Incidence of microvascular dysfunction is increased in hyperlipidemic mice, reducing cerebral blood flow and impairing remote memory.

Introduction: The development of cognitive dysfunction is not necessarily associated with diet-induced obesity. We hypothesized that cognitive dysfunction might require additional vascular damage, for example, in atherosclerotic mice. Methods: We induced atherosclerosis in male C57BL/6N mice by injecting AAV-PCSK9DY (2x1011 VG) and feeding them a cholesterol-rich Western diet. After 3 months, mice were examined for cognition using Barnes maze procedure and for cerebral blood flow. Cerebral vascular morphology was examined by immunehistology. Results: In AAV-PCSK9DY-treated mice, plaque burden, plasma cholesterol, and triglycerides are elevated. RNAseq analyses followed by KEGG annotation show increased expression of genes linked to inflammatory processes in the aortas of these mice. In AAV-PCSK9DY-treated mice learning was delayed and long-term memory impaired. Blood flow was reduced in the cingulate cortex (-17%), caudate putamen (-15%), and hippocampus (-10%). Immunohistological studies also show an increased incidence of string vessels and pericytes (CD31/Col IV staining) in the hippocampus accompanied by patchy blood-brain barrier leaks (IgG staining) and increased macrophage infiltrations (CD68 staining). Discussion: We conclude that the hyperlipidemic PCSK9DY mouse model can serve as an appropriate approach to induce microvascular dysfunction that leads to reduced blood flow in the hippocampus, which could explain the cognitive dysfunction in these mice.

The AT1 Receptor Blocker Telmisartan Reduces Intestinal Mucus Thickness in Obese Mice.

The angiotensin II (type 1) (AT1) receptor blocker telmisartan (TEL) is beneficial for the treatment of individuals suffering from metabolic syndrome. As we have shown that TEL has an impact on gut microbiota, we investigated here whether TEL influences gut barrier function. C57BL/6N mice were fed with chow or high-fat diet (HFD) and treated with vehicle or TEL (8 mg/kg/day). Mucus thickness was determined by immunohistochemistry. Periodic Acid-Schiff staining allowed the number of goblet cells to be counted. Using western blots, qPCR, and immunohistochemistry, factors related to mucus biosynthesis (Muc2, St6galnac), proliferation (Ki-67), or necroptosis (Rip3) were measured. The influence on cell viability was determined in vitro by using losartan, as the water solubility of TEL was too low for in vitro experiments. Upon HFD, mice developed obesity as well as leptin and insulin resistance, which were prevented by TEL. Mucus thickness upon HFD-feeding was diminished. Independent of feeding, TEL additionally reduced mucus thickness. Numbers of goblet cells were not affected by HFD-feeding and TEL. St6galnac expression was increased by TEL. Rip3 was increased in TEL-treated and HFD-fed mice, while Ki-67 decreased. Cell viability was diminished by using >1 mM losartan. The anti-obese effect of TEL was associated with a decrease in mucus thickness, which was likely not related to a lower expression of Muc2 and goblet cells. A decrease in Ki-67 and increase in Rip3 indicates lower cell proliferation and increased necroptosis upon TEL. However, direct cell toxic effects are ruled out, as in vivo concentrations are lower than 1 mM.

Telmisartan induces a specific gut microbiota signature which may mediate its antiobesity effect.

Telmisartan prevents diet-induced obesity (DIO) in rodents. Given that the precise underlying mechanism is not known, we examined whether a gut-related mechanism might be involved. Sprague-Dawley rats received cafeteria diet (CD) for 3 months to develop DIO and were administered either telmisartan (8 mg/kgbw) or vehicle. In addition, pair-fed (PF) rats received CD adjusted to TEL and control rats (CON) only received chow. Stool samples were analysed by 16 S rRNA gene amplicon sequencing. CD-fed rats became obese while TEL, PF and CON rats remained lean. Alpha diversity analyses indicated that bacterial diversity was similar before the study but changed over time. Beta diversity revealed a time-, CD- and telmisartan-dependent effect. The Firmicutes/Bacteroidetes ratio and the abundance of Blautia, Allobaculum and Parasutterella were higher in DIO and PF than in CON, but not in TEL. Enterotype (ET)-like clustering analyses, Kleinberg's hub network scoring and random forest analyses also indicated that telmisartan induced a specific signature of gut microbiota. In response to stool transfer from telmisartan-pre-treated donor to high-fat fed acceptor mice, body weight gain was slightly attenuated. We attribute the anti-obesity action of telmisartan treatment to diet-independent alterations in gut microbiota as the microbiota from telmisartan-treated, CD-fed rats clearly differed from those of DIO and PF rats. ET-like clustering network, random forest classification and the higher stability in bacterial co-occurrence network analyses indicate that there is more than one indicator species for telmisartan's specific signature, which is further strengthened by the fact that we cannot identify a single indicator species.

Telmisartan prevents high-fat diet-induced neurovascular impairments and reduces anxiety-like behavior.

Angiotensin II receptor blockers (telmisartan) prevent rodents from diet-induced obesity and improve their metabolic status. Hyperglycemia and obesity are associated with reduced cerebral blood flow and neurovascular uncoupling which may lead to behavioral deficits. We wanted to know whether a treatment with telmisartan prevents these changes in obesity.We put young mice on high-fat diet and simultaneously treated them with telmisartan. At the end of treatment, we performed laser speckle imaging and magnetic resonance imaging to assess the effect on neurovascular coupling and cerebral blood flow. Different behavioral tests were used to investigate cognitive function.Mice developed diet-induced obesity and after 16, not 8 weeks of high-fat diet, however, the response to whisker pad stimulation was about 30% lower in obese compared to lean mice. Simultaneous telmisartan treatment increased the response again by 10% compared to obese mice. Moreover, telmisartan treatment normalized high-fat diet-induced reduction of cerebral blood flow and prevented a diet-induced anxiety-like behavior. In addition to that, telmisartan affects cellular senescence and string vessel formation in obesity.We conclude, that telmisartan protects against neurovascular unit impairments in a diet-induced obesity setting and may play a role in preventing obesity related cognitive deficits in Alzheimer's disease.

CCL21-expression and accumulation of CCR7+ NK cells in livers of patients with primary sclerosing cholangitis.

The pathogenesis of primary sclerosing cholangitis (PSC), an autoimmune liver disease, remains unknown. The aim of this study was to characterize peripheral blood and intrahepatic NK cells from patients with PSC. Peripheral blood samples from patients with PSC, other autoimmune liver diseases, and from healthy control individuals were used, as well as liver tissues from PSC patients undergoing liver transplantation. Multiparameter flow cytometry showed that peripheral blood NK cells from PSC patients were significantly enriched for CCR7+ and CXCR3+ cells, and CCR7+ but not CXCR3+ cells were also significantly increased within intrahepatic NK cells. PSC patients undergoing liver transplantation furthermore had significantly higher plasma levels of the CCR7-ligand CCL21, and the CXCR3-ligands CXCL10 and CXCL11, and significantly higher levels of CCL21, but not CXCL10, were detected in liver tissues. CCR7+ and CXCR3+ NK cells from PSC patients exhibited significantly higher functional capacity in peripheral blood, but not liver tissues, consistent with chronic activation of these NK cells in the inflamed liver. These data show that PSC is characterized by intrahepatic CCL21 expression and accumulation of CCR7+ NK cells in the inflamed liver tissue.

Systems Vaccinology Identifies an Early Innate Immune Signature as a Correlate of Antibody Responses to the Ebola Vaccine rVSV-ZEBOV.

Predicting vaccine efficacy remains a challenge. We used a systems vaccinology approach to identify early innate immune correlates of antibody induction in humans receiving the Ebola vaccine rVSV-ZEBOV. Blood samples from days 0, 1, 3, 7, and 14 were analyzed for changes in cytokine levels, innate immune cell subsets, and gene expression. Integrative statistical analyses with cross-validation identified a signature of 5 early innate markers correlating with antibody titers on day 28 and beyond. Among those, IP-10 on day 3 and MFI of CXCR6 on NK cells on day 1 were independent correlates. Consistently, we found an early gene expression signature linked to IP-10. This comprehensive characterization of early innate immune responses to the rVSV-ZEBOV vaccine in humans revealed immune signatures linked to IP-10. These results suggest correlates of vaccine-induced antibody induction and provide a rationale to explore strategies for augmenting the effectiveness of vaccines through manipulation of IP-10.

Enzymatic treatment of duck hepatitis B virus: topology of the surface proteins for virions and noninfectious subviral particles.

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1--preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2--phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3--iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes.

BACKGROUND/AIMS: Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS: Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS: Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS: uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.