PD-1 analysis on CD28(-) CD27(-) CD4 T cells allows stimulation-independent assessment of CMV viremic episodes in transplant recipients.

Expression of the inhibitory receptor programmed death 1 (PD-1) on cytomegalovirus (CMV)-specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28(-) CD27(-) double negativity is known as a typical phenotype of CMV-specific CD4 T cells. Therefore, the co-expression of inhibitory receptors on CD28(-) CD27(-) CD4 T cells was assessed as a rapid, stimulation-independent parameter for monitoring CMV complications after transplantation. Ninety-three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross-sectional and longitudinal manner. CMV-specific CD4 T cell levels quantified after stimulation were compared to levels of CD28(-) CD27(-) CD4 T cells. PD-1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression on CD28(-) CD27(-) CD4 T cells were related to viremia. A percentage of ≥0.44% CD28(-) CD27(-) CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV-specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD-1 expression levels on CD28(-) CD27(-) CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA-4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD-1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD-1 expression on CD28(-) CD27(-) CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation-independent parameter to guide duration of antiviral therapy.

In vitro mapping of 1H ultrashort T2* and T2 of porcine menisci.

In this study, mapping of ultrashort T2 and T2* of acutely isolated porcine menisci at B0 = 9.4 T was investigated. Maps of T2 were measured from a slice through the pars intermedia with a spin echo-prepared two-dimensional ultrashort-TE T2 mapping technique published previously. T2* mapping was performed by two-dimensional ultrashort-TE MRI with variable acquisition delay. The measured signal decays were fitted by monoexponential, biexponential and Gaussian-exponential fitting functions. The occurrence of Gaussian-like signal decays is outlined theoretically. The quality of the curve fits was visualized by mapping the value δ = abs(1 - χ(2) red). For T2 mapping, the Gaussian-exponential fit showed the best performance, whereas the monoexponential and biexponential fits showed regionally high values of δ (δ > 20). Interpretation of the Gaussian-exponential parameter maps was found to be difficult, because a Gaussian signal component can be related to mesoscopic (collagen texture) or macroscopic (slice profile, shim, sample geometry) magnetic field inhomogeneities and/or residual (1) H dipole-dipole couplings. It seems likely that an interplay of these effects yielded the observed signal decays. Modulation of the T2* signal decay caused by chemical shift was observed and addressed to fat protons by means of histology. In the T2 measurements, no modulation of the signal decay was observed and the biexponential and Gaussian-exponential fits showed the best performance with comparable values of δ. Our results suggest that T2 mapping provides the more robust method for the characterization of meniscal tissue by means of MRI relaxometry. However, mapping of ultrashort T2, as performed in this study, is time consuming and provides less signal-to-noise ratio per time than the mapping of T2*. If T2* mapping is used, pixel-wise monitoring of the fitting quality based on reduced χ(2) should be employed and great care should be taken when interpreting the parameter maps of the fits.

Nano-HPLC-MS analysis of phospholipids in cerebrospinal fluid of Alzheimer's disease patients--a pilot study.

There is emerging evidence that lipids play an important role in many neurodegenerative processes, for example in Alzheimer's disease (AD). Although different lipid alterations in the AD brain have been reported, there have only been very few investigations of lipid changes in the cerebrospinal fluid (CSF). Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrixes. In this study we developed an on-line HPLC-MS method for phospholipid profiling in the CSF based on nano-HPLC separation using an Amide column and detection with electrospray (ESI) quadrupole-time of flight (QTOF) MS. We achieved good separation, reproducibility, and sensitivity in monitoring of the major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), and sphingomyelin (SM) in CSF. To emphasize the applicability of the method, a pilot study was performed on a group of CSF samples (N = 16) from individuals with probable AD and non-demented controls. We observed a statistically significant increase of SM levels (24.3 ± 2.4%) in CSF from probable AD individuals vs. controls. Our findings indicate that SM levels in the CSF could potentially provide a new lead in AD biomarker research, and show the potential of the method for disease-associated CSF phospholipid screening.

[Functional biostructure of colonic microbiota (central fermenting area, germinal stock area and separating mucus layer) in healthy subjects and patients with diarrhea treated with Saccharomyces boulardii]. / Biostructure fonctionnelle du microbiote colique(zone de fermentation centrale, zone de réservegerminale et couche de mucus séparatrice) chez lessujets sains et chez les patients atteints de diarrhéetraités par Saccharomyces boulardii.

The colonic content can be compared to a spatially structured high output bioreactor composed of three functionally different regions: a separating mucus layer, a germinal stock area, and a central fermenting area. The stool mirrors this structure and can be used for diagnosis in health and disease. In a first part, we introduce a novel method based on fluorescence in situ hybridization (FISH) of sections of punched-out stool cylinders, which allows quantitatively monitor microbiota in the mucus, the germinal stock and the central fermenting areas. in a second part, we demonstrate the practical implementation of this method, describing the biostructure of stool microbiota in healthy subjects and patients with chronic idiopathic diarrhea treated with Saccharomyces boulardii. Punched stool cylinders from 20 patients with chronic idiopathic diarrhea and 20 healthy controls were investigated using fluorescence in situ hybridization. Seventy-three bacterial groups were evaluated. Fluctuations in assembly of 11 constitutive bacterial groups were monitored weekly for 3 weeks prior to, 3 weeks during, and 3 weeks after oral Saccharomyces boulardii supplementation. Typical findings in healthy subjects were a 5-60 µm mucus separating layer; homogeneous distribution and fluorescence, high concentrations (>10 × 10(10) bacterial/mL) of the three habitual bacterial groups: Bacteroides, Roseburia and Faecalibacterium prausnitzii; and low concentrations of the occasional bacterial groups. The diarrhea could be described in terms of increased separating effort, purging, decontamination, bacterial substitution. Typical findings in diarrhea were: increased thickness of the protective mucus layer, its incorporation in the stool, absolute reduction in concentrations of the habitual bacterial groups, suppression of bacterial metabolism in the central fermenting area (hybridization silence), stratification of the stool structure by watery ingredients, and substitutive increase in the concentrations of occasional bacterial groups. The microbial and clinical symptoms of diarrhea were reversible with Saccharomyces boulardii therapy. The structure-functional analysis of stool microbiota allows to quantitatively monitor colonic malfunction and its response to therapy. Saccharomyces boulardii significantly improves the stool biostructure in patients with chronic idiopathic diarrhea and has no influence on the stool microbiota in healthy subjects.

A non-human primate BAC resource to study interchromosomal segmental duplications.

Segmental duplications (SDs) are involved in the reshaping and evolutionary development of primate genome architecture. Their intrinsic property to promote genomic instability facilitates genome rearrangements, thereby contributing to karyotype diversity in primates. However, comparative analyses of SDs based on whole-genome shotgun assemblies of primate genomes may lead to a distorted view of their evolutionary dynamics as this method will incorrectly assemble or simply not represent these regions. Therefore high-quality sequences of chromosomally assigned SDs are indispensable for unraveling the amplification and dispersal pattern of SDs during primate evolution. Here, we use an updated version of the ancestral duplicon state of the non-palindromic SDs of all 4 human Y-chromosome euchromatin/heterochromatin transition regions to perform a survey of duplicons genome-wide across 7 primate species. By adjusting experimental conditions to the mean nucleotide sequence divergence to human we identified 11,075 BAC clones carrying primate orthologs or paralogs of human Y chromosome-derived duplicons. Preliminary results indicate lineage-specific amplification of duplicons in prosimians and gibbons. This BAC-based framework represents the first complete set of a defined number of duplicons over 60 million years of primate evolution. Comparative sequence analysis of this genetic resource can contribute to our deeper understanding of the impact of segmental duplications on primate genome evolution.

Presymptomatic treatment of neonatal guanidinoacetate methyltransferase deficiency.

Prospective observation in a neonate with guanidinoacetate methyltransferase deficiency (GAMT-D), a severe neurometabolic disorder, revealed increased guanidinoacetate levels at birth. After 14-month treatment with creatine, high-dose ornithine, benzoate, and an arginine-restricted diet, the patient's development is normal and she does not present any symptoms of GAMT-D. The authors' observation indicates that early detection of GAMT-D is possible in the neonatal period, and presymptomatic treatment may prevent its manifestation.

Type 1 diabetes and the OAS gene cluster: association with splicing polymorphism or haplotype?

BACKGROUND: The 2',5'-oligoadenylate synthetase genes (OAS1, OAS2, and OAS3) map to human chromosome 12q24 and encode a family of enzymes pivotal to innate antiviral defence. Recently, the minor allele of an OAS1 single nucleotide polymorphism (SNP) that alters splicing (rs10774671) was found to be associated with increased enzymatic activity and, in a case-sibling control study, with type 1 diabetes (T1D). METHODS: We have confirmed this T1D association in 784 nuclear families (two parents and at least one affected offspring) by the transmission disequilibrium test (TDT; G:A = 386:329, p = 0.033). However, because of linkage disequilibrium within OAS1 and with the other two OAS genes, functional attribution of the association to this SNP cannot be assumed. To help answer this question, we also genotyped two non-synonymous SNPs in OAS1 exons 3 and 7. RESULTS: All three SNPs showed significant transmission distortion. Three of the eight possible haplotypes accounted for 98.4% of parental chromosomes and two of them carried the non-predisposing A allele at rs10774671. Parents heterozygous for these two haplotypes showed significant transmission distortion (p = 0.009) despite being homozygous at rs10774671. CONCLUSIONS: We confirm the T1D association with rs10774671, but we conclude that it cannot be attributed (solely) to the splicing variant rs10774671. A serine/glycine substitution in OAS1 exon 3 is more likely a functional variant.

[Apoplexy--current status of diagnostics and therapy]. / Aktuelle Empfehlungen zur Therapie des Schlaganfalls. Vor und nach dem Insult--so versorgen Sie lhren Patienten optimal.

During the past years increasingly stricter criteria have been applied to the primary prevention of ischemic stroke. This applies especially to the treatment of asymptomatic carotid stenosis. An operation is indicated for a blockage of 60% and higher, including symptom-free patients under 75 years of age. At the moment, a final conclusion on the preferred operative procedure--thromboendarterectomy or stent implantation--cannot be made. For the secondary prevention of apoplexy, the highest relative risk reduction for vascular accidents using thrombocyte aggregation inhibitors was achieved with the combination ASA plus dipyridamole. Diuretics, calcium antagonists, ACE inhibitors and angiotensin receptor blockers (ARB) are equally suitable for the reduction of blood pressure after apoplectic insult. Moreover, the latter appear to have advantages for the prevention of a renewed apoplexy. The benefit of statins in the secondary prevention of apoplexy has been substantiated by the Heart Protection Study. Simvastatin has the best evidence for its effectiveness in patients without CHD; in contrast, atorvastatin has possibly more benefits for patients with clinically evident CHD. The direct thrombin inhibitor, ximelagatran, will be available as an alternative to the oral anticoagulant marcumar in the foreseeable future.

Evolutionary breakpoint analysis on Y chromosomes of higher primates provides insight into human Y evolution.

Comparative FISH mapping of PAC clones covering almost 3 Mb of the human AZFa region in Yq11.21 to metaphases of human and great apes unravels breakpoints that were involved in species-specific Y chromosome evolution. An astonishing clustering of evolutionary breakpoints was detected in the very proximal region on the long arm of the human Y chromosome in Yq11.21. These breakpoints were involved in deletions, one specific for the human and another for the orang-utan Y chromosome, in a duplicative translocation/transposition specific for bonobo and chimpanzee Y chromosomes and in a pericentric inversion specific for the gorilla Y chromosome. In addition, our comparative results allow the deduction of a model for the human Y chromosome evolution.

The evolution of the azoospermia factor region AZFa in higher primates.

Clones of a PAC contig encompassing the human AZFa region in Yq11.21 were comparatively FISH mapped to great ape Y chromosomes. While the orthologous AZFa locus in the chimpanzee, the bonobo and the gorilla maps to the long arm of their Y chromosomes in Yq12.1-->q12.2, Yq13.1-->q13.2 and Yq11.2, respectively, it is found on the short arm of the orang-utan subspecies of Borneo and Sumatra, in Yp12.3 and Yp13.2, respectively. Regarding the order of PAC clones and genes within the AZFa region, no differences could be detected between apes and man, indicating a strong evolutionary stability of this non-recombining region.

Accuracy assessment protocols for electromagnetic tracking systems.

Electromagnetic tracking systems have found increasing use in medical applications during the last few years. As with most non-trivial spatial measurement systems, the complex determination of positions and orientations from their underlying raw sensor measurements results in complicated, non-uniform error distributions over the specified measurement volume. This makes it difficult to unambiguously determine accuracy and performance assessments that allow users to judge the suitability of these systems for their particular needs. Various assessment protocols generally emphasize different measurement aspects that typically arise in clinical use. This can easily lead to inconclusive or even contradictory conclusions. We examine some of the major issues involved and discuss three useful calibration protocols. The measurement accuracy of a system can be described in terms of its 'trueness' and its 'precision'. Often, the two are strongly coupled and cannot be easily determined independently. We present a method that allows the two to be disentangled, so that the resultant trueness properly represents the systematic, non-reducible part of the measurement error, and the resultant precision (or repeatability) represents only the statistical, reducible part. Although the discussion is given largely within the context of electromagnetic tracking systems, many of the results are applicable to measurement systems in general.

The Azoospermia region AZFa: an evolutionar y view.

Compared to other regions on the human Y chromosome, the genomic segment encompassing the functionally defined AZFa locus has undergone higher X-Y sequence divergence, which is detectable by fluorescence in-situ hybridisation. This allows an evolutionary definition of an interval enclosing AZFa with a size of about 1.1 Mb. The region includes the genes USP9Y, DBY and UTY and is limited by evolutionary breakpoints within the PAC clones 41L06 and 46M11. These breakpoints restrict an area of possible male specific evolution that may have resulted in the acquisition of male specific functions, including a role in spermatogenesis.

Depletion and sizes of motor units in spinal muscular atrophy.

Motor unit number estimation (MUNE) was applied to the biceps brachii muscles of 13 young patients (age 5--24 years) with spinal muscular atrophy (SMA) and the results compared with those of healthy control subjects matched for age and gender. In the SMA patients, all motor unit (MU) estimates fell below the control range, and there was good correspondence between the values for the two arms in the same subject. No correlation could be found between the MUNEs and the severity of the weakness. This unexpected result was attributed to the presence of small and normal-sized MUs in the muscles of patients, in addition to MUs that appeared to be considerably enlarged. The threefold mean increase in MU potential size was insufficient to compensate for the MU loss. In addition, the study confirmed that there are, on average, approximately 130 MUs in the healthy biceps brachii muscle.

A novel tracking technique for the continuous precise measurement of tumour positions in conformal radiotherapy.

Changing tumour positions induced by organ motion can impede the full exploitation of the strengths of conformal radiotherapy. The unnecessary irradiation of healthy tissue surrounding the target volume can be the consequence. To overcome this, one should measure tumour positions directly and continuously with high resolution in space and time. We have developed a novel tracking technique which will allow this. The method can also be used to survey and monitor the patient positioning. The proper functioning of our method has been technically demonstrated at PSI with the help of phantom irradiation with protons. Implementation into the clinical environment is now beginning.

FISH deletion mapping defines a single location for the Y chromosome stature gene, GCY.

At least 1 in 1000 males lacks part of the long arm of the Y chromosome. This chromosomal aberration is often associated with short stature and infertility. Deletion mapping and genotype-phenotype analysis have previously defined two non-overlapping critical regions for growth controlling gene(s), GCY(s), on the euchromatic portion of the Y chromosome long arm. These initial mapping assignments were based on the analysis of patients carrying a pure 46,XYq- karyotype as defined by classical cytogenetic karyotyping. Four genes have been assigned to the distal one of the two critical regions. To determine whether one or both of these two critical regions harbours GCY and whether one of the four genes assigned to the distal region is involved in determination of stature, nine adult patients with Yq chromosomal abnormalities were studied in detail. By PCR and FISH analysis, we showed that all patients with a previously defined pure 46,XYq- karyotype are actually mosaics with cells containing an idic(Y) or ring(Y) chromosome in association with 45,X0 cells. This leads us to conclude that (1) FISH is an absolute prerequisite for the correct identification of Y chromosomal rearrangements and (2) only patients with interstitial Y deletions are reliable predictors for the physical location of stature gene(s) on Yq. Our molecular analyses of chromosomes from patients with interstitial Yq deletions finally establishes the proximal interval between markers DYZ3 and DYS11 as the only GCY critical interval. No functional gene has so far been identified in this region adjacent to the centromere.

A member of a gene family on Xp22.3, VCX-A, is deleted in patients with X-linked nonspecific mental retardation.

X-linked nonspecific mental retardation (MRX) has a frequency of 0.15% in the male population and is caused by defects in several different genes on the human X chromosome. Genotype-phenotype correlations in male patients with a partial nullisomy of the X chromosome have suggested that at least one locus involved in MRX is on Xp22.3. Previous deletion mapping has shown that this gene resides between markers DXS1060 and DXS1139, a region encompassing approximately 1.5 Mb of DNA. Analyzing the DNA of 15 males with Xp deletions, we were able to narrow this MRX critical interval to approximately 15 kb of DNA. Only one gene, VCX-A (variably charged, X chromosome mRNA on CRI-S232A), was shown to reside in this interval. Because of a variable number of tandem 30-bp repeats in the VCX-A gene, the size of the predicted protein is 186-226 amino acids. VCX-A belongs to a gene family containing at least four nearly identical paralogues on Xp22.3 (VCX-A, -B, -B1, and -C) and two on Yq11.2 (VCY-D, VCY-E), suggesting that the X and Y copies were created by duplication events. We have found that VCX-A is retained in all patients with normal intelligence and is deleted in all patients with mental retardation. There is no correlation between the presence or absence of VCX-B1, -B, and VCX-C and mental status in our patients. These results suggest that VCX-A is sufficient to maintain normal mental development.

Mosaic rearrangement of chromosome 18: characterization by FISH mapping and DNA studies shows trisomy 18p and monosomy 18p both of paternal origin.

Structural abnormalities of chromosome 18p mainly consist of isochromosomes of the short arm, which result in tetrasomy 18p. Trisomy 18p is much rarer, and less well characterized. We report on a 12-year-old girl with minor facial anomalies, delayed development, abnormal hands, atopic dermatitis, and hearing loss. She was mosaic for two abnormal cell lines in peripheral blood. In 90% of cells, a dicentric chromosome with duplication of the whole short arm of chromosome 18 resulted in trisomy 18p; 10% of cells had monosomy 18p, arising from a t(14;18)(p11;q11). FISH mapping, with multiple region specific and locus specific probes from the short and long arm of chromosome 18, showed that the structure of the dicentric chromosome 18 was 18pter-->18q23::18q11-->18pter. DNA polymorphisms for chromosome 18 showed that the abnormalities of chromosome 18 were paternal in origin. Combining all results, we could link the trisomy 18p and monosomy 18p to a common origin via a complex series of events in an early mitosis.