Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses.     

<Emphasis Type="Italic">Vegf-A</Emphasis> mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation

Aims/hypothesis

The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment.

Methods

Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice.

Results

At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean?±?SEM: 0.118?±?0.01 [n?=?3] in Vegf-A mRNA transfected group (VEGF) vs 0.056?±?0.01 [n?=?3] in no RNA [p?<?0.05] vs 0.063?±?0.02 [n?=?4] in Gfp mRNA transfected group (GFP) [p?<?0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085?±?0.02 [n?=?4] in VEGF vs 0.030?±?0.004 [n?=?4] in no RNA [p?<?0.05] vs 0.034?±?0.01 [n?=?5] in GFP [p?<?0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048?±?0.013 [n?=?3] in VEGF vs 0.015?±?0.0051 [n?=?4] in no RNA [p?<?0.01] vs 0.013?±?0.0046 [n?=?4] in GFP [p?<?0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049?±?0.0042 [n?=?8] in VEGF vs 0.029?±?0.0052 [n?=?5] in no RNA [p?<?0.05] vs 0.027?±?0.0056 [n?=?4] in GFP [p?<?0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292?±?0.0032 μl [n?=?7] in VEGF vs 0.0178?±?0.0021 μl [n?=?5] in no RNA [p?<?0.01] vs 0.0129?±?0.0012 μl [n?=?4] in GFP [p?<?0.001]).

Conclusions/interpretation

Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.

     

Is There a Weekly Pattern for Health Searches on Wikipedia and Is the Pattern Unique to Health Topics?

Background

Online health information–seeking behaviors have been reported to be more common at the beginning of the workweek. This behavior pattern has been interpreted as a kind of “healthy new start” or “fresh start” due to regrets or attempts to compensate for unhealthy behavior or poor choices made during the weekend. However, the observations regarding the most common health information–seeking day were based only on the analyses of users’ behaviors with websites on health or on online health-related searches. We wanted to confirm if this pattern could be found in searches of Wikipedia on health-related topics and also if this search pattern was unique to health-related topics or if it could represent a more general pattern of online information searching—which could be of relevance even beyond the health sector.

Objective

The aim was to examine the degree to which the search pattern described previously was specific to health-related information seeking or whether similar patterns could be found in other types of information-seeking behavior.

Methods

We extracted the number of searches performed on Wikipedia in the Norwegian language for 911 days for the most common sexually transmitted diseases (chlamydia, gonorrhea, herpes, human immunodeficiency virus [HIV], and acquired immune deficiency syndrome [AIDS]), other health-related topics (influenza, diabetes, and menopause), and 2 nonhealth-related topics (footballer Lionel Messi and pop singer Justin Bieber). The search dates were classified according to the day of the week and ANOVA tests were used to compare the average number of hits per day of the week.

Results

The ANOVA tests showed that the sexually transmitted disease queries had their highest peaks on Tuesdays (P<.001) and the fewest searches on Saturdays. The other health topics also showed a weekly pattern, with the highest peaks early in the week and lower numbers on Saturdays (P<.001). Footballer Lionel Messi had the highest mean number of hits on Tuesdays and Wednesdays, whereas pop singer Justin Bieber had the most hits on Tuesdays. Both these tracked search queries also showed significantly lower numbers on Saturdays (P<.001).

Conclusions

Our study supports prior studies finding an increase in health information searching at the beginning of the workweek. However, we also found a similar pattern for 2 randomly chosen nonhealth-related terms, which may suggest that the search pattern is not unique to health-related searches. The results are potentially relevant beyond the field of health and our preliminary findings need to be further explored in future studies involving a broader range of nonhealth-related searches.     

Extracellular superoxide dismutase is important for hippocampal neurogenesis and preservation of cognitive functions after irradiation

Cranial irradiation is widely used in cancer therapy, but it often causes cognitive defects in cancer survivors. Oxidative stress is considered a major cause of tissue injury from irradiation. However, in an earlier study mice deficient in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD KO) showed reduced sensitivity to radiation-induced defects in hippocampal functions. To further dissect the role of EC-SOD in neurogenesis and in response to irradiation, we generated a bigenic EC-SOD mouse model (OE mice) that expressed high levels of EC-SOD in mature neurons in an otherwise EC-SOD–deficient environment. EC-SOD deficiency was associated with reduced progenitor cell proliferation in the subgranular zone of dentate gyrus in KO and OE mice. However, high levels of EC-SOD in the granule cell layer supported normal maturation of newborn neurons in OE mice. Following irradiation, wild-type mice showed reduced hippocampal neurogenesis, reduced dendritic spine densities, and defects in cognitive functions. OE and KO mice, on the other hand, were largely unaffected, and the mice performed normally in neurocognitive tests. Although the resulting hippocampal-related functions were similar in OE and KO mice following cranial irradiation, molecular analyses suggested that they may be governed by different mechanisms: whereas neurotrophic factors may influence radiation responses in OE mice, dendritic maintenance may be important in the KO environment. Taken together, our data suggest that EC-SOD plays an important role in all stages of hippocampal neurogenesis and its associated cognitive functions, and that high-level EC-SOD may provide protection against irradiation-related defects in hippocampal functions.Cranial irradiation is widely used as a treatment modality for patients with primary or metastatic brain tumors (1–3), and is also used as a prophylactic treatment to prevent metastases of high-risk tumors to the nervous system (4). Although effective, cranial irradiation is associated with various complications or side effects in cancer survivors (1–3). One of the severe complications is neurocognitive impairment, which can include defects in executive functions and learning and memory (3). Neurocognitive impairments occur in both adults and children and are generally associated with higher doses and younger age (3). The pathogenesis of radiation-induced neurocognitive impairment is not completely understood, but recent studies suggest that suppressed hippocampal neurogenesis (5, 6), increased hippocampal neuronal apoptosis (7, 8), and reduced growth hormone secretion (9, 10) may be involved.The production of reactive oxygen species (ROS) is considered a major cause of radiation-induced tissue damage (11). Ionizing irradiation not only results in the acute generation of short-lived ROS, it also results in a persistent state of oxidative stress that extends up to several months or even years after irradiation (12, 13). Accordingly, animal and cell models with altered antioxidant capacities have been used to investigate the biochemical pathways involved in radiation-induced tissue and cell injuries, and experimental antioxidant-based therapies have been designed to protect normal tissues during radiation treatments (14).Hippocampal neurogenesis is important for hippocampal-dependent functions of learning and memory and the process is exquisitely sensitive to suppression by various stressors, including radiation and oxidative stress (5, 6, 15). To determine if altered redox balance in the hippocampal microenvironment affects hippocampal neurogenesis and the associated functions of learning and memory, we used a knockout mouse model (KO) deficient in the extracellular antioxidant enzyme, EC-superoxide dismutase (EC-SOD), in an earlier study with cranial irradiation (13, 16). When examined at 3–4 mo of age, EC-SOD deficiency was associated with a significant suppression of baseline neurogenesis and impaired hippocampal-dependent cognitive functions (13, 16). Unexpectedly, EC-SOD deficiency also rendered the process less sensitive to radiation-induced changes. The underlining mechanism for this paradoxical finding was not clear, but preliminary studies ruled out up-regulation of major antioxidant enzymes (13). The results suggested that the interaction between redox balance and irradiation and their effects on hippocampal functions can be complex, and understanding how these elements work in concert may be a key to identifying strategies for radioprotection.To manipulate redox balance in the hippocampus, we generated a mouse model with inducible EC-SOD transgenes (17). In the current study, we combined the inducible transgenes with EC-SOD KO and generated a bigenic mouse model, designated as the overexpressor (OE), with high levels of EC-SOD expressed only in Ca/calmodulin-dependent protein kinase- (CaMKII) positive neurons in an otherwise EC-SOD–deficient environment (17). Hippocampal neurogenesis generates new granule cells that are functionally integrated into the hippocampal network (18). Because granule cells are CaMKII-positive neurons and are the principal excitatory neurons in the dentate gyrus, the manipulation leads to an estimated four- to fivefold increase in EC-SOD activity in the hippocampal formation in OE mice (17). The OE mice were used to investigate the effects of altered EC-SOD levels at different stages of hippocampal neurogenesis and the functional consequences of learning and memory. Comparison between WT, OE, and KO mice revealed the importance of EC-SOD in progenitor cell proliferation, dendritic development, and long-term survival of newborn neurons. The study results also suggested that maintenance of the dendritic system following cranial irradiation was important for preservation of neurocognitive functions.     

Sleep apnea and risk of deep vein thrombosis: a non-randomized, pair-matched cohort study

BackgroundPatients with sleep apnea have been reported to be associated with increased prevalence of deep vein thrombosis (DVT) in some papers, which were criticized for either a small sample size or lack of a prospective control. Our study strived to explore the relationship of sleep apnea and the subsequent development of DVT using a nationwide, population-based database.MethodsFrom 2000 to 2007, we identified a study cohort consisting of newly diagnosed sleep apnea cases in the National Health Insurance Research Database. A control cohort without sleep apnea, matched for age, sex, comorbidities, major operation, and fractures, was selected for comparison. The 2 cohorts were followed-up, and we observed the occurrence of DVT by registry of DVT diagnosis.ResultsOf the 10,185 sampled patients (5680 sleep apnea patients vs. 4505 control), 40 (0.39%) cases developed DVT during a mean follow-up period of 3.56 years, including 30 (0.53%) from the sleep apnea cohort and 10 (0.22 %) from the control group. Subjects with sleep apnea experienced a 3.113-fold (95% confidence interval, 1.516-6.390; P = .002) increase in incident DVT, which was independent of age, sex, and comorbidities. Kaplan-Meier analysis also revealed the tendency of sleep apnea patients toward DVT development (log-rank test, P = .001). The risk of DVT was even higher in sleep apnea cases who needed continuous positive airway pressure treatment (hazard ratio 9.575; 95% confidence interval, 3.181-28.818; P <.001).ConclusionSleep apnea may be an independent risk factor for DVT.