Identification and quantification of phosphodiesterase 4 subtypes in CD4 and CD8 lymphocytes from healthy and asthmatic subjects.
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ABSTRACT:
SUBMITTER: Landells LJ
PROVIDER: S-EPMC1572832 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Asthma is a heterogeneous disease characterized by abnormal airway pathophysiology and susceptibility to different stimuli, as exemplified by a subset of patients with exercise-induced bronchoconstriction. Induced sputum provides a noninvasive method to sample airway biofluids that are enriched in proteins.We hypothesized that novel mechanisms in the pathogenesis of asthma might be revealed by studying the patterns of protein expression in induced sputum.We used shotgun proteomics to analyze induced sputum from 5 healthy subjects and 10 asthmatic patients, including 5 with exercise-induced bronchoconstriction. Differential protein expression among asthmatic patients, asthma subphenotypes, and control subjects was determined by using spectral counting and computational methods.Using Gene Ontology analysis, we defined the functional landscape of the induced sputum proteome and applied network analysis to construct a protein interaction map for this airway compartment. Shotgun proteomics analysis identified a number of proteins the differential enrichment or depletion of which robustly distinguished asthmatic patients from healthy control subjects and captured the effects of exercise on induced sputum proteome. Functional and network analysis identified key processes, including proteolytic activity, that are known contributors to airway remodeling. Importantly, this approach highlighted previously unrecognized roles for differentially expressed proteins in pathways implicated in asthma, such as modulation of phospholipase A(2) by secretoglobin, a putative role for S100A8/9 in human asthma, and selective upregulation of complement component 3a in response to exercise in asthmatic patients.Computationally intensive analysis of induced sputum proteome is a powerful approach to understanding the pathophysiology of asthma and a promising methodology to investigating other diseases of the airways.
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Project description:The onset and development of many airway pathologies affect sound propagation throughout the respiratory system; changes in respiratory sounds are detected primarily by auscultation, which is highly skill dependent. The aim of the present study was to compare healthy and asthmatic pulmonary acoustics by applying a 1D model of wave propagation on CT-based patient-specific geometries. High-resolution CT lung images were acquired in five healthy volunteers and five asthmatic patients at total lung capacity (TLC) and functional residual capacity (FRC). Tracheobronchial trees were reconstructed from CT images. Acoustic pressure, impedance and wall radial velocity were measured by simulating acoustic wave propagation of two external, acoustic pressure waves (1 Pa, 200 and 600 Hz) from the trachea level to the 4th generation. In asthmatic patients, acoustic pressure averaged across the last three generations showed a reduction equal to 29.7% (p<0.01) at FRC, at 200 Hz; input and terminal impedance were 34.5% (p<0.05) higher both at FRC and TLC; wall radial velocity was more than 80% (p<0.05) lower in higher generations both at FRC and TLC. Airway differences in asthma alter acoustic parameters at FRC and TLC, with the greatest difference at FRC and 200 Hz. Acoustic wave propagation analysis represents a quantitative approach that has potential to objectively characterize airway differences in individuals with diseases such as asthma.
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Project description:BACKGROUND: T lymphocyte infiltration has been detected in glioma, although its significance remains unclear. The purpose of the present study was to explore the prognostic value of CD4(+) and CD8(+) tumour-infiltrating lymphocytes (TILs) in glioma, and the prognostic value of infiltrating Forkhead box protein 3 (FoxP3(+)) regulatory T cells were also investigated. METHODS: CD4(+), FoxP3(+) and CD8(+) TILs were assessed by immunohistochemical staining of tissue microarray cores from 284 gliomas. Kaplan-Meier analysis and Cox proportional hazards models were used to examine the survival function of these TILs in 90 glioblastoma patients. RESULTS: The number of CD8(+) TILs was inversely correlated with tumour grade (P=0.025), whereas the number of CD4(+) TILs was positively correlated with tumour grade (P=0.002). FoxP3(+) TILs were only observed in glioblastomas, but not in low-grade astrocytomas or oligodendroglial tumours. Among patients with glioblastoma, none of CD4(+) TILs, FoxP3(+) TILs and CD8(+) TILs alone was significantly associated with patient prognosis. However, the presence of high CD4(+) and low CD8(+) TIL levels was an independent predictor of poor progress-free survival (multivariate hazard ratio (HR) 1.618, 95% confidence interval (CI) 1.245-2.101, P<0.001) and poor overall survival (multivariate HR 1.508, 95% CI 1.162-1.956, P=0.002). Moreover, pseudoprogression was more often found in patients with high CD4(+) TILs and high CD8(+) TILs. CONCLUSIONS: The combination of CD4(+) and CD8(+) TILs is a predictor of clinical outcome in glioblastoma patients, and a high level of CD4(+) TILs combined with low CD8(+) TILs was associated with unfavourable prognosis.
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Project description:Persistent small-cell lymphocytosis in dogs with a concurrent mediastinal mass has been associated with both thymoma and small-cell lymphoma. In thymomas, neoplastic thymic epithelial cells induce overproduction and release of polyclonal lymphocytes, whereas thymic lymphoma results in thymic effacement by a clonal expansion of neoplastic lymphocytes and subsequent leukemic phase of lymphoma. Flow cytometry has been used to differentiate these 2 entities by immunophenotyping mediastinal mass aspirates. It has been reported that cases with mediastinal masses in which ≥ 10% of the associated small-cell lymphocytes were double positive for CD4 and CD8 were thymomas, whereas masses associated with < 10% were suggestive of lymphoma. We report a unique case of thymoma-associated lymphocytosis lacking the classic CD4+CD8+ immunophenotype. Our findings suggest that there may be more diversity in the thymoma-associated lymphocyte immunophenotype than has been identified previously; immunophenotyping alone might not be sufficient to differentiate thymic small-cell lymphoma from thymoma-associated lymphocytosis. In dogs with mediastinal masses and peripheral lymphocytosis, employing a variety of testing modalities to avoid misdiagnosis is prudent. These modalities include cytologic and/or histologic evaluation, immunophenotyping, and clonality assessment.
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Project description:LY2775240 is a highly selective, potent and orally-administered inhibitor of phosphodiesterase 4 (PDE4), and is being investigated as a treatment option for inflammatory disorders, such as psoriasis. LY2775240 was investigated in rodent and rhesus monkey nonclinical models. Treatment with LY2775240 led to significant reductions in TNFα production, a marker of PDE4 engagement upon immune activation, in both nonclinical models. In the first part of a 2-part first-in-human randomized study, a wide dose range of LY2775240 was safely evaluated and found to be well-tolerated with common adverse events (AEs) of nausea, diarrhea, and headache. No serious AEs were reported. The pharmacokinetic profile of LY2775240 was well-characterized, with a half-life that can support once-a-day dosing. An ex vivo pharmacodynamic (PD) assay demonstrated dose-dependent PDE4 target engagement as assessed by reduction in TNFα production. A 20 mg dose of LY2775240 led to near-maximal TNFα inhibition in this PD assay in the first part of the study and was selected for comparison with the clinical dose of apremilast (30 mg) in the crossover, second part of this study. The 20 mg dose of LY2775240 demonstrated sustained maximal (50%-80%) inhibition of TNFα over all timepoints over the 24-h duration. The comparator apremilast achieved peak inhibition of ~ 50% at only 4 h postdose with a return to about 10% inhibition within 12 h of dosing. In summary, the nonclinical data and safety, tolerability, and PK/PD data in healthy subjects supports further investigation of LY2775240 in inflammatory indications. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Phosphodiesterase 4 (PDE4) inhibitors, such as apremilast, are currently approved to treat autoimmune disorders, such as psoriasis. LY2775240 is an oral PDE4 inhibitor being developed for treatment of a variety of inflammatory disorders. The degree of enzymatic inhibition achieved by PDE4 inhibitors clinically is poorly understood. WHAT QUESTION DID THIS STUDY ADDRESS? This study investigated single ascending doses of LY2775240, a highly selective oral PDE4 inhibitor, in healthy subjects. LY2775240 was well-tolerated over the dose range evaluated, and pharmacokinetic/pharmacodynamic (PD) profiles were well-characterized. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? This study evaluated different doses of LY2775240 and subsequently compared a selected LY2775240 dose with the clinical dose of apremilast with an ex vivo assay. This information builds a connection between target engagement and clinical efficacy. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? This is the first report of an ex vivo PD assay that has been systematically implemented in a PDE4 inhibitor Phase 1 study. Early investigation of exposure-response relationships versus a comparator can support evaluation of clinically meaningful doses of investigational agents.
| S-EPMC8212710 | biostudies-literature
Project description:Mechanisms underlying the development of virus-induced asthma exacerbations remain unclear. To investigate if epigenetic mechanisms could be involved in virus-induced asthma exacerbations, we undertook DNA methylation profiling in asthmatic and healthy nasal epithelial cells (NECs) during Human Rhinovirus (HRV) infection in vitro.Global and loci-specific methylation profiles were determined via Alu element and Infinium Human Methylation 450 K microarray, respectively. Principal components analysis identified the genomic loci influenced the most by disease-status and infection. Real-time PCR and pyrosequencing were used to confirm gene expression and DNA methylation, respectively.HRV infection significantly increased global DNA methylation in cells from asthmatic subjects only (43.6% to 44.1%, p?=?0.04). Microarray analysis revealed 389 differentially methylated loci either based on disease status, or caused by virus infection. There were disease-associated DNA methylation patterns that were not affected by HRV infection as well as HRV-induced DNA methylation changes that were unique to each group. A common methylation locus stood out in response to HRV infection in both groups, where the small nucleolar RNA, H/ACA box 12 (SNORA12) is located. Further analysis indicated that a relationship existed between SNORA12 DNA methylation and gene expression in response to HRV infection.We describe for the first time that Human rhinovirus infection causes DNA methylation changes in airway epithelial cells that differ between asthmatic and healthy subjects. These epigenetic differences may possibly explain the mechanism by which respiratory viruses cause asthma exacerbations.
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Project description:BACKGROUND: The enumeration of absolute CD4 counts is of primary importance for many medical conditions especially HIV infection where therapeutic initiation depends on the count. These ranges tend to vary across populations. However, these ranges have not been comprehensively established in the Kenyan population. Therefore, this study aimed at establishing the reference ranges for the CD4 and CD8 T-lymphocytes in normal healthy individuals in Kenya. METHODS: A total of 315 individuals of the ages between 16 and 60 years old, in 5 different regions of the country, were recruited into the study. They were screened for diseases that potentially cause lymphocyte homeostasis perturbation. CD4/CD8 Counts were performed by use of a FACSCalibur flow cytometer (Becton-Dickinson, NJ) equipped with automated acquisition and analysis software. Results were analysed according to age, sex and region. RESULTS: Results were presented as means and ranges (in parenthesis) generated non parametrically as 2.5 and 97.5 percentiles as follows; In general population; CD3 1655 (614-2685 cells/?L ), CD4 920 (343-1493 cells/?L), and CD8 646 (187-1139 cells/?L), while according to sex, females; CD3 1787 (697-2841 cells/?L), CD4 1010 (422-1572 cells/?L), CD8 659 (187-1180 cells/?L); males; CD3 1610 (581-2641 cells/?L), CD4 889(320-1459 cells/?L) and CD8 644 (185-1140 cells/?L). The general reference ranges for CD4/CD8 ratios were as follows; general population 1.57(0.50-2.74), males 1.51(0.49-2.64) and females 1.69(0.55-2.95). CONCLUSION: The lymphocyte reference ranges for the Kenyan population are fairly comparable to those established in other African populations. The ranges also differ appreciably from those established in Germany, Italy and Switzerland. Furthermore, the study reported significant differences in the ranges of different population clusters within Kenya, as well us between males and females.
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Project description:IntroductionAlthough evidence suggests that physical exercise reduces systemic inflammation, at the plasma level, there are still contradictions in chronic obstructive pulmonary disease (COPD). In this sense, analysis of intracellular cytokines could clear off the effect of physical exercise on the inflammatory profile of these subjects.AimThe aim was to evaluate the effect of physical training on cytokine expression in CD4+ T lymphocytes from subjects with COPD.MethodsThis is a randomized controlled trial. Subjects with stable COPD were grouped into two groups, exercise and control. In total, 23 subjects with stable COPD were evaluated, of which 15 underwent aerobic strength training [physical exercise group (PEG)] and 8 underwent breathing exercises [respiratory physiotherapy group (RPG)]. Intracellular cytokines [interleukin (IL)-8, IL-13, IL-17, IL-6, IL-2, IL-10, and tumor necrosis factor alpha (TNF-α)] from CD4+ T lymphocytes were analyzed from peripheral blood through flow cytometry, before and after 8 weeks of intervention.ResultsThe PEG and RPG groups had a mean age of 68 ± 5.96 and 72.25 ± 6.86 years and predicted forced expiratory volume in the first second (FEV1) of 58.6 ± 15.99% and 39.75 ± 10.39%, respectively. It was possible to detect a significant reduction in IL-8 (p = 0.0125) and an increase in IL-13 (p = 0.0014) and an increase in TNF-α (p < 0.001) in both groups.ConclusionEight weeks of physical training, both peripheral and respiratory, were able to reduce concentrations of IL-8 and to increase IL-13, and TNF-α in CD4+ T lymphocytes in subjects with stable COPD. The findings reinforce the benefits of interventions in subjects with COPD, revealing data not previously investigated.
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Project description:Mechanisms driving acute graft-versus-host disease (aGVHD) onset in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are still poorly understood. To provide a detailed characterization of tissue-infiltrating T lymphocytes (TL) and search for eventual site-specific specificities, we developed a xenogeneic model of aGVHD in immunodeficient mice. Phenotypic characterization of xenoreactive T lymphocytes (TL) in diseased mice disclosed a massive infiltration of GVHD target organs by an original CD4+CD8+ TL subset. Immunophenotypic and transcriptional profiling shows that CD4+CD8+ TL comprise a major PD1+CD62L-/+ transitional memory subset (>60%) characterized by low level expression of cytotoxicity-related transcripts. CD4+CD8+ TL produce high IL-10 and IL-13 levels, and low IL-2 and IFN-γ, suggestive of regulatory function. In vivo tracking of genetically labeled CD4+ or CD8+ TL subsequently found that CD4+CD8+ TL mainly originate from chronically activated cytotoxic TL (CTL). On the other hand, phenotypic profiling of CD3+ TL from blood, duodenum or rectal mucosa in a cohort of allo-HSCT patients failed to disclose abnormal expansion of CD4+CD8+ TL independent of aGVHD development. Collectively, our results show that acquisition of surface CD4 by xenoreactive CD8+ CTL is associated with functional diversion toward a regulatory phenotype, but rule out a central role of this subset in the pathogenesis of aGVHD in allo-HSCT patients.
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Project description:Epitope-specific CD4+ T lymphocytes were magnetically enriched using ferromagnetic Ni and Fe-Au nanowires coated with a monomer containing a major histocompatibility complex class II-bound peptide epitope (pMHCII). The enriched lymphocytes were subsequently quantified using fluorescence-activated cell sorting (FACS). This was the first use of magnetic nanowires for cell sorting using FACS, and improvements in both specificity and fluorescent signal strength were predicted due to higher particle moments and lengths than conventional paramagnetic beads. Three different types of nanowires (Ni, Fe with Au tip and Fe-Au multilayers) were made by electrodeposition. Ni nanowires separated fewer T cells than Au tipped Fe nanowires, likely because Ni has a lower magnetic moment than Fe. Fe-Au multilayer nanowires separated more T cells than Au-tipped Fe nanowires because there was more monomer per nanowire. Also, increasing the amount of monomer increased the number of CD4+ cells separated. Compared to conventional paramagnetic beads, the nanowires had lower specificity for CD4+ T cells, but had stronger fluorescent signals due to more fluorophores per particle. This results in broader FACS baseline separation between the positive and negative cells, which is useful to detect T cells, even those with lower binding affinity for pMHCII ligands.
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