Ryung S. Kim

Limited immune restoration after 3 years' suppression of HIV-1 replication in patients with moderately advanced disease.

Objective: To describe the magnitude of immune restoration after long-term control of HIV-1 replication. Design: Prospective study of immune restoration in patients starting highly active antiretroviral therapy (HAART). Methods: Patients with moderately advanced HIV-1 infection (CD4 cells between 100 × 106 and 300 × 106/l) who enrolled in a trial of HAART and who had suppression of HIV-1 replication during 3 years of therapy were evaluated for phenotypic and functional indices of immune restoration. Results: Almost all immune restoration achieved occurred during the first year. The median CD4 lymphocyte count increased by 159 × 106 cells/l during the first year (P < 0.001); CD4 lymphocyte rises during the second and third years were not significant. Most decreases in activation antigen expression (CD38/HLA-DR) on CD4 and CD8 lymphocytes occurred during the first year, and after 3 years, patient lymphocytes were still abnormally activated. The proportion of CD4 lymphocytes expressing CD28 increased during the first and second years, but even after 3 years, CD28 expression on CD4 cells remained abnormally low. Lymphocyte proliferative responses to Candida normalized during the first 12 weeks of HAART while responses to tetanus increased only after immunization and enhanced responses to HIV-1 p24 antigen were not observed. Conclusions: Immune restoration was incomplete in patients who started HAART with moderately advanced HIV-1 disease and most changes occurred during the first year. These data suggest that this degree of suppression of HIV-1 replication alone will not suffice to restore immune competence. The clinical significance of incomplete reconstitution of CD4 lymphocyte number, phenotype, and proliferative function in HIV-1 infection remains to be determined.

The RhoGAP Protein DLC-1 Functions as a Metastasis Suppressor in Breast Cancer Cells

The identification of molecular signatures characteristic of tumor cells that are capable of metastatic spread is required for the development of therapeutic interventions to abrogate this lethal process. To facilitate this, we have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 are spontaneously occurring sublines of the MDA-MB-435 cell breast tumor cell line that exhibit phenotypic differences in growth, invasion, and metastatic efficiency in athymic mice. In this study, transcriptional profiles of these cell lines were created using oligonucleotide microarrays representing over 12,000 genes. Intensity modeling and hierarchical clustering analysis identified a 171-gene expression signature that correlated with metastatic phenotype and highlighted several GTPase signaling components. Restoration of one of these GTPases, deleted in liver cancer-1 (DLC-1), in metastatic M4A4 cells to levels observed in the nonmetastatic NM2C5 cell line resulted in the inhibition of migration and invasion in vitro and a significant reduction in the ability of these cells to form pulmonary metastases in athymic mice. These studies show the utility of expression profiling, in an appropriate experimental system, to identify genetic determinants of metastatic sufficiency. The finding that DLC-1 can act as a metastasis-suppressor gene supports an influential role for GTPase signaling in tumor progression.

Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer.

Breast cancers can recur after removal of the primary tumor and treatment to eliminate remaining tumor cells. Recurrence may occur after long periods of time during which there are no clinical symptoms. Tumor cell dormancy may explain these prolonged periods of asymptomatic residual disease and treatment resistance. We generated a dormancy gene signature from published experimental models and applied it to both breast cancer cell line expression data as well as four published clinical studies of primary breast cancers. We found that estrogen receptor (ER) positive breast cell lines and primary tumors have significantly higher dormancy signature scores (P<0.0000001) than ER- cell lines and tumors. In addition, a stratified analysis combining all ER+ tumors in four studies indicated 2.1 times higher hazard of recurrence among patients whose tumors had low dormancy scores (LDS) compared to those whose tumors had high dormancy scores (HDS) (p<0.000005). The trend was shown in all four individual studies. Suppression of two dormancy genes, BHLHE41 and NR2F1, resulted in increased in vivo growth of ER positive MCF7 cells. The patient data analysis suggests that disseminated ER positive tumor cells carrying a dormancy signature are more likely to undergo prolonged dormancy before resuming metastatic growth. Furthermore, genes identified with this approach might provide insight into the mechanisms of dormancy onset and maintenance as well as dormancy models using human breast cancer cell lines.

Role of Epidermal Growth Factor Receptor Signaling in RAS-Driven Melanoma

ABSTRACT The identification of essential genetic elements in pathways governing the maintenance of fully established tumors is critical to the development of effective antioncologic agents. Previous studies revealed an essential role for H-RASV12G in melanoma maintenance in an inducible transgenic model. Here, we sought to define the molecular basis for RAS-dependent tumor maintenance through determination of the H-RASV12G-directed transcriptional program and subsequent functional validation of potential signaling surrogates. The extinction of H-RASV12G expression in established tumors was associated with alterations in the expression of proliferative, antiapoptotic, and angiogenic genes, a profile consistent with the observed phenotype of tumor cell proliferative arrest and death and endothelial cell apoptosis during tumor regression. In particular, these melanomas displayed a prominent RAS-dependent regulation of the epidermal growth factor (EGF) family, leading to establishment of an EGF receptor signaling loop. Genetic complementation and interference studies demonstrated that this signaling loop is essential to H-RASV12G-directed tumorigenesis. Thus, this inducible tumor model system permits the identification and validation of alternative points of therapeutic intervention without neutralization of the primary genetic lesion.

Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.

Immunophenotypic Markers and Antiretroviral Therapy (IMART): T Cell Activation and Maturation Help Predict Treatment Response

To determine whether markers of T cell activation and maturation are independently predictive of the response to potent antiretroviral therapy, the Immunophenotypic Markers and Antiretroviral Therapy study applied a novel data-sharing strategy across 5 Adult AIDS Clinical Trial Group trials that counted naive and activated CD4(+) and CD8(+) T cells in 324 subjects. Regression models--adjustment for baseline CD4 cell count, human immunodeficiency virus (HIV) RNA, and study--revealed that high pretreatment CD8(+) T cell activation predicted virologic failure (P=.046). Additional models showed the greatest increase in CD4(+) T cell counts in subjects with highest pretreatment naive CD4(+) T cell counts (P<.0001), which was enhanced by high CD4(+) and low CD8(+) T cell activation. Total lymphocyte count also predicted a subsequent CD4(+) T cell change. These results document the utility of T cell markers in predicting treatment outcome and their potential value for the study and management of HIV-1 infection.

Body Mass Index, Sex, and Cardiovascular Disease Risk Factors Among Hispanic/Latino Adults: Hispanic Community Health Study/Study of Latinos

Background All major Hispanic/Latino groups in the United States have a high prevalence of obesity, which is often severe. Little is known about cardiovascular disease (CVD) risk factors among those at very high levels of body mass index (BMI). Methods and Results Among US Hispanic men (N=6547) and women (N=9797), we described gradients across the range of BMI and age in CVD risk factors including hypertension, serum lipids, diabetes, and C‐reactive protein. Sex differences in CVD risk factor prevalences were determined at each level of BMI, after adjustment for age and other demographic and socioeconomic variables. Among those with class II or III obesity (BMI ≥35 kg/m2, 18% women and 12% men), prevalences of hypertension, diabetes, low high‐density lipoprotein cholesterol level, and high C‐reactive protein level approached or exceeded 40% during the fourth decade of life. While women had a higher prevalence of class III obesity (BMI ≥40 kg/m2) than did men (7% and 4%, respectively), within this highest BMI category there was a >50% greater relative prevalence of diabetes, hypertension, and hyperlipidemia in men versus women, while sex differences in prevalence of these CVD risk factors were ≈20% or less at other BMI levels. Conclusions Elevated BMI is common in Hispanic/Latino adults and is associated with a considerable excess of CVD risk factors. At the highest BMI levels, CVD risk factors often emerge in the earliest decades of adulthood and they affect men more often than women.

Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction

Background. The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. Methods. Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. Results. Immunoglobulin G (IgG) responses to AM increased significantly 4–8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. Conclusions. Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans.

Prevalence of Hepatitis C Virus Infection in US Hispanic/Latino Adults: Results From the NHANES 2007–2010 and HCHS/SOL Studies

Prevalence of hepatitis C virus (HCV) antibody has been reported in Mexican Americans, but its prevalence in other US Hispanic/Latino groups is unknown. We studied 2 populations of US Hispanic/Latino adults; 3210 from the National Health and Nutrition Examination Survey (NHANES) 2007-2010 and 11 964 from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL). Age-standardized prevalence of HCV antibody was similar in NHANES 2007-2010 (1.5%) and HCHS/SOL (2.0%) but differed significantly by Hispanic/Latino background in HCHS/SOL (eg, 11.6% in Puerto Rican men vs 0.4% in South American men). These findings suggest that the HCV epidemic among US Hispanics/Latinos is heterogeneous.

Dietary patterns and breast cancer risk: a study in 2 cohorts

BACKGROUND Evidence for a role of dietary risk factors in the cause of breast cancer has been inconsistent. The evaluation of overall dietary patterns instead of foods in isolation may better reflect the nature of true dietary exposure in a population. OBJECTIVE We used 2 cohort studies to identify and confirm associations between dietary patterns and breast cancer risk. DESIGN Dietary patterns were derived by using a principal components factor analysis in 1097 breast cancer cases and an age-stratified subcohort of 3320 women sampled from 39,532 female participants in the Canadian Study of Diet, Lifestyle and Health (CSDLH). We conducted a confirmatory factor analysis in 49,410 subjects in the National Breast Screening Study (NBSS) in whom 3659 cases of incident breast cancer developed. Cox regression models were used to estimate HRs for the association between derived dietary factors and risk of breast cancer in both cohorts. RESULTS The following 3 dietary factors were identified from the CSDLH: healthy, ethnic, and meat and potatoes. In the CSDLH, the healthy dietary pattern was associated with reduced risk of breast cancer (HR for high compared with low quintiles: 0.73; 95% CI: 0.58, 0.91; P-trend = 0.001), and the meat and potatoes dietary pattern was associated with increased risk in postmenopausal women only (HR for high compared with low quintiles: 1.26; 95% CI: 0.92, 1.73; P-trend = 0.043). In the NBSS, the association between the meat and potatoes pattern and postmenopausal breast cancer risk was confirmed (HR: 1.31; 95% CI: 0.98, 1.76; P-trend = 0.043), but there was no association between the healthy pattern and risk of breast cancer. CONCLUSION Adherence to a plant-based diet that limits red meat intake may be associated with reduced risk of breast cancer, particularly in postmenopausal women.