SciCombinator

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Concept: PBMC

272

One of the most common integrative medicine (IM) modalities is yoga and related practices. Previous work has shown that yoga may improve wellness in healthy people and have benefits for patients. However, the mechanisms of how yoga may positively affect the mind-body system are largely unknown. Here we have assessed possible rapid changes in global gene expression profiles in the peripheral blood mononuclear cells (PBMCs) in healthy people that practiced either a comprehensive yoga program or a control regimen. The experimental sessions included gentle yoga postures, breathing exercises, and meditation (Sudarshan Kriya and Related Practices - SK&P) compared with a control regimen of a nature walk and listening to relaxing music. We show that the SK&P program has a rapid and significantly greater effect on gene expression in PBMCs compared with the control regimen. These data suggest that yoga and related practices result in rapid gene expression alterations which may be the basis for their longer term cell biological and higher level health effects.

Concepts: DNA, Medicine, Gene, Genetics, Cell nucleus, Gene expression, Organism, PBMC

210

Global HIV-1 treatment would benefit greatly from safe herbal medicines with scientifically validated novel anti-HIV-1 activities. The root extract from the medicinal plant Pelargonium sidoides (PS) is licensed in Germany as the herbal medicine EPs®7630, with numerous clinical trials supporting its safety in humans. Here we provide evidence from multiple cell culture experiments that PS extract displays potent anti-HIV-1 activity. We show that PS extract protects peripheral blood mononuclear cells and macrophages from infection with various X4 and R5 tropic HIV-1 strains, including clinical isolates. Functional studies revealed that the extract from PS has a novel mode-of-action. It interferes directly with viral infectivity and blocks the attachment of HIV-1 particles to target cells, protecting them from virus entry. Analysis of the chemical footprint of anti-HIV activity indicates that HIV-1 inhibition is mediated by multiple polyphenolic compounds with low cytotoxicity and can be separated from other extract components with higher cytotoxicity. Based on our data and its excellent safety profile, we propose that PS extract represents a lead candidate for the development of a scientifically validated herbal medicine for anti-HIV-1 therapy with a mode-of-action different from and complementary to current single-molecule drugs.

Concepts: Immune system, Pharmacology, Medicine, Ayurveda, Alternative medicine, Herbalism, PBMC, Pelargonium sidoides

168

BACKGROUND: Expression profile of the toll like receptors (TLRs) on PBMCs is central to the regulation of proinflammatory markers. An imbalance in the TLRs expression may lead to several types of inflammatory disorders. Furthermore, the dynamic regulation of inflammatory activity and associated impaired production of cytokines by peripheral blood mononuclear cells (PBMCs) in obese individulas remain poorly understood. Therefore, we determined the perturbation in TLRs (TLR2 and TLR4), their adaptor proteins (MyD88, IRAK1 and TRAF6) expression in PBMCs/subcutaneous adipose tissue (AT) as well as inflammatory cytokines changes in obese individuals. METHODS: mRNA expression levels of TLR2, TLR4, IL-6, TNF-alpha and adaptor proteins were determined by RT-PCR. TLR2, TLR4 and adaptor proteins expression in AT was determined by immunohistochemistry. RESULTS: Obese and overweight individuals showed significantly increased expression of TLR2, TLR4 and MyD88 in both PBMCs and AT as compared with lean individuals (P < 0.05). Interestingly, we found a remarkably higher expression of TLRs in obese and overweight individuals with type 2 diabetes (P < 0.05). Increased expression of TLR2, TLR4, MyD88 and IRAK1 correlated with body mass index (BMI) (TLR2: r = 0.91; TLR4: r = 0.88, P <0.0001; MyD88: r = 0.95, P < 0.0001; IRAK1 r = 0.78, P < 0.002). TLRs' expression was also correlated with fasting blood glucose (FBG) (TLR2: r = 0.61, P < 0.002; TLR4: r = 0.52, P < 0.01) and glycated haemoglobin (HbA1c) ( TLR2: r = 0.44, P <0.03; TLR4: r = 0.48, P < 0.03). Transcript levels of IL-6 and TNF-alpha were highly elevated in obese subjects compared to lean subjects. There was a strong association of TLRs' expression in PBMCs with TNF-alpha (TLR2: r = 0.92; TLR4: r = 0.92; P < 0.0001) and IL-6 (TLR2: r = 0.91, P < 0.0001; TLR4: r = 0.81; P < 0.001). Similarly adaptor proteins were significantly correlated with TNF-alpha (MyD88: r = 0.9, P < 0.0001; IRAK1: r = 0.86; P < 0.0002) and IL-6 (MyD88: r = 0.91, P < 0.0001; IRAK1: 0.77; P < 0.002). CONCLUSIONS: TLRs and adapter proteins were overexpressed in PBMCs from obese subjects, which correlated with increased expression of TNF-alpha and IL-6. This association may explain a potential pathophysiological link between obesity and inflammation leading to insulin resistance.

Concepts: Inflammation, Diabetes mellitus, Obesity, Toll-like receptor, Insulin resistance, Adipose tissue, Body mass index, PBMC

165

BACKGROUND: Leprosy is a contagious and chronic systemic granulomatous disease caused by Mycobacterium leprae. In the pathogenesis of leprosy, granulomas play a key role, however, the mechanisms of the formation and maintenance of M. leprae granulomas are still not clearly understood. METHODS: To better understand the molecular physiology of M. leprae granulomas and the interaction between the bacilli and human host cells, we developed an in vitro model of human granulomas, which mimicked the in vivo granulomas of leprosy. Macrophages were differentiated from human monocytes, and infected with M. leprae, and then cultured with autologous human peripheral blood mononuclear cells (PBMCs). RESULTS: Robust granuloma-like aggregates were obtained only when the M. leprae infected macrophages were co-cultured with PBMCs. Histological examination showed M. leprae within the cytoplasmic center of the multinucleated giant cells, and these bacilli were metabolically active. Macrophages of both M1 and M2 types co-existed in the granuloma like aggregates. There was a strong relationship between the formation of granulomas and changes in the expression levels of cell surface antigens on macrophages, cytokine production and the macrophage polarization. The viability of M. leprae isolated from granulomas indicated that the formation of host cell aggregates benefited the host, but the bacilli also remained metabolically active. CONCLUSIONS: A simple in vitro model of human M. leprae granulomas was established using human monocyte-derived macrophages and PBMCs. This system may be useful to unravel the mechanisms of disease progression, and subsequently develop methods to control leprosy.

Concepts: Immune system, Monocyte, Tuberculosis, Mycobacterium, PBMC, Granuloma, Leprosy, Mycobacterium leprae

163

A microfluidic device was developed to separate heterogeneous particle or cell mixtures in a continuous flow using acoustophoresis. In this device, two identical surface acoustic waves (SAWs) generated by interdigital transducers (IDTs) propagated toward a microchannel, which accordingly built up a standing surface acoustic wave (SSAW) field across the channel. A numerical model, coupling a piezoelectric effect in the solid substrate and acoustic pressure in the fluid, was developed to provide a better understanding of SSAW-based particle manipulation. It was found that the pressure nodes across the channel were individual planes perpendicular to the solid substrate. In the separation experiments, two side sheath flows hydrodynamically focused the injected particle or cell mixtures into a very narrow stream along the centerline. Particles flowing through the SSAW field experienced an acoustic radiation force that highly depends on the particle properties. As a result, dissimilar particles or cells were laterally attracted toward the pressure nodes at different magnitudes, and were eventually switched to different outlets. Two types of fluorescent microspheres with different sizes were successfully separated using the developed device. In addition, E. coli bacteria pre-mixed in peripheral blood mononuclear cells (PBMCs) were also efficiently isolated using the SSAW-base separation technique. Flow cytometric analysis on the collected samples found that the purity of separated E. coli bacteria was 95.65%.

Concepts: Protein, Bacteria, Escherichia coli, Acoustics, PBMC, Microphone, Transducers, Surface acoustic wave

148

The presence of circulating tumor cells (CTCs) serves as a prognostic marker and indicator of disease relapse, as well as a means of evaluating treatment efficacy in human and canine lymphoma patients. As an extension of our previous study for the construction of clinically useful GeneScan system, we utilized the GeneScan system for detecting CTCs in canine lymphoma patients. Samples from the primary lesion and peripheral blood mononuclear cells (PBMCs) were obtained from 32 dogs with lymphoma at initial diagnosis. All samples were subjected to polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) followed by GeneScan analysis. Common clonal rearrangements with identical amplified fragments were detected in both the primary lesion and PBMCs in 19 of the 32 dogs (59.4%). However, the detection rate of CTCs varied among the anatomical classification of lymphoma studied. GeneScan analysis following PARR would facilitate studies on determining the clinical significance of CTCs in canine lymphoma patients.

Concepts: DNA, Protein, Gene expression, Cancer, Polymerase chain reaction, Molecular biology, PBMC, Thermus aquaticus

146

The relationship between age, vitamin D status, expression and functionality of the vitamin D receptor (VDR), and key genes in the vitamin D pathway in immune cells is unclear. We enrolled adults 50 to 69 years old (20 subjects) and 70+ (20 subjects) and measured: 1) 25(OH)D levels by liquid chromatography/mass spectrometry; and 2) mRNA expression of VDR, 1α-OHase, 1,25D3-MARRS, TREM-1, cathelicidin, RIG-I, and interferon-β by qRT-PCR. Mean serum 25(OH)D was 30 ± 4 ng/mL and was not associated with age. Baseline expression of VDR, 1α-OHase, 1,25D3-MARRS, TREM-1, and RIG-I also did not differ by age; IFN-β expression, however, was higher in the 70+ year old group. 25(OH)D3- and 1,25(OH)2D3-induced VDR, TREM-1 and cathelicidin expression were similar between age groups, as was LPS-induced expression of VDR and of 1α-OHase. Ligand-induced 1,25D3-MARRS expression was higher in subjects ≥ 70 years. Serum 25(OH)D was inversely associated with LPS-stimulated VDR expression and with baseline or vitamin D-induced TREM-1 expression, adjusting for age, self-rated health, and functional status. In healthy adults ≥ 50 years, the expression and functionality of the VDR, 1α-OHase and key vitamin D pathway genes were not consistently associated with age.

Concepts: Immune system, DNA, Protein, Vitamin D, Genetics, Gene expression, Blood, PBMC

139

For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods. All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values<30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.

Concepts: Immune system, Antibody, Comparison, Blood, Comparisons, PBMC, Database normalization, Cocktail

84

Repeated nucleotide sequences combined with proteins called telomeres cover chromosome ends and dictate cells lifespan. Many factors can modify telomere length, among them are: nutrition and smoking habits, physical activities and socioeconomic status measured by education level. The aim of the study was to determine the influence of above mentioned factors on peripheral blood mononuclear cells telomere length.

Concepts: DNA, Protein, Gene, Nutrition, DNA replication, Telomere, PBMC, Pork

59

Chronic fatigue syndrome (CFS) is a highly debilitating disease of unknown aetiology. Abnormalities in bioenergetic function have been cited as one possible cause for CFS. Preliminary studies were performed to investigate cellular bioenergetic abnormalities in CFS patients. A series of assays were conducted using peripheral blood mononuclear cells (PBMCs) from CFS patients and healthy controls. These experiments investigated cellular patterns in oxidative phosphorylation (OXPHOS) and glycolysis. Results showed consistently lower measures of OXPHOS parameters in PBMCs taken from CFS patients compared with healthy controls. Seven key parameters of OXPHOS were calculated: basal respiration, ATP production, proton leak, maximal respiration, reserve capacity, non-mitochondrial respiration, and coupling efficiency. While many of the parameters differed between the CFS and control cohorts, maximal respiration was determined to be the key parameter in mitochondrial function to differ between CFS and control PBMCs due to the consistency of its impairment in CFS patients found throughout the study (p≤0.003). The lower maximal respiration in CFS PBMCs suggests that when the cells experience physiological stress they are less able to elevate their respiration rate to compensate for the increase in stress and are unable to fulfil cellular energy demands. The metabolic differences discovered highlight the inability of CFS patient PBMCs to fulfil cellular energetic demands both under basal conditions and when mitochondria are stressed during periods of high metabolic demand.

Concepts: Metabolism, Adenosine triphosphate, Mitochondrion, Oxidative phosphorylation, Cellular respiration, Citric acid cycle, PBMC, Bioenergetics