Fluidity of HIV-1-Specific T-Cell Responses during Acute and Early Subtype C HIV-1 Infection and Associations with Early Disease Progression

Deciphering immune events during early stages of human immunodeficiency virus type 1 (HIV-1) infection is critical for understanding the course of disease. We characterized the hierarchy of HIV-1-specific T-cell gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses during acute subtype C infection in 53 individuals and associated temporal patterns of responses with disease progression in the first 12 months. There was a diverse pattern of T-cell recognition across the proteome, with the recognition of Nef being immunodominant as early as 3 weeks postinfection. Over the first 6 months, we found that there was a 23% chance of an increased response to Nef for every week postinfection (P = 0.0024), followed by a nonsignificant increase to Pol (4.6%) and Gag (3.2%). Responses to Env and regulatory proteins appeared to remain stable. Three temporal patterns of HIV-specific T-cell responses could be distinguished: persistent, lost, or new. The proportion of persistent T-cell responses was significantly lower (P = 0.0037) in individuals defined as rapid progressors than in those progressing slowly and who controlled viremia. Almost 90% of lost T-cell responses were coincidental with autologous viral epitope escape. Regression analysis between the time to fixed viral escape and lost T-cell responses (r = 0.61; P = 0.019) showed a mean delay of 14 weeks after viral escape. Collectively, T-cell epitope recognition is not a static event, and temporal patterns of IFN-γ-based responses exist. This is due partly to viral sequence variation but also to the recognition of invariant viral epitopes that leads to waves of persistent T-cell immunity, which appears to associate with slower disease progression in the first year of infection.For more than a decade, there has been a wealth of evidence to show that human immunodeficiency virus (HIV)-specific cytotoxic T-cell (CTL) responses play a role in the control of HIV-1 and simian immunodeficiency virus (SIV) infection. In humans, the first appearance of CTL in primary HIV-1 infection coincides with the decline of peak viremia (7, 27), while depletion of CD8⁺ T cells in SIV infection resulted in elevated viremia (45). Additionally, polymorphisms in HLA class I-restricted CTL responses are associated with differential HIV-1 disease outcomes (25), and the emergence of viral escape within CTL epitopes during acute and chronic SIV or HIV-1 infection demonstrates the effectiveness of CD8⁺ T cells to exert viral selection pressure (21). Dissecting the specificity of HIV-1-specific CD8⁺ T-cell responses that associate with the control of viral replication during acute/early infection is thought to be critical for the design of vaccines and potential immunotherapeutic strategies aimed at stimulating these responses.Preferential targeting of class I-restricted CTL epitopes in Gag during early and chronic HIV-1 infection has been associated with lower viral loads (15, 25, 34, 48, 55), whereas Env- and Nef-specific CD8⁺ T-cell responses have been associated with higher viremia (15, 34, 55). Increasing evidence suggests that patterns of immunodominant HIV-specific CD8⁺ T-cell responses restricted by specific HLA alleles are major determinants of the viral set point (47). In addition, Goonetilleke et al. (17) have provided insight into the rapidity of early escape and the contribution of the first HIV-specific CD8⁺ T-cell responses to the transmitted/founder virus in control of acute viremia. The restriction of CTL epitopes by HLA-B*5801, for example, has also been associated with better viral control (16, 24). However, the temporal nature of epitope-specific responses that associate with viral control has not been explored. Recently, we found no association between the magnitude and breadth of gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses at a static 3-month time point with the viral set point at 12 months (22). The unpredictability of early T-cell responses with later viral control could be a result of HIV variability resulting in epitope escape from humoral and T-cell pressure (1, 8). For example, the impact of CTL pressure on shaping viral diversity at a human population level has been observed through HLA imprinting (6, 9, 44), and several studies have shown that certain selected escape mutations can compromise viral fitness (10, 29, 33, 39). Other studies have also demonstrated that the selection of escape variants in chronic HIV-1 and SIV infection can result in the loss of immune control and disease progression (3, 20). Assessing the nature of T-cell responses longitudinally and relating the patterns of contemporaneous viral recognition with viral diversity may represent alternative insights into factors associated with set point and disease progression.As the global AIDS epidemic continues to expand in sub-Saharan Africa, and South Africa in particular, the need to implement a preventive vaccine through the public health sector remains paramount. To date, several prototype antibody and T-cell-based candidate vaccine trials have been completed worldwide (37), and the recent failure of a phase IIb Ad5-Gag-Pol-Nef HIV-1 vaccine trial has emphasized the challenge of producing an effective T-cell-based vaccine against HIV. Data from the recent ALVAC and AIDSVAX (RV144) trials in Thailand have provided modest efficacy of a vaccine regimen in reducing HIV infection (42), and while the immune mechanisms for this are as yet unclear, these findings have created a platform for identifying immune responses that correlate with protection.The identification of the earliest targets of T cells during acute HIV-1 infection would be helpful in understanding the evolution of immunity when a host first encounters the virus and also would provide insight into the host-pathogen interplay when there is a rapidly changing target. We describe some of the earliest T-cell responses that occur during acute subtype C HIV-1 infection, how these change over time and associate with early disease progression, as well as the kinetics of these changes in relation to autologous viral escape.     

Methods of nonlinear dynamics in estimation of electroencephalograms of healthy people and of patients with epilepsy

A possibility is discussed of use of methods of nonlinear dynamics for analysis of spontaneous EEG and of the EEG caused by low acoustic stimuli in healthy people and in patients with epilepsy. The use of methods of nonlinear dynamics—the fractal dimension of EEG—in clinical and scientific practice is described.     

Exploring the potential of the bacterial carotene desaturase CrtI to increase the beta-carotene content in Golden Rice

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.     

Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo

Self-specific CD8 T cells, which are selected by high-affinity interactions with self-Ags, develop into a lineage distinct from conventional CD8 T cells. We have previously shown that these self-specific cells acquire phenotypic and functional similarities to cells of the innate immune system including the expression of functional receptors associated with NK cells. In this study, we show that these self-specific cells have the ability to produce large amounts of IFN-gamma in response to infection with Listeria monocytogenes in a bystander fashion. The rapid production of IFN-gamma is associated with a dramatic reduction in the number of viable bacteria at the peak of infection. Self-specific CD8 T cells provide only marginal innate protection in the absence of self-Ag; however, the presence of self-Ag dramatically increases their protective ability. Exposure to self-Ag is necessary for the maintenance of the memory phenotype and responsiveness to inflammatory cytokines such as IL-15. Significantly, self-specific CD8 T cells are also more efficient in the production of IFN-gamma and TNF-alpha, thus providing more cytokine-dependent protection against bacterial infection when compared with NK cells. These findings illustrate that self-reactive CD8 T cells can play an important innate function in the early defense against bacterial infection.     

The role of non-classical supramolecular interactions in the structures of 2-amino-4,6-dimethylpyridinium tetrahalocuprate (II) salts

The compounds [2-amino-4,6-dimethylpyridinium]₂CuCl₄ (1) and [2-amino-4,6-dimethylpyridinium]₂CuBr₄ (2) were prepared from acidic ethanolic media containing CuX₂ and [2-amino-4,6-dimethylpyridine] in the molar ratio 1:1. The compounds were characterized by IR and single-crystal X-ray diffraction and found to be isomorphous in the space group with V = 991.2(10) Å³ for (1) and 1059.26(12) Å³ for (2) . There is no significant difference in the non-classical N-H?X hydrogen bonding between (1) and (2). The anions show essentially the same extent of distortion from tetrahedral geometry with max./min. values for the X-Cu-X bond angles of 139.72(6)°/96.78(6)° for (1) and 139.43(4)°/96.64(3)° for (2). Each [CuX₄]²⁻ anion is hydrogen bonded nonsymmetrically to four cations. In this manner, ladder chains are formed that run along the b-axis, with planar cations falling parallel to the (2, 0, 1) plane. Weaker π-π interactions exist between cations from different chains with centroid to centroid distance of 4.07 Å in (1) and a long 4.594 Å in (2). The X-π electrostatic interactions are surprisingly stronger in (2) than in (1) with a Br to centroid of pyridinic ring distance of 3.890 Å compared with 3.996 Å for the chloride analogue.     

Substrates induce conformational changes in human anion exchanger 1 (hAE1) as observed by fluorescence resonance energy transfer

The one-for-one exchange of Cl(-) and HCO(3)(-) ions is catalyzed by human erythrocyte anion exchanger 1 (hAE1) through a ping-pong mechanism whereby the protein exists in two main conformations, with the single anion-binding site exposed at either the cytoplasmic (inner) side (E(i)) or the extracellular side (E(o)), with interconversion between the two states being possible only after anion binding. Steady-state and time-resolved resonance energy transfer (FRET) techniques were used to determine the distance of the binding site for diTBA (bis-(1,3-diethylthiobarbituric acid)trimethine oxonol), a high affinity fluorescent oxonol inhibitor of hAE1, from a benchmark site (probably Lys-430) labeled by external fluorescein maleimide (FM). Using red cell ghost membranes, energy transfer distances were measured in media containing different anions between FM as the donor, covalently attached to one monomer, and diTBA as the acceptor, reversibly bound to the adjacent monomer of a hAE1 dimer. Energy transfer increased significantly in chloride or bicarbonate buffers relative to conditions where no transportable anions were present, that is, in citrate buffer. These differences in transfer efficiencies were interpreted in light of the conformational distributions of hAE1 in various buffers and the possible effects of diTBA itself on the distribution. The analysis indicates that the diTBA binding site comes closer to the FM site by approximately 7 A in chloride buffer as compared to that in citrate (or equivalent changes in diTBA orientation occur) because of the effects of anion binding. This provides the first direct physical evidence for structural changes in hAE1 induced by substrates.     

Alcohol consumption and the risk of incident atrial fibrillation among people with cardiovascular disease

Background:

Moderate alcohol consumption may reduce cardiovascular events, but little is known about its effect on atrial fibrillation in people at high risk of such events. We examined the association between moderate alcohol consumption and the risk of incident atrial fibrillation among older adults with existing cardiovascular disease or diabetes.

Methods:

We analyzed data for 30 433 adults who participated in 2 large antihypertensive drug treatment trials and who had no atrial fibrillation at baseline. The patients were 55 years or older and had a history of cardiovascular disease or diabetes with end-organ damage. We classified levels of alcohol consumption according to median cut-off values for low, moderate and high intake based on guidelines used in various countries, and we defined binge drinking as more than 5 drinks a day. The primary outcome measure was incident atrial fibrillation.

Results:

A total of 2093 patients had incident atrial fibrillation. The age- and sex-standardized incidence rate per 1000 person-years was 14.5 among those with a low level of alcohol consumption, 17.3 among those with a moderate level and 20.8 among those with a high level. Compared with participants who had a low level of consumption, those with higher levels had an increased risk of incident atrial fibrillation (adjusted hazard ratio [HR] 1.14, 95% confidence interval [CI] 1.04–1.26, for moderate consumption; 1.32, 95% CI 0.97–1.80, for high consumption). Results were similar after we excluded binge drinkers. Among those with moderate alcohol consumption, binge drinkers had an increased risk of atrial fibrillation compared with non–binge drinkers (adjusted HR 1.29, 95% CI 1.02–1.62).

Interpretation:

Moderate to high alcohol intake was associated with an increased incidence of atrial fibrillation among people aged 55 or older with cardiovascular disease or diabetes. Among moderate drinkers, the effect of binge drinking on the risk of atrial fibrillation was similar to that of habitual heavy drinking.A trial fibrillation is associated with an increased risk of stroke and a related high burden of mortality and morbidity, both in the general public and among patients with existing cardiovascular disease.^1,2 The prevalence of atrial fibrillation increases steadily with age, as do the associated risks, and atrial fibrillation accounts for up to 23.5% of all strokes among elderly people.³Moderate alcohol consumption has been reported to be associated with a reduced risk of cardiovascular disease and all-cause death,^1,2 whereas heavy alcohol intake and binge drinking have been associated with an increased risk of stroke,⁴ cardiovascular disease and all-cause death.^5,6 Similarly, heavy drinking and binge drinking are associated with an increased risk of incident atrial fibrillation in the general population.⁷ However, the association between moderate drinking and incident atrial fibrillation is less consistent and not well understood among older people with existing cardiovascular disease.In this analysis, we examined whether drinking moderate quantities of alcohol, and binge drinking, would be associated with an increased risk of incident atrial fibrillation in a large cohort of people with existing cardiovascular disease or diabetes with end-organ damage who had been followed prospectively in 2 long-term antihypertensive drug treatment trials.     

Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex

Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.     

Staurosporines Disrupt Phosphatidylserine Trafficking and Mislocalize Ras Proteins

Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.