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Fulltext Antiproliferative activity of xanthones isolated from Artocarpus obtusus

Hashim NM, Rahmani M, Ee GC, Sukari MA, Yahayu M, Oktima W, et al.

J Biomed Biotechnol, 2012;2012:130627.
PMID: 21960741 DOI: 10.1155/2012/130627

An investigation of the chemical constituents in Artocarpus obtusus species led to the isolation of three new xanthones, pyranocycloartobiloxanthone A (1), dihydroartoindonesianin C (2), and pyranocycloartobiloxanthone B (3). The compounds were subjected to antiproliferative assay against human promyelocytic leukemia (HL60), human chronic myeloid leukemia (K562), and human estrogen receptor (ER+) positive breast cancer (MCF7) cell lines. Pyranocycloartobiloxanthone A (1) consistently showed strong cytotoxic activity against the three cell lines compared to the other two with IC(50) values of 0.5, 2.0 and 5.0 μg/mL, respectively. Compound (1) was also observed to exert antiproliferative activity and apoptotic promoter towards HL60 and MCF7 cell lines at respective IC(50) values. The compound (1) was not toxic towards normal cell lines human nontumorigenic breast cell line (MCF10A) and human peripheral blood mononuclear cells (PBMCs) with IC(50) values of more than 30 μg/mL.

Matched MeSH terms: Leukocytes, Mononuclear/drug effects
Repetitive cryotherapy attenuates the in vitro and in vivo mononuclear cell activation response

Lindsay A, Othman MI, Prebble H, Davies S, Gieseg SP

Exp Physiol, 2016 07 01;101(7):851-65.
PMID: 27094349 DOI: 10.1113/EP085795

What is the central question of this study? Acute and repetitive cryotherapy are routinely used to accelerate postexercise recovery, although the effect on resident immune cells and repetitive exposure has largely been unexplored and neglected. What is the main finding and its importance? Using blood-derived mononuclear cells and semi-professional mixed martial artists, we show that acute and repetitive cryotherapy reduces the in vitro and in vivo T-cell and monocyte activation response whilst remaining independent of the physical performance of elite athletes. We investigated the effect of repetitive cryotherapy on the in vitro (cold exposure) and in vivo (cold water immersion) activation of blood-derived mononuclear cells following high-intensity exercise. Single and repeated cold exposure (5°C) of a mixed cell culture (T cells and monocytes) was investigated using in vitro tissue culture experimentation for total neopterin production (neopterin plus 7,8-dihydroneopterin). Fourteen elite mixed martial art fighters were also randomly assigned to either a cold water immersion (15 min at 10°C) or passive recovery protocol, which they completed three times per week during a 6 week training camp. Urine was collected and analysed for neopterin and total neopterin three times per week, and perceived soreness, fatigue, physical performance (broad jump, push-ups and pull-ups) and training performance were also assessed. Single and repetitive cold exposure significantly (P

Matched MeSH terms: Leukocytes, Mononuclear/metabolism; Leukocytes, Mononuclear/physiology*
Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii expressed in the yeast Pichia pastoris

Lau YL, Thiruvengadam G, Lee WW, Fong MY

Parasitol Res, 2011 Sep;109(3):871-8.
PMID: 21455621 DOI: 10.1007/s00436-011-2315-6

In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P

Matched MeSH terms: Leukocytes, Mononuclear/immunology
Novel riboflavin/VE-TPGS modified universal dentine adhesive with superior dentine bond strength and self-crosslinking potential

Daood U, Sauro S, Pichika MR, Omar H, Liang Lin S, Fawzy AS

Dent Mater, 2020 01;36(1):145-156.
PMID: 31818524 DOI: 10.1016/j.dental.2019.11.003

OBJECTIVE: To modify a universal dentine adhesive with different concentrations of riboflavin and D-Alpha 1000 Succinate polyethylene (VE-TPGS) as a chemical enhancer and to assess the micro-tensile bond strength (24h/12 months), determine resin penetration, measurement of intermolecular interactions and cytotoxicity.
MATERIALS AND METHODS: An experimental adhesive system based on bis-GMA, HEMA and hydrophobic monomer was doped with RF0.125 (RF - Riboflavin) or RF/VE-TPGS (0.25/0.50) and submitted to μTBS evaluation. Resin dentine slabs were prepared and examined using SEM and TEM. Adhesion force was analysed on ends of AFM cantilevers deflection. Quenched peptide assays were performed using fluorescence scanner and wavelengths set to 320nm and 405nm. Cytotoxicity was assessed using human peripheral blood mononuclear cell line. Molecular docking studies were carried out using Schrödinger small-molecule drug discovery suite 2018-2. Data from viable cell results was analyzed using one-way ANOVA. Bond strength values were analysed by two-way ANOVA. Nonparametric results were analyzed using a Kruskal-Wallis test at a 0.05 significance level.
RESULTS: RF/VE-TPGS0.25 groups showed highest bond strength results after 24-h storage in artificial saliva (p<0.05). RF/VE-TPGS0.50 groups showed increased bond strength after 12-months of ageing. RF/VE-TPGS modified adhesives showed appreciable presence of a hybrid layer. Packing fraction indicated solid angle profiles describing well sized density and topology relations for the RF/VE-TPGS adhesives, in particular with the RF/VE-TPGS0.50 specimens. Qualitative analysis of the phenotype of macrophages was prominently CD163+ in the RF/VE-TPGS0.50. Both the compounds showed favourable negative binding energies as expressed in terms of 'XP GScore'.
CONCLUSION: New formulations based on the incorporation of RF/VE-TPGS in universal adhesives may be of significant potential in facilitating penetration, distribution and uptake of riboflavin within the dentine surface.

Matched MeSH terms: Leukocytes, Mononuclear
How the immune mousetrap works: Structural evidence for the immunomodulatory action of a peptide from influenza NS1 protein

Zabrodskaya Y, Tsvetkov V, Shurygina AP, Vasyliev K, Shaldzhyan A, Gorshkov A, et al.

Biophys Chem, 2024 Apr;307:107176.
PMID: 38219420 DOI: 10.1016/j.bpc.2024.107176

One of the critical stages of the T-cell immune response is the dimerization of the intramembrane domains of T-cell receptors (TCR). Structural similarities between the immunosuppressive domains of viral proteins and the transmembrane domains of TCR have led several authors to hypothesize the mechanism of immune response suppression by highly pathogenic viruses: viral proteins embed themselves in the membrane and act on the intramembrane domain of the TCRalpha subunit, hindering its functional oligomerization. It has also been suggested that this mechanism is used by influenza A virus in NS1-mediated immunosuppression. We have shown that the peptide corresponding to the primary structure of the potential immunosuppressive domain of NS1 protein (G51) can reduce concanavalin A-induced proliferation of PBMC cells, as well as in vitro, G51 can affect the oligomerization of the core peptide corresponding to the intramembrane domain of TCR, using AFM and small-angle neutron scattering. The results obtained using in cellulo and in vitro model systems suggest the presence of functional interaction between the NS1 fragment and the intramembrane domain of the TCR alpha subunit. We have proposed a possible scheme for such interaction obtained by computer modeling. This suggests the existence of another NS1-mediated mechanism of immunosuppression in influenza.

Matched MeSH terms: Leukocytes, Mononuclear/metabolism
Fulltext Eurycomanone and eurycomanol from Eurycoma longifolia Jack as regulators of signaling pathways involved in proliferation, cell death and inflammation

Hajjouli S, Chateauvieux S, Teiten MH, Orlikova B, Schumacher M, Dicato M, et al.

Molecules, 2014 Sep 16;19(9):14649-66.
PMID: 25230121 DOI: 10.3390/molecules190914649

Eurycomanone and eurycomanol are two quassinoids from the roots of Eurycoma longifolia Jack. The aim of this study was to assess the bioactivity of these compounds in Jurkat and K562 human leukemia cell models compared to peripheral blood mononuclear cells from healthy donors. Both eurycomanone and eurycomanol inhibited Jurkat and K562 cell viability and proliferation without affecting healthy cells. Interestingly, eurycomanone inhibited NF-κB signaling through inhibition of IκBα phosphorylation and upstream mitogen activated protein kinase (MAPK) signaling, but not eurycomanol. In conclusion, both quassinoids present differential toxicity towards leukemia cells, and the presence of the α,β-unsaturated ketone in eurycomanone could be prerequisite for the NF-κB inhibition.

Matched MeSH terms: Leukocytes, Mononuclear/drug effects
Styryl-lactone goniothalamin inhibits TNF-α-induced NF-κB activation

Orlikova B, Schumacher M, Juncker T, Yan CC, Inayat-Hussain SH, Hajjouli S, et al.

Food Chem Toxicol, 2013 Sep;59:572-8.
PMID: 23845509 DOI: 10.1016/j.fct.2013.06.051

(R)-(+)-Goniothalamin (GTN), a styryl-lactone isolated from the medicinal plant Goniothalamus macrophyllus, exhibits pharmacological activities including cytotoxic and anti-inflammatory effects. In this study, GTN modulated TNF-α induced NF-κB activation. GTN concentrations up to 20 μM showed low cytotoxic effects in K562 chronic myelogenous leukemia and in Jurkat T cells. Importantly, at these concentrations, no cytotoxicity was observed in healthy peripheral blood mononuclear cells. Our results confirmed that GTN inhibited tumor necrosis factor-α (TNF-α)-induced NF-κB activation in Jurkat and K562 leukemia cells at concentrations as low as 5 μM as shown by reporter gene assays and western blots. Moreover, GTN down-regulated translocation of the p50/p65 heterodimer to the nucleus, prevented binding of NF-κB to its DNA response element and reduced TNF-α-activated interleukin-8 (IL-8) expression. In conclusion, GTN inhibits TNF-α-induced NF-κB activation at non-apoptogenic concentrations in different leukemia cell models without presenting toxicity towards healthy blood cells underlining the anti-leukemic potential of this natural compound.

Matched MeSH terms: Leukocytes, Mononuclear/cytology; Leukocytes, Mononuclear/drug effects
Application of Ti3 C2 MXene Quantum Dots for Immunomodulation and Regenerative Medicine

Rafieerad A, Yan W, Sequiera GL, Sareen N, Abu-El-Rub E, Moudgil M, et al.

Adv Healthc Mater, 2019 08;8(16):e1900569.
PMID: 31265217 DOI: 10.1002/adhm.201900569

Inflammation is tightly linked to tissue injury. In regenerative medicine, immune activation plays a key role in rejection of transplanted stem cells and reduces the efficacy of stem cell therapies. Next-generation smart biomaterials are reported to possess multiple biologic properties for tissue repair. Here, the first use of 0D titanium carbide (Ti3 C2 ) MXene quantum dots (MQDs) for immunomodulation is presented with the goal of enhancing material-based tissue repair after injury. MQDs possess intrinsic immunomodulatory properties and selectively reduce activation of human CD4+ IFN-γ+ T-lymphocytes (control 87.1 ± 2.0%, MQDs 68.3 ± 5.4%) while promoting expansion of immunosuppressive CD4+ CD25+ FoxP3+ regulatory T-cells (control 5.5 ± 0.7%, MQDs 8.5 ± 0.8%) in a stimulated lymphocyte population. Furthermore, MQDs are biocompatible with bone marrow-derived mesenchymal stem cells and induced pluripotent stem cell-derived fibroblasts. Finally, Ti3 C2 MQDs are incorporated into a chitosan-based hydrogel to create a 3D platform with enhanced physicochemical properties for stem cell delivery and tissue repair. This composite hydrogel demonstrates increased conductivity while maintaining injectability and thermosensitivity. These findings suggest that this new class of biomaterials may help bridge the translational gap in material and stem cell-based therapies for tissue repair and treatment of inflammatory and degenerative diseases.

Matched MeSH terms: Leukocytes, Mononuclear/drug effects; Leukocytes, Mononuclear/metabolism
Differential expression of aldolase, alpha tubulin and thioredoxin peroxidase in peripheral blood mononuclear cells from dengue fever and dengue hemorrhagic fever patients

Thayan R, Huat TL, See LL, Khairullah NS, Yusof R, Devi S

Southeast Asian J. Trop. Med. Public Health, 2009 Jan;40(1):56-65.
PMID: 19323035

We determined the differential expression levels of proteins in peripheral blood mononuclear cells of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Proteins were subjected to two-dimensional electrophoresis, mass spectrometry and Western blot analysis. We identified 8 proteins that were 2-fold or more up-regulated in patients compared to healthy control, three of which, aldolase, thioredoxin peroxidase and alpha tubulin, were related to dengue infection. Both thioredoxin peroxidase and alpha tubulin were over-expressed 4.9 and 3.3 times respectively in DHF compared to DF patients while aldolase was up-regulated 2.2 times in DF compared to DHF patients. Alpha tubulin and thioredoxin peroxidase have the potential to be utilized as biomarkers for DHF.

Matched MeSH terms: Leukocytes, Mononuclear/immunology
Safety and immunogenicity of a prefusion non-stabilized spike protein mRNA COVID-19 vaccine: a phase I trial

Gatechompol S, Kittanamongkolchai W, Ketloy C, Prompetchara E, Thitithanyanont A, Jongkaewwattana A, et al.

Nat Microbiol, 2022 Dec;7(12):1987-1995.
PMID: 36376393 DOI: 10.1038/s41564-022-01271-0

Effective mRNA SARS-CoV-2 vaccines are available but need to be stored in freezers, limiting their use to countries that have appropriate storage capacity. ChulaCov19 is a prefusion non-stabilized SARS-CoV-2 spike-protein-encoding, nucleoside-modified mRNA, lipid nanoparticle encapsulated vaccine that we report to be stable when stored at 2-8 °C for up to 3 months. Here we report safety and immunogenicity data from a phase I open-label, dose escalation, first-in-human trial of the ChulaCov19 vaccine (NCT04566276). Seventy-two eligible volunteers, 36 of whom were aged 18-55 (adults) and 36 aged 56-75 (elderly), were enroled. Two doses of vaccine were administered 21 d apart at 10, 25 or 50 μg per dose (12 per group). The primary outcome was safety and the secondary outcome was immunogenicity. All three dosages of ChulaCov19 were well tolerated and elicited robust dose-dependent and age-dependent B- and T-cell responses. Transient mild/moderate injection site pain, fever, chills, fatigue and headache were more common after the second dose. Four weeks after the second dose, in the adult cohort, MicroVNT-50 geometric mean titre against wild-type SARS-CoV-2 was 848 (95% CI, 483-1,489), 736 (459-1,183) and 1,140 (854-1,522) IU ml-1 at 10, 25 and 50 μg doses, respectively, versus 285 (196-413) IU ml-1 for human convalescent sera. All dose levels elicited 100% seroconversion, with geometric mean titre ratios 4-8-fold higher than for human convalescent sera (P mononuclear cells. The 50 μg dose induced better cross-neutralization against Alpha, Beta, Gamma and Delta variants than lower doses. ChulaCov19 at 50 μg is well tolerated and elicited higher neutralizing antibodies than human convalescent sera, with strong T-cell responses. These antibodies cross-neutralized four variants of concern. ChulaCov19 has proceeded to phase 2 clinical trials. We conclude that the mRNA vaccine expressing a prefusion non-stabilized spike protein is safe and highly immunogenic.

Matched MeSH terms: Leukocytes, Mononuclear
The inhibition of human T cell proliferation by the caspase inhibitor z-VAD-FMK is mediated through oxidative stress

Rajah T, Chow SC

Toxicol Appl Pharmacol, 2014 Jul 15;278(2):100-6.
PMID: 24768707 DOI: 10.1016/j.taap.2014.04.014

The caspase inhibitor benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and l-cysteine, whereas d-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH.

Matched MeSH terms: Leukocytes, Mononuclear/drug effects; Leukocytes, Mononuclear/immunology; Leukocytes, Mononuclear/metabolism
Fulltext Newcastle disease virus strain AF2240 as an oncolytic virus: A review

Kalyanasundram J, Hamid A, Yusoff K, Chia SL

Acta Trop, 2018 Jul;183:126-133.
PMID: 29626432 DOI: 10.1016/j.actatropica.2018.04.007

The discovery of tumour selective virus-mediated apoptosis marked the birth of an alternative cancer treatment in the form of oncolytic viruses. Even though, its oncolytic efficiency was demonstrated more than 50 years ago, safety concerns which resulted from mild to lethal side effects hampered the progress of oncolytic virus research. Since the classical oncolytic virus studies rely heavily on its natural oncolytic ability, virus manipulation was limited, thereby, restricted efforts to improve its safety. In order to circumvent such restriction, experiments involving non-human viruses such as the avian Newcastle disease virus (NDV) was conducted using cultured cells, animal models and human subjects. The corresponding reports on its significant tumour cytotoxicity along with impressive safety profile initiated immense research interest in the field of oncolytic NDV. The varying degree of oncolytic efficiency and virulency among NDV strains encouraged researchers from all around the world to experiment with their respective local NDV isolates in order to develop an oncolytic virus with desirable characteristics. Such desirable features include high tumour-killing ability, selectivity and low systemic cytotoxicity. The Malaysian field outbreak isolate, NDV strain AF2240, also currently, receives significant research attention. Apart from its high cytotoxicity against tumour cells, this strain also provided fundamental insight into NDV-mediated apoptosis mechanism which involves Bax protein recruitment as well as death receptor engagement. Studies on its ability to selectively induce apoptosis in tumour cells also resulted in a proposed p38 MAPK/NF-κB/IκBα pathway. The immunogenicity of AF2240 was also investigated through PBMC stimulation and macrophage infection. In addition, the enhanced oncolytic ability of this strain under hypoxic condition signifies its dynamic tumour tropism. This review is aimed to introduce and discuss the aforementioned details of the oncolytic AF2240 strain along with its current challenges which outlines the future research direction of this virus.

Matched MeSH terms: Leukocytes, Mononuclear
Fulltext Conformational IgE Epitope Mapping of Der p 2 and the Evaluations of Two Candidate Hypoallergens for Immunotherapy

Reginald K, Chew FT

Sci Rep, 2018 02 21;8(1):3391.
PMID: 29467434 DOI: 10.1038/s41598-018-21792-1

Epitope mapping of Der p 2, a clinically important dust-mite allergen is the first step in designing immunotherapy hypoallergen vaccine candidates. Twenty-one single alanine mutants of Der p 2 were generated and their secondary structure was analysed using circular dichroism spectra. Only one mutant, K96A resulted in a misfolded protein. All mutants were tested for serum IgE reactivity using serum from dust mite allergic individuals by immuno dot-blots. Mutations to five residues, N10, E25, K77, K96 and E102 consistently showed reduced IgE reactions compared to wild-type Der p 2, and therefore these residues constitute the major IgE epitopes of Der p 2. Two mutants with consistent low IgE binding, K96A and E102A, were subsequently evaluated as hypoallergen candidates. IgG antibodies raised in mice against both mutants could inhibit human IgE-binding to WT Der p 2. Both mutants had intact T-cell epitopes as they were able to stimulate peripheral blood mononuclear cell proliferation similar to WT Der p 2. However, a switch in Th1:Th2 cytokine profile was not observed. In summary, we have identified the major conformational epitopes of Der p 2, and evaluated two Der p 2 hypoallergen vaccine candidates for immunotherapy.

Matched MeSH terms: Leukocytes, Mononuclear/immunology
Fulltext Functional Polymorphisms of the Arachidonic Acid Pathway Associate with Risks and Clinical Outcomes of Allergic Diseases

Sio YY, Shi P, Matta SA, Fok YTR, Chiang WC, Say YH, et al.

Int Arch Allergy Immunol, 2023;184(6):609-623.
PMID: 37231900 DOI: 10.1159/000530393

INTRODUCTION: The arachidonic acid (AA) pathway plays a crucial role in allergic inflammatory diseases; however, the functional roles of allergy-associated single nucleotide polymorphisms (SNPs) in this pathway remain incompletely illustrated.
METHODS: This study belongs to a part of an ongoing Singapore/Malaysia cross-sectional genetics and epidemiological study (SMCSGES). We performed population genotyping on n = 2,880 individuals from the SMCSGES cohort to assess the associations of SNPs in the AA pathway genes with asthma and allergic rhinitis (AR). Spirometry assessments were performed to identify associations between SNPs and lung function among n = 74 pediatric asthmatic patients from the same cohort. Allergy-associated SNPs were functionally characterized using in vitro promoter luciferase assay, along with DNA methylome and transcriptome data of n = 237 peripheral blood mononuclear cell (PBMC) samples collected from a subset of the SMCSGES cohort.
RESULTS: Genetic association analysis showed 5 tag-SNPs from 4 AA pathway genes were significantly associated with asthma (rs689466 at COX2, rs35744894 at hematopoietic PGD2 synthase (HPGDS), rs11097414 at HPGDS, rs7167 at CRTH2, and rs5758 at TBXA2R, p < 0.05), whereas 3 tag-SNPs from HPGDS (rs35744894, rs11097414, and rs11097411) and 2 tag-SNPs from PTGDR (rs8019916 and rs41312470) were significantly associated with AR (p < 0.05). The asthma-associated rs689466 regulates COX2 promoter activity and associates with COX2 mRNA expression in PBMC. The allergy-associated rs1344612 was significantly associated with poorer lung function, increased risks of asthma and AR, and increased HPGDS promoter activity. The allergy-associated rs8019916 regulates PTGDR promoter activity and DNA methylation levels of cg23022053 and cg18369034 in PBMC. The asthma-associated rs7167 affects CRTH2 expression by regulating the methylation level of cg19192256 in PBMC.
CONCLUSIONS: The present study identified multiple allergy-associated SNPs that modulate the transcript expressions of key genes in the AA pathway. The development of a "personalized medicine" approach with consideration of genetic influences on the AA pathway may hopefully result in efficacious strategies to manage and treat allergic diseases.

Matched MeSH terms: Leukocytes, Mononuclear
Intracellular antigen determination by flow cytometry in leukaemic cells

Chin SF, Cheong SK

Malays J Pathol, 1994 Jun;16(1):69-73.
PMID: 16329579

Several fixation and permeabilization techniques that enable the flow cytometric analysis of the cell contents have been introduced in recent years. These methods allow sensitive detection of intracellular antigens that facilitates the diagnosis of certain diseases. We have undertaken in this study to evaluate a simple method of fixation and permeabilization using 2% paraformaldehyde and Tween 20. Intracellular antigens in three different leukaemia cases were analysed. We found that the method was reliable and easy. Intracellular kappa light chains were found in abundance in a case of plasma cell leukaemia. CD3 and CD22 were found in greater amount intracellularly than on the surface in pre-T-ALL and pre-pre B-ALL respectively.

Matched MeSH terms: Leukocytes, Mononuclear/immunology; Leukocytes, Mononuclear/pathology
Fulltext CD4+CD25hiCD127low regulatory T cells are increased in oral squamous cell carcinoma patients

Lim KP, Chun NA, Ismail SM, Abraham MT, Yusoff MN, Zain RB, et al.

PLoS One, 2014;9(8):e103975.
PMID: 25153698 DOI: 10.1371/journal.pone.0103975

Regulatory T cells (Tregs), a subset of CD4+ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-β and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4+CD25hiCD127low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-β and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8+ cytotoxic T cells and an increase in Tregs (CD4+CD25hiCD127low) were observed. Further, the ratio of CD8+ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-β secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-β. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.

Matched MeSH terms: Leukocytes, Mononuclear/immunology; Leukocytes, Mononuclear/metabolism
Fulltext Dendritic cells pulsed with generated tumor cell lysate from Phyllanthus amarus Schum. & Thonn. induces anti-tumor immune response

Mohamed SIA, Jantan I, Nafiah MA, Seyed MA, Chan KM

BMC Complement Altern Med, 2018 Aug 06;18(1):232.
PMID: 30081891 DOI: 10.1186/s12906-018-2296-4

BACKGROUND: Dendritic cells (DCs) are unique antigen presenting cells (APC) which play a pivotal role in immunotherapy and induction of an effective immune response against tumors. In the present study, 80% ethanol extract of Phyllanthus amarus was used to generate tumor lysate (TLY) derived from HCT 116 and MCF-7 cancer cell lines via induction of apoptosis. Monocyte-derived DCs were generated ex vivo from the adherent population of peripheral blood mononuclear cells (PBMCs). The generated TLY were used to impulse DCs to investigate its effect on their cellular immune functions including antigen presentation capacity, phagocytic activity, chemotaxis capacity, T-cell proliferation and cytokines release.
METHODS: The effect of P. amarus-generated TLY on DCs maturation was evaluated by determination of MHC class I, II and CD 11c expression as well as the co-stimulatory molecules CD 83 and 86 by using flow cytometry. The phagocytic capacity of TLY-pulsed DCs was investigated through FITC-dextran uptake by using flow cytometry. The effect on the cytokines release including IL-12, IL-6 and IL-10 was elucidated by using ELISA. The migration capacity and T cell proliferation activity of pulsed DCs were measured. The relative gene expression levels of cytokines were determined by using qRT-PCR. The major constituents of P. amarus extract were qualitatively and quantitatively analyzed by using validated reversed-phase high performance liquid chromatography (HPLC) methods.
RESULTS: P. amarus-generated TLY significantly up-regulated the expression levels of MHC class I, CD 11 c, CD 83 and 86 in pulsed DCs. The release of interleukin IL-12 and IL-6 was enhanced by TLY-DCs at a ratio of 1 DC: 3 tumor apoptotic bodies (APO), however, the release of IL-10 was suppressed. The migration ability as well as allogeneic T-cell proliferation activities of loaded DCs were significantly enhanced, but their phagocytic capacity was highly attenuated. The gene expression profiles for IL-12 and IL-6 of DCs showed increase in their mRNA gene expression in TLY pulsed DCs versus unloaded and LPS-treated only DCs.
CONCLUSION: The effect of P. amarus-generated TLY on the immune effector mechanisms of DCs verified its potential to induce an in vitro anti-tumor immune response against the recognized tumor antigen.

Matched MeSH terms: Leukocytes, Mononuclear/drug effects; Leukocytes, Mononuclear/immunology
Fulltext Epigenome-wide association study of adiposity and future risk of obesity-related diseases

Campanella G, Gunter MJ, Polidoro S, Krogh V, Palli D, Panico S, et al.

Int J Obes (Lond), 2018 Dec;42(12):2022-2035.
PMID: 29713043 DOI: 10.1038/s41366-018-0064-7

BACKGROUND: Obesity is an established risk factor for several common chronic diseases such as breast and colorectal cancer, metabolic and cardiovascular diseases; however, the biological basis for these relationships is not fully understood. To explore the association of obesity with these conditions, we investigated peripheral blood leucocyte (PBL) DNA methylation markers for adiposity and their contribution to risk of incident breast and colorectal cancer and myocardial infarction.
METHODS: DNA methylation profiles (Illumina Infinium® HumanMethylation450 BeadChip) from 1941 individuals from four population-based European cohorts were analysed in relation to body mass index, waist circumference, waist-hip and waist-height ratio within a meta-analytical framework. In a subset of these individuals, data on genome-wide gene expression level, biomarkers of glucose and lipid metabolism were also available. Validation of methylation markers associated with all adiposity measures was performed in 358 individuals. Finally, we investigated the association of obesity-related methylation marks with breast, colorectal cancer and myocardial infarction within relevant subsets of the discovery population.
RESULTS: We identified 40 CpG loci with methylation levels associated with at least one adiposity measure. Of these, one CpG locus (cg06500161) in ABCG1 was associated with all four adiposity measures (P = 9.07×10-8 to 3.27×10-18) and lower transcriptional activity of the full-length isoform of ABCG1 (P = 6.00×10-7), higher triglyceride levels (P = 5.37×10-9) and higher triglycerides-to-HDL cholesterol ratio (P = 1.03×10-10). Of the 40 informative and obesity-related CpG loci, two (in IL2RB and FGF18) were significantly associated with colorectal cancer (inversely, P

Matched MeSH terms: Leukocytes, Mononuclear/chemistry
Fulltext Phenotypic, transcriptomic and functional profiling reveal reduced activation thresholds of CD8+ T cells in giant cell arteritis

Reitsema RD, van der Geest KSM, Sandovici M, Jiemy WF, Graver JC, Abdulahad WH, et al.

Rheumatology (Oxford), 2022 Dec 23;62(1):417-427.
PMID: 35460236 DOI: 10.1093/rheumatology/keac250

OBJECTIVES: Evidence from temporal artery tissue and blood suggests involvement of CD8+ T cells in the pathogenesis of GCA, but their exact role is poorly understood. Therefore, we performed a comprehensive analysis of circulating and lesional CD8+ T cells in GCA patients.
METHODS: Circulating CD8+ T cells were analysed for differentiation status (CD45RO, CCR7), markers of activation (CD69 and CD25) and proliferation (Ki-67) in 14 newly diagnosed GCA patients and 18 healthy controls by flow cytometry. Proliferative capacity of CD8+ T cells upon anti-CD3 and anti-CD3/28 in vitro stimulation was assessed. Single-cell RNA sequencing of peripheral blood mononuclear cells of patients and controls (n = 3 each) was performed for mechanistic insight. Immunohistochemistry was used to detect CD3, CD8, Ki-67, TNF-α and IFN-γ in GCA-affected tissues.
RESULTS: GCA patients had decreased numbers of circulating effector memory CD8+ T cells but the percentage of Ki-67-expressing effector memory CD8+ T cells was increased. Circulating CD8+ T cells from GCA patients demonstrated reduced T cell receptor activation thresholds and displayed a gene expression profile that is concurrent with increased proliferation. CD8+ T cells were detected in GCA temporal arteries and aorta. These vascular CD8+ T cells expressed IFN-γ but not Ki-67.
CONCLUSION: In GCA, circulating effector memory CD8+ T cells demonstrate a proliferation-prone phenotype. The presence of CD8+ T cells in inflamed arteries seems to reflect recruitment of circulating cells rather than local expansion. CD8+ T cells in inflamed tissues produce IFN-γ, which is an important mediator of local inflammatory responses in GCA.

Matched MeSH terms: Leukocytes, Mononuclear/metabolism
Fulltext N-glycosylation and expression in human tissues of the orphan GPR61 receptor

Kozielewicz P, Alomar H, Yusof S, Grafton G, Cooper AJ, Curnow SJ, et al.

FEBS Open Bio, 2017 12;7(12):1982-1993.
PMID: 29226084 DOI: 10.1002/2211-5463.12339

A number of members of the G protein-coupled receptor class of cell surface receptors are 'orphans' with no known endogenous ligand. One of these orphan receptors is GPR61; there are little data about its expression in human cells and tissues. In this study, we investigated the post-translational modification of GPR61 by N-glycosylation at an identified consensus N-glycosylation site (N12) and the impact of this modification upon the subcellular expression of the protein. The N-glycosylation inhibitor tunicamycin reduced the apparent molecular weight of immunoreactivity associated with myc-tagged GPR61 by 1-2 kDa, which was comparable to the evident molecular weight of the myc-tagged N12S GPR61 mutant with disrupted consensus N-glycosylation site. Analysis of GPR61 expression demonstrated that tunicamycin treatment reduced considerably heterologous expression of GPR61 in the cell membrane despite the N12S GPR61 mutant being readily expressed at the cell surface. These results demonstrate that GPR61 is subject to N-glycosylation but suggest this is not a prerequisite for cell surface expression, although N-glycosylation of other proteins may be important for cell membrane expression of GPR61. Expression of GPR61 protein was demonstrated at the cellular level in human hippocampus and human peripheral blood mononuclear cells. In the latter, there was a significantly higher expression of GPR61 in the Th17 cell subset in comparison with resting CD4+ cells, which may point toward a potential role for the GPR61 receptor in autoimmune diseases. This is the first report that GPR61 protein is subject to post-translational modification and is expressed in immune cell subsets and the hippocampus. These findings will help guide studies to investigate the function of GPR61.

Matched MeSH terms: Leukocytes, Mononuclear

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