Absence of calcium‐independent phospholipase A2β impairs platelet‐activating factor production and inflammatory cell recruitment in Trypanosoma cruzi‐infected endothelial cells

Both acute and chronic phases of Trypanosoma cruzi (T. cruzi) infection are characterized by tissue inflammation, mainly in the heart. A key step in the inflammatory process is the transmigration of inflammatory cells across the endothelium to underlying infected tissues. We observed increased arachidonic acid release and platelet‐activating factor (PAF) production in human coronary artery endothelial cells (HCAEC) at up to 96 h of T. cruzi infection. Arachidonic acid release is mediated by activation of the calcium‐independent phospholipase A(2) (iPLA(2)) isoforms iPLA(2)β and iPLA(2)γ, whereas PAF production was dependent upon iPLA(2)β activation alone. Trypanosoma cruzi infection also resulted in increased cell surface expression of adhesion molecules. Increased adherence of inflammatory cells to T. cruzi‐infected endothelium was blocked by inhibition of endothelial cell iPLA(2)β or by blocking the PAF receptor on inflammatory cells. This suggests that PAF, in combination with adhesion molecules, might contribute to parasite clearing in the heart by recruiting inflammatory cells to the endothelium

Mice with genetic deletion of group VIA phospholipase A2β exhibit impaired macrophage function and increased parasite load in Trypanosoma cruzi-induced myocarditis

Trypanosoma cruzi infection, which is the etiological agent of Chagas disease, is associated with intense inflammation during the acute and chronic phases. The pathological progression of Chagas disease is influenced by the infiltration and transmigration of inflammatory cells across the endothelium to infected tissues, which are carefully regulated processes involving several molecular mediators, including adhesion molecules and platelet-activating factor (PAF). We have shown that PAF production is dependent upon calcium-independent group VIA phospholipase A(2)β (iPLA(2)β) following infection of human coronary artery endothelial cells (HCAECs) with T. cruzi, suggesting that the absence of iPLA(2)β may decrease the recruitment of inflammatory cells to the heart to manage parasite accumulation. Cardiac endothelial cells isolated from iPLA(2)β-knockout (iPLA(2)β-KO) mice infected with T. cruzi demonstrated decreased PAF production compared to that by cells isolated from wild-type (WT) mice but demonstrated increases in adhesion molecule expression similar to those seen in WT mice. Myocardial inflammation in iPLA(2)β-KO mice infected with T. cruzi was similar in severity to that in WT mice, but the iPLA(2)β-KO mouse myocardium contained more parasite pseudocysts. Upon activation, macrophages from iPLA(2)β-KO mice produced significantly less nitric oxide (NO) and caused less T. cruzi inhibition than macrophages from wild-type mice. Thus, the absence of iPLA(2)β activity does not influence myocardial inflammation, but iPLA(2)β is essential for T. cruzi clearance

The absence of myocardial calcium-independent phospholipase a2γ results in impaired prostaglandin e2 production and decreased survival in mice with acute trypanosoma cruzi infection

Cardiomyopathy is a serious complication of Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi. The parasite often infects cardiac myocytes, causing the release of inflammatory mediators, including eicosanoids. A recent study from our laboratory demonstrated that calcium-independent phospholipase A(2)γ (iPLA(2)γ) accounts for the majority of PLA(2) activity in rabbit ventricular myocytes and is responsible for arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) release. Thus, we hypothesized that cardiac iPLA(2)γ contributes to eicosanoid production in T. cruzi infection. Inhibition of the isoform iPLA(2)γ or iPLA(2)β, with the R or S enantiomer of bromoenol lactone (BEL), respectively, demonstrated that iPLA(2)γ is the predominant isoform in immortalized mouse cardiac myocytes (HL-1 cells). Stimulation of HL-1 cells with thrombin, a serine protease associated with microthrombus formation in Chagas' disease and a known activator of iPLA(2), increased AA and PGE(2) release, accompanied by platelet-activating factor (PAF) production. Similarly, T. cruzi infection resulted in increased AA and PGE(2) release over time that was inhibited by pretreatment with (R)-BEL. Further, T. cruzi-infected iPLA(2)γ-knockout (KO) mice had lower survival rates and increased tissue parasitism compared to wild-type (WT) mice, suggesting that iPLA(2)γ-KO mice were more susceptible to infection than WT mice. A significant increase in iPLA(2) activity was observed in WT mice following infection, whereas iPLA(2)γ-KO mice showed no alteration in cardiac iPLA(2) activity and produced less PGE(2). In summary, these studies demonstrate that T. cruzi infection activates cardiac myocyte iPLA(2)γ, resulting in increased AA and PGE(2) release, mediators that may be essential for host survival during acute infection. Thus, these studies suggest that iPLA(2)γ plays a cardioprotective role during the acute stage of Chagas' disease

Highly efficient transfection of human induced pluripotent stem cells using magnetic nanoparticles.

PurposeThe delivery of transgenes into human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) represents an important tool in cardiac regeneration with potential for clinical applications. Gene transfection is more difficult, however, for hiPSCs and hiPSC-CMs than for somatic cells. Despite improvements in transfection and transduction, the efficiency, cytotoxicity, safety, and cost of these methods remain unsatisfactory. The objective of this study is to examine gene transfection in hiPSCs and hiPSC-CMs using magnetic nanoparticles (NPs).MethodsMagnetic NPs are unique transfection reagents that form complexes with nucleic acids by ionic interaction. The particles, loaded with nucleic acids, can be guided by a magnetic field to allow their concentration onto the surface of the cell membrane. Subsequent uptake of the loaded particles by the cells allows for high efficiency transfection of the cells with nucleic acids. We developed a new method using magnetic NPs to transfect hiPSCs and hiPSC-CMs. HiPSCs and hiPSC-CMs were cultured and analyzed using confocal microscopy, flow cytometry, and patch clamp recordings to quantify the transfection efficiency and cellular function.ResultsWe compared the transfection efficiency of hiPSCs with that of human embryonic kidney (HEK 293) cells. We observed that the average efficiency in hiPSCs was 43%±2% compared to 62%±4% in HEK 293 cells. Further analysis of the transfected hiPSCs showed that the differentiation of hiPSCs to hiPSC-CMs was not altered by NPs. Finally, robust transfection of hiPSC-CMs with an efficiency of 18%±2% was obtained.ConclusionThe difficult-to-transfect hiPSCs and hiPSC-CMs were efficiently transfected using magnetic NPs. Our study offers a novel approach for transfection of hiPSCs and hiPSC-CMs without the need for viral vector generation

Charge ordering and antiferromagnetic transitions in Nd<SUB>x</SUB>Ca<SUB>1-x</SUB>MnO<SUB>3</SUB> (x= 0.2, 0.3) manganites

In this paper we present transport, magnetic and electron paramagnetic resonance (EPR) studies of electron doped manganites Nd<SUB>x</SUB>Ca<SUB>1-x</SUB>MnO<SUB>3</SUB>(x=0.2,0.3) with a view to compare and contrast their properties with those of the hole doped Nd<SUB>0.5</SUB>Ca<SUB>0.5</SUB>MnO<SUB>3</SUB> and other manganites. The samples were prepared by the solid-state reaction method and the composition was verified using X-ray and EDAX measurements. Magnetization measurements on the x=0.2 sample (NCMO 0.2) show a peak at 155 K accompanied by a sharp increase in the resistivity. In the case of x=0.3 sample (NCMO 0.3) a peak in the magnetization along with an increase in the resistivity is observed at 220 K. From a comparison of the EPR results in the two compounds, we conclude that the transition in NCMO 0.2 is an antiferromagnetic transition which is likely to be a charge ordering transition as well and that in NCMO 0.3 is only a charge ordering transition at which long-range magnetic order is not established. We infer that in this compound the long-range antiferromagnetic order sets in at a much lower temperature below which the EPR signal disappears due to the opening of an antiferromagnetic gap

Charge ordering and antiferromagnetic transitions in NdxCa1−xMnO3(x=0.2,0.3)Nd_xCa_{1-x}MnO_3(x = 0.2, 0.3) manganites

In this paper we present transport, magnetic and electron paramagnetic resonance (EPR) studies of electron doped manganites $Nd_xCa_{1-x}MnO_3(x = 0.2, 0.3)$ with a view to compare and contrast their properties with those of the hole doped $Nd_{0.5}Ca_{0.5}MnO_3$ and other manganites. The samples were prepared by the solid-state reaction method and the composition was verified using X-ray and EDAX measurements. Magnetization measurements on the x = 0.2 sample (NCMO 0.2) show a peak at 155 K accompanied by a sharp increase in the resistivity. In the case of x = 0.3 sample (NCMO 0.3) a peak in the magnetization along with an increase in the resistivity is observed at 220 K. From a comparison of the EPR results in the two compounds, we conclude that the transition in NCMO 0.2 is an antiferromagnetic transition which is likely to be a charge ordering transition as well and that in NCMO 0.3 is only a charge ordering transition at which long-range magnetic order is not established. We infer that in this compound the long-range antiferromagnetic order sets in at a much lower temperature below which the EPR signal disappears due to the opening of an antiferromagnetic gap

SARS-CoV-2 Variants of Concern Infect the Respiratory Tract and Induce Inflammatory Response in Wild-Type Laboratory Mice

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern pose a major threat to public health, due to possible enhanced virulence, transmissibility and immune escape. These variants may also adapt to new hosts, in part through mutations in the spike protein. In this study, we evaluated the infectivity and pathogenicity of SARS-CoV-2 variants of concern in wild-type C57BL/6 mice. Six-week-old mice were inoculated intranasally with a representative virus from the original B.1 lineage, or the emerging B.1.1.7 and B.1.351 lineages. We also infected a group of mice with a mouse-adapted SARS-CoV-2 (MA10). Viral load and mRNA levels of multiple cytokines and chemokines were analyzed in the lung tissues on day 3 after infection. Our data show that unlike the B.1 virus, the B.1.1.7 and B.1.351 viruses are capable of infecting C57BL/6 mice and replicating at high concentrations in the lungs. The B.1.351 virus replicated to higher titers in the lungs compared with the B.1.1.7 and MA10 viruses. The levels of cytokines (IL-6, TNF-&alpha;, IL-1&beta;) and chemokine (CCL2) were upregulated in response to the B.1.1.7 and B.1.351 infection in the lungs. In addition, robust expression of viral nucleocapsid protein and histopathological changes were detected in the lungs of B.1.351-infected mice. Overall, these data indicate a greater potential for infectivity and adaptation to new hosts by emerging SARS-CoV-2 variants

Differential Pathogenesis of SARS-CoV-2 Variants of Concern in Human ACE2-Expressing Mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current pandemic, resulting in millions of deaths worldwide. Increasingly contagious variants of concern (VoC) have fueled recurring global infection waves. A major question is the relative severity of the disease caused by previous and currently circulating variants of SARS-CoV-2. In this study, we evaluated the pathogenesis of SARS-CoV-2 variants in human ACE-2-expressing (K18-hACE2) mice. Eight-week-old K18-hACE2 mice were inoculated intranasally with a representative virus from the original B.1 lineage or from the emerging B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta), or B.1.1.529 (omicron) lineages. We also infected a group of mice with the mouse-adapted SARS-CoV-2 (MA10). Our results demonstrate that B.1.1.7, B.1.351 and B.1.617.2 viruses are significantly more lethal than the B.1 strain in K18-hACE2 mice. Infection with the B.1.1.7, B.1.351, and B.1.617.2 variants resulted in significantly higher virus titers in the lungs and brain of mice compared with the B.1 virus. Interestingly, mice infected with the B.1.1.529 variant exhibited less severe clinical signs and a high survival rate. We found that B.1.1.529 replication was significantly lower in the lungs and brain of infected mice in comparison with other VoC. The transcription levels of cytokines and chemokines in the lungs of B.1- and B.1.1.529-infected mice were significantly less when compared with those challenged with other VoC. Together, our data provide insights into the pathogenesis of previous and circulating SARS-CoV-2 VoC in mice