Tuesday 10 October 2017

An article published this year in the “Wiley Online Library, International Journal of Laboratory Hematology” using one of our antibodies “APC anti-mouse CD3e clone 145-2C11”, by our customers from Città della Salute e della Scienza di Torino, Italy, in the analysis of Relevance of sample preparation for flow cytometry. Congrats and Thanks.


Summay:
Flow cytometry is a useful tool for diagnosis and minimal residual disease (MRD) study of hematological diseases. Standard sample preparation protocols are characterized by stain-lyse-wash (SLW). To prevent nonspecific bindings and achieve high sensitivity in MRD studies, lyse-wash-stain-wash (LWSW) is required. To our knowledge, no comparison between the two methods has been performed.

Bland-Altman analysis of percentages of cells positive for 104 antibodies in samples simultaneously prepared with LWSW and SLW methods

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APC anti-mouse CD3e clone 145-2C11

Tuesday 3 October 2017

An article published this year in “European Jounal of Immunology” using our “Peptide Production Service (MOG peptide production)”, by our customers from Centro Regional de Investigaciones Biomédicas (CRIB), Albacete, Spain, in the analysis of how Absence of Notch1 in murine myeloid cells attenuates the development of experimental autoimmune encephalomyelitis by affecting Th1 and Th17 priming. Congrats and Thanks.


Summay:
Inhibition of Notch signalling in T cells attenuates the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Growing evidence indicates that myeloid cells are also key players in autoimmune processes. Thus, the present study evaluates the role of the Notch1 receptor in myeloid cells on the progression of myelin oligodendrocyte glycoprotein (MOG)35-55-induced EAE, using mice with a myeloid-specific deletion of the Notch1 gene (MyeNotch1KO). We found that EAE progression was less severe in the absence of Notch1 in myeloid cells. Thus, histopathological analysis revealed reduced pathology in the spinal cord of MyeNotch1KO mice, with decreased microglia/astrocyte activation, demyelination and infiltration of CD4+ T cells. Moreover, these mice showed lower Th1 and Th17 cell infiltration and expression of IFN-γ and IL-17 mRNA in the spinal cord. Accordingly, splenocytes from MyeNotch1KO mice reactivated in vitro presented reduced Th1 and Th17 activation, and lower expression of IL-12, IL-23, TNF-α, IL-6, and CD86. Moreover, reactivated wild-type splenocytes showed increased Notch1 expression, arguing for a specific involvement of this receptor in autoimmune T cell activation in secondary lymphoid tissues. In summary, our results reveal a key role of the Notch1 receptor in myeloid cells for the initiation and progression of EAE.

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An article published this year in “Biomedicine & Pharmacotherapy” using our “Apoptosis Detection Kit FITC”, by our customers from Chandigarh, India.,  in the analysis of how Autophagy as a potential therapeutic target during epithelial to mesenchymal transition in renal cell carcinoma: An in vitro study. Congrats and Thanks.


Summay:
Cancer progression toward invasive and metastatic disease is aided by reactivation of epithelial–mesenchymal transition (EMT), involving transdifferentiation of epithelial cellsinto mesenchymal phenotype. This leads to increased migratory and stem cell-like features in the cells. These EMT cells are more resistant to chemotherapy and it is hypothesized that the phenomenon of autophagy induces resistance, providing a survival strategy for cells.
In the present study, we induced EMT-like phenotype in renal carcinoma cells and identified corresponding higher autophagy flux in these cells. The EMT transformed cells may be a representative of the resistant cancer stem cell(CSC)-like phenotype. Autophagy was identified as a potential mechanism of cell survival in these cells thus implying that autophagy inhibition can lead to enhanced cell death. We also observed that tumor cellsespecially EMT transformed cells, have been ‘primed’ to undergo autophagy by mTOR inhibition. We observed that combined use of autophagy inhibitor and temsirolimus (TEM) improved antitumor activity against RCC in EMT transformed metastatic cells. One of the approaches for inhibiting autophagy was the use of lysosomotropic anti-malarial drugchloroquine (CQ) and we explored the therapeutic potential of combination of CQ and the mTOR inhibitor, TEM. EMT transformed cells showed increased cell cytotoxicity when autophagy was impaired by addition CQ with TEM. This led us to conclude that inhibition of autophagy with the current therapeutic regimen could be useful in targeting the EMT transformed cells along with the bulk tumor cells in RCC.

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Apoptosis Detection Kit FITC
http://www.immunostep.com/apoptosis/3727-anxvkf-100t.html

An article published this year in “Front. Cell. Infect. Microbiol” using our our “Apoptosis Detection Kit FITC”, by our customers from SaBio, Instituto de Investigación en Recursos Cinegéticos, Ciudad Real, Spain,  in the analysis of how Vaccinomics Approach to the Identification of Candidate Protective Antigens for the Control of Tick Vector Infestations and Anaplasma phagocytophilum Infection. Congrats and Thanks.


Summay:
Anaplasma phagocytophilum is an emerging tick-borne pathogen causing human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) in small ruminants, and other forms of anaplasmosis in different domestic and wild animals. The main vectors of this pathogen are Ixodes tick species, particularly I. scapularis in the United States and I. ricinus in Europe. One of the main limitations for the development of effective vaccines for the prevention and control of A. phagocytophiluminfection and transmission is the identification of effective tick protective antigens. The objective of this study was to apply a vaccinomics approach to I. scapularis-A. phagocytophilum interactions for the identification and characterization of candidate tick protective antigens for the control of vector infestations and A. phagocytophilum infection. The vaccinomics pipeline included the use of quantitative transcriptomics and proteomics data from uninfected and A. phagocytophilum-infected I. scapularis ticks for the selection of candidate protective antigens based on the variation in tick mRNA and protein levels in response to infection, their putative biological function, and the effect of antibodies against these proteins on tick cell apoptosis and pathogen infection. The characterization of selected candidate tick protective antigens included the identification and characterization of I. ricinus homologs, functional characterization by different methodologies including RNA interference, immunofluorescence, gene expression profiling, and artificial tick feeding on rabbit antibodies against the recombinant antigens to select the candidates for vaccination trials. The vaccinomics pipeline developed in this study resulted in the identification of two candidate tick protective antigens that could be selected for future vaccination trials. The results showed that I. scapularis lipocalin (ISCW005600) and lectin pathway inhibitor (AAY66632) and I. ricinus homologs constitute candidate protective antigens for the control of vector infestations and A. phagocytophilum infection. Both antigens are involved in the tick evasion of host defense response and pathogen infection and transmission, but targeting different immune response pathways. The vaccinomics pipeline proposed here could be used to continue the identification and characterization of candidate tick protective antigens for the development of effective vaccines for the prevention and control of HGA, TBF, and other forms of anaplasmosis caused by A. phagocytophilum.
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Apoptosis Detection Kit FITC

An article published this year in “Human Molecular Genetics” using our “FITC AnnexinV” and our “PI/RNAsesolution” by our customers from the University of Coimbra, Portugal, in the analysis of how High-throughput screening uncovers miRNAs enhancing glioblastoma cell susceptibility to tyrosine kinase inhibitors. Congrats and Thanks.


Summay:
Glioblastoma (GBM) is a deadly and therapy resistant malignant brain tumour, characterized by an aggressive and diffuse growth pattern, which prevents complete surgical resection. Despite advances in the identification of genomic and molecular alterations that fuel the tumour, average patient survival post-diagnosis remains very low (14.6-months). In addition to being highly heterogeneous, GBM tumour cells exhibit high adaptive capacity to targeted molecular therapies owing to an established network of signalling cascades with functional redundancy, which provides them with robust compensatory survival mechanisms. Here, we investigated whether a multimodal strategy combining multitargeted tyrosine kinase inhibitors (MTKIs) and microRNA (miRNA) modulation could overcome the signalling pathway redundancy in GBM and, hence, promote tumour cell death. By performing a high-throughput screening, we identified a myriad of miRNAs, including those belonging to the miR-302-3p/372-3p/373-3p/520-3p family, which coordinately act with the MTKI sunitinib to decrease GBM cell viability. Two members of this family, hsa-miRNA-302a-3p and hsa-miRNA-520 b, were found to modulate the expression of receptor tyrosine kinase mediators (including AKT1, PIK3CA and SOS1) in U87 and DBTRG human GBM cells. Importantly, administration of mimics of these miRNAs with sunitinib or axitinib resulted in decreased tumour cell proliferation and enhanced cell death, whereas no significant effect was observed when coupling miRNA modulation with temozolomide, the first-line drug for GBM therapy. Overall, our results provide evidence that combining the ‘horizontal’ inhibition of signalling pathways promoted by MTKIs with the ‘vertical’ inhibition of the downstream signalling cascade promoted by hsa-miR-302a-3p and hsa-miR-520 b constitutes a promising approach towards GBM treatment.

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FITC AnnexinV

PI/RNAse Solution

An article published this year in “Cell Death and Disease” using our “Apoptosis Detection Kit PE”, by our customers from the Instituto de Salud Carlos III (ISCIII), Majadahonda, Madrid, Spain,  in the analysis of how Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses. Congrats and Thanks.


Summay:
Aging has a strong impact on the activity of the immune system, enhancing susceptibility to pathogens and provoking a predominant pre-inflammatory status, whereas dampening responses to vaccines in humans and mice. Here, we demonstrate a loss of marginal zone B lymphocytes (MZ, CD19+ CD45R+ CD21++CD23lo) and a decrease of naive B cells (CD19+ IgD+ ), whereas there is an enhancement of a CD19+ CD45Rlo innate-like B cell population (B1REL) and the so-called aged B cell compartment (ABC, CD45R+ CD21loCD23loCD5−CD11b−) in aged senescence-accelerated (SAMP8) mice but not in aged senescence-resistant (SAMR1) mice. These changes in aged SAMP8 mice were associated with lower IgG isotype levels, displaying low variable gene usage repertoires of the immunoglobulin heavy chain (VH) diversity, with a diminution on IgG1-memory B cells (CD11b−Gr1−CD138− IgM−IgD−CD19+ CD38+ IgG1+ ), an increase in T follicular helper (TFH, CD4+ CXCR5+ PD1+ ) cell numbers, and an altered MOMA-1 (metallophilic macrophages) band in primary follicles. LPS-mediated IgG1 responses were impaired in the B1REL and ABC cell compartments, both in vitro and in vivo. These data demonstrate the prominent changes to different B cell populations and in structural follicle organization that occur upon aging in SAMP8 mice. These novel results raise new questions regarding the importance of the cellular distribution in the B cell layers, and their effector functions needed to mount a coordinated and effective humoral response.
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Apoptosis Detection Kit PE

An article published this year in “Frontiers in Cellular and Infection Microbiology” using our “Apoptosis Detection Kit FITC”, by our customers from France, Czechia, Spain, Brazil and US, in the analysis of how Ixodes scapularis Tick Cells Control Anaplasma phagocytophilum Infection by Increasing the Synthesis of Phosphoenolpyruvate from Tyrosine. Congrats and Thanks.


Summay:
The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host–pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis, the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophiluminfection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum. The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

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Apoptosis Detection Kit FITC


An article published this year in “The FASEB Journal” using our “Apoptosis Detection Kit FITC”, by our customers from the University-Hospital of Verona, Italy, in the analysis of how Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury. Congrats and Thanks.


Summay:
Heparanase (HPSE) is part of the biologic network triggered by ischemia/reperfusion (I/R) injury, a complication of renal transplantation and acute kidney injury. During this period, the kidney or graft undergoes a process of macrophages recruitment and activation. HPSE may therefore control these biologic effects. We measured the ability of HPSE and its inhibitor, SST0001, to regulate macrophage polarization and the crosstalk between macrophages and HK-2 renal tubular cells during in vitro hypoxia/reoxygenation (H/R). Furthermore, we evaluated in vivo renal inflammation, macrophage polarization, and histologic changes in mice subjected to monolateral I/R and treated with SST0001 for 2 or 7 d. The in vitro experiments showed that HPSE sustained M1 macrophage polarization and modulated apoptosis, the release of damage associated molecular patterns in post-H/R tubular cells, the synthesis of proinflammatory cytokines, and the up-regulation of TLRs on both epithelial cells and macrophages. HPSE also regulated M1 polarization induced by H/R-injured tubular cells and the partial epithelial–mesenchymal transition of these epithelial cells by M1 macrophages. All these effects were prevented by inhibiting HPSE. Furthermore, the inhibition of HPSE in vivo reduced inflammation and M1 polarization in mice undergoing I/R injury, partially restored renal function and normal histology, and reduced apoptosis. These results show for the first time that HPSE regulates macrophage polarization as well as renal damage and repair after I/R. HPSE inhibitors could therefore provide a new pharmacologic approach to minimize acute kidney injury and to prevent the chronic profibrotic damages induced by I/R.—Masola, V., Zaza, G., Bellin, G., Dall’Olmo, L., Granata, S., Vischini, G., Secchi, M. F., Lupo, A., Gambaro, G., Onisto, M. Heparanase regulates the M1 polarization of renal macrophages and their crosstalk with renal epithelial tubular cells after ischemia/reperfusion injury.

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Apoptosis Detection Kit FITC
http://www.immunostep.com/apoptosis/3727-anxvkf-100t.html

An article published this year in “Mechanisms of Ageing and Development” using  our “Fixation & Permeabilization Solution, Intracell”, by our customers from the  Yeditepe University & Maltepe University Hospital, Istanbul, Turkey in the analysis of Mitochondrial depletion in CD4+ and CD19+  peripheral lymphocytes in early stage Alzheimer’s disease. Congrats and Thanks.


Summay:

 Alzheimer's disease (AD) may be associated with mitochondrial defects. The aim of the present study was to investigate changes in mitochondrial abundance in peripheral lymphocytes of early and late stage AD patients. We analysed levels of mitochondrial DNA (mtDNA) and mean fluorescence intensity (MFI) of the mitochondria-specific antibody 113-1 in CD4+, CD8+, CD19+ and CD56+ peripheral lymphocytes of early and late stage AD by quantitative real-time PCR and flow cytometry, respectively. In early stage AD, the levels of mtDNA were significantly decreased in CD4+, CD19+ and CD56+ peripheral lymphocytes while the MFI of 113-1 staining was significantly decreased in CD4+ and CD19+ cells. Thus, CD4+ and CD19+ peripheral lymphocytes of early stage AD patients exhibit mitochondrial depletion, as seen both at the level of DNA and protein.






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INTRACELL

Monday 2 October 2017

An article published this year in “Toxicology In Vitro” using our “Apoptosis Detection Kit FITC”, by our customers from Torino, Italy,  in the analysis of how Effects of the rare elements lanthanum and cerium on the growth of colorectal and hepatic cancer cell lines. Congrats and Thanks.

Summay:
Human HT-29 and HepG2 cell lines were employed to test the effects of increasing concentrations of two rare earth elements (REEs), namely cerium (Ce) and lanthanum (La), alone or in combination. Effects on cell proliferation were measured using MTT assay, luciferase-based assays and proliferating cell nuclear antigen expression, while cell mortality and type of cell death was determined by Annexin V-FTC test using flow cytometry. Modulation of 84 genes involved in oxidative stress pathways was also studied using RT-PCR based arrays. Major alterations in selected genes compared to basal expression levels of respective control groups were found in the cells exposed to 600 µM Ce for 48 h. In HepG2 cells, 51 out of 84 genes were significantly up- or down-regulated, while in HT-29 cells only 16 genes were significantly up- or down-regulated. Dosage of REEs seems to be the pivotal factor for switching the biological effects from down- to up-regulation of cell growth; thus, low concentrations promoted cell survival and proliferation, but when concentrations increased, REEs exerted anti-proliferative and cytostatic/cytotoxic effects. The molecular mechanisms underlying these effects are still not well-defined and further analysis of the mechanisms that result in inhibition or induction of cell proliferation are crucially important.

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Apoptosis Detection Kit FITC