Mutations in a dominant Nef epitope of simian immunodeficiency virus diminish TCR:epitope peptide affinity but not epitope peptide:MHC class I binding.

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.

Red cell distribution width and all-cause mortality in critically ill patients.

OBJECTIVE: Red cell distribution width is a predictor of mortality in the general population. The prevalence of increased red cell distribution width and its significance in the intensive care unit are unknown. The objective of this study was to investigate the association between red cell distribution width at the initiation of critical care and all cause mortality. DESIGN: Multicenter observational study. SETTING: Two tertiary academic hospitals in Boston, MA. PATIENTS: A total of 51,413 patients, aged ≥ 18 yrs, who received critical care between 1997 and 2007. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The exposure of interest was red cell distribution width as a predictor of mortality in the general population. The prevalence of increased red cell distribution width and its significance in the intensive care unit are unknown and categorized a priori in quintiles as ≤ 13.3%, 13.3% to 14.0%, 14.0% to 14.7%, 14.7% to 15.8%, and >15.8%. Logistic regression examined death by days 30, 90, and 365 postcritical care initiation, inhospital mortality, and bloodstream infection. Adjusted odds ratios were estimated by multivariable logistic regression models. Adjustment included age, sex, race, Deyo-Charlson index, coronary artery bypass grafting, myocardial infarction, congestive heart failure, hematocrit, white blood cell count, mean corpuscular volume, blood urea nitrogen, red blood cell transfusion, sepsis, and creatinine. Red cell distribution width was a particularly strong predictor of all-cause mortality 30 days after critical care initiation with a significant risk gradient across red cell distribution width quintiles after multivariable adjustment: red cell distribution width 13.3% to 14.0% (odds ratio [OR], 1.19; 95% confidence interval [CI], 1.08-1.30; p <.001); red cell distribution width 14.0% to 14.7% (OR, 1.28; 95% CI, 1.16-1.42; p <.001); red cell distribution width 14.7% to 15.8% (OR, 1.69; 95% CI, 1.52-1.86; p <.001); red cell distribution width >15.8% (OR, 2.61; 95% CI, 2.37-2.86; p <.001), all relative to patients with red cell distribution width ≤ 13.3%. Similar significant robust associations postmultivariable adjustments are seen with death by days 90 and 365 postcritical care initiation as well as inhospital mortality. In a subanalysis of patients with blood cultures drawn (n = 18,525), red cell distribution width at critical care initiation was associated with the risk of bloodstream infection and remained significant after multivariable adjustment. The adjusted risk of bloodstream infection was 1.40- and 1.44-fold higher in patients with red cell distribution width values in the 14.7% to 15.8% and >15.8% quintiles, respectively, compared with those with red cell distribution width ≤ 13.3%. Estimating the receiver operating characteristic area under the curve shows that red cell distribution width has moderate discriminative power for 30-day mortality (area under the curve = 0.67). CONCLUSION: Red cell distribution width is a robust predictor of the risk of all-cause patient mortality and bloodstream infection in the critically ill. Red cell distribution width is commonly measured, inexpensive, and widely available and may reflect overall inflammation, oxidative stress, or arterial underfilling in the critically ill.

Neighborhood poverty rate and mortality in patients receiving critical care in the academic medical center setting.

BACKGROUND: Poverty is associated with increased risk of chronic illness but its contribution to critical care outcome is not well defined. METHODS: We performed a multicenter observational study of 38,917 patients, aged ≥ 18 years, who received critical care between 1997 and 2007. The patients were treated in two academic medical centers in Boston, Massachusetts. Data sources included 1990 US census and hospital administrative data. The exposure of interest was neighborhood poverty rate, categorized as < 5%, 5% to 10%, 10% to 20%, 20% to 40% and > 40%. Neighborhood poverty rate is the percentage of residents below the federal poverty line. Census tracts were used as the geographic units of analysis. Logistic regression examined death by days 30, 90, and 365 post-critical care initiation and in-hospital mortality. Adjusted ORs were estimated by multivariable logistic regression models. Sensitivity analysis was performed for 1-year postdischarge mortality among patients discharged to home. RESULTS: Following multivariable adjustment, neighborhood poverty rate was not associated with all-cause 30-day mortality: 5% to 10% OR, 1.05 (95% CI, 0.98-1.14; P = .2); 10% to 20% OR, 0.96 (95% CI, 0.87-1.06; P = .5); 20% to 40% OR, 1.08 (95% CI, 0.96-1.22; P = .2); > 40% OR, 1.20 (95% CI, 0.90-1.60; P = .2); referent in each is < 5%. Similar nonsignificant associations were noted at 90-day and 365-day mortality post-critical care initiation and in-hospital mortality. Among patients discharged to home, neighborhood poverty rate was not associated with 1-year-postdischarge mortality. CONCLUSIONS: Our study suggests that there is no relationship between the neighborhood poverty rate and mortality up to 1 year following critical care at academic medical centers.

Elevation of blood urea nitrogen is predictive of long-term mortality in critically ill patients independent of "normal" creatinine.

OBJECTIVE: We hypothesized that elevated blood urea nitrogen can be associated with all-cause mortality independent of creatinine in a heterogeneous critically ill population. DESIGN: Multicenter observational study of patients treated in medical and surgical intensive care units. SETTING: Twenty intensive care units in two teaching hospitals in Boston, MA. PATIENTS: A total of 26,288 patients, age ≥ 18 yrs, hospitalized between 1997 and 2007 with creatinine of 0.80-1.30 mg/dL. INTERVENTIONS: None. MEASUREMENTS: Blood urea nitrogen at intensive care unit admission was categorized as 10-20, 20-40, and >40 mg/dL. Logistic regression examined death at days 30, 90, and 365 after intensive care unit admission as well as in-hospital mortality. Adjusted odds ratios were estimated by multivariable logistic regression models. MAIN RESULTS: Blood urea nitrogen at intensive care unit admission was predictive for short- and long-term mortality independent of creatinine. Thirty days following intensive care unit admission, patients with blood urea nitrogen of >40 mg/dL had an odds ratio for mortality of 5.12 (95% confidence interval, 4.30-6.09; p < .0001) relative to patients with blood urea nitrogen of 10-20 mg/dL. Blood urea nitrogen remained a significant predictor of mortality at 30 days after intensive care unit admission following multivariable adjustment for confounders; patients with blood urea nitrogen of >40 mg/dL had an odds ratio for mortality of 2.78 (95% confidence interval, 2.27-3.39; p < .0001) relative to patients with blood urea nitrogen of 10-20 mg/dL. Thirty days following intensive care unit admission, patients with blood urea nitrogen of 20-40 mg/dL had an odds ratio of 2.15 (95% confidence interval, 1.98-2.33; <.0001) and a multivariable odds ratio of 1.53 (95% confidence interval, 1.40-1.68; p < .0001) relative to patients with blood urea nitrogen of 10-20 mg/dL. Results were similar at 90 and 365 days following intensive care unit admission as well as for in-hospital mortality. A subanalysis of patients with blood cultures (n = 7,482) demonstrated that blood urea nitrogen at intensive care unit admission was associated with the risk of blood culture positivity. CONCLUSION: Among critically ill patients with creatinine of 0.8-1.3 mg/dL, an elevated blood urea nitrogen was associated with increased mortality, independent of serum creatinine.

Use of molecular beacons for rapid, real-time, quantitative monitoring of cytotoxic T-lymphocyte epitope mutations in simian immunodeficiency virus.

Immune pressure on lentiviruses exerted by cytotoxic T lymphocytes (CTL) selects for virus CTL epitope mutations. Currently employed methods for monitoring emerging CTL epitope mutations rely on the labor-intensive and time-consuming techniques of virus population or clonal sequencing. Here we describe the development of a high-throughput quantitative reverse transcription-PCR assay that facilitates large-scale CTL epitope monitoring. This approach utilizes both sequence-specific molecular beacons and the sequence-independent double-stranded DNA binding dye Sybr Green. We show that this assay detects single-nucleotide mutations in an immunodominant CTL epitope in viral RNA isolated from both viral culture supernatants and plasma samples from simian immunodeficiency virus (SIV)-infected rhesus monkeys. Furthermore, mutant viruses can be detected even when they represent as few as 500 mutant copies in a sample containing 10,000 total copies. This real-time PCR technique for evaluating CTL epitope mutations may prove to be a useful tool for monitoring the genetic drift of human immunodeficiency virus and SIV in infected individuals.

Fitness costs limit viral escape from cytotoxic T lymphocytes at a structurally constrained epitope.

The intense selection pressure exerted by virus-specific cytotoxic T lymphocytes (CTL) on replicating human immunodeficiency virus and simian immunodeficiency virus results in the accumulation of CTL epitope mutations. It has been assumed that fitness costs can limit the evolution of CTL epitope mutations. However, only a limited number of studies have carefully examined this possibility. To explore the fitness costs associated with viral escape from p11C, C-M-specific CTL, we constructed a panel of viruses encoding point mutations at each position of the entire p11C, C-M epitope. Amino acid substitutions at positions 3, 4, 5, 6, 7, and 9 of the epitope significantly impaired virus replication by altering virus production and Gag protein expression as well as by destabilizing mature cores. Amino acid substitutions at position 2 of the epitope were tolerated but required reversion or additional compensatory mutations to generate replication-competent viruses. Finally, while amino acid substitutions at positions 1 and 8 of the p11C, C-M epitope were functionally tolerated, these substitutions were recognized by p11C, C-M-specific CTL and therefore provided no selection advantage for the virus. Together, these data suggest that limited sequence variation is tolerated by the region of the capsid encoding the p11C, C-M epitope and therefore that only a very limited number of mutations can allow successful viral escape from the p11C, C-M-specific CTL response.

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope.

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.