Single Lysophosphatidylcholine Components Exhibit Adjuvant Activities In Vitro and In Vivo

ABSTRACT Improving vaccine immunogenicity by developing new adjuvant formulations has long been a goal of vaccinologists. It has previously been shown that a natural mix of lysophosphatidylcholine (LPC) from chicken eggs promotes mature dendritic cell (DC) generation in vitro and primes antigen-specific immune responses in mice. In the present study, we dissected the adjuvant potentials of five individual LPC components found in the chicken egg mixture. In vitro analyses of the impact of the individual components on the maturation of human DCs were performed by means of phenotypic analysis, chemokine secretion analysis, and analysis of the ability of mature DC to stimulate T lymphocytes. Two components, C16:0-LPC and C18:0-LPC, were identified to be capable of the upregulation of expression of CD86, HLA-DR, and CD40 on in vitro-cultured monocyte-derived DCs from healthy donors. Both induced the release of chemokines to high concentrations (macrophage inflammatory protein 1, monocyte chemoattractant protein 1) or moderate concentrations (interleukin-8 [IL-8], gamma interferon-inducible protein 10). In addition, C16:0-LPC engaged naïve T cells to produce gamma interferon. This suggests that C16:0-LPC and C18:0-LPC have the capacity to promote, at least in vitro, a Th1-oriented response. The intravenous injection of C16:0-LPC or C18:0-LPC into mice resulted in the detectable secretion of IL-6 and IL-5 in sera. Both LPC components were tested for their capacities to act as adjuvants for two selected immunogens: the hepatitis B virus surface antigen and the hepatitis C virus NS3 helicase. The secretion of specific IgG1 was observed with either or both C16:0-LPC and C18:0-LPC, depending on the immunogen tested, and was observed at an efficiency comparable to that of alum. These data identify C16:0-LPC and C18:0-LPC as the active components of the LPC natural mixture. Although discrepancies between the results of the in vitro and in vivo analyses existed, studies with animals suggest that these components can trigger significant and specific humoral-mediated immunity.

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CXCL10/Gamma Interferon-Inducible Protein 10-Mediated Protection against Leishmania amazonensis Infection in Mice

Infection and Immunity ◽

10.1128/iai.01073-06 ◽

2006 ◽

Vol 74 (12) ◽

pp. 6769-6777 ◽

Cited By ~ 38

Author(s):

Rene E. Vasquez ◽

Lynn Soong

Keyword(s):

Cell Function ◽

Gamma Interferon ◽

Leishmania Amazonensis ◽

Interleukin 4 ◽

Dependent Manner ◽

Monocyte Chemoattractant Protein 1 ◽

Inducible Protein ◽

Interferon Inducible Protein

ABSTRACT Leishmania amazonensis can cause progressive disease in most inbred strains of mice. We have previously shown that L. amazonensis-infected C57BL/6 mice have profound impairments in expression of proinflammatory cytokines and chemokines and in activation of antigen-specific CD4+ T cells. These impairments are independent of interleukin-4 (IL-4) but partially due to IL-10 production. The precise mechanism of pathogenesis associated with L. amazonensis infection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and for the eventual control of the infection. In this study, we examined the roles of CXCL10/gamma interferon-inducible protein 10 (IP-10) and CCL2/monocyte chemoattractant protein 1 (MCP-1) in the activation of the macrophage effector function in vitro and their efficacy in ameliorating infection in vivo. Bone marrow-derived macrophages of both BALB/c and C57BL/6 mice were treated with increasing concentrations of recombinant chemokines prior to infection with either stationary-phase promastigotes or tissue-derived amastigotes. We found that treatment with IP-10 or MCP-1 significantly reduced parasite burdens, in a dose-dependent manner, and triggered nitric oxide production. When susceptible C57BL/6 mice were injected locally with IP-10 following L. amazonensis infection, there was a significant delay in lesion development and a reduction in parasite burdens, accompanied by 7- and 3.5-fold increases in gamma interferon and IL-12 secretion, respectively, in restimulated lymph node cells. This study confirms that IP-10 plays a protective role in promoting the reduction of intracellular parasites and thereby opens new avenues for therapeutic control of nonhealing cutaneous leishmaniasis in the New World.

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Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

RNA ◽

10.1261/rna.078814.121 ◽

2022 ◽

pp. rna.078814.121

Author(s):

Anna Ender ◽

Nadine Grafl ◽

Tim Kolberg ◽

Sven Findeiss ◽

Peter F. Stadler ◽

...

Keyword(s):

Homo Sapiens ◽

Rnase P ◽

Single Stranded Region ◽

Domains Of Life ◽

The Individual ◽

The Impact ◽

Individual Enzyme ◽

Trna Precursor

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

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Knockdown of Leptin Receptor Affects Macrophage Phenotype in the Tumor Microenvironment Inhibiting Breast Cancer Growth and Progression

Cancers ◽

10.3390/cancers12082078 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2078

Author(s):

Luca Gelsomino ◽

Giuseppina Daniela Naimo ◽

Rocco Malivindi ◽

Giuseppina Augimeri ◽

Salvatore Panza ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Microenvironment ◽

Leptin Receptor ◽

Macrophage Phenotype ◽

In Vivo Models ◽

Monocyte Chemoattractant Protein 1 ◽

Arginase 1 ◽

The Impact

Aberrant leptin (Ob) signaling, a hallmark of obesity, has been recognized to influence breast cancer (BC) biology within the tumor microenvironment (TME). Here, we evaluated the impact of leptin receptor (ObR) knockdown in affecting BC phenotype and in mediating the interaction between tumor cells and macrophages, the most abundant immune cells within the TME. The stable knockdown of ObR (ObR sh) in ERα-positive and ERα-negative BC cells turned the tumor phenotype into a less aggressive one, as evidenced by in vitro and in vivo models. In xenograft tumors and in co-culture experiments between circulating monocytes and BC cells, the absence of ObR reduced the recruitment of macrophages, and also affected their cytokine mRNA expression profile. This was associated with a decreased expression and secretion of monocyte chemoattractant protein-1 in ObR sh clones. The loss of Ob/ObR signaling modulated the immunosuppressive TME, as shown by a reduced expression of programmed death ligand 1/programmed cell death protein 1/arginase 1. In addition, we observed increased phagocytic activity of macrophages compared to control Sh clones in the presence of ObR sh-derived conditioned medium. Our findings, addressing an innovative role of ObR in modulating immune TME, may open new avenues to improve BC patient health care.

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Role of endothelial MCP-1 in monocyte adhesion to inflamed human endothelium under physiological flow

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00349.2002 ◽

2002 ◽

Vol 283 (6) ◽

pp. H2584-H2591 ◽

Cited By ~ 44

Author(s):

U. Maus ◽

S. Henning ◽

H. Wenschuh ◽

K. Mayer ◽

W. Seeger ◽

...

Keyword(s):

Flow Chamber ◽

Monocyte Adhesion ◽

Monocyte Chemoattractant Protein 1 ◽

Physiological Flow ◽

Translational Arrest ◽

Epithelial Barriers ◽

Human Pulmonary Artery ◽

The Impact

Monocyte chemoattractant protein-1 (MCP-1) is an essential chemokine involved in monocyte traffic across endo- and epithelial barriers both in vitro and in vivo. However, the contribution of endothelial MCP-1 signaling via its CCR2 receptor in monocyte adhesion to inflamed endothelium under flow is incompletely understood. A sensitive flow chamber assay was used to assess monocyte adhesion to TNF-α-activated primary human pulmonary artery endothelial cells (HPAEC) during physiological shear stress. Monocyte adhesion was markedly reduced (∼45%) when HPAEC-derived MCP-1 was either neutralized with anti-MCP-1 mAb or inhibited by translational arrest of MCP-1 mRNA transcripts with MCP-1 antisense oligomers. Corresponding efficacy was observed for blockade of monocyte CCR2 receptor function by anti-CCR2 mAb or MCP-1 antagonists (9–76 analog). The impact of endothelial MCP-1 on monocyte-HPAEC adhesion occurred via β2-integrin but not via β1-integrin adhesion pathways. In this line, pretreatment of monocytes with MCP-1 but not RANTES provoked a rapid and transient neoepitope 24 expression on β2-integrin α-chains, as analyzed by increased reporter mAb24 binding. Collectively, our data show an important cross talk of endothelial MCP-1 with monocyte CCR2 effecting monocyte firm adhesion to inflamed HPAEC under physiological flow conditions.

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The Impact of the Antigenic Composition of Chimeric Proteins on Their Immunoprotective Activity against Chronic Toxoplasmosis in Mice

Vaccines ◽

10.3390/vaccines7040154 ◽

2019 ◽

Vol 7 (4) ◽

pp. 154 ◽

Cited By ~ 3

Author(s):

Justyna Gatkowska ◽

Katarzyna Dzitko ◽

Bartłomiej Tomasz Ferra ◽

Lucyna Holec-Gąsior ◽

Malwina Kawka ◽

...

Keyword(s):

Recombinant Proteins ◽

Chimeric Proteins ◽

Protective Capacity ◽

Antigenic Composition ◽

Specific Igg ◽

The Individual ◽

Immunogenic Properties ◽

The Impact

Toxoplasmosis may pose a serious threat for individuals with weakened or undeveloped immune systems. However, to date, there is no specific immunoprophylaxis for humans. Thus, the aim of this study was to evaluate the immunogenicity of three trivalent—SAG2-GRA1-ROP1L (SGR), SAG1L-MIC1-MAG1 (SMM), and GRA1-GRA2-GRA6 (GGG)—and two tetravalent—SAG2-GRA1-ROP1-GRA2 (SGRG) and SAG1-MIC1-MAG1-GRA2 (SMMG)—chimeric T. gondii proteins, as well as their protective potential against chronic toxoplasmosis in laboratory mice. All three trivalent recombinant proteins possessed immunogenic properties, as defined by specific humoral and cellular responses in vaccinated mice characterized by the synthesis of specific IgG (IgG1/IgG2a) antibodies in vivo and the release of Th1/Th2 cytokines by stimulated splenocytes in vitro. Immunization with all three recombinant proteins provided partial protection against toxoplasmosis, although the protective capacity strongly depended on the individual antigenic composition of each preparation. The antigens providing the highest (86%) and lowest (45%) protection, SGR and SMM, respectively, were supplemented with GRA2 antigen fragment, to form the tetravalent chimeric proteins SGRG and SMMG. Further study revealed that the tetravalent preparations exhibited high immunogenic potential; however, the addition of another antigen to the recombinant protein structure had distinct effects on the protection generated, compared to that of the trivalent counterparts, depending on the antigen tested.

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The impact of Zataria multiflora Boiss extract on in vitro and in vivo Th1/Th2 cytokine (IFN-γ/IL4) balance

Journal of Ethnopharmacology ◽

10.1016/j.jep.2013.10.003 ◽

2013 ◽

Vol 150 (3) ◽

pp. 1024-1031 ◽

Cited By ~ 35

Author(s):

Mohammad Hossein Boskabady ◽

Sakine Shahmohammadi Mehrjardi ◽

Abadorrahim Rezaee ◽

Houshang Rafatpanah ◽

Sediqeh Jalali

Keyword(s):

Zataria Multiflora ◽

Th2 Cytokine ◽

Ifn Γ ◽

The Impact

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Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.965-975.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 965-975 ◽

Cited By ~ 105

Author(s):

Tom S. Kim ◽

Cynthia Heinlein ◽

Robert C. Hackman ◽

Peter S. Nelson

Keyword(s):

Serine Protease ◽

Serine Proteases ◽

Biological Activities ◽

Type Ii ◽

Phenotypic Analysis ◽

Normal Prostate ◽

Prostate Hyperplasia ◽

Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

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The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

Scientific Reports ◽

10.1038/s41598-021-84117-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroaki Kanzaki ◽

Tetsuhiro Chiba ◽

Junjie Ao ◽

Keisuke Koroki ◽

Kengo Kanayama ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Progression Free Survival ◽

Erk Signaling ◽

Enzyme Linked Immunosorbent Assay ◽

Antitumor Effects ◽

The Impact

AbstractFGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

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Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium

Animals ◽

10.3390/ani11051414 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1414

Author(s):

Josep M. Cambra ◽

Emilio A. Martinez ◽

Heriberto Rodriguez-Martinez ◽

Maria A. Gil ◽

Cristina Cuello

Keyword(s):

Culture Medium ◽

Embryo Quality ◽

Transcriptional Profiling ◽

Defined Medium ◽

Platelet Factor ◽

Defined Media ◽

Defined Culture ◽

The Impact

The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

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Preclinical Testing of Radiopharmaceuticals for the Detection and Characterization of Osteomyelitis: Experiences from a Porcine Model

Molecules ◽

10.3390/molecules26144221 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4221

Author(s):

Aage Kristian Olsen Alstrup ◽

Svend Borup Jensen ◽

Ole Lerberg Nielsen ◽

Lars Jødal ◽

Pia Afzelius

Keyword(s):

Animal Model ◽

Animal Models ◽

Porcine Model ◽

Animal Experiments ◽

Radioactive Tracers ◽

The Individual ◽

Selection Of

The development of new and better radioactive tracers capable of detecting and characterizing osteomyelitis is an ongoing process, mainly because available tracers lack selectivity towards osteomyelitis. An integrated part of developing new tracers is the performance of in vivo tests using appropriate animal models. The available animal models for osteomyelitis are also far from ideal. Therefore, developing improved animal osteomyelitis models is as important as developing new radioactive tracers. We recently published a review on radioactive tracers. In this review, we only present and discuss osteomyelitis models. Three ethical aspects (3R) are essential when exposing experimental animals to infections. Thus, we should perform experiments in vitro rather than in vivo (Replacement), use as few animals as possible (Reduction), and impose as little pain on the animal as possible (Refinement). The gain for humans should by far exceed the disadvantages for the individual experimental animal. To this end, the translational value of animal experiments is crucial. We therefore need a robust and well-characterized animal model to evaluate new osteomyelitis tracers to be sure that unpredicted variation in the animal model does not lead to a misinterpretation of the tracer behavior. In this review, we focus on how the development of radioactive tracers relies heavily on the selection of a reliable animal model, and we base the discussions on our own experience with a porcine model.

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