The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics

Gene expression microarray analyses of mixtures of cells approximate a weighted average of the gene expression profiles (GEPs) of each cell type according to its relative abundance in the overall cell sample being analyzed. If the targeted subpopulation of cells is in the minority, or the expected perturbations are marginal, then such changes will be masked by the GEP of the normal/unaffected cells. We show that the GEP of a minor cell subpopulation is often lost when that cell subpopulation is of a frequency less than 30 percent. The GEP is almost always masked by the other cell subpopulations when that frequency drops to 10 percent or less. On the basis of these results one should always assume that the GEP of a given cell subpopulation is probably seriously affected by, the presence of significant numbers of other "contaminating" cell types. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. If a cell subpopulation of interest is small, very high-throughput cell separation technologies are needed to separate enough cells for conventional microarrays. However, high-throughput flow cytometry/cell sorting overcomes many restrictions of experimental enrichment conditions. This technology can also be used to sort smaller numbers of cells of specific cell subpopulations and subsequently amplify their mRNAs before microarray analyses. When purification techniques are applied to unfixed samples, the potential for changes in gene levels during the process of collection is an additional concern. Since RNA rapidly degrades, and specific mRNAs turn over in minutes or hours, the cell separation process must be very rapid. Hence, high-throughput cell separation (HTS) technologies are needed that can process the necessary number of cells expeditiously in order to avoid such uncontrolled changes in the target cells GEP. In cases where even the use of HTS yields only a small number of cells, the mRNAs (after reverse transcription to cDNA's) must be amplified to yield enough material for conventional microarray analyses. However, the problem of using "microamplification" PCR methods to expand the amount of cDNAs (from mRNAs) is that it is very difficult to amplify equally all of the mRNAs. Unequal amplification leads to a distorted gene expression profile on the microarray. Linear amplifications is difficult to achieve. Unfortunately, present-day gene-chips need to be about 100 times more sensitive than they are now to be able to do many biologically and biomedically meaningful experiments and clinical tests

Naturally Rehearsing Passwords

We introduce quantitative usability and security models to guide the design of password management schemes --- systematic strategies to help users create and remember multiple passwords. In the same way that security proofs in cryptography are based on complexity-theoretic assumptions (e.g., hardness of factoring and discrete logarithm), we quantify usability by introducing usability assumptions. In particular, password management relies on assumptions about human memory, e.g., that a user who follows a particular rehearsal schedule will successfully maintain the corresponding memory. These assumptions are informed by research in cognitive science and validated through empirical studies. Given rehearsal requirements and a user's visitation schedule for each account, we use the total number of extra rehearsals that the user would have to do to remember all of his passwords as a measure of the usability of the password scheme. Our usability model leads us to a key observation: password reuse benefits users not only by reducing the number of passwords that the user has to memorize, but more importantly by increasing the natural rehearsal rate for each password. We also present a security model which accounts for the complexity of password management with multiple accounts and associated threats, including online, offline, and plaintext password leak attacks. Observing that current password management schemes are either insecure or unusable, we present Shared Cues--- a new scheme in which the underlying secret is strategically shared across accounts to ensure that most rehearsal requirements are satisfied naturally while simultaneously providing strong security. The construction uses the Chinese Remainder Theorem to achieve these competing goals

Quality of T-cell responses versus reduction in viral load: results from an exploratory phase II clinical study of Vacc-4x, a therapeutic HIV vaccine

Background Immunization with Vacc-4x, a peptide-based therapeutic vaccine for HIV-1, has shown a statistically significant reduction in viral load set point compared to placebo during treatment interruption in an exploratory phase II clinical study enrolling 135 subjects (NCT00659789). This vaccine aims to induce sustained cell-mediated immune responses to conserved domains on HIV p24. Methods After 6 immunizations on ART over 28 weeks, treatment was interrupted for up to 24 weeks (Vacc-4x n=88; placebo n=38). Immunological analyses (ELISPOT, proliferation, intracellular cytokine staining (ICS)) to HIV p24 were carried out at central laboratories. The HLA class I profile (Vacc-4x n=73, placebo n=32) was also determined. Results For subjects that remained off ART until week 52 (Vacc-4x n=56, placebo n=25), there was a log 0.44 reduction in viral load set point between the Vacc-4x and placebo groups (p=0.0397). There was a similar distribution of HLA class I alleles in the two treatment arms, with the exception of the B35 allele (27% of Vacc-4x subjects versus 8% placebo subjects). The viral load of ELISPOT positive Vacc-4x subjects was significantly lower than that of placebo subjects (p=0.023). There was no significant difference in T-cell proliferation responses between Vacc-4x and placebo groups, however, the percentage of subjects showing proliferative CD4 and CD8 T-cell responses to Vacc-4x peptides increased over time only for the Vacc-4x group. ICS analysis showed a predominance of CD8-mediated T-cell responses to p24 that were significantly increased from baseline for the Vacc-4x group (p<0.043) but not for the placebo group(p>0.05). There was also a trend towards higher numbers of polyfunctional T-cells in the Vacc-4x group compared to the placebo group (p=0.188). Conclusion These findings suggest Vacc-4x immunization can influence the quality of immune responses to HIV-1 p24 irrespective of HLA status, and contribute to a reduction in viral load

Clinical features, treatment, and outcome of pediatric steroid refractory acute graft-versus-host disease: a multicenter study

Steroid-refractory acute graft-versus-host disease (SR-aGvHD) is a severe complication in pediatric allogeneic hematopoietic stem cell transplantation (HSCT). We aimed to assess clinical course and outcomes of pediatric SR-aGvHD. We performed a retrospective nationwide multicenter cohort study in the Netherlands. All patients aged 0 to 18 years who underwent transplantation between 2010 and 2020 with SR-aGvHD were included. For each patient, weeldy clinical aGvHD grade and stage, immunosuppressive treatment and clinical outcomes were collected. The primary study endpoint was the clinical course of SR-aGvHD over time. As a secondary outcome, factors influencing overall survival and SR-aGvHD remission were identified using a multistate Cox model. 20% of transplanted children developed grade II-IV aGvHD, of which 51% (n = 81) was SR-aGvHD. In these patients, second-line therapy was started at a median of 8 days after initial aGvHD-diagnosis. Forty-nine percent of SR-aGvHD patients received 3 or more lines of therapy. One year after start of second-line therapy, 34 patients (42%) were alive and in remission of aGvHD, 14 patients (17%) had persistent GvHD, and 33 patients (41%) had died. SR-aGvHD remission rate was lower in cord blood graft recipients than in bone marrow (BM) or peripheral blood stem cell (PBSC) recipients (hazard ratio [HR] = 0.51, 0.27-0.94, P = .031). Older age was associated with higher mortality (HR = 2.62, 1.04-6.60, P = .04, fourth quartile [aged 13.9-17.9] versus first quartile [aged 0.175-3.01]). In BM/PBSC recipients older age was also associated with lower remission rates (HR = 0.9, 0.83-0.96, P = .004). Underlying diagnosis, donor matching or choice of second-line therapy were not associated with outcome. Respiratory insufficiency caused by pulmonary GvHD was a prominent cause of death (26% of deceased). Our study demonstrates that SR-aGvHD confers a high mortality risk in pediatric HSCT. Older age and use of CB grafts are associated with an unfavorable outcome. Multicenter studies investigating novel treatment strategies to prevent pediatric SR-aGvHD and inclusion of children in ongoing trials, together with timely initiation of second-line interventions are pivotal to further reduce GvHD-related mortality. (C) 2022 The American Society for Transplantation and Cellular Therapy.Transplantation and immunomodulatio

Modeling influencing factors in B-cell reconstitution after hematopoietic stem cell transplantation in children

Reduced total and memory B-cell numbers in peripheral blood long term after hematopoietic stem cell transplantation (HSCT) are associated with an increased incidence of infections and immune complications. Using novel modelling strategies, baseline factors influencing B-cell reconstitution can be comprehensively studied. This study aims to investigate the numerical total and memory B-cell reconstitution in children and the association with baseline determinants 0.5-2 years after allogeneic HSCT. Eligible for inclusion were children transplanted in our center between 2004-2017 who received a first HSCT for malignant or non-malignant disorders. The continuous absolute counts of total and memory B-cells were evaluated as outcome measure. Exploratory analysis at one year was done to identify possible determinants. Linear mixed effect modelling was used to analyze the association of these determinants with total and memory B-cell reconstitution 0.5-2 years after HSCT. In a cohort of 223 evaluable patients analyzed at 1-year after HSCT donor age, stem cell source, donor type, recipient age and conditioning were identified as significant determinants for total and memory B-cell numbers. Multivariable analysis revealed that both donor and recipient age were inversely correlated with the size of total and memory B-cell reconstitution. In contrast, no correlation was found with stem cell source, donor type and conditioning. Making use of linear mixed modelling both stem cell donor and recipient age were identified as independent determinants of total and memory B-cell reconstitution 0.5-2 years after HSCT.Development and application of statistical models for medical scientific researc

Effects of Combined CCR5/Integrase Inhibitors-Based Regimen on Mucosal Immunity in HIV-Infected Patients Naïve to Antiretroviral Therapy: A Pilot Randomized Trial

Whether initiation of antiretroviral therapy (ART) regimens aimed at achieving greater concentrations within gut associated lymphoid tissue (GALT) impacts the level of mucosal immune reconstitution, inflammatory markers and the viral reservoir remains unknown. We included 12 HIV- controls and 32 ART-naïve HIV patients who were randomized to efavirenz, maraviroc or maraviroc+raltegravir, each with fixed-dose tenofovir disoproxil fumarate/emtricitabine. Rectal and duodenal biopsies were obtained at baseline and at 9 months of ART. We performed a comprehensive assay of T-cell subsets by flow cytometry, T-cell density in intestinal biopsies, plasma and tissue concentrations of antiretroviral drugs by high-performance liquid chromatography/mass spectroscopy, and plasma interleukin-6 (IL-6), lipoteichoic acid (LTA), soluble CD14 (sCD14) and zonulin-1 each measured by ELISA. Total cell-associated HIV DNA was measured in PBMC and rectal and duodenal mononuclear cells. Twenty-six HIV-infected patients completed the follow-up. In the duodenum, the quadruple regimen resulted in greater CD8+ T-cell density decline, greater normalization of mucosal CCR5+CD4+ T-cells and increase of the naïve/memory CD8+ T-cell ratio, and a greater decline of sCD14 levels and duodenal HIV DNA levels (P = 0.004 and P = 0.067, respectively), with no changes in HIV RNA in plasma or tissue. Maraviroc showed the highest drug distribution to the gut tissue, and duodenal concentrations correlated well with other T-cell markers in duodenum, i.e., the CD4/CD8 ratio, %CD4+ and %CD8+ HLA-DR+CD38+ T-cells. Maraviroc use elicited greater activation of the mucosal naïve CD8+ T-cell subset, ameliorated the distribution of the CD8+ T-cell maturational subsets and induced higher improvement of zonulin-1 levels. These data suggest that combined CCR5 and integrase inhibitor based combination therapy in ART treatment naïve patients might more effectively reconstitute duodenal immunity, decrease inflammatory markers and impact on HIV persistence by cell-dependent mechanisms, and show unique effects of MVC in duodenal immunity driven by higher drug tissue penetration and possibly by class-dependent effects

Clinical Assessment of a Recombinant Simian Adenovirus ChAd63: A Potent New Vaccine Vector

Background. Vaccine development in human Plasmodium falciparum malaria has been hampered by the exceptionally high levels of CD8+ T cells required for efficacy. Use of potently immunogenic human adenoviruses as vaccine vectors could overcome this problem, but these are limited by preexisting immunity to human adenoviruses

Interferon-Alpha Administration Enhances CD8+ T Cell Activation in HIV Infection

Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006). These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy). As plasma HIV levels fell with interferon therapy, this was correlated with a "paradoxical" increase in CD8+ T cell activation (p<0.001).Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection

Multisystem inflammatory syndrome in children in Western Countries? Decreasing Incidence as the pandemic progresses?: An observational multicenter international cross-sectional study

Background: SARS-CoV-2 variations as well as immune protection after previous infections and/or vaccination may have altered the incidence of multisystemic inflammatory syndrome in children (MIS-C). We aimed to report an international time-series analysis of the incidence of MIS-C to determine if there was a shift in the regions or countries included into the study. Methods: This is a multicenter, international, cross-sectional study. We collected the MIS-C incidence from the participant regions and countries for the period July 2020 to November 2021. We assessed the ratio between MIS-C cases and COVID-19 pediatric cases in children Transplantation and immunomodulatio