of sera in I-ELISA with CEF. C-ELISA confirmed also the individual variability of the antibody responses of infected sheep to protein antigens. Antibody responses to O-PS C and M epitopes were detected in all experimentally infected sheep and in half of the naturally infected sheep, but these responses were also heterogeneous in relation to their intensities. Antibody responses to R-LPS epitopes detected by use of C-ELISA with the anti-R-LPS MAbs were low or negative in most of the infected animals. Despite antibody response heterogeneity for CEF antigens, immunoblot and C-ELISA results indicated that, among the CEF antigens, the O-PS epitopes (C and M epitopes) and epitopes of the major 25- to 27- and 31- to 34-kDa outer membrane proteins seem to be the most promising for detecting B. melitensis infection in sheep.     

Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries

Summary Lipoprotein(a), as an atherogenic particle, represents an independent risk factor for coronary heart disease. In the present study the morphological distribution of apoprotein (a) and apoprotein B within the arterial wall is described. Apoprotein B, a constituent of very low-density lipoprotein, low-density lipoprotein and lipoprotein(a) has previously been demonstrated in atheromatous lesions. Lipoprotein(a) possesses an additional protein, designated apoprotein (a). Autopsy material (n=74) from the left coronary artery and from the thoracic aorta has been examined by means of immunohistochemistry and both apoprotein (a) and apoprotein B were detected, primarily associated with the extracellular matrix and accumulating in lesions in the arterial wall. The staining pattern for both antigens was almost always found to be congruent, suggesting that the detection of (a)-antigen has to be attributed at least in part to the presence of lipoprotein(a). It is concluded that both low-density lipoprotein and lipoprotein(a) have an important role in the pathogenesis of atherosclerosis.     

Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay.

A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.     

Characterization of heat, oxidative, and acid stress responses in Brucella melitensis

Brucella melitensis is a facultative intracellular pathogen which is able to survive and replicate within phagocytic cells. Therefore, it has to adapt to a range of different hostile environments. In order to understand the mechanisms of intracellular survival employed by virulent B. melitensis 16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses by two-dimensional (2-D) polyacrylamide gel electrophoresis was used. Depending on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis. On the basis of N-terminal sequence analysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified. Some of them were previously reported for Brucella, such as BvrR, DnaK, GroEL, and Cu-Zn superoxide dismutase (SOD). Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydrogenase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 component beta subunit. Results indicated that B. melitensis could bring specific regulatory mechanisms into play in response to stress conditions. For example, the ribosome releasing factor in B. melitensis appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for Escherichia coli, was strongly down-regulated in B. melitensis in response to heat stress. Some of the identified proteins and their potential specific regulation could be required for the adaptation of B. melitensis to environmental stresses encountered in phagocytic cells and possibly for bacterial virulence.