Symptomatic type 1 protein C deficiency caused by a de novo Ser270Leu mutation in the catalytic domain

Heterozygosity for a C8524T transition in the protein C gene converting Ser270(TCG) to Leu(TTG) in the protease domain was identified in a family with venous thrombosis. The mutation was associated with parallel reduction in plasma levels of protein C anticoagulant activity and protein C antigen, which is consistent with a type 1 deficiency. Transient expression of mutant protein C cDNA in human kidney 293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein compared with wild-type protein was reduced by at least 97% while the intracellular content of mutant and wild-type protein was similar. Northern blot analysis of total mRNA from transfected cells showed no reduction of the mutant protein C mRNA compared with wild-type protein C mRNA. Collectively, these results indicate that the Ser270Leu mutation in the affected family caused the plasma protein C deficiency and are consistent with a disease mechanism that involves synthesis of mutant protein followed by intracellular degradation before its secretion into the extracellular space. The mutation was not present in the parents of the proband, suggesting a de novo mutation. Non-paternity was excluded after the analysis of three intragenic protein C polymorphisms and six dinucleotide repeat allele sets located in five different chromosomes.     

Simultaneous measurement of 25 inflammatory markers and neurotrophins in neonatal dried blood spots by immunoassay with xMAP technology

BACKGROUND: Inflammatory reactions and other events in early life may be part of the etiology of late-onset diseases, including cerebral palsy, autism, and type 1 diabetes. Most neonatal screening programs for congenital disorders are based on analysis of dried blood spot samples (DBSS), and stored residual DBSS constitute a valuable resource for research into the etiology of these diseases. The small amount of blood available, however, limits the number of analytes that can be determined by traditional immunoassay methodologies. METHODS: We used new multiplexed sandwich immunoassays based on flowmetric Luminex xMAP technology to measure inflammatory markers and neutrophins in DBSS. RESULTS: The high-capacity 25-plex multianalyte method measured 23 inflammatory and trophic cytokines, triggering receptor expressed on myeloid cells-1 (TREM-1), and C-reactive protein in two 3.2-mm punches from DBSS. It also measured 26 cytokines and TREM-1 in serum. Standards Recovery in the 25-plex method were 90%-161% (mean, 105%). The low end of the working range for all 25 analytes covered concentrations found in DBSS from healthy newborns. Mean recovery of exogenous analytes added at physiologic concentrations in DBSS models was 174%, mean intra- and interassay CVs were 6.2% and 16%, respectively, and the mean correlation between added and measured analytes was r2 = 0.91. In DBSS routinely collected on days 5-7 from 8 newborns with documented inflammatory reactions at birth, the method detected significantly changed concentrations of inflammatory cytokines. Measurements on DBSS stored at -24 degrees C for >20 years showed that most cytokines are detectable in equal concentrations over time. CONCLUSIONS: The method can reliably measure 25 inflammatory markers and growth factors in DBSS. It has a large potential for high-capacity analysis of DBSS in epidemiologic case-control studies and, with further refinements, in neonatal screening.     

Saliva cortisol measurement: simple and reliable assessment of the glucocorticoid replacement therapy in Addison's disease

No ideal parameter is available for assessment of the glucocorticoid replacement therapy in Addison's disease. Serum cortisol day-curves can be used to monitor the therapy, but this technique is cumbersome and expensive. We evaluated the potential for saliva cortisol measurement in this setting. We found excellent correlation between serum and saliva cortisol after oral intake of cortisone acetate (no. 7) or iv administration of hydrocortisone (no. 4) (Pearson's R=0.83-0.98, p<0.002). A morning dose of 12.5 mg cortisone acetate yielded wide interindividual variations in cortisol levels in saliva. Saliva cortisol measurements were successfully adopted to evaluate and adjust doses in outpatients. We conclude that cortisol measurement in saliva is practical and reliable, and is preferable to serum cortisol measurement in the assessment of the glucocorticoid replacement therapy. Our results confirm that only a minority of patients require more than 12.5 mg of cortisone acetate (equivalent to 10 mg hydrocortisone) in the morning to have sufficient cortisol levels during the first part of the day.     

Immune responses of a meningococcal A + W outer membrane vesicle (OMV) vaccine with and without aluminium hydroxide adjuvant in two different mouse strains

Meningococci (Neisseria meningiditis) of serogroups A and W have caused large epidemics of meningitis in sub‐Saharan Africa for decades, and affordable and multivalent vaccines, effective in all age groups, are needed. A bivalent serogroup A and W (A + W) meningococcal vaccine candidate consisting of deoxycholate‐extracted outer membrane vesicles (OMV) from representative African disease isolates was previously found to be highly immunogenic in outbred mice when formulated with the adjuvant aluminium hydroxide (AH). OMV has been shown to have inherent adjuvant properties. In order to study the importance of AH and genetical differences between mice strains on immune responses, we compared the immunogenicity of the A + W OMV vaccine when formulated with or without AH in inbred C57BL/6J and BALB/cJ mice (Th1 and Th2 dominant strains, respectively). The immunogenicity of the vaccine was found to be comparable in the two mice strains despite their different immune profiles. Adsorption to AH increased anti‐OMV IgG levels and serum bactericidal activity (SBA). The immune responses were increased by each dose for the adsorbed vaccine, but the third dose did not significantly improve the immunogenicity further. Thus, a vaccine formulation with the A and W OMV will likely benefit from including AH as adjuvant.     

Growth of whaleworm (<Emphasis Type="Italic">Anisakis simplex</Emphasis>, Nematodes,Ascaridoidea, Anisakidae) third-stage larvae in paratenic fish hosts

The growth of Anisakis simplex L3 larvae in the three paratenic hosts saithe ( Pollachius virens), cod (Gadus morhua) and redfish (Sebastes marinus) was studied based on material collected off the island of Vega on the west coast of Norway over a period of 1 year. In all three fish species, a positive correlation between the length of larvae and the age of the fish was shown. The number of large larvae, here defined as L3>28 mm, increases with increasing age of the host. These findings clearly indicate an accumulation of large L3 larvae and larval growth in the fish host. The larvae were additionally grouped according to the tissue--muscle, liver or other viscera--they were located in. When the average lengths of the three groups of larvae were compared, the gadoids saithe and cod had somewhat higher values for L3 larvae found in the liver. Redfish, on the other hand, which store fat to a relatively higher degree in the viscera, had the highest average length of larvae in this tissue. The lowest average values in all three species were found in the musculature. These trends suggest that the growth of A. simplex L3 larvae is positively correlated with the fat content of the tissue in which it is encapsulated. Of the three host species included in this study, saithe seemed to be the best suited for A. simplex L3, as the growth rate and average length of the parasite was greatest in this species. The growth and average length of L3 was least pronounced in redfish while cod held an intermediate position.     

'Vanishing embryo syndrome' in IVF/ICSI

BACKGROUND: In a Danish population-based cohort study assessing the risk of cerebral palsy in children born after IVF, we made some interesting observations regarding 'vanishing co-embryos'. METHODS and RESULTS: All live-born children born in Denmark from 1 January 1995 to 31 December 2000 were included in this analysis. The children conceived by IVF/ICSI (9444) were identified through the IVF Register, the children conceived without IVF/ICSI (395 025) were identified through The Danish Medical Birth Register. Main outcome measure was the incidence of cerebral palsy. Within the IVF/ICSI children we found indications of an increased risk of cerebral palsy in those children resulting from pregnancies, where the number of embryos transferred was higher than the number of children born. CONCLUSIONS: The association between vanishing embryo syndrome and incidence of cerebral palsy following IVF requires further investigation in larger, adequately powered, studies.     

Identification of the Complement Regulatory Protein CD59 in Human Colostrum and Milk

PROBLEM: Complement lytic activity has been demonstrated, and a potential for its activation is present in human colostrum and milk. This necessitates the presence of regulatory mechanisms protecting epithelial cells in the oropharynx and the gastrointestinal tract of the infant, the milk cellular elements, and bacteria colonizing the oropharynx and the gastrointestinal tract. Lactoferrin and C1 inhibitor have been attributed such a role. However, it is likely that additional protection against the cytolytic activity of the membrane attack complex is required. This has lead us to investigate the presence of the complement regulatory protein CD59 in human colostrum and milk, and to further characterize the source of secretion. METHOD: Samples of human colostrum and milk were obtained from volunteers at different stages of lactation, and separated into fat, skim milk, and milk cellular elements by centrifugation. Normal human mammary gland tissues were obtained from patients undergoing biopsy for benign conditions. SDS-PAGE and Western blotting, and an immuno dot-blot assay were used to identify CD59 in human milk. Immunohistochemistry was performed on all tissue samples and cytospins of the milk cellular elements, using monoclonal antibodies to CD59. RESULTS: CD59 was present in cell-free colostrum and milk as a 19–25 kDa glycoprotein. No variation in CD59 levels was detected between colostrum and milk. CD59 was present in great amounts in the cytoplasm and was highly expressed on the surface membrane on mammary gland acinar and ductal epithelial cells, while the milk cellular elements contained CD59 mainly in their cytoplasm. CONCLUSION: The complement regulatory protein CD59 present in cell-free human colostrum and milk may exert its effects both in the mammary gland and in the oropharynx and gastrointestinal tract of the infant. The lobuloalveolar epithelial cells in the mammary gland are the likely source of secretion.     

Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer

Gudlaugsson E, Skaland I, Janssen E A M, Smaaland R, Shao Z, Malpica A, Voorhorst F & Baak J P A
(2012) Histopathology
Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer Aims: The proliferation factor Ki67 is prognostic in breast cancer and included in international therapy guidelines, but measurement procedures differ between laboratories. We compared the reproducibility and prognostic value of different Ki67 sampling and measurement methods. Methods and results: In 237 T_1,2N₀M₀ breast cancers without adjuvant systemic treatment, strictly standardized section thickness, automated antigen retrieval and immunohistochemistry were used. The percentages of Ki67‐positive nuclei were assessed using (i) a ‘quick‐scan rapid estimate’, (ii) ocular‐square‐guided counts by independent pathologists, (iii) computerized point‐grid‐sampling interactive morphometry (CIM) and (iv) automated digital image analysis (DIA). Quick‐scan rapid estimates were poorly reproducible. The optimal prognostic thresholds of Ki67 counts by two pathologists differed greatly (4%, 14%; kappa: 0.36), with many therapeutic differences. CIM‐Ki67 and DIA‐Ki67 were strongly prognostic (P < 0.0001) and reproducible. DIA‐Ki67 (threshold: 6.5%) was the strongest and most robust prognosticator (the threshold could vary from 4 to 15% without significant prognostic loss). Ki67 was prognostically strongest in the periphery of the tumour. Conclusion: In node‐negative breast cancer without adjuvant systemic treatment, Ki67% by DIA, but not subjective counts, is reproducible and prognostically strong. This casts serious doubt on therapeutic guidelines using subjective counts of Ki67.     

Effects of high dose of simvastatin on levels of dopamine and its reuptake in prefrontal cortex and striatum among SD rats

Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin increased dopamine receptors in the rat prefrontal cortex [Q. Wang, W.L. Ting, H. Yang, P.T. Wong, High doses of simvastatin upregulate dopamine D₁ and D₂ receptor expression in the rat prefrontal cortex: possible involvement of endothelial nitric oxide synthase, Br. J. Pharmacol. 144 (2005) 933–939] and restored its downregulation in a model of Parkinson's disease (PD) [Q. Wang, P.H. Wang, C. McLachlan, P.T. Wong, Simvastatin reverses the downregulation of dopamine D1 and D2 receptor expression in the prefrontal cortex of 6-hydroxydopamine-induced Parkinsonian rats, Brain Res. 1045 (2005) 229–233]. Here we explore the effects of simvastatin treatment on tissue dopamine content and reuptake. Sprague–Dawley rats were given simvastatin (1 and 10 mg kg⁻¹ day⁻¹, p.o.) for 4 weeks. Brain tissue from prefrontal cortex and striatum were taken out for dopamine content and its reuptake. Using high-performance liquid chromatographic-mass spectrometer (HPLC-MS), simvastatin (10 mg kg⁻¹ day⁻¹) was found to increase dopamine content by 110% in the striatum but decreased by 76% in the prefrontal cortex compared with the saline treated group. Dopamine (DA) reuptake was unchanged in both brain regions. These results suggest that chronic treatment with high dose of simvastatin may affect DA tissue level in prefrontal cortex and striatum without changing on DA reuptake. This may have important clinical implications in psychiatric and striatal dopaminergic disorders.     

Differential effects of cancer rehabilitation depending on diagnosis and patients' cognitive coping style

OBJECTIVE: The major aim was to explore the extent to which the Miller Behavioral Style Scale (MBSS) can be used to differentiate cancer patients who are likely to benefit from rehabilitation efforts with a strong information component from those who are not. METHODS: Newly diagnosed patients with breast, gastrointestinal, or prostate cancer (N = 442) were included in a randomized, prospective study of the effects (on anxiety, depression, intrusion, avoidance) of rehabilitation approximately 4 months after diagnosis as compared with control patients. Patients were classified as "monitors" or "blunters" on the basis of the MBSS (368 patients, 83%, completed the MBSS). RESULTS: The expected interaction at postintervention between coping style and experimental condition (ie, rehabilitation or control) was found only for avoidance among breast and prostate cancer patients. Assignment to the rehabilitation or control condition was of no importance for outcome among blunters. Among monitors, the response pattern differed between breast and prostate cancer patients. Prostate cancer monitors seemed to benefit from rehabilitation on all outcome measures, whereas intrusion and avoidance were reduced among breast cancer patients in the control condition. This interaction of diagnosis with condition (rehabilitation or control) among monitors is suggested to be due to demands for diagnosis-specific information during diagnostic work, in the period just after diagnosis, and before treatment decision. CONCLUSIONS: Only the monitor concept seems useful for predicting response to cancer rehabilitation with a strong information component. However, whether rehabilitation is of benefit depends also on other factors.