In Depth Analysis on the Binding Sites of Adamantane Derivatives in HCV (Hepatitis C Virus) p7 Channel Based on the NMR Structure

Background

The recently solved solution structure of HCV (hepatitis C virus) p7 ion channel provides a solid structure basis for drug design against HCV infection. In the p7 channel the ligand amantadine (or rimantadine) was determined in a hydrophobic pocket. However the pharmocophore (−NH₂) of the ligand was not assigned a specific binding site.

Results

The possible binding sites for amino group of adamantane derivatives is studied based on the NMR structure of p7 channel using QM calculation and molecular modeling. In the hydrophobic cavity and nearby three possible binding sites are proposed: His17, Phe20, and Trp21. The ligand binding energies at the three binding sites are studied using high level QM method CCSD(T)/6–311+G(d,p) and AutoDock calculations, and the interaction details are analyzed. The potential application of the binding sites for rational inhibitor design are discussed.

Conclusions

Some useful viewpoints are concluded as follows. (1) The amino group (−NH₂) of adamantane derivatives is protonated (−NH₃ ⁺), and the positively charged cation may form cation-π interactions with aromatic amino acids. (2) The aromatic amino acids (His17, Phe20, and Trp21) are the possible binding sites for the protonated amino group (−NH₃ ⁺) of adamantane derivatives, and the cation-π bond energies are 3 to 5 times stronger than the energies of common hydrogen bonds. (3) The higher inhibition potent of rimantadine than amantadine probably because of its higher pK_a value (pK_a = 10.40) and the higher positive charge in the amino group. The potential application of p7 channel structure for inhibitor design is discussed.     

Theoretical studies on protein-nucleic acid interactions. I. Interaction of positively charged amino acids with nucleic acid fragments

Coulombic interactions between the side chains of charged amino acids (Arg⁺, Lys⁺, and His⁺) and negatively charged phosphate groups of nucleic acid fragments have been studied theoretically. Diribose monophosphate and dideoxyribose monophosphate are chosen as model systems for single-stranded RNA and DNA, respectively. The interaction energies have been calculated by second-order perturbation theory using simplified formulas for individual terms. The interaction energy in this formalism is a sum of electrostatic, polarization, dispersion, and repulsive energies. Our results show that about 90% of the total interaction energy is contributed by the electrostatic term alone. Contribution from the repulsive term exceeds that from the dispersion term. Calculated interaction energies suggest that Lys⁺ and His⁺ form more stable complexes with RNA than with single-stranded DNA. On the other hand, Arg⁺ has a higher affinity for DNA than for RNA. The affinity of nucleic acids for the three amino acids is in the order Lys⁺ > His⁺ > Arg⁺. Further, the basic amino acid residues form more stable complexes with A-DNA than with B-DNA. The role of the Coulombic interactions in the specific recognition of nucleic acids by proteins is discussed.     

Decreased Frequency of Intestinal Regulatory CD5+ B Cells in Colonic Inflammation

Background

CD5⁺ B cells are a type of regulatory immune cells, though the involvement of this B cell subset in intestinal inflammation and immune regulation is not fully understood.

Methods

We examined the distribution of CD5⁺ B cells in various mouse organs. Expression levels of CD11b, IgM, and toll-like receptor (TLR)-4 and -9 in B cells were evaluated. In vitro, TLR-stimulated IL-10 production by colonic lamina propria (LP) CD5⁺ and CD5^- B cells was measured. In vivo, mice with acute or chronic dextran sulfate sodium (DSS)-induced colonic injury were examined, and the frequency of colonic LP CD5⁺ B cells in those was assessed by flow cytometry.

Results

The expression level of TLR9 was higher in colonic LP CD5⁺ B cells as compared to CD5^- B cells. Colonic LP CD5⁺ B cells produced greater amounts of IL-10 following stimulation with TLR ligands, especially TLR9, as compared with the LP CD5^- B cells. Acute intestinal inflammation transiently decreased the frequency of colonic LP CD5⁺ B cells, while chronic inflammation induced a persistent decrease in colonic LP CD5⁺ B cells and led to a CD5^- B cell-dominant condition.

Conclusion

A persistent altered mucosal B cell population caused by chronic gut inflammation may be involved in the pathogenesis of inflammatory bowel diseases.     

Post-Prandial Protein Handling: You Are What You Just Ate

Background

Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults.

Objective

To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein.

Design

12 healthy young males ingested 20 g intrinsically [1-¹³C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-²H₅]-phenylalanine, L-[ring-²H₂]-tyrosine, and L-[1-¹³C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment.

Results

55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min^-1·100 mL leg volume^-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h^-1 based upon the muscle protein bound L-[ring-²H₅]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-¹³C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE).

Conclusion

Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis.

Trial Registration

trialregister.nl 3638     

Comparative Metabolite Profiling of Two Rice Genotypes with Contrasting Salt Stress Tolerance at the Seedling Stage

Background

Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level.

Methods

Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated.

Results

We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na⁺ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment.

Conclusions

Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress.     

Dynamic Changes in Amino Acid Concentration Profiles in Patients with Sepsis

Objectives

The goal of this work was to explore the dynamic concentration profiles of 42 amino acids and the significance of these profiles in relation to sepsis, with the aim of providing guidance for clinical therapies.

Methods

Thirty-five critically ill patients with sepsis were included. These patients were further divided into sepsis (12 cases) and severe sepsis (23 cases) groups or survivor (20 cases) and non-survivor (15 cases) groups. Serum samples from the patients were collected on days 1, 3, 5, 7, 10, and 14 following intensive care unit (ICU) admission, and the serum concentrations of 42 amino acids were measured.

Results

The metabolic spectrum of the amino acids changed dramatically in patients with sepsis. As the disease progressed further or with poor prognosis, the levels of the different amino acids gradually increased, decreased, or fluctuated over time. The concentrations of sulfur-containing amino acids (SAAs), especially taurine, decreased significantly as the severity of sepsis worsened or with poor prognosis of the patient. The serum concentrations of SAAs, especially taurine, exhibited weak negative correlations with the Sequential Organ Failure Assessment (SOFA) (r=-0.319) and Acute Physiology and Chronic Health Evaluation (APACHE) II (r=-0.325) scores. The areas under the receiver operating characteristic curves of cystine, taurine, and SAA levels and the SOFA and APACHE II scores, which denoted disease prognosis, were 0.623, 0.674, 0.678, 0.86, and 0.857, respectively.

Conclusions

Critically ill patients with disorders of amino acid metabolism, especially of SAAs such as cystine and taurine, may provide an indicator of the need for the nutritional support of sepsis in the clinic.

Trial Registration

ClinicalTrial.gov identifier NCT01818830.     

Comparative Validation of Five Quantitative Rapid Test Kits for the Analysis of Salt Iodine Content: Laboratory Performance,User- and Field-Friendliness

Background

Iodine deficiency has important health and development consequences and the introduction of iodized salt as national programs has been a great public health success in the past decades. To render national salt iodization programs sustainable and ensure adequate iodization levels, simple methods to quantitatively assess whether salt is adequately iodized are required. Several methods claim to be simple and reliable, and are available on the market or are in development.

Objective

This work has validated the currently available quantitative rapid test kits (quantRTK) in a comparative manner for both their laboratory performance and ease of use in field settings.

Methods

Laboratory performance parameters (linearity, detection and quantification limit, intra- and inter-assay imprecision) were conducted on 5 quantRTK. We assessed inter-operator imprecision using salt of different quality along with the comparison of 59 salt samples from across the globe; measurements were made both in a laboratory and a field setting by technicians and non-technicians. Results from the quantRTK were compared against iodometric titration for validity. An ‘ease-of-use’ rating system was developed to identify the most suitable quantRTK for a given task.

Results

Most of the devices showed acceptable laboratory performance, but for some of the devices, use by non-technicians revealed poorer performance when working in a routine manner. Of the quantRTK tested, the iCheck^® and I-Reader^® showed most consistent performance and ease of use, and a newly developed paper-based method (saltPAD) holds promise if further developed.

Conclusions

User- and field-friendly devices are now available and the most appropriate quantRTK can be selected depending on the number of samples and the budget available.     

MzPIP2;1: An Aquaporin Involved in Radial Water Movement in Both Water Uptake and Transportation,Altered the Drought and Salt Tolerance of Transgenic Arabidopsis

Background

Plants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature, which results in water deficit and even death. Water uptake and transportation play a critical role in response to these stresses. Many aquaporin proteins, localized at different tissues, function in various transmembrane water movements. We targeted at the key aquaporin in charge of both water uptake in roots and radial water transportation from vascular tissues through the whole plant.

Results

The MzPIP2;1 gene encoding a plasma membrane intrinsic protein was cloned from salt-tolerant apple rootstock Malus zumi Mats. The GUS gene was driven by MzPIP2;1 promoter in transgenic Arabidopsis. It indicated that MzPIP2;1 might function in the epidermal and vascular cells of roots, parenchyma cells around vessels through the stems and vascular tissues of leaves. The ectopically expressed MzPIP2;1 conferred the transgenic Arabidopsis plants enhanced tolerance to slight salt and drought stresses, but sensitive to moderate salt stress, which was indicated by root length, lateral root number, fresh weight and K⁺/Na⁺ ratio. In addition, the possible key cis-elements in response to salt, drought and cold stresses were isolated by the promoter deletion experiment.

Conclusion

The MzPIP2;1 protein, as a PIP2 aquaporins subgroup member, involved in radial water movement, controls water absorption and usage efficiency and alters transgenic plants drought and salt tolerance.     

Pediatric Primitive Neuroectodermal Tumors of the Central Nervous System Differentially Express Granzyme Inhibitors

Background

Central nervous system (CNS) primitive neuroectodermal tumors (PNETs) are malignant primary brain tumors that occur in young infants. Using current standard therapy, up to 80% of the children still dies from recurrent disease. Cellular immunotherapy might be key to improve overall survival. To achieve efficient killing of tumor cells, however, immunotherapy has to overcome cancer-associated strategies to evade the cytotoxic immune response. Whether CNS-PNETs can evade the immune response remains unknown.

Methods

We examined by immunohistochemistry the immune response and immune evasion strategies in pediatric CNS-PNETs.

Results

Here, we show that CD4⁺, CD8⁺, γδ-T-cells, and Tregs can infiltrate pediatric CNS-PNETs, although the activation status of cytotoxic cells is variable. Pediatric CNS-PNETs evade immune recognition by downregulating cell surface MHC-I and CD1d expression. Intriguingly, expression of SERPINB9, SERPINB1, and SERPINB4 is acquired during tumorigenesis in 29%, 29%, and 57% of the tumors, respectively.

Conclusion

We show for the first time that brain tumors express direct granzyme inhibitors (serpins) as a potential mechanism to overcome cellular cytotoxicity, which may have consequences for cellular immunotherapy.     

Ambulatory Blood Pressure Monitoring in Individuals with HIV: A Systematic Review and Meta-Analysis

Introduction

Abnormal diurnal blood pressure (BP) rhythms may contribute to the high cardiovascular disease risk in HIV-positive (HIV⁺) individuals. To synthesize the current literature on ambulatory BP monitoring (ABPM) in HIV⁺ individuals, a systematic literature review and meta-analysis were performed.

Methods

Medical databases were searched through November 11, 2015 for studies that reported ABPM results in HIV⁺ individuals. Data were extracted by 2 reviewers and pooled differences between HIV⁺ and HIV-negative (HIV^-) individuals in clinic BP and ABPM measures were calculated using random-effects inverse variance weighted models.

Results

Of 597 abstracts reviewed, 8 studies with HIV⁺ cohorts met the inclusion criteria. The 420 HIV⁺ and 714 HIV^- individuals in 7 studies with HIV^- comparison groups were pooled for analyses. The pooled absolute nocturnal systolic and diastolic BP declines were 3.16% (95% confidence interval [CI]: 1.13%, 5.20%) and 2.92% (95% CI: 1.64%, 4.19%) less, respectively, in HIV⁺ versus HIV^- individuals. The pooled odds ratio for non-dipping systolic BP (nocturnal systolic BP decline <10%) in HIV⁺ versus HIV^- individuals was 2.72 (95% CI: 1.92, 3.85). Differences in mean clinic, 24-hour, daytime, or nighttime BP were not statistically significant. I² and heterogeneity chi-squared statistics indicated the presence of high heterogeneity for all outcomes except percent DBP dipping and non-dipping SBP pattern.

Conclusions

An abnormal diurnal BP pattern may be more common among HIV⁺ versus HIV^- individuals. However, results were heterogeneous for most BP measures, suggesting more research in this area is needed.     

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

Background

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As⁺³) and cadmium (Cd⁺²)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd⁺²-and As⁺³-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.

Methods

Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As⁺³-and Cd⁺²-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.

Results

It was shown that the As⁺³-and Cd⁺²-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.

Conclusions

Tumor initiating cells isolated from SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.     

Changes in Non-Enzymatic Antioxidants in the Blood Following Anaerobic Exercise in Men and Women

Purpose

The aim of this study was to compare changes in total oxidative status (TOS), total antioxidative capacity (TAC) and the concentration of VitA, VitE, VitC, uric acid (UA), reduced (GSH) and oxidized glutathione (GSSG) in blood within 24 hours following anaerobic exercise (AnEx) among men and women.

Methods

10 women and 10 men performed a 20-second bicycle sprint (AnEx). Concentrations of oxidative stress indicators were measured before AnEx and 3, 15 and 30 minutes and 1 hour afterwards. UA, GSH and GSSH were also measured 24 hours after AnEx. Lactate and H⁺ concentrations were measured before and 3 minutes after AnEx.

Results

The increase in lactate and H⁺ concentrations following AnEx was similar in both sexes. Changes in the concentrations of all oxidative stress indicators were significant and did not differ between men and women. In both sexes, TOS, TAC, TOS/TAC and VitA and VitE concentrations were the highest 3 minutes, VitC concentration was the highest 30 minutes, and UA concentration was the highest 1 hour after AnEx. GSH concentration was significantly lower than the initial concentration from 15 minutes to 24 hour after AnEx. GSSG concentration was significantly higher, while the GSH/GSSG ratio was significantly lower than the initial values 1 hour and 24 hour after AnEx.

Conclusions

With similar changes in lactate and H⁺ concentrations, AnEx induces the same changes in TAC, TOS, TOS/TAC and non-enzymatic antioxidants of low molecular weight in men and women. Oxidative stress lasted at least 24 hours after AnEx.     

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma

Background

Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied.

Methods

We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry.

Results

Subjects with severe asthma had higher PAR-2 expression on CD14⁺⁺CD16⁺ monocytes (intermediate monocytes) and also higher percentage of CD14⁺⁺CD16⁺PAR-2⁺ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14⁺⁺CD16⁺PAR-2⁺ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14⁺⁺CD16⁺ PAR-2⁺ cells in peripheral blood compared to subjects without exacerbations.

Conclusions

PAR-2 expression is increased on CD14⁺⁺CD16⁺ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14⁺⁺CD16⁺ monocytes may play a role in the pathogenesis of severe asthma.     

Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer

Purpose

While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model.

Experimental Design

Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma.

Results

Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8⁺ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8⁺ effector T cells: FoxP3⁺ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8⁺ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8⁺ T cell-mediated antitumor immunity.

Conclusion

Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.     

The Role of Regulatory CD4 T Cells in Maintaining Tolerance in a Mouse Model of Autoimmune Hepatitis

Background

The role of regulatory CD4 T cells (Treg) in immune-mediated liver disease is still under debate. It remains disputed whether Treg suppress T cell-mediated hepatitis in vivo and whether hepatic regulatory T cells are functional in patients with autoimmune hepatitis.

Methods

We used TF-OVA mice, which express ovalbumin in hepatocytes, to investigate the impact of Treg in a model of autoimmune hepatitis. Treg isolated from inflamed livers of TF-OVA mice were tested for their functionality in vitro. By employing double transgenic TF-OVAxDEREG (DEpletion of REGulatory T cells) mice we analyzed whether Treg-depletion aggravates autoimmune inflammation in the liver in vivo.

Results

CD25⁺Foxp3⁺ CD4 T cells accumulated in the liver in the course of CD8 T cell-mediated hepatitis. Treg isolated from inflamed livers were functional to suppress CD8 T-cell proliferation in vitro. Depletion of Treg in TF-OVAxDEREG mice dramatically amplified T cell-mediated hepatitis. Repeated administration of antigen-specific CD8 T cells led to a second wave of inflammation only after depletion of Treg.

Conclusion

Our data add to the evidence for an important role of Treg in autoimmune hepatitis and show that Treg reduce the severity of T-cell mediated hepatitis in vivo. They constitute a key immune cell population that actively maintains a tolerogenic milieu in the liver and protects the liver against repeated inflammatory challenges.     

Lipophilic Triphenylphosphonium Cations Inhibit Mitochondrial Electron Transport Chain and Induce Mitochondrial Proton Leak

Background

The lipophilic positively charged moiety of triphenylphosphonium (TPP⁺) has been used to target a range of biologically active compounds including antioxidants, spin-traps and other probes into mitochondria. The moiety itself, while often considered biologically inert, appears to influence mitochondrial metabolism.

Methodology/Principal Findings

We used the Seahorse XF flux analyzer to measure the effect of a range of alkylTPP⁺ on cellular respiration and further analyzed their effect on mitochondrial membrane potential and the activity of respiratory complexes. We found that the ability of alkylTPP⁺ to inhibit the respiratory chain and decrease the mitochondrial membrane potential increases with the length of the alkyl chain suggesting that hydrophobicity is an important determinant of toxicity.

Conclusions/Significance

More hydrophobic TPP⁺ derivatives can be expected to have a negative impact on mitochondrial membrane potential and respiratory chain activity in addition to the effect of the biologically active moiety attached to them. Using shorter linker chains or adding hydrophilic functional groups may provide a means to decrease this negative effect.     

CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel

Background

Chronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection.

Methodology

CD4⁺CD25^hiFOXP3⁺ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE.

Principal Findings

S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only.

Conclusions

Schistosoma-associated CD4⁺CD25^hiFOXP3⁺Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.     

Persistence of Pathological Distribution of NK Cells in HIV-Infected Patients with Prolonged Use of HAART and a Sustained Immune Response

Objective

A prospective analysis of the distribution of NK subsets and natural cytotoxicity receptors (NKp30/NKp46) in HIV patients with long-term HAART use and sustained virological and immunological response.

Methods

The main inclusion criteria were: at least 3 years’ receipt of HAART; current CD4⁺ count ≥ 500 cells/mm³; undetectable viral load for at least 24 months; no hepatotropic virus co-infection. Percentages of CD56^dim, CD56^bright NK cells and CD56^neg CD16⁺ cells were obtained. Expression of the NCRs, NKp30 and NKp46 was analysed in CD56⁺ cells. Thirty-nine infected patients and sixteen healthy donors were included in the study.

Results

The percentages of total CD56⁺ and CD56^dim NK cells were significantly lower in HIV-infected patients than in healthy donors (70.4 vs. 50.3 and 80.9 vs. 66.1 respectively). The percentage of total CD56⁺ NK cells expressing NCR receptors was lower in HIV patients than in healthy donors (NKp30: 25.20 vs. 58.63; NKp46: 24.8 vs. 50.59). This was also observed for CD56^dim and CD56^bright NK cells. Length of time with undetectable HIV viral load was identified as an independent factor associated with higher expression of NKp30 and NKp46.

Conclusion

Despite the prolonged and effective use of HAART, HIV-infected patients do not fully reconstitute the distribution of NK cells. Length of time with an undetectable viral load was related to greater recovery of NKp30/NKp46 receptors.