Normal Human Leukocytes Research Articles

Bimetallic Gold–Iron Oxide Nanoparticles as Carriers of Methotrexate: Perspective Tools for Biomedical Applications

Bimetallic nanoparticles (BMNPs) combine unique and synergistic properties of two metals, allowing new specific applications. In this study, bimetallic AuFe nanoparticles and their conjugates with methotrexate (MTX) were obtained with an environmentally safe method of metal-vapor synthesis. The composition and electronic structure of the particles were investigated with X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray (EDX) spectrum and X-ray absorption spectroscopy (XANES and EXAFS). The effects of BMNP-MTX conjugates on human primary cells and tumor cell lines were evaluated with neutral red uptake and MTT in vitro cytotoxicity assays. Bright-field microscopy analyses of tumor spheroid size and evaluations of tumor spheroid vitality based on SFDA AM staining were carried out. In vitro assays for an antibacterial activity evaluation of the generated samples were performed. The influence of BMNP-MTX on cytokine production with normal leukocytes was assessed using ELISA. X-ray analyses of the samples demonstrated that gold was in the ground state Au0 as well as Au+ and Au3+ states are present in small quantities, whereas iron existed as a mixture of non-histometric oxides with states close to Fe2+ and Fe3+. The modification of the AuFe system with MTX is accompanied by a threefold increase in the relative proportion of the Au+ state. BMNP-MTX conjugates demonstrated significant antitumor activity compared to the drug alone, which proves the ability of the generated nanoconjugates to improve the effectiveness of MTX therapy. This was confirmed by a marked reduction in the size and vitality of AuFe-MTX-treated 3D tumor spheroids. In addition to their selective antitumor activity, AuFe-MTX exhibited moderate antibacterial activity and induced sample-specific cytokine production with normal human leukocytes—which points to an immunostimulatory potential. The present findings indicate important and diverse biological properties of BMNP-MTX conjugates and thus highlight perspectives for their biomedical applications and new immune-specific abilities.

ANALYSIS OF DOXORUBICIN-INDUCED MITOCHONDRIAL DNA DELETIONS IN HUMAN BLOOD LEUKOCYTES AND THP-1 CELL LINE BY PCR

Doxorubicin (DOX) is an antineoplastic drug with potent cardiotoxic activity due to selective accumulation and disruption of mitochondrial functioning in cardiomyocytes. Accumulating evidence indicates that DOX can also induce genotoxic side effects in other cells. The mutagenic effects of DOX in human peripheral blood leukocytes and THP-1 leukemia cells were analyzed using PCR. It was revealed that DOX could induce deletions of different sizes and loci of mitogenomes of normal human leukocytes and THP-1 leukemic cells.

007 Antibodies to the BP180 C-terminus cause autoimmune blistering disease through activation of the C5a/C5aR1 axis and Fcγ receptors

Bullous pemphigoid (BP) is a rare autoimmune blistering disease characterized by autoantibodies targeting BP180 with the non-collagenous 16A (NC16A) domain representing the immunodominant site. The role of additional extracellular targets of the BP180 C-terminus has not yet been unequivocally demonstrated. Here, we showed that BP180 ectodomain-reactive sera depleted for NC16A IgG induced dermal-epidermal separation in control skin after incubation with normal human leukocytes indicating the pathogenic potential of anti-BP180 C-terminal IgG.

In Vitro Induction of Apoptosis in Isolated Acute Myeloid Leukemia Cells: The Role of Anastatica hierochuntica Methanolic Extract.

Anastatica hierochuntica L. (Cruciferae) has been known in Egyptian folk medicine as a remedy for gastrointestinal disorders, diabetes and heart diseases. Despite the wide usage, A. hierochuntica research provides insufficient data to support its traditional practice. The cytotoxicity of A. hierochuntica methanolic extract was investigated on acute myeloid leukemia blasts (AML) and normal human peripheral leucocytes (NHPL). The phytochemical identification of bioactive compounds using 1H-NMR and LC-ESI-MS was also performed. A. hierochuntica extract caused non-significant cytotoxicity on NHPL, while the cytotoxicity on AML was significant (IC50: 0.38 ± 0.02 μg/mL). The negative expression of p53, upregulation of Caspase-3 and increase in the BAX/BCL-2 ratio were reported at the protein and mRNA levels. The results suggest that A. hierochuntica extract induced AML cell death via the p53-independent mitochondrial intrinsic pathway and further attention should be paid to this plant as a promising natural anticancer agent.

Synthesis and Biological Evaluation of Benzo[f]indole-4,9-diones N-Linked to Carbohydrate Chains as New Type of Antitumor Agents

In this work, we report the synthesis of three series of carbohydrate-based benzo[f]indole-4,9-diones and amino-1,4-naphthoquinone derivatives and evaluated their cytotoxic activity against eight human cancer cell lines. Several compounds showed a promising cytotoxic activity toward the tumor cell lines (half maximal inhibitory concentration (IC50) < 10.0 μM). The importance of the substitution pattern of the quinone derivatives on the antitumor activity was also discussed. 3-Carboethoxy-2-methyl-benzo[f]indole-4,9-dione derivatives were more cytotoxic than their parent compounds and amino-1,4-naphthoquinones. Unlike clinically useful anticancer agent doxorubicin, the majority of synthesized compounds did not exhibit any lytic effects against erythrocytes or normal human leukocytes.

In Memoriam: Marvin A. Van Dilla: 1919-2019.

© 2020 International Society for Advancement of Cytometry.

Synthesis and evaluation of anticancer activity of new 9-acridinyl amino acid derivatives

A series of eleven 9-acridinyl amino acid derivatives were synthesized using a two-step procedure. Cytotoxicity was tested on the K562 and A549 cancer cell lines and normal diploid cell line MRC5 using the MTT assay. Compounds 6, 7, 8 and 9 were the most active, with IC50 values comparable to or lower than that of chemotherapeutic agent amsacrine. 8 and 9 were especially effective in the A549 cell line (IC50 ≈ 6 μM), which is of special interest since amsacrine is not sufficiently active in lung cancer patients. Cell cycle analysis revealed that 7 and 9 caused G2/M block, amsacrine caused arrest in the S phase, while 6 and 8 induced apoptotic cell death independently of the cell cycle regulation. In comparison to amsacrine, 6, 7, 8, and 9 showed similar inhibitory potential towards topoisomerase II, whereas only 7 showed DNA intercalation properties. In contrast to amsacrine, 6, 7, 8 and 9 showed a lack of toxicity towards unstimulated normal human leucocytes.

Idarubicin-bromelain combination sensitizes cancer cells to conventional chemotherapy.

Objective(s):The primary cytotoxic effects of anticancer drugs like idarubicin, a chemotherapeutic agent, are not limited to neoplastic cells; they also produce similar effects in normal cells. In this study, we hypothesized that the combination of idarubicin-bromelain could make cancer cells more susceptible to cytotoxicity and genotoxicity.Materials and Methods:To test our hypothesis, the optimal concentrations of idarubicin and bromelain were combined and incubated in the HL-60 cancer cell line and normal human mononuclear leukocytes (PBMC) for 24, 48, and 72 hr. Cytotoxicity and genotoxicity were evaluated by measurement of ATP cell viability test, DNA damage, Caspase-3, Acridine orange/ethidium bromide (AO/EB), and DAPI fluorescent dyes in both cell types.Results:The combination of idarubicin-bromelain significantly reduced cell proliferation in the more potent HL-60 compared to PBMC in all incubation times (P<0.05). DNA damage and Caspase-3 levels (except for 24 hr) were also higher in the HL-60 cell line in comparison with PBMC and were statistically significant (P<0.05). The percentages of apoptotic images obtained by DAPI and AO / EB morphological examination were increased in both cells, depending on the combination dose.Conclusion:Based on these results, it can be concluded that idarubicin combined with bromelain produces more cytotoxic effects in low concentrations in comparison with when it was used per se in the HL-60 cells. Conversely, it was found that this combination in PBMC caused less cytotoxicity and less genotoxicity. Taken together, it can be said that this new combination makes cancer cells more sensitive to conventional therapy.

505P - High NAMPT expression and anti-tumour activity of NAMPT inhibitor in adult T-cell leukemia/lymphoma

BackgroundAdult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes induced by human T-cell leukemia virus-1, and has a poor outcome. New molecular targets for the prevention and treatment of ATL are urgently needed. We previously reported that Sirtuin 1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, is highly expressed in primary acute-type ATL cells. NAD+ biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) modulates Sirtuin 1 activity. Here, we examined the expression and effects of inhibiting NAMPT, a rate-limiting. MethodsPeripheral blood mononuclear cells from ATL patients were carried out in accordance with the guidelines of the Committees for Ethical Review of Research involving Human Subjects at Kagoshima University Hospital. Cell viability was evaluated in the S1T cell line derived from an ATL patient, MT-2 cell line derived from normal human leukocytes transformed by leukemic T-cells from an ATL patient, and primary ATL cells. Animal experiments were approved by the Animal Care and Use Committee of Rakuno Gakuen University in accordance with the Guide for the Care and Use of Laboratory Animals. ResultsPeripheral blood mononuclear cells from acute-type ATL patients expressed significantly higher NAMPT protein levels than cells from healthy controls. FK866, a NAMPT inhibitor, induced apoptosis in fresh ATL cells ex vivo and HTLV-1-infected T-cell lines in vitro, which was accompanied by NAD+ depletion, activation of caspases, DNA fragmentation, and disruption of mitochondrial transmembrane potential. A pan-caspase inhibitor failed to prevent the FK866-induced cell death, while FK866 increased endonuclease G, a caspase-independent cell death mediator. Intriguingly, FK866 activated autophagy, revealed by increased LC3-II protein levels and autophagic flux. Thus, FK866 simultaneously activated apoptosis and autophagy. Finally, FK866 treatment markedly decreased human ATL tumor xenograft growth in immunodeficient mice. ConclusionsThese results demonstrate that NAMPT inhibition induces autophagy and caspase-dependent and -independent cell death in ATL cells, suggesting a novel therapeutic strategy for patients with this fatal disease. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Abstract 4422: Molecular and functional characterization of alpha satellite non-coding RNAs

Abstract Eukaryotic cell cycle progression requires the coordination of multiple molecular events in space and time to bring about proper transition from one phase to the next, and to ensure faithful genomic inheritance to daughter cells. The roles of coding transcription, translation, and RNA/protein degradation in cell cycle regulation are straightforward, since they control the abundance of proteins necessary for its progression. However, the contribution of non-coding transcription to this process is poorly understood. Non-coding transcription is notoriously interesting in mitosis for two reasons: First, transcription is repressed in most of the genome, but it is active in the centromere itself during mitosis; second, centromeric transcription produces non-coding RNA molecules which are integral components of the centromere and kinetochore and have functional roles in chromosome segregation. Here, we characterized the centromeric human alpha satellite non-coding RNAs (cencRNAs). We detected that the centromere produces high-molecular-weight (&amp;gt5 kb) alpha satellite transcripts in sense and antisense orientation in several cancer cell lines and normal human leukocytes. These RNAs have a half-life of ~ 30 min and vary in abundance, but not in size, throughout the cell cycle. By analyzing their abundance under various conditions that promote mitotic arrest, we discovered a new association between proteasome inhibition and cencRNAs overexpression in G2/M HCT116 cells. We also show that their transcriptional inhibition mitigates the mitotic arrest induced by proteasome inhibitors. Furthermore, we demonstrate that exogenous expression of cencRNAs can slightly increase the mitotic index of SW480 cells. Using mass spectrometry-coupled RNA pulldown, we discovered that these RNAs interact with proteins essential for chromosome segregation. Our data show that centromeric transcription is regulated by proteasomes. Proteasomal inhibition promotes abnormal mitotic progression owing to the overexpression of non-coding RNAs at the onset of mitosis and the presumed impairment of their partner proteins functions in mitosis. Citation Format: Rodrigo E. Cáceres, Marco A. Andonegui, Diego A. Oliva, Rodrigo González, Fernando Luna, Cristian G. Arriaga, Alejandro López, Diddier G. Prada, Clementina Castro, Paulina Molina, Carlos De la Rosa, José L. Reyes, Sabrine Hédouin, Florent Hubé, Claire Francastel, Luis A. Herrera. Molecular and functional characterization of alpha satellite non-coding RNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4422.

Overview of the Biological Activities of a Methanol Extract from Wild Red Belt Conk, Fomitopsis pinicola (Agaricomycetes), Fruiting Bodies from Central Italy.

Fomitopsis pinicola (Sw.) P. Karst. (Fomitopsidaceae) is a medicinal mushroom with a variety of healthy properties. In this study we tested the radical scavenging activity and antimicrobial and anticancer potential of methanol extracts of F. pinicola from central Italy. Molecular identification confirmed that the samples were F. pinicola; a Basic Local Alignment Search Tool search showed a close match (99% sequence identity) with European isolates of this species. The free radical scavenging capacities, measured by DPPH assay, showed that the extract activity was 3.5% that of Trolox. The MTT test, evaluated after 72 hours of treatment with increasing doses of extract (5-500 μg · mL-1), considerably inhibited proliferation in a dose-dependent manner in 2 human tumor cell lines. This reduction was coupled with a relevant induction of apoptosis in the human leukemia THP-1 cell line after 24 hours of treatment, but a relevant toxic effect occurred in the human colon adenocarcinoma HT29 cell line. The genotoxic potential of the methanol extracts was studied by single-cell gel electrophoresis of normal human leukocytes exposed to 20 μg extract at 37°C for 30 minutes; no DNA damage was observed. The F. pinicola methanol extract was found to have varying degrees of antifungal effects against the pathogenic fungi tested (minimum inhibitory concentration from 23.63 to 66.81 μg · mL-1). The results show that the tested F. pinicola extract has strong antimicrobial and chemo-preventive activities, but is a poor antioxidant.

Abstract 2650: Radioimmunotherapy for acute myeloid leukemia targeting human aspartyl (asparaginyl) β-hydroxylase

Abstract Treatment options for acute myeloid leukemia (AML) are limited and have modest impact on 5-year survival rates in patients &amp;lt;60 years of age (~35-40%) and on median survival of older patients (&amp;lt; 1 year). The incidence of AML has been rising, with an anticipated 19,950 new cases in 2016. Thus, the need for novel, more efficacious and better tolerated therapy for AML is urgent. Human aspartyl (asparaginyl) β-hydroxylase (HAAH) is an embryonic/developmental protein, which is down-regulated in normal cells after birth but overexpressed on the surface of many malignant cells, where it has been demonstrated to be sufficient to induce cancer cell proliferation, motility and invasiveness. We hypothesized that HAAH is selectively overexpressed on leukemia cells and thus targeting it with a fully human monoclonal antibody (mAb) conjugated to the short half-life α-emitter 213Bi could provide a novel radioimmunotherapy (RIT) treatment option with a reasonable therapeutic index. Using immunocytochemistry and flow cytometry with anti-HAAH mouse mAbs as well as a fully human mAb, PAN-622, we investigated the expression of HAAH on a variety of cytogenetically- and mutationally heterogeneous leukemia cell lines, including MV4-11, MOLM-14, HL-60, KG1α, Kasumi-1, K562, J45.1, and CCRF-CEM, as well as on normal human leukocytes. PAN-622 was further modified with DTPA-maleimide to allow chelation of radionuclides, specifically 213Bi, to the antibody. The 213Bi-DTPA-PAN-622 was then employed in killing assays to measure selective dose-dependent killing of leukemic cells. HAAH was highly expressed, as detected by flow cytometry, on all tested leukemia cell lines with KG1α and HL-60 demonstrating the highest levels of expression amongst the AML lines. Binding was observed by both PAN-622 and the modified DTPA-PAN-622 conjugate. Interestingly, expression of HAAH was not detected on CD45+ normal human leukocytes, including in subpopulations of monocytes (CD14+), B-cells (CD19+), T-cells (CD3+) or hematopoietic stem cells (CD34+). To determine whether PAN-622 could selectively target leukemia cells in a background of normal human blood, flow cytometry was performed on leukemia cells spiked into whole blood. Only leukemia cells were stained with the PAN-622 antibody as indicated by simultaneous staining for surface markers specific to the various leukocyte subpopulations. Selective killing of leukemia cell lines was demonstrated following incubation with 213Bi-DTPA-PAN-622 which induced dose dependent specific killing over a 6.7-670 pM concentration range. No killing was detected when using 213Bi conjugated to an isotype-matched control antibody at a similar specific activity and dose range. Thus, HAAH is a novel oncogenic target expressed on leukemia cells, which can be selectively targeted for RIT via the fully-human PAN-622 mAb. Experiments targeting primary leukemia cells in patient-derived bone marrow aspirates are ongoing. Citation Format: Michael S. Lebowitz, Ekaterina Revskaya, Kanam Malhotra, Amir Shahlaee, Steven Fuller, Maria R. Baer, Noa G. Holtzman, Ashkan Emadi, Ekaterina Dadachova, Hossein A. Ghanbari. Radioimmunotherapy for acute myeloid leukemia targeting human aspartyl (asparaginyl) β-hydroxylase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2650. doi:10.1158/1538-7445.AM2017-2650

Prognostic impact of programmed death-ligand 1 expression in correlation with human leukocyte antigen class I expression status in stage I adenocarcinoma of the lung

ObjectiveThe study objective was to investigate the prognostic impact of programmed death-ligand 1 expression in correlation with human leukocyte antigen class I expression on tumor cells in early-stage adenocarcinoma of the lung, because both programmed death-ligand 1 and human leukocyte antigen class I molecules play important roles in cancer immunity. MethodsNinety-four patients with completely resected pathologic stage I lung adenocarcinoma were retrospectively reviewed. Programmed death-ligand 1 expression on tumor cells was evaluated with immunohistochemistry in correlation with several clinicopathologic and molecular features, including human leukocyte antigen class I expression on tumor cells. ResultsFifteen patients (16.0%) had tumor with positive programmed death-ligand 1 expression (percentage of tumor cells expressing programmed death-ligand 1, ≥5%), and the incidence was significantly higher in poorly differentiated tumors. There was no significant correlation between human leukocyte antigen class I expression and programmed death-ligand 1 expression. Programmed death-ligand 1 positivity was a significant factor to predict a poor survival (5-year survival, 66.7% vs 85.9%; P = .049), which was enhanced in tumors with normal human leukocyte antigen class I expression (P = .029) but was not evident in tumors with reduced human leukocyte antigen class I expression (P = .552). ConclusionsThe prognostic impact of programmed death-ligand 1 expression on tumor cells in early-stage lung adenocarcinoma may be distinct according to concurrent human leukocyte antigen class I expression.

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris

&lt;em&gt;β&lt;/em&gt;-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by &lt;em&gt;α&lt;/em&gt;- and/or &lt;em&gt;β&lt;/em&gt;-subunits encoded by HEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant &lt;em&gt;β&lt;/em&gt;-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast &lt;em&gt;Pichia pastoris&lt;/em&gt; &lt;em&gt;GS115&lt;/em&gt;. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14.606 U.mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at&lt;br /&gt;4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates &lt;em&gt;P. pastoris GS115&lt;/em&gt; potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant &lt;em&gt;β&lt;/em&gt;-hexosaminidases produced within the same host.

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Screening of coastal soil samples from Ramnad district, Tamil Nadu, South India, revealed the presence of a novel non-haemolytic and non-insecticidal Bacillus thuringiensis strain that contained spherical inclusion body and exerted cytotoxic action on cultured human cancer cells. When protoplasmic extract of the B.t. LDC 501 isolate digest with proteinase K exerted significant toxicity on cell lines such as U937, HL-60 and primary leukemic cells. It also had mild influence on Jurkat (T cell lymphoma) but nontoxic to normal human leukocytes. Majority of the biochemical tests of the strain revealed similar characteristics like that of insecticidal strain B. thuringiensis (BGSC-4Q2) with slight variation. Moreover, antibiotic sensitivity assay discriminated the strain from B. anthracis due to its ability to resist ampicillin. Phylogenetic analysis revealed the distinctive nature of the strain as it forms a separate clade from other Bacillus strains.

A Human-Specific α7-Nicotinic Acetylcholine Receptor Gene in Human Leukocytes: Identification, Regulation and the Consequences of CHRFAM7A Expression.

The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A overexpression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5'-untranslated region (UTR) identified a unique 1-kb sequence that independently regulates CHRFAM7A gene expression. Because overexpression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (for example, FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte transepithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells upregulated CHRNA7, which, in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin, on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation.

Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra.

We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210(BCR/ABL) (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800-850 cm(-1) frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones. In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of patients to be proposed in the clinical setting of leukemia.

Characterization of MT-2 cells as a human regulatory T cell-like cell line.

The paucity of naturally occurring CD4+FoxP3+ regulatory T cells (Tregs) and difficulties in the identification and isolation of such cells are major obstacles in the study of human Tregs. The availability of human Treg-like cell lines is likely to facilitate a better understanding of the molecular basis of Treg function and aid the discovery of pharmacological reagents to regulate Treg activity in a disease state. In this study, we examined the Treg phenotype and the immune suppressive effect of a human T-cell leukemia virus type 1 (HTLV-1) infected cell line, MT-2. The results clearly demonstrated that the MT-2 cell line has the phenotypic and functional characteristics of human Tregs and is a useful tool in the study of human Tregs. The MT-2 cell line was derived from normal human cord leukocytes of a healthy donor by co-cultivation with leukemic cells from an adult T-cell leukemia (ATL) patient.1 It has been shown that MT-2 cells express FoxP3, and when mixed at a ratio of 1∶2 (suppressors/responders), MT-2 cells inhibited the proliferative response of purified human peripheral blood (PB) CD4+CD25− cells stimulated with dendritic cells and anti-CD3 Ab.2 However, the Treg phenotypic markers and relative suppressive activity of MT-2 cells on both proliferation and cytokine production of cocultured responder cells need to be further evaluated.

Gammaglobulins, autoantibodies, therapeutic antibodies and anti-drug antibodies in rheumatoid arthritis.

Gammaglobulins, autoantibodies, therapeutic antibodies and anti-drug antibodies in rheumatoid arthritis.

V‑set and transmembrane domain‑containing 1 is silenced in human hematopoietic malignancy cell lines with promoter methylation and has inhibitory effects on cell growth.

Numerous leukocyte differentiation antigens act as important markers for research, diagnosis, triage and eventually treatment targets for hematopoietic malignancies. V‑set and transmembrane domain‑containing 1 (VSTM1) was identified by immunogenomic analysis as a potential leukocyte differentiation antigen gene. VSTM1 is located at 19q13.4 on human chromosomes, an important genomic region prone to genetic and epigenetic modifications in numerous hematopoietic malignancies. VSTM1‑v1, a primary splicing form encoded by VSTM1, is a type I transmembrane molecule with an extracellular immunoglobulin V‑like domain and two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. In the present study, VSTM1 expression was examined in normal human peripheral leukocytes and hematopoietic tumor cell lines; in addition, the aberrant methylation of the VSTM1 gene was evaluated using methylation‑specific polymerase chain reaction (MSP). The results of the present study demonstrated that VSTM1 was widely expressed in normal human peripheral blood leukocytes, including granulocytes and monocytes, in concurrence with previous studies, as well as lymphocytes; in addition, the molecular size and expression levels of VSTM1 varied considerably between leukocytes. However, VSTM1 was undetectable in numerous hematopoietic tumor cell lines following promoter hypermethylation. The effects of pharmacologically‑induced demethylation of the VSTM1 gene and promoter region were analyzed using MSP and biosulfite genomic sequencing, and the results revealed that VSTM1 expression was restored in methylation‑silenced Jurkat cells. In addition, CKK‑8 assays revealed that VSTM1‑v1 overexpression in Jurkat cells resulted in growth suppression. Furthermore, the inhibitory effect on cell growth was enhanced following antibody‑induced cross‑linking of VSTM1‑v1. In conclusion, the results of the present study indicated that promoter methylation silenced VSTM1 and negatively regulated cell growth in human hematopoietic malignancy cell lines.