Spatial genome organization during T-cell differentiation.

The spatial organization of genomes within the mammalian cell nucleus is non-random. The functional relevance of spatial genome organization might be in influencing gene expression programs as cells undergo changes during development and differentiation. To gain insight into the plasticity of genomes in space and time and to correlate the activity of specific genes with their nuclear position, we systematically analyzed the spatial genome organization in differentiating mouse T-cells. We find significant global reorganization of centromeres, chromosomes and gene loci during the differentiation process. Centromeres were repositioned from a preferentially internal distribution in undifferentiated cells to a preferentially peripheral position in differentiated CD4+ and CD8+ cells. Chromosome 6, containing the differentially expressed T-cell markers CD4 and CD8, underwent differential changes in position depending on whether cells differentiated into CD4+ or CD8+ thymocytes. Similarly, the two marker loci CD4 and CD8 showed distinct behavior in their position relative to the chromosome 6 centromere at various stages of differentiation. Our results demonstrate that significant spatial genome reorganization occurs during differentiation and indicate that the relationship between dynamic genome topology and single gene regulation is highly complex.

Comparison of viral load and human leukocyte antigen statistical and neural network predictive models for the rate of HIV-1 disease progression across two cohorts of homosexual men.

We compared the performance of HIV-1 RNA and models based on human leukocyte antigen (HLA) in predicting the rate of HIV-1 disease progression using both linear regression and neural network models across two different cohorts of homosexual men. In all, 139 seroconverters from the Multicenter AIDS Cohort Study were used as the training set and 97 seroconverters from the District of Columbia Gay (DCG) cohort were used for validation to assess the generalizability of trained predictive models. Both viral load and HLA markers were strongly predictive of disease progression (p < .0001 and p = .001, respectively), with viral load superior to HLA (change in -2 log likelihood [-2LL] 26.7 and 10.2, respectively, in proportional hazards models). Consideration of both HLA markers and viral load offered no significant predictive advantage over viral load alone in most cases; however, HLA-based predictions obtained from neural networks modeling improved the discrimination among patients with high viral load (p = .02). Viral load, HLA scores, and rapid disease progression were moderately correlated (p < .01 for all three pairs of these variables). The median viral load was 10(3.70) copies/ml among DCG patients who had more favorable than unfavorable HLA markers and 10(4.66) copies/ml among patients with more unfavorable than favorable HLA markers. Viral load is a simpler, stronger predictor of disease progression than early developed HLA models, but neural network methods and further refined HLA models may offer additional prognostic information, especially for rapid progressors. The correlation between viral load and HLA markers suggests a possible HLA effect on setting viral load levels.

Use of neural networks to model complex immunogenetic associations of disease: human leukocyte antigen impact on the progression of human immunodeficiency virus infection.

Complex immunogenetic associations of disease involving a large number of gene products are difficult to evaluate with traditional statistical methods and may require complex modeling. The authors evaluated the performance of feed-forward backpropagation neural networks in predicting rapid progression to acquired immunodeficiency syndrome (AIDS) for patients with human immunodeficiency virus (HIV) infection on the basis of major histocompatibility complex variables. Networks were trained on data from patients from the Multicenter AIDS Cohort Study (n = 139) and then validated on patients from the DC Gay cohort (n = 102). The outcome of interest was rapid disease progression, defined as progression to AIDS in <6 years from seroconversion. Human leukocyte antigen (HLA) variables were selected as network inputs with multivariate regression and a previously described algorithm selecting markers with extreme point estimates for progression risk. Network performance was compared with that of logistic regression. Networks with 15 HLA inputs and a single hidden layer of five nodes achieved a sensitivity of 87.5% and specificity of 95.6% in the training set, vs. 77.0% and 76.9%, respectively, achieved by logistic regression. When validated on the DC Gay cohort, networks averaged a sensitivity of 59.1% and specificity of 74.3%, vs. 53.1% and 61.4%, respectively, for logistic regression. Neural networks offer further support to the notion that HIV disease progression may be dependent on complex interactions between different class I and class II alleles and transporters associated with antigen processing variants. The effect in the current models is of moderate magnitude, and more data as well as other host and pathogen variables may need to be considered to improve the performance of the models. Artificial intelligence methods may complement linear statistical methods for evaluating immunogenetic associations of disease.

Thermal modeling of laser capture microdissection.

A first-order thermal analysis is applied to Laser Capture Microdissection (LCM), a new microscope technique for routine targeting and extraction of specific cells from tissue sections for subsequent multiplex molecular analysis. In LCM a polymer film placed in contact with the tissue is focally activated by a pulsed IR laser beam and is melted and bonded to adjacent targeted cells. A three-dimensional finite-element model is used to predict the thermal transients within the polymer, the captured tissue, and its macromolecules. The simulations allow a comparison of models for the physical process of LCM with the experimental data on the dependence of the transfer spot size on laser power. The validated physical model and the thermal simulations permit optimization of the complex LCM parameter space for a wide variety of configurations and applications.

Incorporating global information into secondary structure prediction with hidden Markov models of protein folds.

Here we propose an approach to include global structural information in the secondary structure prediction procedure based on hidden Markov models (HMMs) of protein folds. We first identify the correct fold or 'topology' of a protein by means of the HMMs of topology families of proteins. Then the most likely structural model for that protein is used to modify the sequence of secondary structure states previously obtained with a prediction algorithm. Our goal is to investigate the effect on the prediction accuracy of including global structural information in the secondary structure prediction scheme, by means of the HMMs. We find that when the HMM of the predicted topology of a protein is used to adjust the secondary structure sequence predicted originally with the Quadratic-Logistic method, the cross-validated prediction accuracy (Q3) improves by 3%. The topology is correctly predicted in 68% of the cases. We conclude that this HMM based approach is a promising tool for effectively incorporating global structural information in the secondary structure prediction scheme.

Changes in biologic phenotype of human immunodeficiency virus during treatment of patients with didanosine.

Progression to AIDS in patients harboring human immunodeficiency virus type-1 (HIV-1) isolates expressing a syncytium-inducing (SI) phenotype is faster than in those in whom the virus expresses a non-SI (NSI) phenotype. Zidovudine monotherapy does not appear to alter this outcome. To examine the role of didanosine (ddI) monotherapy in phenotype expression, HIV-1 isolates from 73 patients receiving ddI for up to 72 weeks were analyzed. After 12 weeks, the number of isolates expressing an NSI phenotype was 29% higher than at the start of treatment. Patients receiving high-dose ddI (375 mg twice daily) were significantly more likely to express the NSI phenotype at 12 weeks than patients who received low-dose ddI (100 mg twice daily), even after adjustment for phenotype and CD4 cell count at baseline, suggesting that ddI may be selective against the faster-replicating virus. ddI at 375 mg twice daily significantly increases the probability of an NSI phenotype over the short term in patients with advanced HIV disease.

Primary structure of the V3 region of gp120 from sequential human immunodeficiency virus type 1 isolates obtained from patients from the time of seroconversion.

V3 loop sequences were compared from 5 human immunodeficiency virus type 1 (HIV-1)-infected patients over time. Three patients remained asymptomatic and 2 became symptomatic with large decrease in CD4 cell counts. The patient isolates were previously evaluated for phenotypic and antigenic properties and had different sensitivities to serum neutralization and changes in phenotype. This study showed a number of amino acid changes for the 2 symptomatic patients, each of whom progressed to AIDS during the study. The only amino acid substitution consistently associated with reduced CD4 cell counts, cytopathic effect, and progression to AIDS was Arg at position 11. Specific amino acid changes could not be correlated with increasing serum neutralization resistance or cytotropism changes. Increased loop charge was associated with a switch from macrophage to T cell tropism and a decrease in the number of CD4 cells. The study shows the importance of naturally occurring mutations in the V3 loop in controlling the biologic properties of HIV-1.

Productive in vitro infection of human umbilical vein endothelial cells and three colon carcinoma cell lines with HIV-1.

The objective of this study was to assess the ability of HIV-1 to establish an in vitro infection of primary human umbilical vein endothelial cells (HUVEC). The HUVEC and colon carcinoma cell lines were inoculated with different isolates of HIV-1 (HIV-1SF2, HIV-1Mck and HIV-1LAI) and productive viral infection was assessed by both the detection of p24 core antigen in the culture supernatants and the presence of specific spliced HIV mRNA. The infection which was detected in the inoculated HUVEC and all the colon carcinoma cell lines could not be blocked using an antibody targeted against the CD4 receptor. Furthermore, the HIV-inoculated HUVEC secreted elevated levels of IL-6 and this increase was found to be proportional to the size of the viral inoculum. No changes in the production of IL-1 beta, TNF-alpha, IFN-alpha and IFN-gamma were detected following HIV infection. The colon carcinoma cells, however, did not secrete increased levels of these cytokines following HIV-1 inoculation. These results confirm that non-CD4 expressing cells, such as endothelial cells and certain colon epithelial cells, serve as targets and reservoirs for HIV. Moreover, the production of IL-6 by HIV-infected endothelial cells may be a contributing factor to the aberrant immunoregulation associated with HIV infection in vivo.

Neutralizing antibodies against sequential autologous human immunodeficiency virus type 1 isolates after seroconversion.

The emergence of human immunodeficiency virus type 1 (HIV-1) variants with different sensitivities to serum neutralization and biologic phenotype was studied for 2-5 years after primary HIV-1 infection in 5 subjects. In 3 subjects, the initial virus isolate from seroconversion could be neutralized by autologous serum, but isolates obtained at two subsequent times exhibited reduced sensitivity to serum neutralization, decreased replication in primary macrophages, and increased ability to induce syncytia. Two of these 3 subjects progressed to AIDS and died. Sequential virus isolates from the other 2 subjects showed variability in sensitivity to serum neutralization or biologic features. These patients remained relatively stable in clinical status. Thus, viruses isolated at seroconversion appear to be either non-syncytium-inducing, strong macrophage-tropic, serum neutralization-sensitive phenotypes with stable clinical status or to have escaped neutralization by autologous sera over time, have reduced macrophage tropism and increased syncytia formation, and be associated with disease progression.

Identification of HIV-1 in semen following primary HIV-1 infection.

OBJECTIVE: To determine whether HIV could be identified in semen samples during the first few weeks after infection. DESIGN: A series of three homosexual men with symptomatic primary HIV-1 infection. METHODS: Each subject provided a series of semen samples that was examined for HIV-1 by virus culture, polymerase chain reaction (PCR) and transmission electron micrography. RESULTS: The first samples obtained for each subject (17, 22 and 24 days following onset of primary HIV-1 infection) were all positive by PCR and negative by viral culture. Of 13 samples obtained during the first 80 days after onset of primary HIV-1 infection and analysed by PCR, 10 were positive. Only one of these samples was virus culture-positive. Four semen samples obtained from two subjects during treatment with zidovudine were PCR-positive. Eight samples were examined for presence of HIV-1 by electron microscopy and one was found to be positive. CONCLUSIONS: These results indicate that men with HIV-1 infection are potentially infectious through sexual transmission during the first few weeks after infection. The findings emphasize that individuals in all stages of HIV-1 infection should practise safer sex to reduce transmission of HIV-1.

Any questions.

The South Oxfordshire Branch extend our thanks to Trevor Clay for taking part as a panel member on the BBC Radio 4 Any Questions programme, broadcast from the John Rat-cliffe Hospital, Oxford on March 18.

Prevalence of major mental retardation and associated disabilities in the Canadian Maritime Provinces.

A cross-sectional survey of the prevalence of major mental retardation in 7- to 10-year-olds in the three Canadian Maritime provinces was carried out in 1980. Case ascertainment from multiple sources was considered to be virtually complete and resulted in a study group of 307 children, for whom 72% of the parents consented to personal data collection. The overall regional prevalence of major mental retardation was 3.65/1,000. Variation in county prevalence rate (based on parental residence at the time of birth) was associated, by regression analysis, with maternal age, areas of greater population concentrations, and number of physicians per capita. These associations were still present when children with Down syndrome were excluded from the analysis. Speech and behavior disorders and epilepsy were the major associated disabilities.

Causal origins of major mental handicap in the Canadian Maritime provinces.

From a retrospective epidemiological survey, the prevalence of major mental handicap among seven- to 10-year-old children in the Maritime region of Canada in 1980 was estimated to be 36.5 per 10,000 children. Based on information obtained for 221 of the 307 children ascertained by the survey, prenatal origins dominated (58 per cent), followed by perinatal (10 per cent) and postnatal (4 per cent). No specific cause could be determined for 27 per cent of the children, but 41 per cent of this group had epilepsy and/or cerebral palsy in addition to major mental handicap. Significant differences were found in the causal spectrum between the Maritime region of Canada and regions studied in other developed countries. The results of this survey have implications for planning prevention programs, and for epidemiological surveillance and monitoring of adverse reproductive outcomes.