Association of oxidized ApoB and oxidized ApoA-I with high-risk coronary plaque features in cardiovascular disease.

BACKGROUND: Oxidized apolipoprotein B (oxLDL) and oxidized ApoA-I (oxHDL) are proatherogenic. Their prognostic value for assessing high-risk plaques by coronary computed tomography angiography (CCTA) is missing. METHODS: In a prospective, observational study, 306 participants with cardiovascular disease (CVD) had extensive lipoprotein profiling. Proteomics analysis was performed on isolated oxHDL, and atherosclerotic plaque assessment was accomplished by quantitative CCTA. RESULTS: Patients were predominantly White, overweight men (58.5%) on statin therapy (43.5%). Increase in LDL-C, ApoB, small dense LDL-C (P < 0.001 for all), triglycerides (P = 0.03), and lower HDL function were observed in the high oxLDL group. High oxLDL associated with necrotic burden (NB; ß = 0.20; P < 0.0001) and fibrofatty burden (FFB; ß = 0.15; P = 0.001) after multivariate adjustment. Low oxHDL had a significant reverse association with these plaque characteristics. Plasma oxHDL levels better predicted NB and FFB after adjustment (OR, 2.22; 95% CI, 1.27-3.88, and OR, 2.80; 95% CI, 1.71-4.58) compared with oxLDL and HDL-C. Interestingly, oxHDL associated with fibrous burden (FB) change over 3.3 years (ß = 0.535; P = 0.033) when compared with oxLDL. Combined Met136 mono-oxidation and Trp132 dioxidation of HDL showed evident association with coronary artery calcium score (r = 0.786; P < 0.001) and FB (r = 0.539; P = 0.012) in high oxHDL, whereas Met136 mono-oxidation significantly associated with vulnerable plaque in low oxHDL. CONCLUSION: Our findings suggest that the investigated oxidized lipids are associated with high-risk coronary plaque features and progression over time in patients with CVD. CLINICALTRIALS: gov NCT01621594. FUNDING: National Heart, Lung, and Blood Institute at the NIH Intramural Research Program.

Changes in nasal, pharyngeal and salivary secretory IgA levels in patients with COVID-19 and the possibility of correction of their secretion using combined intranasal and oral administration of a pharmaceutical containing antigens of opportunistic microorganisms.

Background: Although extensive research has been conducted on the role of local immunity in patients with SARS-CoV-2, little is known about the production and concentrations of secretory IgA (SIgA) in different mucosal compartments. This article aims to assess the secretion of SIgA in the nasal and pharyngeal compartments and saliva of patients with COVID-19 and to investigate the possibility and efficiency of correction of their secretion using combined intranasal and oral administration of a pharmaceutical containing antigens of opportunistic microorganisms. Methods: This study included 78 inpatients, aged between 18 and 60 years, who had confirmed COVID-19 with moderate lung involvement. The control group (n=45) received basic therapy, and the treatment group (n=33) was additionally administered the bacteria-based pharmaceutical Immunovac VP4 from day 1 to day 10 of hospitalization. SIgA levels were measured by ELISA at baseline and on days 14 and 30. Results: No systemic or local reactions associated with Immunovac VP4 were reported. We observed a statistically significant reduction in the duration of fever and hospitalization in patients who received Immunovac VP4 compared with those from the control group (p=0.03 and p=0.05, respectively). Changes over time in SIgA levels in nasal swabs were found to be significantly different in the two treatment groups (F=7.9, p[78.0]<0.001). On day 14 of observation, patients in the control group showed a statistically significant reduction in SIgA levels from baseline (p=0.02), whereas patients in the Immunovac VP4 group had stable SIgA levels (p=0.07). On day 30 after the start of treatment, there was a statistically significant increase in SIgA levels in the Immunovac VP4 group compared with baseline (from 77.7 (40.5-98.7) µg/L to 113.4 (39.8-156.7) µg/L; p=0.05) and the levels measured on day 14 (from 60.2 (23.3-102.9) µg/L to 113.4 (39.8-156.7) µg/L; p=0.03). The control group showed a statistically significant decrease in levels of nasal SIgA (to 37.3) on day 30 (p=0.007 for comparison with baseline values and p=0.04 for comparison with levels measured on day 14). Changes over time in SIgA levels measured in pharyngeal swabs were also different between the two treatment groups, and this difference reached statistical significance (F=6.5, p[73.0]=0.003). In the control group, this parameter did not change throughout the study (p=0.17 for a comparison between the levels measured on day 14 and the baseline values, and p=0.12 for a comparison between the levels measured on day 30 and the baseline values). In the Immunovac VP4 group, there was a statistically significant increase from baseline in SIgA levels on study day 30: from 1.5 (0.2-16.5) µg/L to 29.8 (3.6-106.8) µg/L (p=0.02). Changes over time in salivary SIgA did not show a significant difference between study groups (F=0.3, p[66.3]=0.75). Conclusion: As part of combination therapy, the bacteria-based immunostimulant agent Immunovac VP4 increases SIgA levels in the nasal and pharyngeal compartments and induces clinical improvement. Induced mucosal immunity is central to the prevention of respiratory infections, particularly in patients with post-COVID-19 syndrome.

Mucosal immunity in health care workers' respiratory tracts in the post-COVID-19 period.

Coronavirus disease (COVID-19) has generated interest in the assessment of systemic immune status, but existing knowledge about mucosal immunity is clearly insufficient to understand the full pathogenetic mechanisms of the disease. The aim of this study was to evaluate the long-term effects of novel coronavirus infection on mucosal immunity in the postinfection period among health care workers (HCWs). A total of 180 health care workers with and without a history of COVID-19 who ranged in age from 18 to 65 years were enrolled in this one-stage, cross-sectional study. The study subjects completed the 36-Item Short Form (36) Health Survey (SF-36) and the Fatigue Assessment Scale. Secretory immunoglobulin A (sIgA) and total immunoglobulin G (IgG) levels were quantified in saliva samples, induced sputum samples, and nasopharyngeal and oropharyngeal scrapings by an enzyme-linked immunosorbent assay. Specific anti-SARS-CoV-2 IgG antibodies were quantified in serum samples by chemiluminescence immunoassay. Analysis of the questionnaire data showed that all HCWs with a history of COVID-19 reported health problems that limited their daily activities and negative changes in their emotional health three months after the disease, regardless of its severity. The following shifts were detected in the adaptive arm of the immune response in different mucosal compartments. Among subjects who had severe or moderate-to-severe COVID-19, salivary sIgA levels were significantly higher than those in the control group (p < 0.05 and p < 0.005, respectively). Compared to the subjects in the control group, all subjects with prior COVID-19 had significantly higher levels of total IgG in induced sputum. In the group of patients who had had severe infection, total IgG in saliva was also higher (p < 0.05). A direct statistically significant correlation was also detected between the levels of total IgG in all studied samples and the levels of specific IgG antibodies against SARS-CoV-2 in the serum. A significant correlation was observed between total IgG levels and the parameters of physical and social activities, mental health, and fatigue levels. Our study demonstrated long-term changes in the humoral mucosal immune response, which were most pronounced in health care workers with a history of severe or moderate-to-severe COVID-19, and an association of these changes with certain clinical signs of post-COVID-19 syndrome.

APOBEC3-induced mutation of the hepatitis virus B DNA genome occurs during its viral RNA reverse transcription into (-)-DNA.

APOBEC3s are innate single-stranded DNA cytidine-to-uridine deaminases that catalyze mutations in both pathogen and human genomes with significant roles in human disease. However, how APOBEC3s mutate a single-stranded DNA that is available momentarily during DNA transcription or replication in vivo remains relatively unknown. In this study, utilizing hepatitis B virus (HBV) viral mutations, we evaluated the mutational characteristics of individual APOBEC3s with reference to the HBV replication process through HBV whole single-strand (-)-DNA genome mutation analyses. We found that APOBEC3s induced C-to-T mutations from the HBV reverse transcription start site continuing through the whole (-)-DNA transcript to the termination site with variable efficiency, in an order of A3B >> A3G > A3H-II or A3C. A3B had a 3-fold higher mutation efficiency than A3H-II or A3C with up to 65% of all HBV genomic cytidines being converted into uridines in a single mutation event, consistent with the A3B localized hypermutation signature in cancer, namely, kataegis. On the other hand, A3C expression led to a 3-fold higher number of mutation-positive HBV genome clones, although each individual clone had a lower number of C-to-T mutations. Like A3B, A3C preferred both 5'-TC and 5'-CC sequences, but to a lesser degree. The APOBEC3-induced HBV mutations were predominantly detected in the HBV rcDNA but were not detectable in other intermediates including HBV cccDNA and pgRNA by primer extension of their PCR amplification products. These data demonstrate that APOBEC3-induced HBV genome mutations occur predominantly when the HBV RNA genome was reversely transcribed into (-)-DNA in the viral capsid.

Class B Scavenger Receptors BI and BII Protect against LPS-Induced Acute Lung Injury in Mice by Mediating LPS.

Recent studies suggest an anti-inflammatory protective role for class B scavenger receptor BI (SR-BI) in endotoxin-induced inflammation and sepsis. Other data, including ours, provide evidence for an alternative role of SR-BI, facilitating bacterial and endotoxin uptake and contributing to inflammation and bacterial infection. Enhanced endotoxin susceptibility of SR-BI-deficient mice due to their anti-inflammatory glucocorticoid deficiency complicates the understanding of SR-BI's role in endotoxemia/sepsis, calling for the use of alternative models. In this study, using human SR-BI (hSR-BI) and hSR-BII transgenic mice, we found that SR-BI and, to a lesser extent, its splicing variant SR-BII protect against LPS-induced lung damage. At 20 h after intratracheal LPS instillation, the extent of pulmonary inflammation and vascular leakage was significantly lower in hSR-BI and hSR-BII transgenic mice than in wild-type mice. Higher bronchoalveolar lavage fluid (BALF) inflammatory cell count and protein content and lung tissue neutrophil infiltration found in wild-type mice were associated with markedly (2 to 3 times) increased proinflammatory cytokine production compared to these parameters in transgenic mice following LPS administration. The markedly lower endotoxin levels detected in BALF of transgenic versus wild-type mice and the significantly increased BODIPY-LPS uptake observed in lungs of hSR-BI and hSR-BII mice 20 h after the i.t. LPS injection suggest that hSR-BI- and hSR-BII-mediated enhanced LPS clearance in the airways could represent the mechanism of their protective role against LPS-induced acute lung injury.

SR-BI mediates neutral lipid sorting from LDL to lipid droplets and facilitates their formation.

SR-BI binds various lipoproteins, including HDL, LDL as well as VLDL, and mediates selective cholesteryl ester (CE) uptake. HDL derived CE accumulates in cellular lipid droplets (LDs), which also store triacylglycerol (TAG). We hypothesized that SR-BI could significantly facilitate LD formation, in part, by directly transporting LDL derived neutral lipids (NL) such as CE and TAG into LDs without lipolysis and de novo lipid synthesis. SR-BI overexpression greatly increased LDL uptake and LD formation in stably transfected HeLa cells (SR-BI-HeLa). LDs isolated from SR-BI-HeLa contained 4- and 7-times more CE and TAG, respectively, than mock-transfected HeLa (Mock-HeLa). In contrast, LDL receptor overexpression in HeLa (LDLr-HeLa) greatly increased LDL uptake, degradation with moderate 1.5- and 2-fold increases of CE and TAG, respectively. Utilizing CE and TAG analogs, BODIPY-TAG (BP-TAG) and BODIPY-CE (BP-CE), for tracking LDL NL, we found that after initial binding of LDL to SR-BI-HeLa, apoB remained at the cell surface, while BP-CE and BP-TAG were sorted and simultaneously transported together to LDs. Both lipids demonstrated limited internalization to lysosomes or endoplasmic reticulum in SR-BI-HeLa. In LDLr-HeLa, NLs demonstrated clear lysosomal sequestration without their sorting to LDs. An inhibition of TAG and CE de novo synthesis by 90-95% only reduced TAG and CE LD content by 45-50%, and had little effect on BP-CE and BP-TAG transport to LDs in SR-BI HeLa. Furthermore, intravenous infusion of 1-2 mg of LDL increased liver LDs in normal (WT) but not in SR-BI KO mice. Mice transgenic for human SR-BI demonstrated higher liver LD accumulation than WT mice. Finally, Electro Spray Infusion Mass Spectrometry (ESI-MS) using deuterated d-CE found that LDs accumulated up to 40% of unmodified d-CE LDL. We conclude that SR-BI mediates LDL-induced LD formation in vitro and in vivo. In addition to cytosolic NL hydrolysis and de novo lipid synthesis, this process includes selective sorting and transport of LDL NL to LDs with limited lysosomal NL sequestration and the transport of LDL CE, and TAG directly to LDs independently of de novo synthesis.

Reactions of radiation-induced electrons with carbon dioxide in inert cryogenic films: matrix tuning of the excess electron interactions in solids.

A single-electron reduction of carbon dioxide is supposed to be an important basic step in various processes, ranging from interstellar chemistry to photocatalytic transformations. In this work, we report an FTIR spectroscopic study on the reactions of carbon dioxide (12CO2 and 13CO2) with the radiation-induced excess electrons in deposited cryogenic matrices with different physical characteristics (Ne, N2, Ar, Xe) occurring at 6 K. The reaction was monitored by the observation of carbon dioxide radical anions. It was found that attachment of excess electrons to CO2 occurred in neon and nitrogen matrices, but not in argon and xenon. In the case of nitrogen, the formation of matrix-related cationic species (N4+˙ and NNCO+˙) was also observed. Since the CO2 molecules have a negative intrinsic electron affinity, it was suggested that the electron attachment to CO2 is controlled by the energy of excess electrons in the solid matrix, which is determined by the value of the corresponding conduction band bottom energy (V0). The implications of the obtained results are discussed.

Heat shock proteins stimulate APOBEC-3-mediated cytidine deamination in the hepatitis B virus.

Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often up-regulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations. Here, using mutational analyses of hepatitis B virus (HBV), we found that Hsp90 stimulates deamination activity of APOBEC-3G (A3G), A3B, and A3C during co-expression in human liver HepG2 cells. Hsp90 directly stimulated A3G deamination activity when the purified proteins were used in in vitro reactions. Hsp40, -60, and -70 also had variable stimulatory effects in the cellular assay, but not in vitro Sequencing analyses further demonstrated that Hsp90 increased both A3G cytosine mutation efficiency on HBV DNA and total HBV mutation frequency. In addition, Hsp90 shifted A3G's cytosine region selection in HBV DNA and increased A3G's 5' nucleoside preference for deoxycytidine (5'-CC). Furthermore, the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin dose dependently inhibited A3G and A3B mutational activity on HBV viral DNA. Hsp90 knockdown by siRNA or by Hsp90 active-site mutation also decreased A3G activity. These results indicate that heat shock proteins, in particular Hsp90, stimulate APOBEC-3-mediated DNA deamination activity, suggesting a potential physiological role in carcinogenesis and viral innate immunity.

Human SR-BII mediates SAA uptake and contributes to SAA pro-inflammatory signaling in vitro and in vivo.

Serum amyloid A (SAA) is an acute phase protein with cytokine-like and chemotactic properties, that is markedly up-regulated during various inflammatory conditions. Several receptors, including FPRL-1, TLR2, TLR4, RAGE, class B scavenger receptors, SR-BI and CD36, have been identified as SAA receptors. This study provides new evidence that SR-BII, splice variant of SR-BI, could function as an SAA receptor mediating its uptake and pro-inflammatory signaling. The uptake of Alexa Fluor488 SAA was markedly (~3 fold) increased in hSR-BII-expressing HeLa cells when compared with mock-transfected cells. The levels of SAA-induced interleukin-8 secretion by hSR-BII-expressing HEK293 cells were also significantly (~3-3.5 fold) higher than those detected in control cells. Moderately enhanced levels of phosphorylation of all three mitogen-activated protein kinases, ERK1/2, and p38 and JNK, were observed in hSR-BII-expressing cells following SAA stimulation when compared with control wild type cells. Transgenic mice with pLiv-11-directed liver/kidney overexpression of hSR-BI or hSR-BII were used to assess the in vivo role of each receptor in SAA-induced pro-inflammatory response in these organs. Six hours after intraperitoneal SAA injection both groups of transgenic mice demonstrated markedly higher (~2-5-fold) expression levels of inflammatory mediators in the liver and kidney compared to wild type mice. Histological examinations of hepatic and renal tissue from SAA-treated mice revealed moderate level of damage in the liver of both transgenic but not in the wild type mice. Activities of plasma transaminases, biomarkers of liver injury, were also moderately higher in hSR-B transgenic mice when compared to wild type mice. Our findings identify hSR-BII as a functional SAA receptor that mediates SAA uptake and contributes to its pro-inflammatory signaling via the MAPKs-mediated signaling pathways.

Synthetic Amphipathic Helical Peptides Targeting CD36 Attenuate Lipopolysaccharide-Induced Inflammation and Acute Lung Injury.

Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema.

Human SR-BI and SR-BII Potentiate Lipopolysaccharide-Induced Inflammation and Acute Liver and Kidney Injury in Mice.

The class B scavenger receptors BI (SR-BI) and BII (SR-BII) are high-density lipoprotein receptors that recognize various pathogens, including bacteria and their products. It has been reported that SR-BI/II null mice are more sensitive than normal mice to endotoxin-induced inflammation and sepsis. Because the SR-BI/II knockout model demonstrates multiple immune and metabolic disorders, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with pLiv-11-directed expression of human SR-BI (hSR-BI) or human SR-BII (hSR-BII). At 6 h after i.p. LPS injection, transgenic hSR-BI and hSR-BII mice demonstrated markedly higher serum levels of proinflammatory cytokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared with wild-type (WT) mice. LPS-stimulated inducible NO synthase expression was 3- to 6-fold higher in the liver and kidney of both transgenic strains, although serum NO levels were similar in all mice. Despite the lower high-density lipoprotein plasma levels, both transgenic strains responded to LPS by a 5-fold increase of plasma corticosterone levels, which were only moderately lower than in WT animals. LPS treatment resulted in MAPK activation in tissues of all mice; however, the strongest response was detected for hepatic extracellular signal-regulated protein kinase 1 and 2 and kidney JNK of both transgenic mice. Histological examination of hepatic and renal tissue from LPS-challenged mice revealed more injury in hSR-BII, but not hSR-BI, transgenic mice versus WT controls. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, significantly increase LPS-induced inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs susceptible to LPS toxicity.

Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease progression in mice independent of blood pressure regulation.

Scavenger receptor CD36 participates in lipid metabolism and inflammatory pathways important for cardiovascular disease and chronic kidney disease (CKD). Few pharmacological agents are available to slow the progression of CKD. However, apolipoprotein A-I-mimetic peptide 5A antagonizes CD36 in vitro. To test the efficacy of 5A, and to test the role of CD36 during CKD, we compared wild-type to CD36 knockout mice and wild-type mice treated with 5A, in a progressive CKD model that resembles human disease. Knockout and 5A-treated wild-type mice were protected from CKD progression without changes in blood pressure and had reductions in cardiovascular risk surrogate markers that are associated with CKD. Treatment with 5A did not further protect CD36 knockout mice from CKD progression, implicating CD36 as its main site of action. In a separate model of kidney fibrosis, 5A-treated wild-type mice had less macrophage infiltration and interstitial fibrosis. Peptide 5A exerted anti-inflammatory effects in the kidney and decreased renal expression of inflammasome genes. Thus, CD36 is a new therapeutic target for CKD and its associated cardiovascular risk factors. Peptide 5A may be a promising new agent to slow CKD progression.

Lipoprotein X Causes Renal Disease in LCAT Deficiency.

Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.

TLR4 mutant mice are protected from renal fibrosis and chronic kidney disease progression.

Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppression. In this study we tested the role of Toll-like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild-type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low-dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low-grade systemic inflammation, splenocyte apoptosis and increased expression of the immune inhibitory receptor PD-1 in the spleen, which were not observed in TLR4 mutant mice. In vitro, endotoxin (LPS) directly upregulated NLRP3 inflammasome expression in renal epithelial cells via TLR4. In summary, TLR4 contributes to renal fibrosis and CKD progression, at least in part, via inflammasome activation in renal epithelial cells, and may also participate in the dysregulated immune response that is associated with CKD.

Hypermutation of ApoB mRNA by rat APOBEC-1 overexpression mimics APOBEC-3 hypermutation.

APOBEC-3 proteins induce C-to-U hypermutations in the viral genome of various viruses and have broad antiviral activity. Generally, only a small proportion of viral genomes (<10(-)(2)) are hypermutated by APOBEC-3s, but often many cytidines (up to 40%) are converted into uridine. The mechanism of this unique selective hypermutation remains unknown. We found that rat APOBEC-1 overexpression had a hypermutation pattern similar to that of APOBEC-3s on its substrate apolipoprotein B (apoB) mRNA. Transient plasmid transfection of rat APOBEC-1 resulted in 0.4% and 1.8% hypermutations with apoB mRNA in HepG2 and McA7777 cells, respectively. The low frequency of hypermutated apoB mRNA targets was enriched by differential DNA denaturation PCR at 72-76 °C, with hypermutation levels increasing up to 67%. Up to 69.6% of cytidines in HepG2 and up to 75.5% of cytidines in McA7777 cells were converted into uridines in the hypermutated apoB mRNA. When rat APOBEC-1 was overexpressed by adenovirus, the hypermutation frequency of apoB mRNA increased from 0.4% to ∼20% and was readily detected by regular PCR. However, this higher expression efficiency only increased the frequency of hypermutation, not the number of affected cytidines in hypermutated targets. Rat APOBEC-1 hypermutation was modulated by cofactors and eliminated by an E181Q mutation, indicating the role of cofactors in hypermutation. The finding of an APOBEC-3 hypermutation pattern with rat APOBEC-1 suggests that cofactors could also be involved in APOBEC-3 hypermutation. Using hepatitis B virus hypermutation, we found that KSRP increased APOBEC-3C and APOBEC-3B hypermutation. These data show that, like rat APOBEC-1 hypermutation, cellular factors may play a regulatory role in APOBEC-3 hypermutation.

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice.

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.

Class B scavenger receptor types I and II and CD36 mediate bacterial recognition and proinflammatory signaling induced by Escherichia coli, lipopolysaccharide, and cytosolic chaperonin 60.

Class B scavenger receptors (SR-B) are lipoprotein receptors that also mediate pathogen recognition, phagocytosis, and clearance as well as pathogen-induced signaling. In this study we report that three members of the SR-B family, namely, CLA-1, CLA-2, and CD36, mediate recognition of bacteria not only through interaction with cell wall LPS but also with cytosolic chaperonin 60. HeLa cells stably transfected with any of these SR-Bs demonstrated markedly (3- to 5-fold) increased binding and endocytosis of Escherichia coli, LPS, and chaperonin 60 (GroEL) as revealed by both FACS analysis and confocal microscopy imaging. Increased pathogen (E. coli, LPS, and GroEL) binding to SR-Bs was also associated with the dose-dependent stimulation of cytokine secretion in the order of CD36 > CLA-2 > CLA-1 in HEK293 cells. Pathogen-induced IL-6-secretion was reduced in macrophages from CD36- and SR-BI/II-null mice by 40-50 and 30-40%, respectively. Intravenous GroEL administration increased plasma IL-6 and CXCL1 levels in mice. The cytokine responses were 40-60% lower in CD36(-/-) relative to wild-type mice, whereas increased cytokine responses were found in SR-BI/II(-/-) mice. While investigating the discrepancy of in vitro versus in vivo data in SR-BI/II deficiency, SR-BI/II(-/-) mice were found to respond to GroEL administration without increases in either plasma corticosterone or aldosterone as normally seen in wild-type mice. SR-BI/II(-/-) mice with mineralocorticoid replacement demonstrated an ∼40-50% reduction in CXCL1 and IL-6 responses. These results demonstrate that, by recognizing and mediating inflammatory signaling of both bacterial cell wall LPS and cytosolic GroEL, all three SR-B family members play important roles in innate immunity and host defense.

Communication: stabilization of radical anions with weakly bound electron in condensed media: a case study of diacetonyl radical anion.

The radical anion resulting from electron capture by diacetonyl molecule has been characterized by EPR and optical absorption spectroscopy in glassy ether matrices at 77 K. In non-polar alkane glasses this species was not observed under the same conditions, which confirms the crucial role of matrix interactions in stabilizing this species. Calculations at the MP2 level show the vertical detachment energy to increase gradually from roughly zero for a bare anion to ∼1 eV for the complex involving six ether molecules.

Hypermutation induced by APOBEC-1 overexpression can be eliminated.

APOBEC-1 overexpression in liver has been shown to effectively reduce apoB-100 levels. However, nonspecific hypermutation and liver tumor formation potentially related to hypermutation in transgenic animals compromise its potential use for gene therapy. In studying apoB mRNA editing regulation, we found that the core editing auxiliary factor ACF dose-dependently increases APOBEC-1 nonspecific hypermutation and specific editing with variable site sensitivity. Overexpression of APOBEC-1 together with ACF in human hepatic HepG2 cells hypermutated apoB mRNAs 20%-65% at sites 6639, 6648, 6655, 6762, 6802, and 6845, in addition to the normal 90% editing at 6666. The hypermutation activity of APOBEC-1 was decreased to background levels by a single point APOBEC-1 mutation of P29F or E181Q, while 50% of wild-type control editing at the normal site was retained. The hypermutations on both apoB and novel APOBEC-1 target 1 (NAT1) mRNA were also decreased to background levels with P29F and E181Q mutants in rat liver primary culture cells. The loss of hypermutation with the mutants was associated with significantly decreased APOBEC-1/ACF interaction. These data suggest that nonspecific hypermutation induced by overexpressing APOBEC-1 can be virtually eliminated by site-specific mutation, while maintaining specific editing activity at the normal site, reopening the potential use of APOBEC-1 gene therapy for hyperlipidemia.