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Atherosclerotic plaques are sites of chronic inflammation with diverse cell contents and complex immune signaling. Plaque progression and destabilization are driven by the infiltration of immune cells and the cytokines that mediate their interactions. Here, we attempted to compare the systemic cytokine profiles in the blood plasma of patients with atherosclerosis and the local cytokine production, using ex vivo plaque explants from the same patients. The developed method of 41-plex xMAP data normalization allowed us to differentiate twenty-two cytokines produced by the plaque that were not readily detectable in free circulation and six cytokines elevated in blood plasma that may have other sources than atherosclerotic plaque. To verify the xMAP data on the putative atherogenesis-driving chemokines MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), and fractalkine (CX3CL1), qPCR was performed. The MIP1A ( CCL3 ), MIP1B ( CCL4 ), FKN ( CX3CL1 ) , and MCP1 ( CCL2 ) genes were expressed at high levels in the plaques, whereas RANTES ( CCL5 ) was almost absent. The expression patterns of the chemokines were restricted to the plaque cell types: the MCP1 ( CCL2 ) gene was predominantly expressed in endothelial cells and monocytes/macrophages, MIP1A ( CCL3 ) in monocytes/macrophages, and MIP1B (CCL4) in monocytes/macrophages and T cells. RANTES ( CCL5 ) was restricted to T cells, while FKN ( CX3CL1 ) was not differentially expressed. Taken together, our data indicate a plaque-specific cytokine production profile that may be a useful tool in atherosclerosis studies.

SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5'-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. CONCLUSION: Alternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Alternative ORFs in-frame with the known genes are challenging to reveal. Yet they may contribute significantly to proteome diversity. Here we focused on the individual expression of the SERPINA1 gene exon 5 leading to direct translation of alpha1-antitrypsin (AAT) C-terminal peptides. The discovery of alternative ways for their production may expand the current understanding of the serpin gene's functioning. We detected short transcripts expressed primarily in hepatocytes. We identified four variants of hepatocyte-specific SERPINA1 short transcripts and individually probed their potential to be translated in living cells. The long mRNA gave the full-length AAT-eGFP fusion, while in case of short transcripts we deduced four active SERPINA1 in-frame alternative ORFs encoding 10, 21, 153 and 169 amino acids AAT C-terminal oligo- and polypeptides. Unlike secretory AAT-eGFP fusion exhibiting classical AAT behavior, truncated AAT-fusions differ by intracellular retention and nuclear enrichment. Immunofluorescence on the endogenous AAT C-terminal epitope showed its accumulation in the cell nucleoli, indicating that short transcripts may be translated in vivo. FANTOM5 CAGE data on SERPINA1 suggest that short transcripts originate from the post-transcriptional cleavage of the spliced mRNA, initiated mainly from the hepatocyte-specific promoter. CONCLUSION: Short SERPINA1 transcripts may represent a source for the direct synthesis of AAT C-terminal peptides with properties uncommon to AAT., (Copyright © 2021. Published by Elsevier B.V.)

Atherosclerotic cardiovascular disease (ASCVD) remains an important cause of morbidity in the general population and risk for ASCVD is increased approximately 2-fold in persons living with HIV infection (PLWH). This risk is linked to elevated CD8 T cell counts that are abundant in atherosclerotic plaques and have been implicated in disease pathogenesis yet the mechanisms driving T cell recruitment to and activation within plaques are poorly defined. Here we investigated the role of CD8 T cells in atherosclerosis in a non-human primate model of HIV infection and in the HIV-uninfected elderly; we sought to identify factors that promote the activation, function, and recruitment to endothelium of CX3CR1+ CD8 T cells. We measured elevated expression of CX3CL1 and IL-15, and increased CD8 T cell numbers in the aortas of rhesus macaques infected with SIV or SHIV, and demonstrated similar findings in atherosclerotic vessels of HIV-uninfected humans. We found that recombinant TNF enhanced the production and release of CX3CL1 and bioactive IL-15 from aortic endothelial cells, but not from aortic smooth muscle cells. IL-15 in turn promoted CX3CR1 surface expression on and TNF synthesis by CD8 T cells, and IL-15-treated CD8 T cells exhibited enhanced CX3CL1-dependent chemoattraction toward endothelial cells in vitro. Finally, we show that CD8 T cells in human atherosclerotic plaques have an activated, resident phenotype consistent with in vivo IL-15 and CX3CL1 exposure. In this report, we define a novel model of CD8 T cell involvement in atherosclerosis whereby CX3CL1 and IL-15 operate in tandem within the vascular endothelium to promote infiltration by activated CX3CR1+ memory CD8 T cells that drive further endothelial activation via TNF. We propose that these interactions are prevalent in aging and in PLWH, populations where circulating activated CX3CR1+ CD8 T cell numbers are often expanded., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: N.T.F serves as a consultant for, and M.M.L. has received competitive funding from Gilead. M.M.L. has consulted for Lilly. The work of L.M. was funded by the NICHD Intramural Program. All other authors have no competing interests to declare.

Cytotoxic CD4 T cells are linked to cardiovascular morbidities and accumulate in both HIV and CMV infections, both of which are associated with increased risk of cardiovascular disease (CVD). In this study, we identify CMV coinfection as a major driver of the cytotoxic phenotype, characterized by elevated CD57 expression and reduced CD28 expression, in circulating CD4 T cells from people living with HIV infection, and investigate potential mechanisms linking this cell population to CVD. We find that human CD57 + CD4 T cells express high levels of the costimulatory receptor CD2 and that CD2/LFA-3 costimulation results in a more robust and polyfunctional effector response to TCR signals, compared with CD28-mediated costimulation. CD57 + CD4 T cells also express the vascular endothelium-homing receptor CX3CR1 and migrate toward CX3CL1-expressing endothelial cells in vitro. IL-15 promotes the cytotoxic phenotype, elevates CX3CR1 expression, and enhances the trafficking of CD57 + CD4 T cells to endothelium and may therefore be important in linking these cells to cardiovascular complications. Finally, we demonstrate the presence of activated CD57 + CD4 T cells and expression of CX3CL1 and LFA-3 in atherosclerotic plaque tissues from HIV-uninfected donors. Our findings are consistent with a model in which cytotoxic CD4 T cells contribute to CVD in HIV/CMV coinfection and in atherosclerosis via CX3CR1-mediated trafficking and CD2/LFA-3-mediated costimulation. This study identifies several targets for therapeutic interventions and may help bridge the gap in understanding how CMV infection and immunity are linked to increased cardiovascular risk in people living with HIV infection., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Recently neutrophil-based nanoparticles (NPs) drug delivery systems have gained considerable attention in cancer therapy. Numerous studies have been conducted to identify optimal NPs parameters for passive tumor targeting, while there is a fundamental dearth of knowledge about the factors governing cell-mediated delivery. Here, by using intravital microscopy and magnetic resonance imaging, we describe accumulation dynamics of 140 nm magnetic cubes and clusters in murine breast cancer (4T1) and colon cancer (CT26) models. Notwithstanding rapid clearance from the blood flow, NPs readily accumulated in tumors at later time points. Both NPs types were captured mostly by intravascular neutrophils immediately after injection, and transmigration of NPs-bound neutrophils through the vessel wall was first shown in real-time. A dramatic drop in NPs accumulation upon Ly6G and Gr1 depletion further confirmed the role of neutrophils as a biocarrier for targeting tumors. Of note, for shorter circulating NPs, a cell-dependent delivery route was more impactful, while the accumulation of longer circulating counterpart was less compromised by neutrophil depletion. Neutrophil-mediated transport was also shown to depend on tumor type, with more efficiency noted in neutrophil-rich tumors. Revealing NPs characteristics and host factors influencing the neutrophil-based tumor targeting will help to rationally design drug delivery systems for improved cancer treatment. STATEMENT OF SIGNIFICANCE: Utilizing host cells as trojan horses for delivery nanodrugs to tumor site is a promising approach for cancer therapy. However, it is not clear yet how nanoparticles characteristics and tumor properties affect the efficiency of cell-based nanoparticles transport. Here, we compare neutrophil-based delivery of different-shaped magnetic nanoparticles (cubes and clusters) in two tumor models. The results suggest that neutrophil-mediated route is more impactful for rapidly cleared cubes, than for longer circulating clusters. The efficiency of cell-based accumulation also correlated with the level of neutrophils recruitment to different tumor types. These finding are important for rationale design of nanocarriers and predicting the efficiency of neutrophil-mediated drug delivery between patients and tumor types., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Enhanced glucose uptake by cancer cells was demonstrated in many studies in vitro and in vivo. Glycolysis is one of the main ways of obtaining energy in hypoxia conditions. However, in addition to energy exchange, carbohydrates are also necessary for the posttranslational modification of the protein molecules. Cancer cells are often characterized by an enhanced expression of different glycoproteides. Correct glycosylation defines the structure and activity of such molecules. We demonstrated that under the same cultivation conditions, the intensity of glycosylation does not depend on the total number of potential O-glycosylation sites in one molecule. As a model for the investigation, the tandem repeat region (region with variable number of tandem repeats) of the human mucin MUC1, in which each of the repeats carries four potential O-glycosylation sites, was used. An increase of the tandem repeat number in the recombinant protein did not lead to a proportional increase in the level of sLe a glycosides. A consequence of this was a reduction in the number of recombinant proteins associated with the cytoplasmic membrane at an overall high expression level. Prolongation of the cultivation duration led to a reduction in the expression level of the recombinant proteins by up to 30% of the initial level, and the intensity of this reduction was in a direct ratio to the number of tandem repeats in the protein molecule., (© 2018 Wiley Periodicals, Inc.)

Successful negative selection of autoreactive T cells requires expression of maximum amount of epitopes representing all possible protein isoforms in the thymus. Absence of some possible protein spliceforms in the thymus due to realization of some, but not all splicing combination, may limit the negative selection. Here we show that about 25% of studied mouse genes with well-described alternative splicing event encode some epitopes hidden from thymus. For five out of 10 randomly selected genes with predicted "hidden" epitopes, namely, Add2, Dst, Golga7, Lmna, Nasp, these findings were confirmed experimentally. Thus, for approximately 10-15% of alternatively expressed genes, their splicing patterns in Thymus may limit the set of epitopes available for negative selection.

Human mucin MUC1 plays an important role in cancer development. The increased level of this molecule expression during cancer cell progression induces metastasis and is associated with poor prognosis for patients. There is a large body of experimental data on the role of various functional domains of human mucin MUC1 in metastasis. While, the cytoplasmic domain determined to play a definitive role, the influence of extracellular domain on cancer cell invasiveness still remains unclear. The present paper reveals that the extracellular domain of MUC1 molecule consists of two functional subdomains-the region of tandem repeats (TR) and the region of irregular repeats (IR). We demonstrate the ability of each of these subdomains to alter the invasiveness of cancer cells. The presence of the MUC1 molecules containing TR subdomain (MUC1-TR) on the surface of low-invasive cancer cells leads to the increase in their transendothelial migration potency, while the addition of the IR subdomain to the MUC1-TR molecule (MUC1-IR-TR) restores their natural low invasiveness. J. Cell. Biochem. 118: 4002-4011, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Normalization of real-time quantitative PCR data to appropriate reference genes is crucial to accurately interpret results. Many genes commonly used as reference standards do not perform as expected, depending on cell type and experimental design. In our previous work, we addressed the issue of suitable reference genes for lymphoid tissue and successfully applied the normalization factor-based approach to discriminate lymphoid malignancies according to their cyclin D1 mRNA level. Here, we addressed the problem of reference gene selection and sufficient number on an enlarged sample set with seven candidate genes. The experimental set included 165 samples of spleens, lymph nodes, and peripheral blood mononuclear cells from patients with different types of non-Hodgkin lymphomas along with non-neoplastic lymphoid specimens. For the first time, we compared all major stability ranking algorithms of Visual Basic for Applications (VBA) applets geNorm, BestKeeper, and NormFinder and tested candidate reference genes on a large and heterogeneous set of fresh clinical lymphoid samples. We concluded that a normalization-based approach using three reference genes (YWHAZ, UBC and ACTB) allows for robust reduction of the variance in real-time PCR results and that the further addition of reference genes does not improve data normalization. This creates a clinically applicable tool for PCR-based lymphoma diagnostics., (© 2015 by the Association of Clinical Scientists, Inc.)

Unlabelled: Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in cocultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents expression of the reporter gene in the transfected cells but permits its expression in target cells after infection. However, the gamma-globin intron is not efficiently removed by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the intron-containing RNA via microRNA 30 (miR30)-based short hairpin RNAs. Using two optimized fluorescent reporter vectors and flow cytometry, we determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cell-free infection; furthermore, this increase was dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify the infection in lymphocytes and in primary cells with a higher sensitivity and allow the detection and quantitation of multiply infected cells, providing better tools to study retroviral cell-mediated infection., Importance: The human-pathogenic retroviruses HTLV-1 and HIV-1 can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current study, we generated vectors that are significantly improved over the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in cells in mixed culture. These luciferase- or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one-step transfection/infection assay., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Increasing evidence of intratumor heterogeneity and its augmentation due to selective pressure of microenvironment and recent achievements in cancer therapeutics lead to the need to investigate and track the tumor subclonal structure. Cell sorting of heterogeneous subpopulations of tumor and tumor-associated cells has been a long established strategy in cancer research. Advancement in lasers, computer technology and optics has led to a new generation of flow cytometers and cell sorters capable of high-speed processing of single cell suspensions. Over the last several years cell sorting was used in combination with molecular biological methods, imaging and proteomics to characterize primary and metastatic cancer cell populations, minimal residual disease and single tumor cells. It was the principal method for identification and characterization of cancer stem cells. Analysis of single cancer cells may improve early detection of tumors, monitoring of circulating tumor cells, evaluation of intratumor heterogeneity and chemotherapeutic treatments. The aim of this review is to provide an overview of major cell sorting applications and approaches with new prospective developments such as microfluidics and microchip technologies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Quantum dots (QD) nanocrystals consisting of CdSe core with ZnS shell are a novel class of fluorophores with tremendous potential in microscopy and cytometry techniques. The unique optical features of Qdots, namely, high photostability and extinction coefficient, wide absorption and narrow emission spectra, and large Stokes shift make them desirable fluorescent tags for diverse biomedical applications. Applications of this novel technology in microscopy and cytometry produce reliable multicolor specimens due to increased photostability, ability for multiplexing and narrow emission spectra of nanocrystals. QD conjugates are available on the market and could be prepared in the laboratory. This paper describes the application of QD-conjugates for immunophenotyping and FISH assessment of cells and tissues, and the requirements for microscope and flow cytometer reengineering for successful use of QD in multiplex fluorescent format. Despite the considerable progress, important methodological issues still need to be solved in terms of QD nanocrystals' size, heterogeneity, functionalization and stability of their conjugates. We discuss practical approaches and challenges that need to be addressed to make QD immunostaining a standard method in biology.

Objective: Cyclin D1, an important component of cell cycle machinery and a protein with known oncogenic potential, is downregulated in normal mature B lymphocytes. Its expression detected in a number of malignancies, including B-cell lymphomas, may be important for oncogenesis., Materials and Methods: In our work, we determined the level of cyclin D1 expression in various B-cell lymphomas (i.e., mantle cell lymphoma, B-cell chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and compared it with normal B cells. For cyclin D1 level evaluation, the real-time quantitative polymerase chain reaction data was normalized. We tested five reference genes for stability on our sample set and using the three most stable ones (YWHAZ, ubiquitin c, and HPRT) obtained rather small intra-group variance for cyclin D1 expression in most lymphomas. This allowed their statistically significant ranking according to cyclin D1 expression level., Results: Median values of normalized cyclin D1 expression determined by real-time quantitative polymerase chain reaction were 1.32 for mantle cell lymphoma, 0.02 for B-cell chronic lymphocytic leukemia, 0.009 for diffuse large B-cell lymphoma, 0.004 for marginal zone lymphoma, 0.002 for follicular lymphoma compared with 0.0003 for reactive lymphoid tissue, and 0.00004 for sorted B cells of healthy donors., Conclusions: Our data demonstrate that mantle cell lymphoma, a lymphoma with t(11;14)(q13;q32) translocation, has the level of cyclin D1 increased by four orders of magnitude, while other B-cell lymphomas without t(11;14)(q13;q32) translocation still have the level of cyclin D1 significantly elevated above that of normal lymphocytes (2 orders for B-cell chronic lymphocytic leukemia and an order for other lymphomas) and suggests more than one method of its upregulation in malignant B cells., (Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)