Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection

Early Intravenous Ibuprofen Decreases Narcotic Requirement And Length Of Stay After Traumatic Rib Fracture

Pain control after traumatic rib fracture is essential to avoid respiratory complications and prolonged hospitalization. Narcotics are commonly used, but adjunctive medications such as nonsteroidal anti-inflammatory drugs may be beneficial. Twenty-one patients with traumatic rib fractures treated with both narcotics and intravenous ibuprofen (IVIb) (Treatment) were retrospectively compared with 21 age- and rib fracture-matched patients who received narcotics alone (Control). Pain medication requirements over the first 7 hospital days were evaluated. Mean daily IVIb dose was 2070 ± 880 mg. Daily intravenous morphine-equivalent requirement was 19 ± 16 vs 32 ± 24 mg (P\u3c0.0001). Daily narcotic requirement was significantly decreased in the Treatment group on Days 3 through 7 (P\u3c0.05). Total weekly narcotic requirement was significantly less among Treatment patients (P = 0.004). Highest and lowest daily pain scores were lower in the Treatment group (P\u3c0.05). Hospital length of stay was 4.4 ± 3.4 versus 5.4 ± 2.9 days (P = 0.32). There were no significant complications associated with IVIb therapy. Early IVIb therapy in patients with traumatic rib fractures significantly decreases narcotic requirement and results in clinically significant decreases in hospital length of stay. IVIb therapy should be initiated in patients with traumatic rib fractures to improve patient comfort and reduce narcotic requirement. Copyright Southeastern Surgical Congress. All rights reserved

Gunshot wound incidence as a persistent, tragic symptom of area deprivation

Background: More than 30,000 Americans die every year of firearm-related injuries. Gun violence is frequently addressed by law enforcement and policing, as opposed to public health interventions that might address poverty or deprivation. Our goal was to evaluate the past 20 years of gunshot wound injury demographics seen at our level I academic trauma center and create a risk map model correlating gunshot wound incidence with area deprivation. Methods: Patients admitted for gunshot wound-related injuries between 1996 and 2017 were identified using our trauma registry. Demographic and injury data were extracted and analyzed. Multivariable logistic regression models were created to identify predictors of mortality. Geographic information system mapping of incident location and home address was completed to identify zip code hot spots of high gunshot wound incidence. Area Deprivation Indices, which reflect local income or poverty, housing, education, and employment were used as a marker of relative economic disadvantage. Spearman rank correlation was used to determine the relationship between Area Deprivation Indices score and gunshot wound rate. Results: A total of 2,413 patients with gunshot wounds were evaluated. The cohort had a mean age of 28.8 ± 11.5 and was 89.6% male. Mean Injury Severity Score was 11 ± 12.5. gunshot wounds were most frequently a result of assault (91.1%), followed by unintentional injury (3.4%). Geographic information systems mapping revealed significant clustering of gunshot wounds. The areas with highest per capita incidence of gunshot wounds was strongly correlated with Area Deprivation Indices (0.594, P \u3c .001). Conclusion: Geographic regions of known lower socioeconomic resources have higher incidence of gunshot wounds in our community. Both Area Deprivation Indices and gunshot wound incidents in these distressed communities remained unchanged throughout the past 20 years, despite law enforcement crime suppression efforts. Gunshot wounds appear to be a symptom of area deprivation, similar to failing schools and poor health outcomes. Efforts to decrease poverty and community capacity-building may help alleviate this area deprivation

A strong initial IFN response is induced an IRF-3 response by TLR3 but is not enough to clear HCV.

<p>A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.</p

Envelope proteins affect the response to non-HCV responses through RIG-I receptor but only temporarily through TLR3.

<p>A) IFNβ gene expression of stably transfected LH86 cells expressing Core, E1E2 or NS3/4A 4 days after HCV infection. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 mRNA expression 7 days after infection of stably transfected LH86 cells carrying the HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. E) RIG-I mRNA expression 7 days after infection of stably transfected LH86 cells carrying HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. D) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL transfected Poly I:C. Expression was calculated as described in part A. E) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL extracellular Poly I:C. Expression determined as described in part A.</p

RIG-I is linked to the expression of TRAIL receptors DR4 and DR5.

<p>A) TRAIL mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) DR4 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in A. C) DR5 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in part A. D) TRAIL mRNA expression 4 days after infection of LH86 cells silenced for RIG-I, TLR3 or TLR7 calculated as described for part A. E) DR4 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. F) DR5 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. G) Phase view (100X magnification) of transfected cells 7 days after infection. Top row are LH86 cells and bottom row Huh7.5 cells. The labels note what each cell was transfected with.</p

IFN response is dependent on viral replication.

<p>A) IFNβ mRNA expression was measured daily from the total RNA of LH86 cells treated with an MOI of 0.1 of HCV/JFH-1. The expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. The “No virus” control indicates cells that were cultured with uninfected Huh7.5 supernatant, “HCV” is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1), “heated HCV” is the same supernatant as “HCV” but heated for 15 minutes at 72°C and “UV-treated HCV” was exposed to UV light for 15 minutes. B) IFNβ mRNA expression of different dilutions of virus (1∶1 MOI = 0.1) from day 0 and day 4 post infection calculated by the ΔΔCt method (determined as in part A). C) IFNβ mRNA expression of LH86 cells electroporated with <i>in vitro</i> transcribed HCV/JFH-1 or its non-replicating counterpart HCV/JFH-GND (day 0 is the day of the electroporation). Expression was calculated as described for part A.</p

The expression of TLR3 and RIG-I is affected by intact virion proteins in hepatocytes

<p>A) IFNβ mRNA expression in Huh7.5 TLR3 or TLR7 stable cell lines co-transfected with either PKR or RIG-I after infection with HCV MOI = 0.1. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 and RIG-I mRNA expression levels 7 days after infection with different HCV dilutions (methodology as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021186#pone-0021186-g001" target="_blank">figure 1C</a>). C) TLR3 and RIG-I gene expression levels 7 days after infection with normal, heated or UV-treated virus calculated as described in part A.</p

Primer sets used for cloning.

<p>Primer sets used for cloning.</p