Tumor necrosis factor-alpha in whole blood cultures of preeclamptic patients and healthy pregnant and nonpregnant women.

OBJECTIVES: Tumor necrosis factor-alpha (TNF-alpha) is recognized as a likely mediator of the excessive endothelial activation and injury that is a key pathogenetic mechanism of preeclampsia. We used whole blood cell cultures from 12 patients with severe preeclampsia and from 12 healthy pregnant and nonpregnant women to determine the release of TNF-alpha by unstimulated leukocytes as a measure of their state of activation, and their response to stimulation with lipopolysaccharide (LPS) as an indicator of their state of priming. METHODS: Blood was cultivated without and with LPS, and TNF-alpha release was measured after six and 24 hours of cultivation by enzyme-linked immunoassays. Differential leukocyte counts were performed, and TNF-alpha values calculated per 10(5) monocytes. RESULTS: In unstimulated whole blood cultures, TNF-alpha release after six hours of cultivation was similar in all three groups; but after 24 hours, TNF-alpha concentrations in culture supernatants from preeclamptic patients were significantly higher than were values obtained in blood from normotensive pregnant women. In LPS-stimulated blood cultures with a maximum of TNF-alpha release at six hours cultivation time, TNF-alpha concentrations were significantly lower in preeclamptic women than they were in both control groups. We showed in an additional experiment that a strong LPS challenge following preactivation with high doses of LPS resulted in reduced release of TNF-alpha compared with release of TNF-alpha following preactivation with low doses of LPS. CONCLUSIONS: The observed high capacity for spontaneous TNF-alpha release by leukocytes in preeclampsia indicates activation of TNF-alpha producing leukocytes by the disease process. Preactivation and exhaustion of leukocytes by leakage of TNF-alpha could lead to the reduced response to TNF-alpha inducer LPS as observed in blood cultures from preeclamptic patients.     

Effectiveness of a Multidisciplinary Education Protocol in Children with Asthma (0-4 Years) in Primary Health Care

This paper describes the results of an asthma self-management protocol delivered to parents of children aged 0-4 years. The protocol was delivered by general practitioners (GPs), community nurses, asthma nurses, and doctors in child health centers. It consisted of 16 educational modules developed after an initial needs assessment of parents and a task analysis of primary care practitioners. The program was evaluated by means of an experimental design. Parents participating in the program had significantly more knowledge, a more favorable attitude toward asthma, and a higher self-efficacy score with respect to performing asthma self-management behaviors. Also, they reported performing self-management behaviors more frequently than parents in the control group. One-year follow-up results, which were collected for parents in the treatment group only, showed that the described changes were sustained. Further, the treatment group was found to have decreased its emergency and nonemergency use of the physician's office and to have a reduction in (reported) asthma severity. Process evaluation indicated that most modules were provided by the GPs to nearly all parents. After parents had read the modules at home, almost all the information was discussed in the next contact. GPs seldom referred patients to the community nurses, although this was suggested in the protocol.     

Probiotics as flourishing benefactors for the human body.

This article provides a comprehensive review of the beneficial effects of various strains of probiotics in preventing and treating certain diseases. Currently, changed lifestyles as well as the increased use of antibiotics are significant factors challenging the preservation of a healthy intestinal microflora. The concept of probiotics is to restore and uphold a microflora advantageous for the human body. Probiotics are found in a number of fermented dairy products, infant formula, and dietary supplements. In the presence of prebiotics, which are nondigestible food ingredients favorable for probiotic growth, their survival in the intestine is ameliorated.     

The effect of the interferon-gamma-inducible processing machinery on the generation of a naturally tumor-associated human cytotoxic T lymphocyte epitope within a wild-type and mutant p53 sequence context

The human wild-type (wt) p53.264-272 peptide is a universal tumor antigen and recognized by HLA-A*0201 (A2.1)-restricted CTL. Generation of this epitope by constitutive 20S proteasomes is prevented by a p53 R to H hotspot mutation at the C-terminal flanking residue 273. We report on the impact of the interferon-gamma (IFN-gamma)-inducible proteasomal activator PA28 (11S regulator) and the immunoproteasome on the in vitro and cellular processing of wt and mutant (mut) p53 substrates. We found that production of the antigenic 264-272 peptide from wt p53 by constitutive as well as immunoproteasomes is accelerated and amplified by the PA28 activator. PA28 and (immuno)proteasomes were not capable to reconvert the resistance of epitope release from mut p53. Maximum and accelerated antigen production in vitro and on the cellular level required the IFN-gamma-inducible interaction of immunoproteasomes and PA28. We conclude that efficient processing of p53.264272 from wt p53 is governed by the proteasome/PA28 complex. These studies have important implications for p53-specific cancer immunotherapy and demonstrate that the effects of the immunoproteasome and PA28 are influenced by the individual epitope and its flanking sequence context.     

Deletion of the ferric uptake regulator Fur impairs the in vitro growth and virulence of Actinobacillus pleuropneumoniae

In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. pleuropneumoniae Deltafur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.     

Overrepresentation of genetic variation in the AnkyrinG interactome is related to a range of neurodevelopmental disorders

Upon the discovery of numerous genes involved in the pathogenesis of neurodevelopmental disorders, several studies showed that a significant proportion of these genes converge on common pathways and protein networks. Here, we used a reversed approach, by screening the AnkyrinG protein-protein interaction network for genetic variation in a large cohort of 1009 cases with neurodevelopmental disorders. We identified a significant enrichment of de novo potentially disease-causing variants in this network, confirming that this protein network plays an important role in the emergence of several neurodevelopmental disorders.Subject terms: Genetics research, Neurological disorders     

Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes

The most common genetic disorder in humans, trisomy, is caused predominantly by errors in chromosome segregation during oogenesis. Isolated mouse oocytes resuming meiosis and progressing to metaphase II in vitro have recently been used to assess targets, aneugenic potential and sensitivity of oocytes to chemical exposures. In order to extend in vitro maturation tests to earlier stages of oogenesis, an in vitro assay with mouse preantral follicle cultures has been established. It permits the identification of direct and also indirect effects of environmental chemicals on the somatic compartment, the follicle and theca cells, that may lead to disturbances of oocyte growth, maturation and chromosome segregation. Early preantral follicles from prepubertal female mice are cultured in microdroplets for 12 days under strictly controlled conditions. The follicle-enclosed oocytes resume maturation, develop to metaphase II and become in vitro ovulated within 16 h after a physiological ovulatory stimulus with recombinant human gonadotrophins and epidermal growth factor. These oocytes grown and matured in vitro possess normal barrel-shaped spindles with well-aligned chromosomes. Their chromosomes segregate with high fidelity during anaphase I. The model aneugen colchicine induced a meiotic arrest and aneuploidy in these in vitro grown, follicle-enclosed oocytes in a dose-dependent manner, comparable to in vivo tests. Therefore, preantral follicle culture appears to provide an effective and reliable method to assess the influences of environmental mutagens, pharmaceutical agents and potentially endocrine disrupting chemicals on the fidelity of female meiosis.     

Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole

Aneuploidy tests are important in evaluating genetic hazards especially when chemical exposures are suspected to affect the fidelity of chromosome segregation in oocytes and embryos. In the current study, a newly established method, mouse preantral follicle culture, was employed to grow oocytes in vitro within follicles. The sensitivity of in vitro grown follicle enclosed oocytes was compared with oocytes maturing in vivo in the ovary. In both the cases, oocytes were exposed to the cytostatic chemical, nocodazole, from the time of hormonally stimulated resumption of meiosis. The in vivo study revealed a significant decrease in the number of ovulated mouse oocytes and an increase in meiosis I-arrested and hyperploid metaphase II oocytes at a single i.p. dose of 70 mg/kg body weight of nocodazole. A significant increase was also observed in the number of meiosis I-arrested and hyperploid mouse oocytes from preantral follicle culture, when they were cultured in the presence of >or=30 nM nocodazole during the final stages of maturation. This concentration is slightly lower than that previously shown to induce nondisjunction in denuded mouse oocytes or in cultured human lymphocytes. The higher sensitivity of the in vitro matured oocytes from preantral follicle culture than that of denuded oocytes may be related to a synergistic adverse influence of nocodazole on the oocyte, on somatic cell integrity and on cell-cell communication, which possibly also affects ovulation in vivo. When expressed in molarity relative to the mouse weight, the effective dose of the acute exposure in vivo is 3-4 orders of magnitude higher than the lowest effective concentration employed continuously in vitro. Reduced bioavailability of nocodazole to the target cells due to its poor water solubility may contribute to this difference. Preantral follicle culture can be helpful in analysing mechanisms in chemically induced aneuploidy in mammalian oogenesis, and in predicting the consequences of chemical exposures in vivo.