Influence of antithyroid antibodies in euthyroid women on in vitro fertilization-embryo transfer outcome

The direct synthesis and subsequent rapid characterisation of multiple antigen peptides (MAPs) for use as immunogens has presented difficulties, partly because of the formation of incomplete or truncated peptide sequences during the synthetic procedure. Therefore, many researchers have resorted to ligation procedures for the synthesis of MAP constructs. This article describes a method to improve the yield of MAP constructs by direct synthesis methods, as well as a general procedure that enables easier characterisation of the synthetic products. In particular, during the synthesis of MAP constructs, a capping procedure was introduced after each amino acid coupling step, thus improving significantly the yield of the desired multi-dendritic peptidic immunogens. Through the use of this capping procedure, problems arising from the incomplete amino acid residue coupling at the point of synthesis were minimised, and any deletion peptides which formed could be eliminated more readily during the subsequent purification procedures. In addition, previous difficulties in purification and characterisation of MAP construct by, e.g. electrospray mass spectroscopy (ES-MS), often led to the multi-dendritic peptidic immunogens being used without full characterisation after dialysis and recovery of the product(s). This article describes an enzymatic (tryptic) digestion method with the MAP construct, followed by characterisation of the enzymatic digest by reversed phase high-performance liquid chromatography-ES-MS. With this method, fragments of the MAP construct cleaved at specific amino acid residue sites (e.g. lysine or arginine) within the sequence of the parent peptide can be readily determined and the kinetics of the digestion easily followed. This enzymatic digestion procedure thus provides a facile approach to confirm that all of the multi-dendritic arms of the purified MAP construct have been equivalently elongated during the peptide synthesis and that consequently the purified construct structure contains the correct peptide sequence.     

Changes of prothrombin fragment 1+2 (F 1+2) as a function of increasing intensity of oral anticoagulation--considerations on the suitability of F 1+2 to monitor oral anticoagulant treatment

Plasma F 1+2 levels, the activation peptide originating from the factor Xa-mediated activation of prothrombin, increase in many clinical conditions associated with hypercoagulability and decrease in patients on oral anticoagulant treatment (OAT). However. the usefulness of F 1+2 measurement to monitor OAT has not yet been investigated in clinical studies. Before those studies are attempted, the plausibility of its implementation in the laboratory control of OAT should be evaluated. In this respect, a thorough investigation of the pattern of changes of F 1+2 as a function of increased intensity of anticoagulation expressed as International Normalized Ratio is essential. One hundred and thirty-two patients on long-term warfarin treatment were recruited to cover 8 ranges of anticoagulation from < 1.5 to 9.0 INR. F 1+2 was measured in batch on frozen plasma and INR was determined on fresh plasma. The relationship of F 1+2 vs. INR showed a hyperbolic pattern with F 1+2 levels decreasing progressively and significantly as a function of increasing INR up to 3.0. A further decrease in F 1+2 levels observed at INR up to 4.0 was not statistically significant. At INR greater than 4.0, F 1+2 reached a plateau, with mean levels not significantly different for patients at increasing INR up to 9.0. Since the risk of bleeding increases at INR greater than 4.5, our results suggest that F 1+2 is of little value to assess the hemorrhagic risk in patients on OAT.     

Down-regulation of hepatic and renal 11 beta-hydroxysteroid dehydrogenase in rats with liver cirrhosis

Twenty-four crossbred primiparous sows were used to investigate the influence of insulin administration after weaning on the intrafollicular insulin-like growth factor i (IGF-I) system. Sows received 0.4 i.u. insulin kg-1 bodyweight or an equivalent volume of saline for 3 days (n = 5 insulin; n = 4 saline) or 5 days (n = 5 insulin; n = 6 saline) after weaning or served as untreated controls on day 1 (n = 4). The number and diameters of ovarian follicles were recorded, and fluid was aspirated from the 20 largest follicles for determination of oestradiol and IGF-I by radioimmunoassay and of insulin-like growth factor-binding proteins (IGFBPs) by western ligand blotting. The walls of the follicles were collected for mRNA analysis by RNase protection assay or granulosa cells were collected for estimation of apoptosis by flow cytometry. Insulin treatment resulted in smaller diameters of all follicles (P < 0.05) and tended (P < 0.07) to increase the number of follicles available on day 5 compared with saline-treated animals (19.8 versus 17.8). The concentration of oestradiol in follicular fluid from large (7-10 mm) follicles on days 3 and 5 was reduced (treatment by size class interaction; P < 0.05) by insulin treatment. Insulin also reduced intrafollicular concentrations of IGF-I at days 3 and 5 after weaning (treatment by day interaction; P < 0.02) while the amounts of IGFBP-3 and IGFBPs of molecular mass 30 and 22 kDa decreased from day 3 to day 5 in saline-treated animals only (treatment by day interaction; P < 0.05). Gene expression for IGF-I increased in saline-treated animals but decreased fourfold in insulin-treated sows from day 3 to day 5 (treatment by day interaction; P < 0.002). Gene expression for IGFBP-d decreased (P < 0.04) from day 3 to day 5, while expression of IGFBP-2 was unaffected by treatment or day. Overall, insulin influenced the IGF-I system in a manner consistent with slowing follicular growth and possibly allowed more follicles to become available for ovulation.     

Predictive value of clinical examination, transrectal ultrasound and magnetic resonance imaging prior to radiotherapy in carcinoma of the cervix

The objectives of this study were to compare tumour staging and volume assessment by examination under anaesthesia (EUA), transrectal ultrasound (TRU) and magnetic resonance imaging (MRI) in patients with invasive carcinoma of the cervix, and to correlate findings with long-term outcome following treatment by radiotherapy. Tumour staging was performed on 60 patients immediately before starting radiotherapy. Clinicians and radiologists performing EUA, TRU or MRI were blinded to the results of other investigations. Tumour stage and dimensions were recorded prospectively for each technique, and analysed for concordance. The relationship between pre-treatment stage, size of tumour and patient outcome after radiotherapy was assessed, using clinical status 5 years after treatment as the truth measure. EUA, TRU and MRI assigned the same tumour stage in only 30% of patients and EUA and MRI agreed tumour stage in a further 27%. In cases of disagreement, the MRI stage correlated better with outcome than the TRU or EUA stage. There was a significant difference between tumour volume obtained from measurements made on MRI and those from TRU. 62% of patients with enlarged lymph nodes on pre-treatment MRI either died, or developed tumour recurrence or metastases. The ability of MRI to assess the full extent of bulky tumours and the presence of lymph node enlargement was an advantage over both EUA and TRU in identifying patients with a poor prognosis.     

Essential hypertension: first reason for persistent hypertension after unilateral adrenalectomy for primary aldosteronism?

OBJECTIVE: To evaluate the performance of two pulse oximeters in the measurement of arterial hemoglobin saturation in hypoxemic children. DESIGN: Prospective, repeated-measures observational study. SETTING: A 16-bed pediatric intensive care unit in a children's tertiary hospital. PATIENTS: Sixty-six patients with arterial saturation of <90%. INTERVENTIONS: Three arterial blood samples were taken from each subject during a 48-hr period. Pulse oximeter measurements of arterial saturation were compared with arterial saturation determined by cooximetry. MEASUREMENTS AND MAIN RESULTS: Arterial saturation was measured using one or both pulse oximeters (SpO2) and compared with the arterial hemoglobin saturation determined by cooximetry (SaO2). Sixty-two subjects were studied, using the Ohmeda pulse oximeter giving 185 data points (78 with saturations <75% [defined by the average of pulse oximeter and cooximeter]); 53 subjects were studied, using the Hewlett-Packard pulse oximeter yielding 155 data points (60 with saturations <75%). SpO2 ranged from 24% to 94%. Bias and precision of the Ohmeda pulse oximeter were -2.8% and 4.8% >75% and -0.8% and 8.0% <75%. Bias and precision of the Hewlett-Packard pulse oximeter were -0.5% and 5.1% >75% and 0.4% and 4.6% <75%. Intrapatient regression coefficient (r) for the differences between pulse oximeter and cooximeter was 0.58 for the Ohmeda and 0.59 for the Hewlett-Packard. Regression coefficients for predicting change in cooximeter value given a change in the Ohmeda pulse oximeter were 0.59 and 0.71 <75% and >75%, respectively. Similar coefficients for the Hewlett-Packard pulse oximeter were 0.50 and 0.70, respectively. CONCLUSION: The performance of the Ohmeda pulse oximeter deteriorated below an SpO2 of 75%. The Hewlett-Packard pulse oximeter performed consistently above and below an SpO2 of 75%. The ability of both pulse oximeters to reliably predict change in SaO2 based on change in pulse oximetry was limited. We recommend measurement of PaO2 or SaO2 for important clinical decisions.     

Glycosphingolipid binding specificities of Neisseria meningitidis and Haemophilus influenzae: detection, isolation, and characterization of a binding-active glycosphingolipid from human oropharyngeal epithelium

The glycosphingolipid binding specificities of Haemophilus influenzae and Neisseria meningitidis were investigated as to the binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby, similar binding profiles, for the binding of the two bacteria to lactosylceramide, isoglobotriaosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, lactotetraosylceramide, neolactotetraosylceramide, and sialylneolactohexaosylceramide, were obtained. On a closer view the binding preferences of the bacteria could be differentiated into three groups. The first specificity is recognition of lactosylceramide. The second specificity is binding to gangliotriaosylceramide and gangliotetraosylceramide, since conversion of the acetamido group of the N-acetylgalactosamine of gangliotriaosylceramide and gangliotetraosylceramide to an amine prevented the binding of the bacteria, and thus the binding to these two glycosphingolipids represents a separate specificity from lactosylceramide recognition. Preincubation of H. influenzae with neolactotetraose inhibited the binding to neolactotetraosylceramide, while the binding to lactosylceramide, gangliotetraosylceramide, or lactotetraosylceramide was unaffected. Thus, the third binding specificity is represented by neolactotetraosylceramide, and involves recognition of other neolacto series glycosphingolipids with linear N-acetyllactosamine chains, such as sialyl-neolactohexaosylceramide. The relevance of the detected binding specificities for adhesion to target cells was addressed as to the binding of the bacteria to glycosphingolipids from human granulocytes, epithelial cells of human nasopharyngeal tonsils and human plexus choroideus. Binding-active neolactotetraosylceramide was thereby detected in human granulocytes and the oropharyngeal epithelium.     

Functional and metabolic evaluation of the athlete's heart by magnetic resonance imaging and dobutamine stress magnetic resonance spectroscopy

BACKGROUND: The question of whether training-induced left ventricular hypertrophy in athletes is a physiological rather than a pathophysiological phenomenon remains unresolved. The purpose of the present study was to detect any abnormalities in cardiac function in hypertrophic hearts of elite cyclists and to examine the response of myocardial high-energy phosphate metabolism to high workloads induced by atropine-dobutamine stress. METHODS AND RESULTS: We studied 21 elite cyclists and 12 healthy control subjects. Left ventricular mass, volume, and function were determined by cine MRI. Myocardial high-energy phosphates were examined by 31P magnetic resonance spectroscopy. There were no significant differences between cyclists and control subjects for left ventricular ejection fraction (59+/-5% versus 61+/-4%), left ventricular cardiac index (3.4+/-0.4 versus 3.4+/-0.4 L x min(-1) x m[-2]), peak early filling rate (562+/-93 versus 535+/-81 mL/s), peak atrial filling rate (315+/-93 versus 333+/-65 mL/s), ratio of early and atrial filling volumes (3.0+/-1.0 versus 2.6+/-0.6), mean acceleration gradient of early filling (5.2+/-1.4 versus 5.8+/-1.9 L/s2), mean deceleration gradient of early filling(-3.1 +/- 0.9 versus -3.2 +/- 0.7 L/s2), mean acceleration gradient of atrial filling (3.6+/-1.8 versus 4.5+/-1.7 L/s2), and atrial filling fraction (0.23+/-0.06 versus 0.26+/-0.04, respectively). Cyclists and control subjects showed similar decreases in the ratio of myocardial phosphocreatine to ATP measured with 31P magnetic resonance spectroscopy during atropine-dobutamine stress (1.41+/-0.20 versus 1.41+/-0.18 at rest to 1.21+/-0.20 versus 1.16+/-0.13 during stress, both P=NS). CONCLUSIONS: Left ventricular hypertrophy in cyclists is not associated with significant abnormalities of cardiac function or metabolism as assessed by MRI and spectroscopy. These observations suggest that training-induced left ventricular hypertrophy in cyclists is predominantly a physiological phenomenon.     

Characterisation of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01

The distribution, function and ontogeny of the mononuclear phagocyte system of the chicken were characterised using the monoclonal antibody (MAb) KUL01. KUL01 specifically recognises chicken monocytes, macrophages and interdigitating cells, as well as activated microglia cells. Its tissue distribution allowed to discriminate KUL01 from all earlier described MAb, reactive with mononuclear phagocytes. The specificity of KUL01 for mononuclear phagocytes was further confirmed in functional assays: KUL01-positive macrophages in spleen and liver actively took up colloidal carbon, while monocytes and spleen and gut macrophages contained non-specific esterase and acid phosphatase activities characteristic for antigen-processing. Further, it was demonstrated that KUL01-reactive peripheral blood monocytes express MHC-II, but not CD4. In all tissues investigated, the same morphological subtypes of macrophages were detected in chicken at similar localisations as in mammals, indicating a high degree of conservation between the mononuclear phagocyte system of the chicken and of mammals.     

Differential effects of high salt intake on neuropeptide Y and adrenergic markers in hearts of Dahl rats

Adrenergic markers and neuropeptide Y (NPY) were examined in Dahl NaCl-sensitive and -resistant outbred male rats, fed either 0.35% or 8% NaCl diets for 8 weeks. The high salt diet caused left ventricular hypertrophy in sensitive rats but not in the resistant strain. Norepinephrine stores were not affected by high salt intake, but tyrosine hydroxylase, and dopamine beta-hydroxylase were elevated in the salt-induced hypertrophied left ventricle in conjunction with increased levels of nerve growth factor and p75 neurotrophin receptor. In contrast, high salt intake reduced ventricular neuropeptide Y in both Dahl salt-resistant and -sensitive rats.     

Studies on the induction of antigen-specific antibody in anti-CD40 cultured human B lymphocytes

Costimulatory signals provided by T cells are required for B cells to produce specific antibody to T-dependent antigen. We have investigated the suitability of using the CD40 culture system for the proliferation and differentiation of Ag-specific human B cells using cytomegalovirus (CMV) or tetanus toxoid (TT) as antigen. We modified the CD40 culture system (CD32-transfected L cells, anti-CD40, and IL-4) by applying a sequential cytokine stimulation and compared total B-cell cultures with antigen-specific B cells preselected by panning. The detection of specific antibody became possible when antigen-selected B cells were cultured for 7 days in the CD40 system to induce clonal expansion, followed by the addition of IL-2 and IL-10 for an additional 7 days to induce plasma-cell differentiation. We conclude that our initial inability to detect specific antibody in the CD40 system is due to overgrowth of nonspecific B-cell clones and that selection of antigen-specific B cells by panning overcomes this problem. Induction of antigen-specific antibody production was found to be optimal when the initial contact with antigen during panning was limited to between 1 to 24 hours.