HIV-Infected Individuals with Low CD4/CD8 Ratio despite Effective Antiretroviral Therapy Exhibit Altered T Cell Subsets,Heightened CD8+ T Cell Activation,and Increased Risk of Non-AIDS Morbidity and Mortality

A low CD4/CD8 ratio in elderly HIV-uninfected adults is associated with increased morbidity and mortality. A subset of HIV-infected adults receiving effective antiretroviral therapy (ART) fails to normalize this ratio, even after they achieve normal CD4+ T cell counts. The immunologic and clinical characteristics of this clinical phenotype remain undefined. Using data from four distinct clinical cohorts and three clinical trials, we show that a low CD4/CD8 ratio in HIV-infected adults during otherwise effective ART (after CD4 count recovery above 500 cells/mm³) is associated with a number of immunological abnormalities, including a skewed T cell phenotype from naïve toward terminally differentiated CD8+ T cells, higher levels of CD8+ T cell activation (HLADR+CD38+) and senescence (CD28− and CD57+CD28−), and higher kynurenine/tryptophan ratio. Changes in the peripheral CD4/CD8 ratio are also reflective of changes in gut mucosa, but not in lymph nodes. In a longitudinal study, individuals who initiated ART within six months of infection had greater CD4/CD8 ratio increase compared to later initiators (>2 years). After controlling for age, gender, ART duration, nadir and CD4 count, the CD4/CD8 ratio predicted increased risk of morbidity and mortality. Hence, a persistently low CD4/CD8 ratio during otherwise effective ART is associated with increased innate and adaptive immune activation, an immunosenescent phenotype, and higher risk of morbidity/mortality. This ratio may prove useful in monitoring response to ART and could identify a unique subset of individuals needed of novel therapeutic interventions.     

Direct measurement of T-cell receptor repertoire diversity with AmpliCot

Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA concentration. The DNA is melted and then reannealed under stringent conditions that allow only homoduplexes to form. The sequence diversity of a sample is proportional to the product of its concentration and the time required for it to anneal. After validating AmpliCot with a library of diverse sequences, we use it to measure the diversity of expressed rearrangements of the gene encoding the T-cell antigen receptor (TCR) beta chain. AmpliCot measurements are in good agreement with previous estimates of murine TCR repertoire diversity that required extensive cloning and sequencing.     

Prospective Antiretroviral Treatment of Asymptomatic,HIV-1 Infected Controllers

The study of HIV-infected “controllers” who are able to maintain low levels of plasma HIV RNA in the absence of antiretroviral therapy (ART) may provide insights for HIV cure and vaccine strategies. Despite maintaining very low levels of plasma viremia, controllers have elevated immune activation and accelerated atherosclerosis. However, the degree to which low-level replication contributes to these phenomena is not known. Sixteen asymptomatic controllers were prospectively treated with ART for 24 weeks. Controllers had a statistically significant decrease in ultrasensitive plasma and rectal HIV RNA levels with ART. Markers of T cell activation/dysfunction in blood and gut mucosa also decreased substantially with ART. Similar reductions were observed in the subset of “elite” controllers with pre-ART plasma HIV RNA levels below conventional assays (<40 copies/mL). These data confirm that HIV replication persists in controllers and contributes to a chronic inflammatory state. ART should be considered for these individuals (ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01025427","term_id":"NCT01025427"}}NCT01025427).     

Design, construction, and validation of a modular library of sequence diversity standards for polymerase chain reaction

Methods to measure the sequence diversity of polymerase chain reaction (PCR)-amplified DNA lack standards for use as assay calibrators and controls. Here we present a general and economical method for developing customizable DNA standards of known sequence diversity. Standards ranging from 1 to 25,000 sequences were generated by directional ligation of oligonucleotide “words” of standard length and GC content and then amplified by PCR. The sequence accuracy and diversity of the library were validated using AmpliCot analysis (DNA hybridization kinetics) and Illumina sequencing. The library has the following features: (i) pools containing tens of thousands of sequences can be generated from the ligation of relatively few commercially synthesized short oligonucleotides; (ii) each sequence differs from all others in the library at a minimum of three nucleotide positions, permitting discrimination between different sequences by either sequencing or hybridization; (iii) all sequences have identical length, GC content, and melting temperature; (iv) the identity of each standard can be verified by restriction digestion; and (v) once made, the ends of the library may be cleaved and replaced with sequences to match any PCR primer pair. These standards should greatly improve the accuracy and reproducibility of sequence diversity measurements.     

A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers

HIV-infected individuals maintaining undetectable viremia in the absence of therapy (HIV controllers) often maintain high HIV-specific T cell responses, which has spurred the development of vaccines eliciting HIV-specific T cell responses. However, controllers also often have abnormally high T cell activation levels, potentially contributing to T cell dysfunction, CD4+ T cell depletion, and non-AIDS morbidity. We hypothesized that a weak T regulatory cell (Treg) response might contribute to the control of viral replication in HIV controllers, but might also contribute to generalized immune activation, contributing to CD4+ T cell loss. To address these hypotheses, we measured frequencies of activated (CD38+ HLA-DR+), regulatory (CD4+CD25+CD127(dim)), HIV-specific, and CMV-specific T cells among HIV controllers and 3 control populations: HIV-infected individuals with treatment-mediated viral suppression (ART-suppressed), untreated HIV-infected "non-controllers" with high levels of viremia, and HIV-uninfected individuals. Despite abnormally high T cell activation levels, controllers had lower Treg frequencies than HIV-uninfected controls (P = 0.014). Supporting the propensity for an unusually low Treg response to viral infection in HIV controllers, we observed unusually high CMV-specific CD4+ T cell frequencies and a strong correlation between HIV-specific CD4+ T cell responses and generalized CD8+ T cell activation levels in HIV controllers (P ≤ 0.001). These data support a model in which low frequencies of Tregs in HIV controllers may contribute to an effective adaptive immune response, but may also contribute to generalized immune activation, potentially contributing to CD4 depletion.     

Discriminating lymphomas and reactive lymphadenopathy in lymph node biopsies by gene expression profiling

Background

Diagnostic accuracy of lymphoma, a heterogeneous cancer, is essential for patient management. Several ancillary tests including immunophenotyping, and sometimes cytogenetics and PCR are required to aid histological diagnosis. In this proof of principle study, gene expression microarray was evaluated as a single platform test in the differential diagnosis of common lymphoma subtypes and reactive lymphadenopathy (RL) in lymph node biopsies.

Methods

116 lymph node biopsies diagnosed as RL, classical Hodgkin lymphoma (cHL), diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL) were assayed by mRNA microarray. Three supervised classification strategies (global multi-class, local binary-class and global binary-class classifications) using diagonal linear discriminant analysis was performed on training sets of array data and the classification error rates calculated by leave one out cross-validation. The independent error rate was then evaluated by testing the identified gene classifiers on an independent (test) set of array data.

Results

The binary classifications provided prediction accuracies, between a subtype of interest and the remaining samples, of 88.5%, 82.8%, 82.8% and 80.0% for FL, cHL, DLBCL, and RL respectively. Identified gene classifiers include LIM domain only-2 (LMO2), Chemokine (C-C motif) ligand 22 (CCL22) and Cyclin-dependent kinase inhibitor-3 (CDK3) specifically for FL, cHL and DLBCL subtypes respectively.

Conclusions

This study highlights the ability of gene expression profiling to distinguish lymphoma from reactive conditions and classify the major subtypes of lymphoma in a diagnostic setting. A cost-effective single platform "mini-chip" assay could, in principle, be developed to aid the quick diagnosis of lymph node biopsies with the potential to incorporate other pathological entities into such an assay.

     

Equations for potential annual direct incident radiation and heat load

Abstract. Estimation of potential annual direct incident radiation has traditionally required numerical integration with simulation models. As an alternative, we present convenient equations for use in spreadsheet, GIS, and database applications. Input variables are latitude, slope, and aspect. The equations apply to 0–60° north latitude, slopes from 0–90°, and all aspects. By transforming aspect, the equations can also be applied as an index of heat load, symmetrical about a northeast to southwest axis.     

IFN-alpha secretion by type 2 predendritic cells up-regulates MHC class I in the HIV-1-infected thymus.

The ability of HIV-1 to evade the host immune response leads to the establishment of chronic infection. HIV-1 has been reported to up-regulate MHC I molecules on the surface of thymocytes from HIV-1-infected thymus. We demonstrate in this study that HIV-1 up-regulates MHC I on both HIV-1-infected and uninfected thymocytes in a manner that is independent of Nef, proportional to viral replication, and entirely mediated by IFN-alpha. IL-3Ralpha+ type 2 predendritic cells (preDC2) resident in the thymic medulla secrete IFN-alpha, which acts on IFN-alphabetaR-expressing immature thymocytes to induce MHC I expression. Furthermore, thymic preDC2 are permissive for HIV-1 infection and positive for intracellular p24. These data demonstrate the ability of IFN-alpha secreted by preDC2 to induce MHC I up-regulation in the HIV-1-infected human thymus.