Benjamas Aiamkitsumrit

Detection of a K-ras mutation in urine of patients with colorectal cancer

We previously demonstrated that human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation, which may be useful in the detection of colorectal cancer. In this report we have determined the stability of DNA in urine and have found that the half-life time interval of this small, fragmented DNA is at least 4 hours post collection. We further compared, in a blinded study, the frequency of detecting mutated K-ras sequence in DNA isolated from plasma and urine derived from individuals who have either a colorectal carcinoma (CRC), or adenomatous polyps that contain a mutation in codon 12 of the K-ras proto-oncogene. There was an 83% concurrence of mutated DNA detected in urine and its corresponding disease tissue from the same individuals, when paired urine and tissue sections from 20 patients with either CRC or adenomatous polyps were analyzed for K-ras mutation. However, only a 56% concurrence was observed when the matched plasma specimens were tested from these 20 patients. These results suggest that urine might be a better resource for detecting K-ras mutation in circulating DNA.

Impact of Tat Genetic Variation on HIV-1 Disease

The human immunodeficiency virus type 1 (HIV-1) promoter or long-terminal repeat (LTR) regulates viral gene expression by interacting with multiple viral and host factors. The viral transactivator protein Tat plays an important role in transcriptional activation of HIV-1 gene expression. Functional domains of Tat and its interaction with transactivation response element RNA and cellular transcription factors have been examined. Genetic variation within tat of different HIV-1 subtypes has been shown to affect the interaction of the viral transactivator with cellular and/or viral proteins, influencing the overall level of transcriptional activation as well as its action as a neurotoxic protein. Consequently, the genetic variability within tat may impact the molecular architecture of functional domains of the Tat protein that may impact HIV pathogenesis and disease. Tat as a therapeutic target for anti-HIV drugs has also been discussed.

Development of co-selected single nucleotide polymorphisms in the viral promoter precedes the onset of human immunodeficiency virus type 1-associated neurocognitive impairment

The long terminal repeat (LTR) regulates gene expression of HIV-1 by interacting with multiple host and viral factors. Cross-sectional studies in the pre-HAART era demonstrated that single nucleotide polymorphisms (SNPs) in peripheral blood-derived LTRs (a C-to-T change at position 3 of C/EBP site I (3T) and at position 5 of Sp site III (5T)) increased in frequency as disease severity increased. Additionally, the 3T variant correlated with HIV-1-associated dementia. LTR sequences derived by longitudinal sampling of peripheral blood from a single patient in the DrexelMed HIV/AIDS Genetic Analysis Cohort resulted in the detection of the 3T and 5T co-selected SNPs before the onset of neurologic impairment, demonstrating that these SNPs may be useful in predicting HIV-associated neurological complications. The relative fitness of the LTRs containing the 3T and/or 5T co-selected SNPs as they evolve in their native patient-derived LTR backbone structure demonstrated a spectrum of basal and Tat-mediated transcriptional activities using the IIIB-derived Tat and colinear Tat derived from the same molecular clone containing the 3T/5T LTR SNP. In silico predictions utilizing colinear envelope sequence suggested that the patient’s virus evolved from an X4 to an R5 swarm prior to the development of neurological complications and more advanced HIV disease. These results suggest that the HIV-1 genomic swarm may evolve during the course of disease in response to selective pressures that lead to changes in prevalence of specific polymorphisms in the LTR, env, and/or tat that could predict the onset of neurological disease and result in alterations in viral function.

Bioinformatic Analysis of HIV-1 Entry and Pathogenesis

The evolution of human immunodeficiency virus type 1 (HIV-1) with respect to co-receptor utilization has been shown to be relevant to HIV-1 pathogenesis and disease. The CCR5-utilizing (R5) virus has been shown to be important in the very early stages of transmission and highly prevalent during asymptomatic infection and chronic disease. In addition, the R5 virus has been proposed to be involved in neuroinvasion and central nervous system (CNS) disease. In contrast, the CXCR4-utilizing (X4) virus is more prevalent during the course of disease progression and concurrent with the loss of CD4(+) T cells. The dual-tropic virus is able to utilize both co-receptors (CXCR4 and CCR5) and has been thought to represent an intermediate transitional virus that possesses properties of both X4 and R5 viruses that can be encountered at many stages of disease. The use of computational tools and bioinformatic approaches in the prediction of HIV-1 co-receptor usage has been growing in importance with respect to understanding HIV-1 pathogenesis and disease, developing diagnostic tools, and improving the efficacy of therapeutic strategies focused on blocking viral entry. Current strategies have enhanced the sensitivity, specificity, and reproducibility relative to the prediction of co-receptor use; however, these technologies need to be improved with respect to their efficient and accurate use across the HIV-1 subtypes. The most effective approach may center on the combined use of different algorithms involving sequences within and outside of the env-V3 loop. This review focuses on the HIV-1 entry process and on co-receptor utilization, including bioinformatic tools utilized in the prediction of co-receptor usage. It also provides novel preliminary analyses for enabling identification of linkages between amino acids in V3 with other components of the HIV-1 genome and demonstrates that these linkages are different between X4 and R5 viruses.

Cocaine alters cytokine profiles in HIV-1-infected African American individuals in the DrexelMed HIV/AIDS genetic analysis cohort.

Background:This study evaluated the relationship between illicit drug use and HIV-1 disease severity in HIV-1–infected patients enrolled in the DREXELMED HIV/AIDS Genetic Analysis Cohort. Because cocaine is known to have immunomodulatory effects, the cytokine profiles of preferential nonusers, cocaine users, and multidrug users were analyzed to understand the effects of cocaine on cytokine modulation and HIV-1 disease severity. Methods:Patients within the cohort were assessed approximately every 6 months for HIV-1 clinical markers and for history of illicit drug, alcohol, and tobacco use. The Luminex human cytokine 30-plex panel was used for cytokine quantitation. Analysis was performed using a newly developed biostatistical model. Results:Substance abuse was common within the cohort. Using the drug screens at the time of each visit, the subjects in the cohort were categorized as preferential nonusers, cocaine users, or multidrug users. The overall health of the nonuser population was better than that of the cocaine users, with peak and current viral loads in nonusers substantially lower than those in cocaine and multidrug users. Among the 30 cytokines investigated, differential levels were established within the 3 populations. The T-helper 2 cytokines, interleukin-4 and -10, known to play a critical role during HIV-1 infection, were positively associated with increasing cocaine use. Clinical parameters such as latest viral load, CD4+ T-cell counts, and CD4:CD8 ratio were also significantly associated with cocaine use, depending on the statistical model used. Conclusions:Based on these assessments, cocaine use seems to be associated with more severe HIV-1 disease.

HIV-1 Genetic Variation Resulting in the Development of New Quasispecies Continues to Be Encountered in the Peripheral Blood of Well- Suppressed Patients.

As a result of antiretroviral therapeutic strategies, human immunodeficiency virus type 1 (HIV-1) infection has become a long-term clinically manageable chronic disease for many infected individuals. However, despite this progress in therapeutic control, including undetectable viral loads and CD4+ T-cell counts in the normal range, viral mutations continue to accumulate in the peripheral blood compartment over time, indicating either low level reactivation and/or replication. Using patients from the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort, whom have been sampled longitudinally for more than 7 years, genetic change was modeled against to the dominant integrated proviral quasispecies with respect to selection pressures such as therapeutic interventions, AIDS defining illnesses, and other factors. Phylogenetic methods based on the sequences of the LTR and tat exon 1 of the HIV-1 proviral DNA quasispecies were used to obtain an estimate of an average mutation rate of 5.3 nucleotides (nt)/kilobasepair (kb)/year (yr) prior to initiation of antiretroviral therapy (ART). Following ART the baseline mutation rate was reduced to an average of 1.02 nt/kb/yr. The post-ART baseline rate of genetic change, however, appears to be unique for each patient. These studies represent our initial steps in quantifying rates of genetic change among HIV-1 quasispecies using longitudinally sampled sequences from patients at different stages of disease both before and after initiation of combination ART. Notably, while long-term ART reduced the estimated mutation rates in the vast majority of patients studied, there was still measurable HIV-1 mutation even in patients with no detectable virus by standard quantitative assays. Determining the factors that affect HIV-1 mutation rates in the peripheral blood may lead to elucidation of the mechanisms associated with changes in HIV-1 disease severity.

HIV-1 Promoter Single Nucleotide Polymorphisms Are Associated with Clinical Disease Severity.

The large majority of human immunodeficiency virus type 1 (HIV-1) markers of disease progression/severity previously identified have been associated with alterations in host genetic and immune responses, with few studies focused on viral genetic markers correlate with changes in disease severity. This study presents a cross-sectional/longitudinal study of HIV-1 single nucleotide polymorphisms (SNPs) contained within the viral promoter or long terminal repeat (LTR) in patients within the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. HIV-1 LTR SNPs were found to associate with the classical clinical disease parameters CD4+ T-cell count and log viral load. They were found in both defined and undefined transcription factor binding sites of the LTR. A novel SNP identified at position 108 in a known COUP (chicken ovalbumin upstream promoter)/AP1 transcription factor binding site was significantly correlated with binding phenotypes that are potentially the underlying cause of the associated clinical outcome (increase in viral load and decrease in CD4+ T-cell count).

Construction of a Herpes Simplex Virus Type 1 Mutant with Only a Three-Nucleotide Change in the Branchpoint Region of the Latency-Associated Transcript (LAT) and the Stability of Its Two-Kilobase LAT Intron

ABSTRACT Previous studies using a eukaryotic expression system indicated that the unusual stability of the latency-associated transcript (LAT) intron was due to its nonconsensus branchpoint sequence (T.-T Wu, Y.-H. Su, T. M. Block, and J. M. Taylor, Virology, 243:140-149, 1998). The present study investigated the role of the branchpoint sequence in the stability of the intron expressed from the herpes simplex virus type 1 (HSV-1) genome and the role of LAT intron stability in the HSV-1 life cycle. A branchpoint mutant called Sy2 and the corresponding rescued viruses, SyRA and SyRB, were constructed. To preserve the coding sequence of the immediate early gene icp0, which overlaps with the branchpoint region of the 2-kb LAT, a 3-nucleotide mutation into the branchpoint region of the 2-kb LAT was introduced, resulting in a branchpoint that is 85% identical to the consensus intron branchpoint sequence of eukaryotic cells. As anticipated, there was a 90- to 96-fold reduction in 2-kb LAT accumulation following productive infection in tissue culture and latent infection in mice with Sy2, as determined by Northern blot analysis. These results clearly suggest that the accumulation of the 2-kb intron in tissue culture and in vivo is, at least in part, due to the nonconsensus branchpoint sequence of the LAT intron. Interestingly, a failure to accumulate LAT was associated with greater progeny production of Sy2 at a low multiplicity of infection (0.01) in tissue culture, but not in mice. However, the ability of mutant Sy2 to reactivate from trigeminal ganglia (TG) derived from latently infected mice was indistinguishable from that of wild-type virus, as assayed in the mouse TG explant reactivation system.

Defining Differential Genetic Signatures in CXCR4- and the CCR5-Utilizing HIV-1 Co-Linear Sequences

The adaptation of human immunodeficiency virus type-1 (HIV-1) to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4) viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5) viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM) algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR) transcription factor sites within co-linear viral protein R (Vpr), trans-activator of transcription (Tat), or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co-linear sequences in HIV-1-infected patients.

Utilization of HIV-1 envelope V3 to identify X4- and R5-specific Tat and LTR sequence signatures

BackgroundHIV-1 entry is a receptor-mediated process directed by the interaction of the viral envelope with the host cell CD4 molecule and one of two co-receptors, CCR5 or CXCR4. The amino acid sequence of the third variable (V3) loop of the HIV-1 envelope is highly predictive of co-receptor utilization preference during entry, and machine learning predictive algorithms have been developed to characterize sequences as CCR5-utilizing (R5) or CXCR4-utilizing (X4). It was hypothesized that while the V3 loop is predominantly responsible for determining co-receptor binding, additional components of the HIV-1 genome may contribute to overall viral tropism and display sequence signatures associated with co-receptor utilization.ResultsThe accessory protein Tat and the HlV-1 long terminal repeat (LTR) were analyzed with respect to genetic diversity and compared by Jensen–Shannon divergence which resulted in a correlation with both mean genetic diversity as well as the absolute difference in genetic diversity between R5- and X4-genome specific trends. As expected, the V3 domain of the gp120 protein was enriched with statistically divergent positions. Statistically divergent positions were also identified in Tat amino acid sequences within the transactivation and TAR-binding domains, and in nucleotide positions throughout the LTR. We further analyzed LTR sequences for putative transcription factor binding sites using the JASPAR transcription factor binding profile database and found several putative differences in transcription factor binding sites between R5 and X4 HIV-1 genomes, specifically identifying the C/EBP sites I and II, and Sp site III to differ with respect to sequence configuration for R5 and X4 LTRs.ConclusionThese observations support the hypothesis that co-receptor utilization coincides with specific genetic signatures in HIV-1 Tat and the LTR, likely due to differing transcriptional regulatory mechanisms and selective pressures applied within specific cellular targets during the course of productive HIV-1 infection.