Endogenous serotonin in human blood platelets: factors that may influence reference values

The endogenous content of serotonin in human platelets has been used in many clinical studies to indicate platelet activation. A decrease in platelet serotonin compared with controls has been regarded as an indicator of platelet activation. However, the results published are difficult to compare, because of huge variations in endogenous serotonin between control groups in different investigations. This is likely to be because of lack of standardization. Several factors that influence the endogenous platelet serotonin content were studied in more than 200 blood donors. The most important factor was the total g force of the centrifugation used to isolate platelets. Also the age and sex of platelet donors, number of platelets in platelet-rich plasma, and mean platelet volume influenced normal serotonin values. Using a standardized centrifugation procedure (2700 g min) the mean endogenous serotonin was 2.80 nmol/10(9) platelets in young women and 2.58 in elderly women, and 2.67 in young men and 2.30 in elderly men. The differences both for age and sex were statistically significant. Endogenous platelet serotonin shows intrapersonal stability over time, since endogenous platelet serotonin did not change on repeated venepuncture for 9 weeks. Factors such as age, sex and isolation procedure must therefore be considered when endogenous platelet serotonin are studied in relation to disease and treatment.     

The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.     

Antibiotic prophylaxis in total hip arthroplastyEffects of antibiotic prophylaxis systemically and in bone cement on the revision rate of 22,170 primary hip replacements followed 0-14 years in the Norwegian Arthroplasty Register

We studied the effects of antibiotic prophylaxis, systemically and in bone cement, on the revision rate of cemented total hip arthroplasties (THAs) in data from the Norwegian Arthroplasty Register during the period 1987-2001. To have comparable groups, only THAs performed because of primary osteoarthritis, using cemented implants with documented good results, and high-viscosity cement were included. If systemic antibiotic prophylaxis had been given, only operations with cephalosporin or penicillin were selected. Cox-estimated survival relative revision risks (RR) are presented with adjustment for differences among groups in gender, age, cement brand, type of systemic antibiotic prophylaxis, type of prosthesis, type of operating room, and duration of the operation. Of 22,170 THAs studied, 696 THAs (3.1%) were revised, 440 (2.0%) for aseptic loosening and 102 (0.5%) for deep infection. We found the lowest risk of revision when the antibiotic prophylaxis was given both systemically and in the cement (15,676 THAs). Compared to this combined regime, patients who received antibiotic prophylaxis only systemically (5,960 THAs) had a 1.4 times higher revision rate with all reasons for revision as endpoint (p= 0.001), 1.3 times higher with aseptic loosening (p= 0.02) and 1.8 times higher with infection as the endpoint (p= 0.01). With the combined antibiotic regime, the results were better if antibiotics were given 4 times on the day of surgery (2,194 THAs), as compared to once (1,424 THAs) (p<0.001), twice (2,680 THAs) (p<0.001), or 3 times (5,522 THAs) (p= 0.02). Those who received systemic prophylaxis a single day 1, 2 or 3 times, as compared to 4 times, had a revision rate 1.8-3.5 times higher with all reasons for revision as endpoint, 1.5-3.1 times higher with aseptic loosening, and 2.7-6.8 times higher with infection. When we compared systemic prophylaxis 4 times in 1 day, no further improvement resulted in those given systemic prophylaxis for 2 days (1,928 THAs) or 3 days (717 THAs). In a subset of data including only the Charnley prosthesis, we obtained similar results. This observational study shows that the best results were recorded when antibiotic prophylaxis was given both systemically and in the bone cement, and if the systemic antibiotic was given 4 times on the day of surgery.     

De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features

Makrythanasis P, Moix I, Gimelli S, Fluss J, Aliferis K, Antonarakis SE, Morris MA, Béna F, Bottani A. De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features. Loss‐of‐function mutations of MECP2 are responsible for Rett syndrome (RTT), an X‐linked neurodevelopmental disorder affecting mainly girls. The availability of MECP2 testing has led to the identification of such mutations in girls with atypical RTT features and the recognition of milder forms. Furthermore, duplication of the entire gene has recently been described in boys with mental retardation and recurrent infections. We describe a girl with a heterozygous de novo MECP2 duplication. The patient, at the age of 19, has mental retardation with no autistic features. She is friendly but gets frequently anxious. She has neither dysmorphic features nor malformations. Her motor development was delayed with walking at 20 months. Speech is fluid with good pronunciation but is simple and repetitive. Diagnosis was made after single‐strand conformation analysis (SSCA) and multiplex ligation‐dependent probe amplification (MLPA) analysis of MECP2. Array comparative genomic hybridization (aCGH) analysis showed a duplication of 29 kb including MECP2 and part of IRAK1. Fluorescent in situ hybridization (FISH) has revealed that the duplicated region is inserted near the telomere of the short arm of chromosome 10. X‐chromosome inactivation in leukocyte DNA was not skewed. We conclude that it is likely that this MECP2 duplication is responsible for the mental retardation in this patient. This case broadens the phenotypic spectrum of MECP2 abnormalities with consequent implication in diagnosis and genetic counselling of girls with non‐syndromic mental retardation.     

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Folate and one-carbon metabolism gene polymorphisms and their associations with oral facial clefts

Folate metabolism plays a critical role in embryonic development. Prenatal folate supplementation reduces the risk of neural tube defects and probably oral facial clefts. Previous studies of related metabolic genes have associated polymorphisms in cystathionine-beta-synthase (CBS) and 5,10-methylenetetrahydrofolate reductase (MTHFR) with cleft risk. We explored associations between genes related to one-carbon metabolism and clefts in a Norwegian population-based study that included 362 families with cleft lip with or without cleft palate (CL/P) and 191 families with cleft palate only (CPO). We previously showed a 39% reduction in risk of CL/P with folic acid supplementation in this population. In the present study we genotyped 12 polymorphisms in nine genes related to one-carbon metabolism and looked for associations of clefting risk with fetal polymorphisms, maternal polymorphisms, as well as parent-of-origin effects, using combined likelihood-ratio tests (LRT). We also stratified by maternal periconceptional intake of folic acid (>400 microg) to explore gene-exposure interactions. We found a reduced risk of CL/P with mothers who carried the CBS C699T variant (rs234706); relative risk was 0.94 with one copy of the T allele (95% CI 0.63-1.4) and 0.50 (95% CI 0.26-0.96) with two copies (P = 0.008). We found no evidence of interaction of this variant with folate status. We saw no evidence of risk from the MTHFR C677T variant (rs1801133) either overall or after stratifying by maternal folate intake. No associations were found between any of the polymorphisms and CPO. Genetic variations in the nine metabolic genes examined here do not confer a substantial degree of risk for clefts.