Interplay of egg white gel pH and intragastric pH: Impact on breakdown kinetics and mass transport processes.

Egg white gels have been utilized as a model system to study protein breakdown kinetics based on physical and biochemical breakdown processes during in vitro gastric digestion. Additionally, the impact of regulating intragastric pH on the breakdown kinetic processes was investigated. The present study evaluated the impact of gel pH (based on the pH of protein dispersion prepared at pH 3, 5 and 7.5) and intragastric pH regulation (with or without adjustment to pH 2 during in vitro gastric digestion) on the effective diffusion of gastric juice components (water and HCl), gel softening kinetics during gastric digestion, microstructural analysis using micro- computed tomography and protein hydrolysis in the liquid and solid fraction of egg white gel digesta. Egg white gels were subjected to 30 s oral digestion and 15, 30, 60, 120, 180 or 240 min gastric digestion in a static in vitro gastric digestion model, with or without gastric pH adjustment to pH 2. The gel pH affected all the properties measured during gastric digestion and each gel pH represented a specific driving mechanism for protein breakdown. A lower gel pH (pH 3) demonstrated a higher diffusion of moisture and acid, resulting in faster softening (p < 0.05). An intermediate pH (pH 5) showed greater protein-protein interactions due to the proximity to the isoelectric point of egg white proteins, resulting in very slow softening during digestion (p < 0.05), and a higher pH (pH 7) resulted in higher acid diffusion, intermediate gel hardness and very slow softening kinetics (p < 0.05). The gastric pH adjustment during digestion of egg protein gels affected (p < 0.05) the equilibrium moisture and acid contents as well as protein hydrolysis. The study confirmed that there is an interplay between initial gel pH and the intragastric pH which affected the breakdown kinetics of egg white gels during the gastric digestion process.

Caecum OX40+CD4 T-cell subset associates with mucosal damage and key markers of disease in treated HIV-infection.

BACKGROUND: Blood OX40-expressing CD4 T-cells from antiretroviral (ART)-treated people living with HIV (PWH) were found to be enriched for clonally-expanded HIV sequences, hence contributing to the HIV reservoir. OX40-OX40L is also a checkpoint regulator of inflammation in multiple diseases. We explored gut mucosal OX40+CD4+ T-cells and their potential significance in HIV disease. METHODS: Biopsies of caecum and terminal-ileum of ART-treated PWH (n = 32) were obtained and mucosal damage and HIV reservoir were assessed. Mucosal OX40+ and Ki67+ CD4 T-cell subsets, as well as several tissue T-cell subsets modulating mucosal integrity and homeostasis (Th17, Th22, Treg, Tc17, Tc22, IL17+TCRγδ, IL22+TCRγδ) were quantified. Inflammatory-related markers, T-cell activation and thymic output were also determined in blood samples. Correlations were explored using Spearman rank test and corrected for multiple comparisons by Benjamini-Hochberg. RESULTS: Compared to healthy controls, a high frequency of mucosal, mainly caecum, CD4 T-cells were OX40+ in PWH. Such frequency strongly correlated with nadir CD4 (r = -0.836; p < 0.0001), CD4/CD8 ratio (r = -0.630; p = 0.002), caecum mucosal damage (r = 0.606; p = 0.008), caecum Th22 (r = -0.635; p = 0.002), caecum Th17 (r = 0.474; p = 0.03) and thymic output (r = -0.686; p < 0.001). It also correlated with Neutrophil-to-Lymphocyte Ratio and blood CD4 T-cell activation and tended to with mucosal HIV reservoir. CONCLUSION: High frequencies of caecum OX40+CD4 T-cells are found in people with HIV (PWH) and successful viral control. Interestingly, this cellular subset reflects key markers of disease and peripheral T-cell activation, as well as HIV-driven mucosal damage. OX40+CD4 T-cells deserve further investigation since they could expand because of T-cell homeostatic proliferation and relate to the Th22/Th17 gut mucosal ratio.

Altered T-cell subset distribution in the viral reservoir in HIV-1-infected individuals with extremely low proviral DNA (LoViReTs).

BACKGROUND: HIV cure strategies aim to eliminate viral reservoirs that persist despite successful antiretroviral therapy (ART). We have previously described that 9% of HIV-infected individuals who receive ART harbor low levels of provirus (LoViReTs). METHODS: We selected 22 LoViReTs matched with 22 controls ART suppressed for more than 3 years with fewer than 100 and more than 100 HIV-DNA copies/106  CD4+ T cells, respectively. We measured HIV reservoirs in blood and host genetic factors. Fourteen LoViReTs underwent leukapheresis to analyze replication-competent virus, and HIV-DNA in CD4+ T-cell subpopulations. Additionally, we measured HIV-DNA in rectum and/or lymph node biopsies from nine of them. RESULTS: We found that LoViReTs harbored not only lower levels of total HIV-DNA, but also significantly lower intact HIV-DNA, cell-associated HIV-RNA, and ultrasensitive viral load than controls. The proportion of intact versus total proviruses was similar in both groups. We found no differences in the percentage of host factors. In peripheral blood, 71% of LoViReTs had undetectable replication-competent virus. Minimum levels of total HIV-DNA were found in rectal and lymph node biopsies compared with HIV-infected individuals receiving ART. The main contributors to the reservoir were short-lived transitional memory and effector memory T cells (47% and 29%, respectively), indicating an altered distribution of the HIV reservoir in the peripheral T-cell subpopulations of LoViReTs. CONCLUSION: In conclusion, LoViReTs are characterized by low levels of viral reservoir in peripheral blood and secondary lymphoid tissues, which might be explained by an altered distribution of the proviral HIV-DNA towards more short-lived memory T cells. LoViReTs can be considered exceptional candidates for future interventions aimed at curing HIV.

Voice, Body Cues and Facial Expression in Emotion Recognition of Spanish Children and Adolescents: The Validation of Bell-Lysaker Emotion Recognition Test

Emotion recognition has been traditionally measured trough the recognition of emotional expressions of static faces. Studies suggest that emotion recognition is progressively acquired from early stages in our infancy. However, the literature regarding other emotional domains such as voice or body movements is scarce. Additionally, the number of tools that integrate several domains is limited, especially in children and adolescents, and none of them tested in Spanish samples. Therefore, this study aimed to define the psychometric properties of the Bell-Lysaker Emotion Recognition Task (BLERT) and a new-designed alternate version providing normative data in Spanish children and adolescents (from 8 to 15 years old corresponding to 3th). Moreover, we aim to describe the emotional acquisition trajectory of children and adolescents with a tool that integrates voice, face expressions and body movements. For that purpose, BLERT was translated into Spanish (BLERT–SI) and an alternate version was created (BLERT–SII). A total of 545 children and adolescents from 8 to 15 year-old participated in the study (250 male/295 female). All participants fulfilled BLERT–SI and BLERT–SII within two weeks of difference. Order of presentation was counterbalanced. Results showed that BLERT–SI and SII have good internal consistency (α = .70 and 71 respectively). Test-retest reliability showed a moderate correlation (r = .45; p < .001). Percentages equivalences per age are provided. Age correlated with BLERT–SI (r = .31; p < .001) and BLERT–SII (r = .21; p < .001), showing a progressive acquisition and development of emotion recognition during this period. BLERT–SI and SII are useful tools when studying the follow-up of children and adolescents. (AU)

Robust HIV-specific CD4+ and CD8+ T-cell responses distinguish elite control in adolescents living with HIV from viremic nonprogressors.

BACKGROUND: Elite controllers are therapy-naive individuals living with HIV capable of spontaneous control of plasma viraemia for at least a year. Although viremic nonprogressors are more common in vertical HIV-infection than in adults' infection, elite control has been rarely characterized in the pediatric population. DESIGN: We analyzed the T-cell immunophenotype and the HIV-specific response by flow cytometry in four pediatric elite controllers (PECs) compared with age-matched nonprogressors (PNPs), progressors and HIV-exposed uninfected (HEUs) adolescents. RESULTS: PECs T-cell populations had lower immune activation and exhaustion levels when compared with progressors, reflected by a more sustained and preserved effector function. The HIV-specific T-cell responses among PECs were characterized by high-frequency Gag-specific CD4+ T-cell activity, and markedly more polyfunctional Gag-specific CD8+ activity, compared with PNPs and progressors. These findings were consistently observed even in the absence of protective HLA-I molecules such as HLA-B∗27/57/81. CONCLUSION: Pediatric elite control is normally achieved after years of infection, and low immune activation in PNPs precedes the increasing ability of CD8+ T-cell responses to achieve immune control of viraemia over the course of childhood, whereas in adults, high immune activation in acute infection predicts subsequent CD8+ T-cell mediated immune control of viremia, and in adult elite controllers, low immune activation is therefore the consequence of the rapid CD8+ T-cell mediated immune control generated after acute infection. This distinct strategy adopted by PECs may help identify pathways that facilitate remission in posttreatment controllers, in whom protective HLA-I molecules are not the main factor.

An HLA-I signature favouring KIR-educated Natural Killer cells mediates immune control of HIV in children and contrasts with the HLA-B-restricted CD8+ T-cell-mediated immune control in adults.

Natural Killer (NK) cells contribute to HIV control in adults, but HLA-B-mediated T-cell activity has a more substantial impact on disease outcome. However, the HLA-B molecules influencing immune control in adults have less impact on paediatric infection. To investigate the contribution NK cells make to immune control, we studied >300 children living with HIV followed over two decades in South Africa. In children, HLA-B alleles associated with adult protection or disease-susceptibility did not have significant effects, whereas Bw4 (p = 0.003) and low HLA-A expression (p = 0.002) alleles were strongly associated with immunological and viral control. In a comparator adult cohort, Bw4 and HLA-A expression contributions to HIV disease outcome were dwarfed by those of protective and disease-susceptible HLA-B molecules. We next investigated the immunophenotype and effector functions of NK cells in a subset of these children using flow cytometry. Slow progression and better plasma viraemic control were also associated with high frequencies of less terminally differentiated NKG2A+NKp46+CD56dim NK cells strongly responsive to cytokine stimulation and linked with the immunogenetic signature identified. Future studies are indicated to determine whether this signature associated with immune control in early life directly facilitates functional cure in children.

VIP-SPOT: an Innovative Assay To Quantify the Productive HIV-1 Reservoir in the Monitoring of Cure Strategies.

Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4+ T cells that produce HIV antigen upon stimulation. The performance of the VIP-SPOT assay was validated in samples from viremic (n = 18) and antiretroviral therapy (ART)-treated subjects (n = 35), and the results were compared with total and intact proviral DNA and plasma viremia. The size of the functional reservoir, measured by VIP-SPOT, correlates with total HIV-1 DNA and, more strongly, with intact proviruses. However, the frequency of HIV antigen-producing cells is 100-fold lower than that of intact proviruses, thus suggesting that most latently infected cells harboring full-length proviruses are not prone to reactivation. Furthermore, VIP-SPOT was useful for evaluating the efficacy of latency reversing agents (LRAs) in primary cells. VIP-SPOT is a novel tool for measuring the size of the functional HIV-1 reservoir in a rapid, sensitive, and precise manner. It might benefit the evaluation of cure strategies based on immune clearance, as these will specifically target this minor fraction of the viral reservoir, and might assist in the identification of novel therapeutic candidates that modulate viral latency. IMPORTANCE Current efforts aimed at finding a definitive cure for HIV-1 infection are hampered mainly by the persistence of a viral reservoir in latently infected cells. While complete viral eradication from the body remains elusive, finding a functional cure to enable control of viremia without the need for continuous treatment is a key goal. As the lower reservoir size increases the likelihood of controlling viremia, new therapeutic strategies aim to reduce the size of this viral reservoir. Evaluating the efficacy of these strategies requires a robust assay to measure the viral reservoir. Currently available options are subject to overestimation or underestimation of the productive reservoir. In order to overcome this limitation, we have developed a novel assay, viral protein spot (VIP-SPOT), to precisely quantify the frequency of infected cells that retain the ability to reactivate and produce viral proteins.

Early Initiation of Antiretroviral Therapy Following In Utero HIV Infection Is Associated With Low Viral Reservoirs but Other Factors Determine Viral Rebound.

BACKGROUND: Early HIV diagnosis allows combination antiretroviral therapy (cART) initiation in the first days of life following in utero (IU) infection. The impact of early cART initiation on infant viral reservoir size in the setting of high-frequency cART nonadherence is unknown. METHODS: Peripheral blood total HIV DNA from 164 early treated (day 0-21 of life) IU HIV-infected South African infants was measured using droplet digital PCR at birth and following suppressive cART. We evaluated the impact of cART initiation timing on HIV reservoir size and decay, and on the risk of subsequent plasma viremia in cART-suppressed infants. RESULTS: Baseline HIV DNA (median 2.8 log10 copies/million peripheral blood mononuclear cells, range 0.7-4.8) did not correlate with age at cART initiation (0-21 days) but instead with maternal antenatal cART use. In 98 infants with plasma viral suppression on cART, HIV DNA half-life was 28 days. However, the probability of maintenance of plasma aviremia was low (0.46 at 12 months) and not influenced by HIV DNA load. Unexpectedly, longer time to viral suppression was associated with protection against subsequent viral rebound. CONCLUSIONS: With effective prophylaxis against mother-to-child transmission, cART initiation timing in the first 3 weeks of life is not critical to reservoir size.

Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain.

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1' position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues.

Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at "canonical" position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1' substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.