Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

A 10-Mb paracentric inversion of chromosome arm 2p inactivates MSH2 and is responsible for hereditary nonpolyposis colorectal cancer in a North-American kindred

Genomic deletions of the MSH2 gene are a frequent cause of hereditary nonpolyposis colorectal cancer (HNPCC), a common hereditary predisposition to the development of tumors in several organs including the gastrointestinal and urinary tracts and endometrium. The mutation spectrum at the MSH2 gene is extremely heterogeneous because it includes nonsense and missense point mutations, small insertions and deletions leading to frameshifts, and larger genomic deletions, the latter representing approximately 25% of the total mutation burden. Here, we report the identification and molecular characterization of the first paracentric inversion of the MSH2 locus known to cause HNPCC. Southern blot analysis and inverse PCR showed that the centromeric and telomeric breakpoints of the paracentric inversion map within intron 7 and to a contig 10 Mb 3' of MSH2, respectively. Pathogenicity of the paracentric inversion was demonstrated by conversion analysis. The patient's lymphocytes were employed to generate somatic cell hybrids to analyze the expression of the inverted MSH2 allele in an Msh2-deficient rodent cellular background. The inversion was shown to abolish MSH2 expression by both northern and western analysis. This study confirms that Southern blot analysis still represents a useful and informative tool to screen for and identify complex genomic rearrangements in HNPCC. Moreover, monoallelic expression analysis represents an attractive approach to demonstrate pathogenicity of unusual mutations in autosomal dominant hereditary conditions.     

Can population differences explain the contrasting results of the Mwanza, Rakai, and Masaka HIV/sexually transmitted disease intervention trials?: A modeling study

OBJECTIVE: To determine whether population differences can explain the contrasting impacts on HIV observed in the Mwanza trial of sexually transmitted disease (STD) syndromic treatment (ST), the Rakai trial of STD mass treatment (MT), and the Masaka trial of information, education, and communication (IEC) with and without ST as well as to predict the effectiveness of each intervention strategy in each population. METHODS: Stochastic modeling of the transmission of HIV and 6 STDs was used with parameters fitted to demographic, sexual behavior, and epidemiological data from the trials and general review of STD/HIV biology. RESULTS: The baseline trial populations could be simulated by assuming higher risk behavior in Uganda compared with Mwanza in the 1980s, followed by reductions in risk behavior in Uganda preceding the trials. In line with trial observations, the projected HIV impacts were larger for the ST intervention in Mwanza than for the MT intervention in Rakai or the IEC and IEC + ST interventions in Masaka. All 4 simulated intervention strategies were more effective in reducing incidence of HIV infection in Mwanza than in either Rakai or Masaka. CONCLUSIONS: Population differences in sexual behavior, curable STD rates, and HIV epidemic stage can explain most of the contrast in HIV impact observed between the 3 trials. This study supports the hypothesis that STD management is an effective HIV prevention strategy in populations with a high prevalence of curable STDs, particularly in an early HIV epidemic.     

A novel case of infantile sacral teratoma and a constitutional t(12;15)(q13;q25) pat

Cytogenetic analysis of peripheral lymphocytes of an infantile patient with a sacral teratoma revealed a constitutional translocation (12;15)(q13;q25) pat. The same translocation was found in four additional relatives. Loss of heterozygosity analysis of the patient's tumor material showed retention of both translocation-derived chromosomes. Since allelic loss in the 12q13 region has been observed in germ cell tumors, we hypothesize that disregulation of genes located at or near the 12q13 breakpoint may be related to the development of this sacral teratoma. As a first step towards the identification of these genes, a 12q13 genomic contig that spans the breakpoint has been constructed.     

Analysis of the genomic sequence of a human metapneumovirus

We recently described the isolation of a novel paramyxovirus from children with respiratory tract disease in The Netherlands. Based on biological properties and limited sequence information the virus was provisionally classified as the first nonavian member of the Metapneumovirus genus and named human metapneumovirus (hMPV). This report describes the analysis of the sequences of all hMPV open reading frames (ORFs) and intergenic sequences as well as partial sequences of the genomic termini. The overall percentage of amino acid sequence identity between APV and hMPV N, P, M, F, M2-1, M2-2, and L ORFs was 56 to 88%. Some nucleotide sequence identity was also found between the noncoding regions of the APV and hMPV genomes. Although no discernible amino acid sequence identity was found between two of the ORFs of hMPV and ORFs of other paramyxoviruses, the amino acid content, hydrophilicity profiles, and location of these ORFs in the viral genome suggest that they represent SH and G proteins. The high percentage of sequence identity between APV and hMPV, their similar genomic organization (3'-N-P-M-F-M2-SH-G-L-5'), and phylogenetic analyses provide evidence for the proposed classification of hMPV as the first mammalian metapneumovirus.     

Symptomatic peripheral arterial disease: the value of a validated questionnaire and a clinical decision rule

BACKGROUND: If a validated questionnaire, when applied to patients reporting with symptoms of intermittent claudication, could adequately discriminate between those with and without peripheral arterial disease, GPs could avoid the diagnostic measurement of the ankle brachial index. AIM: To investigate the Edinburgh Claudication Questionnaire (ECQ) in general practice and to develop a clinical decision rule based on risk factors to enable GPs to easily assess the likelihood of peripheral arterial disease. DESIGN OF STUDY: An observational study. SETTING: General practice in The Netherlands. METHOD: This observational study included patients of > or =55 years visiting their GP for symptoms suggestive of intermittent claudication or with one risk factor. The ECQ and the ankle brachial index were performed. The prevalence of peripheral arterial disease, defined as an ankle brachial index <0.9, was related to risk factors using logistic regression analyses, on which a clinical decision rule was developed and related to the presence of peripheral arterial disease. RESULTS: Of the 4790 included patients visiting their GP with symptoms suggestive of intermittent claudication, 4527 were eligible for analyses. The prevalence of peripheral arterial disease in this group was 48.3%. The sensitivity of the ECQ was only 56.2%. The prevalence of peripheral arterial disease in a clinical decision rule that included age, male sex, smoking, hypertension, hypercholesterolemia, and a positive ECQ, increased from 14% in the lowest to 76% in the highest category. CONCLUSION: This study indicates that the ECQ alone has an inadequate diagnostic value in detecting patients with peripheral arterial disease. The ankle brachial index should be performed to diagnose peripheral arterial disease in patients with complaints suggestive of intermittent claudication, although our clinical decision rule could help to differentiate between extremely high and lower prevalence of peripheral arterial disease.     

How can the evaluation of genetic tests be enhanced? Lessons learned from the ACCE framework and evaluating genetic tests in the United Kingdom.

Advances in genetic technology are increasing the availability of genetic tests, not only for rare single gene disorders, but also for common diseases such as breast and colo-rectal cancer. Before there can be widespread uptake of these tests, they must be evaluated to confirm the benefits of their use. But how should genetic tests be evaluated, given the speed at which new tests are emerging? One highly influential approach is the analytic validity, clinical validity, clinical utility and ethical, legal and social issues (ACCE) framework, which has provided a benchmark for the evaluation of genetic tests. The approach has been adopted and adapted by the United Kingdom Genetic Testing Network, with the help of the Public Health Genetics Unit in Cambridge, to evaluate new genetic tests for use in the National Health Service. We discuss a number of conceptual, methodological, and practical issues concerning the evaluation of genetic tests, based on lessons learned from applying the ACCE framework and from the UK experience, and make a number of recommendations to further strengthen the evaluation of genetic tests.     

Genomic instability occurs in colorectal carcinomas but not in adenomas

Genomic instability, as demonstrated by the presence of additional alleles at short tandemly repeated (STR) loci, has recently been observed in colorectal tumours from individuals with hereditary non-polyposis colorectal cancer (HNPCC), and in some sporadic tumours. These neoplasms have been called replication error positive (RER⁺). In this study, we confirm the presence of genomic instability in a proportion of unselected colorectal carcinomas but find no evidence of instability in adenomas. We further report replication errors in a tetranucleotide sequence, and in STRs within two tumour suppressor genes. 108 colorectal adenocarcinomas and 46 adenomas were analysed for the presence of variant bands at 4–15 microsatellite markers. Seven (6.5%) of carcinomas were RER⁺, four of which originated from the proximal colon. Analysis of the adenomas and of matched adenoma-carcinoma and carcinoma-metastatic samples from four patients suggests that the replication errors may occur during the development of carcinomas but are rare in adenomas. © 1993 Wiley-Liss, Inc.