Multiscale Self-Assembly of Silicon Quantum Dots into an Anisotropic Three-Dimensional Random Network.

Multiscale self-assembly is ubiquitous in nature but its deliberate use to synthesize multifunctional three-dimensional materials remains rare, partly due to the notoriously difficult problem of controlling topology from atomic to macroscopic scales to obtain intended material properties. Here, we propose a simple, modular, noncolloidal methodology that is based on exploiting universality in stochastic growth dynamics and driving the growth process under far-from-equilibrium conditions toward a preplanned structure. As proof of principle, we demonstrate a confined-but-connected solid structure, comprising an anisotropic random network of silicon quantum-dots that hierarchically self-assembles from the atomic to the microscopic scales. First, quantum-dots form to subsequently interconnect without inflating their diameters to form a random network, and this network then grows in a preferential direction to form undulated and branching nanowire-like structures. This specific topology simultaneously achieves two scale-dependent features, which were previously thought to be mutually exclusive: good electrical conduction on the microscale and a bandgap tunable over a range of energies on the nanoscale.

Vaccination With Heterologous HIV-1 Envelope Sequences and Heterologous Adenovirus Vectors Increases T-Cell Responses to Conserved Regions: HVTN 083.

BACKGROUND: Increasing the breadth of human immunodeficiency virus type 1 (HIV-1) vaccine-elicited immune responses or targeting conserved regions may improve coverage of circulating strains. HIV Vaccine Trials Network 083 tested whether cellular immune responses with these features are induced by prime-boost strategies, using heterologous vectors, heterologous inserts, or a combination of both. METHODS: A total of 180 participants were randomly assigned to receive combinations of adenovirus vectors (Ad5 or Ad35) and HIV-1 envelope (Env) gene inserts (clade A or B) in a prime-boost regimen. RESULTS: T-cell responses to heterologous and homologous insert regimens targeted a similar number of epitopes (ratio of means, 1.0; 95% confidence interval [CI], .6-1.6; P = .91), but heterologous insert regimens induced significantly more epitopes that were shared between EnvA and EnvB than homologous insert regimens (ratio of means, 2.7; 95% CI, 1.2-5.7; P = .01). Participants in the heterologous versus homologous insert groups had T-cell responses that targeted epitopes with greater evolutionary conservation (mean entropy [±SD], 0.32 ± 0.1 bits; P = .003), and epitopes recognized by responders provided higher coverage (49%; P = .035). Heterologous vector regimens had higher numbers of total, EnvA, and EnvB epitopes than homologous vector regimens (P = .02, .044, and .045, respectively). CONCLUSIONS: These data demonstrate that vaccination with heterologous insert prime boosting increased T-cell responses to shared epitopes, while heterologous vector prime boosting increased the number of T-cell epitopes recognized. CLINICAL TRIALS REGISTRATION: NCT01095224.

HIV-1 vaccine-induced T-cell responses cluster in epitope hotspots that differ from those induced in natural infection with HIV-1.

Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.

Vaccine-induced gag-specific T cells are associated with reduced viremia after HIV-1 infection.

The contribution of host T-cell immunity and HLA class I alleles to the control of human immunodeficiency virus (HIV-1) replication in natural infection is widely recognized. We assessed whether vaccine-induced T-cell immunity, or expression of certain HLA alleles, impacted HIV-1 control after infection in the Step MRKAd5/HIV-1 gag/pol/nef study. Vaccine-induced T cells were associated with reduced plasma viremia, with subjects targeting ≥3 gag peptides presenting with half-log lower mean viral loads than subjects without Gag responses. This effect was stronger in participants infected proximal to vaccination and was independent of our observed association of HLA-B*27, -B*57 and -B*58:01 alleles with lower HIV-1 viremia. These findings support the ability of vaccine-induced T-cell responses to influence postinfection outcome and provide a rationale for the generation of T-cell responses by vaccination to reduce viremia if protection from acquisition is not achieved. Clinical trials identifier: NCT00095576.

The Migration Pattern of a Short-Tapered Femoral Stem Correlates with the Occurrence of Cortical Hypertrophies: A 10-Year Longitudinal Study Using Ein Bild Röntgen Analyse-Femoral Component Analysis.

Background: Shorter hip stems have shown promising mid-term results but lack long-term data. High rates of distal cortical hypertrophy (CH) have been described, suggesting a more diaphyseal load transmission. This study aimed to determine patient-specific and surgery-related factors influencing CH and their impact on 10-year outcomes. Methods: It included 100 consecutive total hip arthroplasties (THAs) using the Fitmore stem (Zimmer, Warsaw, Indiana), with clinical and radiographic follow-ups at 1, 2, 5, and at least 10 years post-surgery. Results: No revisions were performed due to aseptic loosening after a mean of 11.6 years (range: 10-13.5 years). CH was observed in 26% of hips, primarily in Gruen zones 3 and 5. There was no significant difference in the Harris Hip Score between patients with and without CH. Larger stem sizes and greater axial subsidence significantly correlated with CH occurrence (OD 1.80, (1.13-1.92), p = 0.004; OD 1.47, (1.04-2.08), p = 0.028). The Fitmore stem demonstrated excellent survival rates and favorable outcomes over 10 years. Conclusions: Despite a lower CH rate compared to other studies, significant correlations with stem size and subsidence were identified. This study underscores the importance of patient selection and achieving high primary stability to maintain the metaphyseal anchoring concept.

DNA Karyometry for Automated Detection of Cancer Cells.

Background: Microscopical screening of cytological samples for the presence of cancer cells at high throughput with sufficient diagnostic accuracy requires highly specialized personnel which is not available in most countries. Methods: Using commercially available automated microscope-based screeners (MotiCyte and EasyScan), software was developed which is able to classify Feulgen-stained nuclei into eight diagnostically relevant types, using supervised machine learning. the nuclei belonging to normal cells were used for internal calibration of the nuclear DNA content while nuclei belonging to those suspicious of being malignant were specifically identified. The percentage of morphologically abnormal nuclei was used to identify samples suspected of malignancy, and the proof of DNA-aneuploidy was used to definitely determine the state malignancy. A blinded study was performed using oral smears from 92 patients with Fanconi anemia, revealing oral leukoplakias or erythroplakias. In an earlier study, we compared diagnostic accuracies on 121 serous effusion specimens. In addition, using a blinded study employing 80 patients with prostate cancer who were under active surveillance, we aimed to identify those whose cancers would not advance within 4 years. Results: Applying a threshold of the presence of >4% of morphologically abnormal nuclei from oral squamous cells and DNA single-cell or stemline aneuploidy to identify samples suspected of malignancy, an overall diagnostic accuracy of 91.3% was found as compared with 75.0% accuracy determined by conventional subjective cytological assessment using the same slides. Accuracy of automated screening effusions was 84.3% as compared to 95.9% of conventional cytology. No prostate cancer patients under active surveillance, revealing DNA-grade 1, showed progress of their disease within 4.1 years. Conclusions: An automated microscope-based screener was developed which is able to identify malignant cells in different types of human specimens with a diagnostic accuracy comparable with subjective cytological assessment. Early prostate cancers which do not progress despite applying any therapy could be identified using this automated approach.

Virus-specific CD8+ T-cell responses better define HIV disease progression than HLA genotype.

HLA alleles B57/58, B27, and B35 have the strongest genetic associations with HIV-1 disease progression. The mechanisms of these relationships may be host control of HIV-1 infection via CD8(+) T-cell responses. We examined these immune responses in subjects from the Seattle Primary Infection Cohort with these alleles. CD8(+) T-cell responses to conserved HIV epitopes within B57/58 alleles (TW10 and KF11) and B27 alleles (KK10 and FY10) delayed declines in CD4(+) T-cell counts (4 to 8 times longer), while responses to variable epitopes presented by B35 alleles (DL9 and IL9) resulted in more rapid progression. The plasma viral load was higher in B57/58(+) and B27(+) subjects lacking the conserved B57/58- and B27-restricted responses. The presence of certain B57/58-, B27-, and B35-restricted HIV-specific CD8(+) T-cell responses after primary HIV-1 infection better defined disease progression than the HLA genotype alone, suggesting that it is the HIV-specific CD8(+) T cells and not the presence of a particular HLA allele that determine disease progression. Further, the most effective host CD8(+) T-cell responses to HIV-1 were prevalent within an HLA allele, represented a high total allele fraction of the host CD8(+) T-cell response, and targeted conserved regions of HIV-1. These data suggest that vaccine immunogens should contain only conserved regions of HIV-1.

Peripheral autoreactive CD8 T-cell frequencies are too variable to be a reliable predictor of disease progression of human type 1 diabetes.

OBJECTIVES: The detection of a peripheral immune cell signature that specifically reflects autoimmunity in type 1 diabetes would enable the prediction and staging of disease on an individual basis. However, defining such a signature is technically challenging. Reliable interpretation of immune cell-related biomarkers depends on their inherent variability and, to understand this variability, longitudinal analyses are required. METHODS: We performed a longitudinal observational study in which 40 individuals with elevated genetic risk of type 1 diabetes and persistent islet autoantibodies provided a blood sample every 4-6 weeks for > 1 year. RESULTS: Peripheral immune cell composition (T cells, NK cells and monocytes) was assessed using well-validated flow cytometry panels and demonstrated that, while non-antigen-specific immune cell subsets were stable over time, autoantigen-reactive T-cell frequencies were highly variable in and between individuals. Neither the frequency nor phenotype of non-antigen-specific subsets or autoreactive CD8+ T cells associated with clinical onset of T1D. CONCLUSION: The findings from the Type 1 Diabetes Longitudinal BIomarker Trial underscore the inherent challenge of evaluating changes in peripheral immune cell populations as surrogates of organ-specific disease activity. The variability of peripheral antigen-specific T cells precludes their use as a prognostic marker and clearly demonstrates that a reliable prognostic cell signature remains elusive.

Automated detection of cancer cells in effusion specimens by DNA karyometry.

BACKGROUND: The average sensitivity of conventional cytology for the identification of cancer cells in effusion specimens is only approximately 58%. DNA image cytometry (DNA-ICM), which exploits the DNA content of morphologically suspicious nuclei measured on digital images, has a sensitivity of up to 91% for the detection of cancer cells. However, when performed manually, to our knowledge to date, an expert needs approximately 60 minutes for the analysis of a single slide. METHODS: In the current study, the authors present a novel method of supervised machine learning for the automated identification of morphologically suspicious mesothelial and epithelial nuclei in Feulgen-stained effusion specimens. The authors compared this with manual DNA-ICM and a gold standard cytological diagnosis for 121 cases. Furthermore, the authors retrospectively analyzed whether the amount of morphometrically abnormal mesothelial or epithelial nuclei detected by the digital classifier could be used as an additional diagnostic marker. RESULTS: The presented semiautomated DNA karyometric solution identified more diagnostically relevant abnormal nuclei compared with manual DNA-ICM, which led to a higher sensitivity (76.4% vs 68.5%) at a specificity of 100%. The ratio between digitally abnormal and all mesothelial nuclei was found to identify cancer cell-positive slides at 100% sensitivity and 70% specificity. The time effort for an expert therefore is reduced to the verification of a few nuclei with exceeding DNA content, which to our knowledge can be accomplished within 5 minutes. CONCLUSIONS: The authors have created and validated a computer-assisted bimodal karyometric approach for which both nuclear morphology and DNA are quantified from a Feulgen-stained slide. DNA karyometry thus increases the diagnostic accuracy and reduces the workload of an expert when compared with manual DNA-ICM.

Systematic Assessment of Immune Marker Variation in Type 1 Diabetes: A Prospective Longitudinal Study.

Immune analytes have been widely tested in efforts to understand the heterogeneity of disease progression, risk, and therapeutic responses in type 1 diabetes (T1D). The future clinical utility of such analytes as biomarkers depends on their technical and biological variability, as well as their correlation with clinical outcomes. To assess the variability of a panel of 91 immune analytes, we conducted a prospective study of adults with T1D (<3 years from diagnosis), at 9-10 visits over 1 year. Autoantibodies and frequencies of T-cell, natural killer cell, and myeloid subsets were evaluated; autoreactive T-cell frequencies and function were also measured. We calculated an intraclass correlation coefficient (ICC) for each marker, which is a relative measure of between- and within-subject variability. Of the 91 analytes tested, we identified 35 with high between- and low within-subject variability, indicating their potential ability to be used to stratify subjects. We also provide extensive data regarding technical variability for 64 of the 91 analytes. To pilot the concept that ICC can be used to identify analytes that reflect biological outcomes, the association between each immune analyte and C-peptide was also evaluated using partial least squares modeling. CD8 effector memory T-cell (CD8 EM) frequency exhibited a high ICC and a positive correlation with C-peptide, which was also seen in an independent dataset of recent-onset T1D subjects. More work is needed to better understand the mechanisms underlying this relationship. Here we find that there are a limited number of technically reproducible immune analytes that also have a high ICC. We propose the use of ICC to define within- and between-subject variability and measurement of technical variability for future biomarker identification studies. Employing such a method is critical for selection of analytes to be tested in the context of future clinical trials aiming to understand heterogeneity in disease progression and response to therapy.

Characterization of diplopia in non-demented patients with Parkinson's disease.

INTRODUCTION: Although diplopia is considered a frequent symptom of Parkinson's disease (PD), little is known about its clinical manifestation, associated mechanisms and treatment. Here we characterized binocular diplopia in non-demented PD patients in an interdisciplinary setting. METHODS: PD patients were prospectively screened for diplopia, visual hallucinations, problems with spatial perception, contrast sensitivity, presence of blurred vision, and history of ophthalmological comorbidities via interview. Two groups of PD patients, one with and one without diplopia, underwent clinical and ophthalmological assessment to characterize diplopia in these patients. Clinical features were investigated using the Unified Parkinson's Disease Rating Scale and the Non-Motor Symptoms Scale. RESULTS: The frequency of binocular diplopia was 29.6% (n = 37) in our cohort of 125 Parkinson's disease patients. Related mechanisms were heterogeneous including convergence insufficiency, strabismus, and motor fluctuations, as well as symptoms related to visual hallucinations. Diplopia was associated with other visual disturbances like visual hallucinations, blurred vision and problems with spatial perception. Beyond that, diplopia was found to be a predictive factor (3.2, odds ratio) for the occurrence of visual hallucinations in PD. CONCLUSION: Binocular diplopia represents a frequent and relevant symptom in PD patients. Different subtypes should be considered due to different associated mechanisms including ophthalmic pathology and motor fluctuation, as well as intermediate to higher level visual processes. Diplopia seems to be part of a continuous spectrum of positive visual symptoms in Parkinson's disease.

Clinical correlates of index values in the focus HerpeSelect ELISA for antibodies to herpes simplex virus type 2 (HSV-2).

BACKGROUND: Clinical correlates of HerpeSelect ELISA index values are poorly understood. OBJECTIVES: This study was designed to determine the effects of time of infection, test variability, and antibody avidity on index values. STUDY DESIGN: Sera (N=313) from 81 patients with new HSV-2 infections and 236 sera from 32 patients with long-standing (median 11.3 years) HSV-2 were tested by HerpeSelect HSV-2 ELISA. High positive, low positive and negative controls were run on 42 test plates to establish test variability. RESULTS: Index values tended to rise after infection, peaking a median of 9-10 weeks post-infection (range 8-323 days). Of 32 patients with established HSV-2 infections, 7 (22%) had at least one low index value (>1.1 to < or =3.5), and one had a transient seroreversion event. Test variability of index values was substantially lower than inter- or intra-patient variability. Median antibody avidity was higher in sera with high versus low index values in established infections, but unrelated to index value in patients with early infections. CONCLUSIONS: Index values or index value changes are not absolute indicators of early versus established HSV-2 infection or solely a function of test variability. Low antibody avidity may contribute to low index values once infection is established.

Removing defocused objects from single focal plane scans of cytological slides.

BACKGROUND: Virtual microscopy and automated processing of cytological slides are more challenging compared to histological slides. Since cytological slides exhibit a three-dimensional surface and the required microscope objectives with high resolution have a low depth of field, these cannot capture all objects of a single field of view in focus. One solution would be to scan multiple focal planes; however, the increase in processing time and storage requirements are often prohibitive for clinical routine. MATERIALS AND METHODS: In this paper, we show that it is a reasonable trade-off to scan a single focal plane and automatically reject defocused objects from the analysis. To this end, we have developed machine learning solutions for the automated identification of defocused objects. Our approach includes creating novel features, systematically optimizing their parameters, selecting adequate classifier algorithms, and identifying the correct decision boundary between focused and defocused objects. We validated our approach for computer-assisted DNA image cytometry. RESULTS AND CONCLUSIONS: We reach an overall sensitivity of 96.08% and a specificity of 99.63% for identifying defocused objects. Applied on ninety cytological slides, the developed classifiers automatically removed 2.50% of the objects acquired during scanning, which otherwise would have interfered the examination. Even if not all objects are acquired in focus, computer-assisted DNA image cytometry still identified more diagnostically or prognostically relevant objects compared to manual DNA image cytometry. At the same time, the workload for the expert is reduced dramatically.

Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials.

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7-30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.

Cluster analysis: a useful tool for the analysis of cerebral laser-Doppler scanning data.

Laser-Doppler (LD) fluxmetry (LDF) is a widely used method for the measurement of relative tissue perfusion. Assessing LD-flux at multiple locations using a scanning technique greatly reduces movement artefacts and makes repetitive measurements at the same location possible. However, measurements in brain are often confounded by superficial cortical vessels. Commonly applied strategies to circumvent this problem, such as defining a cut-off point to exclude the high flux data of vessels or calculating the median from multiple locations to estimate regional cerebral blood flow (rCBF) all have specific shortcomings. The aim of this study was to analyse LD-data by mathematically discriminating between parenchymal and vessel data based on the distribution of flux data. Data was obtained by scanning the cortex of 15 male Sprague-Dawley rats using a matrix of 6x10 equidistant (500 microm) points. Standard statistical analysis as well as cluster analysis using the complete linkage algorithm was performed. The LD-data showed a bimodal frequency distribution with low values representing parenchymal and high values representing vessel flux. Parenchyma and vessels were reliably discriminated by cluster analysis. This was shown by mapping the vessel clusters on the scan matrix with the location of the superficial cortical vessels using Chi-square testing (p<0.0001). The parenchymal data followed a Gaussian normal distribution (p<0.851), whereas the vessel data did not (p<0.0001). Thus, cluster analysis is useful to discriminate parenchymal from vessel flux, thereby significantly improving the accuracy of LD-scanning data.

Use of "biokit HSV-2 Rapid Assay" to improve the positive predictive value of Focus HerpeSelect HSV-2 ELISA.

BACKGROUND: Commercially available assays to detect antibodies to the herpes simplex virus type 2 (HSV-2)-specific glycoprotein gG-2 have markedly improved serologic diagnosis of HSV-2 infection. However, even tests with high specificity can have low positive predictive values in low prevalence populations. HSV-2 is a chronic, life-long viral infection that requires both medical attention and potential alterations in health care strategy. As such, the concern for false positive diagnoses is high confirmatory testing is routine for other viral serologies such as HIV and hepatitis C. We evaluated such a strategy for HSV-2 serology by using an easily performed commercial test, biokitHSV-2 rapid test ("Biokit"; Biokit USA, Lexington MA) as a confirmatory test for the widely used gG-2 specific serology ("Focus;" HerpeSelect HSV-2 ELISA; Focus Diagnostics, Cypress CA). METHODS: We tested 782 sera by Focus HSV-2 ELISA, Biokit, and the current gold standard test, Western blot (WB). RESULTS: The positive predictive value of the Focus HSV-2 ELISA increased from 80.5% to 95.6% when Biokit testing was performed on sera that were initially positive by Focus HSV-2 ELISA. Confirmatory testing increased the specificity markedly among sera with Focus EIA values between 1.1 and 3.5: only 35% of low positive (index values 1.1-3.5) Focus HSV-2 ELISA results confirmed as positive by Biokit and WB compared with 92% of those with index values >3.5. Mathematical modeling of the data resulted in expected positive predictive values over 98% for populations with antibody prevalences typical of clinical practices in the US and Europe. CONCLUSION: Confirmatory Biokit testing of positive Focus HSV-2 ELISA results is fast, easy, and effective in reducing falsely positive HSV-2 antibody results. Patients, clinicians, and laboratories could benefit from the enhanced specificity of this simple HSV-2 serologic test combination.

Inaccuracy of certain commercial enzyme immunoassays in diagnosing genital infections with herpes simplex virus types 1 or 2.

Type-specific serologic results may be inaccurate if not based on glycoprotein G (gG). Commercial tests based on crude antigen (Zeus Scientific, Raritan, NJ; Wampole Laboratories, Cranbury, NJ; DiaSorin, Stillwater, MN) and one using gG-1 and gG-2 (Focus Technologies, Cypress, CA) were compared with Western blot on serum samples from patients with culture-documented first symptomatic episodes of genital herpes simplex virus (HSV) type 1 (n = 17) or HSV-2 (n = 49) infection or recurrent genital episodes (HSV-1, 30; HSV-2, 49). Concordance with Western blot results was 56% for Zeus, 63% for Wampole, 52% to 54% for DiaSorin, and 83% for Focus. Sensitivity and specificity, respectively, for HSV-1 were 77% and 53% (Zeus), 91% and 35% (Wampole), 98% and 8% (DiaSorin), 94% and 70% (DiaSorin predominant antibody), and 83% and 90% (Focus); for HSV-2 they were 88% and 81% (Zeus), 92% and 83% (Wampole), 96% and 54% (DiaSorin), 38% and 98% (DiaSorin predominant antibody), and 98% and 96% (Focus). Type-specific serologic testing for HSV should be performed with gG-based tests for accurate diagnosis of symptomatic genital herpes.

Relative cerebral blood flow during the secondary expansion of a cortical lesion in rats.

The size of a cerebral contusion is not finite at the moment of trauma, but liable to secondary increase during the following hours and days. In the present study we investigated whether this phenomenon may be related to changes in cortical blood flow (cCBF). In rats a cortical lesion grew to 140% of its initial volume during the first 24 h after injury. During the time of most rapid lesion expansion (<6 h after the insult) marked hypoperfusion (approximately 30% of baseline) was found in the ipsilateral hemisphere by laser Doppler scanning fluxmetry. In the peri-contusional area cCBF slowly recovered to approximately 80% of baseline, while in the distant brain not affected by delayed cell death, significant hyperperfusion (approximately 160% of baseline) was observed. Thus, early hypoperfusion might be an important mechanism for secondary lesion expansion.

Measuring inhibition of HIV replication by ex vivo CD8⁺ T cells.

HIV replication is unrestrained in vivo in the vast majority of infected subjects, and the ability of some rare individuals to control this virus is poorly understood. Standard immunogenicity assays for detecting HIV-1-specific CD8(+) T-cell responses, such as IFN-γ ELISpot and intracellular cytokine staining, generally fail to correlate with in vivo inhibition of HIV replication. Several viral inhibition assays, which measure the effectiveness of CD8(+) T-cell responses in suppressing HIV replication in vitro, have been described; but most depend on in vitro expansion of CD8(+) T cells, and some show inhibitory activity in HIV-negative individuals. We have optimized an assay to assess the suppressive capability of CD8(+) T cells directly ex vivo, eliminating the potential for altering their function through activation or expansion prior to assay setup, and thereby enhancing the assay's sensitivity by avoiding non-specific inhibition. With this method, the ability of ex vivo CD8(+) T cells to control HIV-1 replication in vitro can be quantified over several orders of magnitude. Specifically, our assay can be used to better define the antiviral function of CD8(+) T cells induced by vaccination, and can provide insight into their ability to control viral replication if HIV infection occurs post-vaccination.