High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

Long-term efficacy of laparoscopic Nissen versus Toupet fundoplication for the management of types III and IV hiatal hernias.

BACKGROUND: Laparoscopic hiatal hernia repair via Toupet or Nissen fundoplication remains the most commonly performed procedures for management of large hiatal hernia. Few studies have compared the procedures' long-term effectiveness. This study sought to characterize the efficacy of laparoscopic Toupet versus Nissen fundoplication for types III and IV hiatal hernia. METHODS: With IRB approval, a review of all laparoscopic hiatal hernia repairs with mesh reinforcement performed over 7 years at a single center by one surgeon was conducted. Hiatal hernias were classified as type III or IV using operative reports and preoperative imaging. Patients with type I, II, or recurrent hiatal hernia and patients receiving concomitant procedures were excluded. The GERD-Health Related Quality of Life Survey was administered by telephone no earlier than 18 months postoperatively. RESULTS: A total of 473 patients underwent laparoscopic fundoplication; 179 having type III or IV hiatal hernia met inclusion criteria; 62 underwent Toupet, 117 underwent Nissen fundoplication. Average patient age was 64 years; 63% of patients were female. Cohorts were similar in demographics, comorbidities, and intraoperative factors. Survey was completed by 77 patients (43%): 50 having Nissen and 27 Toupet. Median time of survey completion after surgery was 54 months (Nissen) and 25 months (Toupet). Median survey responses across all items for both groups were 0 (no symptoms) with no significant variation between groups. Of patients that had Nissen, 26% reported current proton-pump inhibitor use versus 31% of Toupet patients (p = 0.486). Patient-reported satisfaction with current condition was similar between groups (67% Toupet, 72% Nissen, p = 0.351). CONCLUSIONS: Patient-reported symptoms and satisfaction did not vary for patients receiving laparoscopic Nissen versus Toupet fundoplication, which may indicate that patients with large type III and IV hiatal hernia undergoing either procedure have similar long-term postoperative symptom control.

Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function.

Whether influenza virus replication in macrophages is productive or abortive has been a topic of debate. Utilizing a panel of 28 distinct human, avian, and swine influenza viruses, we found that only a small subset can overcome cellular blocks to productively replicate in murine and primary human macrophages. Murine macrophages have two cellular blocks. The first block is during viral entry, where virions with relatively acid-stable hemagglutinin (HA) proteins are rendered incapable of pH-induced triggering for membrane fusion, resulting in lysosomal degradation. The second block is downstream of viral replication but upstream of late protein synthesis. In contrast, primary human macrophages only have one cellular block that occurs after late protein synthesis. To determine the impact of abortive replication at different stages of the viral life cycle or productive replication on macrophage function, we assessed cytotoxicity, nitric oxide or reactive oxygen species production, and phagocytosis. Intriguingly, productive viral replication decreased phagocytosis of IgG-opsonized bioparticles and Fc receptor CD16 and CD32 surface levels, a function, to our knowledge, never before reported for an RNA virus. These data suggest that replication in macrophages affects cellular function and plays an important role in pathogenesis during infection in vivo IMPORTANCE: Macrophages are a critical first line of defense against respiratory pathogens. Thus, understanding how viruses evade or exploit macrophage function will provide greater insight into viral pathogenicity and antiviral responses. We previously showed that only a subset of highly pathogenic avian (HPAI) H5N1 influenza virus strains could productively replicate in murine macrophages through a hemagglutinin (HA)-mediated mechanism. These studies expand upon this work and demonstrate that productive replication is not specific to unique HPAI H5N1 viruses; an H1N1 strain (A/WSN/33) can also replicate in macrophages. Importantly, we identify two cellular blocks limiting replication that can be overcome by an avian-like pH of activation for nuclear entry and a yet-to-be-identified mechanism(s) to overcome a postnuclear entry block. Overcoming these blocks reduces the cell's ability to phagocytose IgG-opsonized bioparticles by decreasing Fc receptor surface levels, a mechanism previously thought to occur during bacterial and DNA viral infections.

Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo.

UNLABELLED: Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-ß]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results in vivo using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis in vivo. IMPORTANCE: Human astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both in vitro and in vivo. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis.