The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

 Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK₁ and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK₁ clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl₂. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds. Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997     

Polyreactivity of human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20-, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells.     

Circulating immune complexes in old people and in diabetics: correlation with autoantibodies.

Circulating immune complexes (CIC) were detected by a solid-phase radioassay in 34% of fifty-three insulin-dependent diabetics (IDD) as compared to 18% of forty-five non-insulin-dependent diabetics (NIDD) and 14% of 173 control subjects. In control subjects, the prevalence of CIC increased with age and was higher in males than in females. In IDD, immune complexes were found with the highest frequency before the age of 30 and after 50. There was no significant difference between the incidence of CIC in old IDD and their age-matched controls. The same sera were also tested for the presence of the following autoantibodies; nuclear , thyroid, gastric, smooth and striated muscle; mitochondria, sub-maxillary and adrenal gland and liver-kidney microsome. Sera containing at least one antibody were found in 16.4% of controls, 55.3% of IDD and 40% of NIDD. The prevalence of autoantibodies increased with age in controls but not in IDD. Islet cell antibodies were present in 28.5% of IDD and 2.9% of control subjects; they were more frequent in young patients. CIC and autoantibodies were statistically associated both in controls and IDD; in the patients, CIC were associated not only with islet cell antibodies but also with other autoantibodies. The possible relation between autoimmunity, degenerative vascular diseases and CIC is discussed.     

Good end-of-life care according to patients and their GPs

BACKGROUND: Most patients prefer to die at home, where a GP provides end-of-life care. A few previous studies have been directed at the GPs' values on good end-of-life care, yet no study combined values of patients and their own GP. AIM: To explore the aspects valued by both patients and GPs in end-of-life care at home, and to reflect upon the results in the context of future developments in primary care. DESIGN OF STUDY: Interviews with patients and their own GP. SETTING: Primary care in the Netherlands. METHOD: Qualitative, semi-structured interviews with 20 GPs and 30 of their patients with a life expectancy of less than 6 months, and cancer, heart failure or chronic obstructive pulmonary disease as underlying disease. RESULTS: Patients and GPs had comparable perceptions of good end-of-life care. Patients and GPs identified four core items that they valued in end-of-life care: availability of the GP for home visits and after office-hours, medical competence and cooperation with other professionals, attention and continuity of care. CONCLUSIONS: Future developments in the organisation of primary care such as the restriction of time for home visits, more part-time jobs and GP cooperatives responsible for care after office hours, may threaten valued aspects in end-of-life care.     

Effects of a feed additive blend on broilers challenged with heat stress

ABSTRACT

We evaluated a blend of medium-chain fatty acids (MCFA), organic acids, and a polyphenol antioxidant on gut integrity. Eighty Ross Broilers were exposed to 20–22°C (control – normothermic) or to 35–39.5°C (heat stress) for eight hours a day for a period of 1 or 5 days. Birds were fed a standard diet, or a diet supplemented with the test blend. Thereafter, birds were euthanized, and intestinal sections were excised for morphological, morphometric and gene expression analyses. Blood samples were collected for glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px) activity and trolox equivalent antioxidant capacity (TEAC) determination. Heart and liver tissues were used to quantify the expression of heat shock proteins 60 and 70 (HSP60 and HSP70, respectively) and inhibitor of kappa light chain gene enhancer in B cells alpha (IKBA). The jejunum was the most sensitive intestinal section, where heat stress modulated the expression of HSP70, of the inflammatory markers IKBA, interleukin 8 (IL-8), interferon gamma (IFNγ), and toll-like receptor 4 (TLR4). Moreover, expression of tight junctions (CLDN1, ZO1 and ZO2) and nutrient transporters (PEPT1 and EAAT3) was modulated especially in the jejunum. In conclusion, the feed additive blend protected intestines during heat stress from the decrease in villus height and crypt depth, and from the increase in villus width. Especially in the jejunum, heat stress played an important role by modulating oxidative stress and inflammation, impairing gut integrity and nutrient transport, and such deleterious effects were alleviated by the feed additive blend.

RESEARCH HIGHLIGHTS

• Jejunum is the most sensitive intestinal segment during heat stress.

• Heat stress affects the expression of tight junctions and nutrient transporters.

• Feed management helps to alleviate the disturbances caused by heat stress.

• A blend of MCFA, organic acids and a polyphenol protects broilers under heat stress.

     

Opsonic antibodies to outer membrane protein P2 of nonencapsulated Haemophilus influenza are strain specific.

The ability of monoclonal antibodies (MAbs) specific for variable and conserved epitopes of outer membrane protein (OMP) P2 (b,c) of nonencapsulated Haemophilus influenza to promote opsonophagocytosis of this bacterium by human polymorphonuclear leucocytes (PMNs) was determined by flow cytometry. MAbs rendering PMNs fluorescent because of association with fluorescein isothiocyanate-labelled bacteria were defined as stimulating opsonophagocytosis. Opsonophagocytosis was dependent on the presence of both antibodies and complement. Of the 14 MAbs directed to the variable parts of OMP P2 (L. van Alphen, P. Eijk, L. Geelen-van den Broek, and J. Dankert, Infect. Immun. 59:247-252, 1991), 9 stimulated opsonophagocytosis. Four of the five nonopsonophagocytic MAbs that were immunoglobulin G1 were unable to cause complement activation. The MAbs promoting opsonophagocytosis included MAbs specific for one or more OMP P2 antigenic variants of H. influenzae strains isolated from patients with chronic bronchitis during persistent infection. MAbs cross-reacting in enzyme-linked immunosorbent assays with nonrelated H. influenzae did not promote opsonophagocytosis of strains from other patients. Opsonophagocytosis was not observed in the presence of three MAbs reacting with OMP P2 epitopes common in H. influenzae. These results indicate that OMP P2-dependent opsonophagocytosis of nonencapsulated H. influenzae is strictly strain specific.     

Diagnostic value of anti-Candida enolase antibodies.

An immunodominant antigen with enolase enzyme activity was purified and used for the development of an assay to detect antibodies directed against this antigen in sera from patients with either invasive candidiasis or Candida colonization. The Au enzyme-linked immunosorbent assay established with the Candida enolase antigen was able to discriminate significantly between invasive candidiasis and colonization in both immunocompetent and immunodeficient groups of patients. The test had a sensitivity of 50% and a specificity of 86% in the immunocompetent patient group. In the immunodeficient patient group, a sensitivity of 53% and a specificity of 78% were established. Antibody levels determined by a counterimmunoelectrophoresis assay with the same set of sera resulted in a better sensitivity for sera from the immunocompetent patient group but a lower specificity, i.e., 80 and 29%, respectively. The counterimmunoelectrophoresis assay of sera from the immunodeficient patient group was not able to discriminate significantly between invasive candidiasis and colonization. With the use of more serum from each patient, the sensitivity of the antibody detection assays increased, while the specificity was maintained. The increase, however, was not statistically significant. Combining the results of the antibody assays with antigen titers obtained by the Cand-Tec assay did not improve the predictive value with respect to invasive candidiasis, as determined by multivariance regression analysis. Furthermore, it was demonstrated by performance of Western blots (immunoblots) that sera from patients as well as a rabbit antiserum cross-reacted with the Candida enolase and baker's yeast enolase enzyme. However, by tandem crossed immunoelectrophoresis it was demonstrated that the antibodies were directed toward different epitopes of the antigen.     

Cationic immune complex arthritis in mice--a new model. Synergistic effect of complement and interleukin-1.

A novel cationic immune-complex-mediated arthritis (ICA) model was developed in mice. The highly cationic protein lysozyme was coupled to poly-L-lysine (PLL) and injected intra-articularly into the knee joint of the mouse, shortly after systemic administration of specific antibodies. A vehement joint inflammation developed, characterized by severe joint swelling and the influx of predominantly polymorphonuclear (PMN) leukocyte. Unique properties were combined in this protein. First, an excellent retention of the antigen in joint structures was found, facilitating sufficient IC formation in the synovial tissue and at the cartilage surface. Secondly, PLL.lysozyme appeared to be a potent inducer of interleukin-1 (IL-1). Similar IL-1 production was measured at 6 hours, in both immune or nonimmune mice. Neutralization with antibodies against either IL-1 alpha or IL-1 beta revealed that IL-1 alpha was the dominant cytokine. Resident cells were responsible for this IL-1 production since a comparable IL-1 signal was measured after intra-articular injection of PLL.lys in neutropenic mice. We further investigated whether IL-1 and complement factors were involved in the onset of this ICA. Neutralizing the IL-1 production with antibodies directed against IL-1 alpha and beta showed a significant decrease in joint swelling. Complement depletion by cobra venom factor also prevented the onset of arthritis for the greater part. Only a minor swelling remained at 6 hours after eliciting arthritis, which was similar to the swelling after injecting the antigen alone and probably reflects IL-1 mediated inflammation. In this study, the authors show a synergistic action of IL-1 and complement in the onset of cationic ICA. Unique properties of the antigen such as excellent retention and its ability to induce IL-1 are combined within one molecule and make this antigen arthritogenic in the presence of antibodies and complement activation.