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1.  Hump-Shaped Density-Dependent Regulation of Mosquito Oviposition Site-Selection by Conspecific Immature Stages: Theory, Field Test with Aedes albopictus, and a Meta-Analysis 
PLoS ONE  2014;9(3):e92658.
Oviposition site selection by gravid females is an important determinant of the distribution, abundance, and dynamics of dipteran hematophagous insects. The presence of conspecific immature stages in a potential oviposition site could, on the one hand, indicate the suitability of that site but on the other hand could indicate the potential for intraspecific competition. In this paper, we present a graphic model suggesting that the trade-off between these two opposing forces could result in a hump-shaped density-dependent relationship between oviposition rate and conspecific immature stage density (hereafter, the “Hump-shaped regulation model”) with positive effects of aggregation prevailing at low densities and negative effect of intraspecific competition prevailing at higher densities. We field-tested the predictions of this model at both the egg- and the larval levels with Aedes albopictus and evaluated if and how these relationships are affected by resource enrichment. We found support for the hump-shaped regulation model at both the larval and the egg levels. Using oviposition cups containing varying numbers of conspecific larvae, we showed that the oviposition activity of Ae. albopictus first increases and then decreases with larvae number. Medium enrichment resulted in higher hatching rate, and demonstrated linear relations for the no-enrichment treatment where larvae density range was low and hump-shaped relationship for the enriched medium that had a wider larvae density range. Using pairs of oviposition cups, we showed that at low egg densities mosquitoes laid more eggs on substrates containing pre-existing eggs. However, at higher egg densities, mosquitoes laid more eggs on a virgin substrate. Based on our results and on a meta-analysis, we suggest that due to study design or methodological shortcomings the hump-shaped regulation model is often left undetected and that it is likely to be more common than currently thought.
PMCID: PMC3969363  PMID: 24681526
2.  AltitudeOmics: The Integrative Physiology of Human Acclimatization to Hypobaric Hypoxia and Its Retention upon Reascent 
PLoS ONE  2014;9(3):e92191.
An understanding of human responses to hypoxia is important for the health of millions of people worldwide who visit, live, or work in the hypoxic environment encountered at high altitudes. In spite of dozens of studies over the last 100 years, the basic mechanisms controlling acclimatization to hypoxia remain largely unknown. The AltitudeOmics project aimed to bridge this gap. Our goals were 1) to describe a phenotype for successful acclimatization and assess its retention and 2) use these findings as a foundation for companion mechanistic studies. Our approach was to characterize acclimatization by measuring changes in arterial oxygenation and hemoglobin concentration [Hb], acute mountain sickness (AMS), cognitive function, and exercise performance in 21 subjects as they acclimatized to 5260 m over 16 days. We then focused on the retention of acclimatization by having subjects reascend to 5260 m after either 7 (n = 14) or 21 (n = 7) days at 1525 m. At 16 days at 5260 m we observed: 1) increases in arterial oxygenation and [Hb] (compared to acute hypoxia: PaO2 rose 9±4 mmHg to 45±4 while PaCO2 dropped a further 6±3 mmHg to 21±3, and [Hb] rose 1.8±0.7 g/dL to 16±2 g/dL; 2) no AMS; 3) improved cognitive function; and 4) improved exercise performance by 8±8% (all changes p<0.01). Upon reascent, we observed retention of arterial oxygenation but not [Hb], protection from AMS, retention of exercise performance, less retention of cognitive function; and noted that some of these effects lasted for 21 days. Taken together, these findings reveal new information about retention of acclimatization, and can be used as a physiological foundation to explore the molecular mechanisms of acclimatization and its retention.
PMCID: PMC3962396  PMID: 24658407
3.  EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy 
Nature Medicine  2011;17(5):589-595.
Hepatitis C virus (HCV) is a major cause of liver disease. Therapeutic options are limited and preventive strategies are absent. Entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen we identified epidermal growth factor receptor and ephrin receptor A2 as host co-factors for HCV entry. Blocking of kinase function by approved inhibitors broadly inhibited HCV infection of all major HCV genotypes and viral escape variants in cell culture and an animal model in vivo. Receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and membrane fusion. These results identify RTKs as novel HCV entry co-factors and uncover that kinase inhibitors have significant antiviral activity. Inhibition of RTK function may constitute a novel approach for prevention and treatment of HCV infection.
PMCID: PMC3938446  PMID: 21516087
Animals; Antigens, CD; physiology; Antigens, CD81; Antiviral Agents; pharmacology; Base Sequence; Cell Line; Claudin-1; Hepacivirus; drug effects; physiology; Hepatitis C; physiopathology; prevention & control; therapy; virology; Host-Pathogen Interactions; physiology; Humans; Ligands; Membrane Proteins; physiology; Mice; Protein Kinase Inhibitors; pharmacology; Quinazolines; pharmacology; RNA Interference; RNA, Small Interfering; genetics; Receptor, EphA2; antagonists & inhibitors; genetics; physiology; Receptor, Epidermal Growth Factor; antagonists & inhibitors; genetics; physiology; Virus Internalization; drug effects; Antiviral / Cell-cell transmission / Liver / Phosphotyrosine kinase / HCV escape variants
4.  Implications of alcohol intoxication at the time of burn and smoke inhalation injury: an epidemiologic and clinical analysis 
Up to 50% of burn patient fatalities have a history of alcohol use, and for those surviving to hospitalization, alcohol intoxication may increase the risk of infection and mortality. Yet, the effect of binge drinking on burn patients specifically with inhalation injuries is not well described. We aimed to investigate the epidemiology and outcomes of this select patient population.
In a prospective study, 53 patients with an inhalation injury and a documented blood alcohol content (BAC) were grouped as: BAC negative (n=37), BAC 1–79 mg/dL (n=4), and BAC ≥ 80 mg/dL (n=12). Those in the latter group were designated as binge drinkers according to National Institute on Alcohol Abuse and Alcoholism criteria.
Binge drinkers with an inhalation injury had considerably smaller % total body surface area (TBSA) burns than did their non-drinking counterparts (mean % TBSA 10.6 vs 24.9; p = 0.065), and significantly lower revised Baux scores (mean 75.9 vs 94.9; p = 0.030). Despite binge-drinkers having smaller injuries, the groups did not differ in terms of outcomes and resource utilization. Finally, those in the binge-drinking group had considerably higher carboxyhemoglobin levels (median 5.2 vs 23.0; p=0.026) than did non-drinkers.
Binge drinkers with inhalation injuries surviving to hospitalization had less severe injuries than did non-drinkers, though their outcomes and burden to the health care infrastructure were similar to the non-drinking patients. Our findings affirm the impact of alcohol intoxication at the time of burn and smoke inhalation injury, placing renewed emphasis on injury prevention and alcohol abuse education.
PMCID: PMC3540156  PMID: 23079566
Alcohol; Burn; Smoke Inhalation
5.  Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study 
Autophagy is the regulated process cells use to recycle non-essential, redundant, or inefficient components and is an adaptive response during times of stress. In addition to its role in enabling the cell to gain vital nutrients in times of stress, autophagy can also be involved in elimination of intracellular microorganisms, tumor suppression, and antigen presentation. Because of difficulty in diagnosing autophagy, few clinical studies have been performed. This study examined whether autophagy occurs in hepatocytes during sepsis. Electron microscopy (EM) was performed on liver samples obtained from both an observational clinical cohort of 6 septic patients and 4 control patients as well as liver specimens from mice with surgical sepsis (via cecal ligation and puncture (CLP)) or sham operation. EM demonstrated increased autophagic vacuoles in septic versus non-septic patients. Randomly selected fields (3,000 square microns) from control and septic patients contained 1.2 ± 1.5 versus 5.3 ± 3.3 (mean ± SD) complex lysosomal/autophagolysosomal structures per image respectively (P<0.001). In rare instances, hepatocytes with autophagic vacuoles appeared to be unequivocally committed to death. Membrane alterations (membrane vacuoles, invagination into adjacent organelles and myelin figure-like changes) occur in a subpopulation of mitochondria in sepsis, but other hepatocyte organelles showed no consistent ultrastructural injury. Findings in murine sepsis paralleled those of patients, with 7.2 ± 1.9 versus 38.7 ± 3.9 lysosomal/autophagolysosomal structures in sham and septic mice, respectively (P =0.002). Quantitative RT-PCR demonstrated that sepsis-induced the upregulation of select apoptosis and cytokine gene expression with minimal changes in the core autophagy genes in liver. In conclusion, hepatocyte autophagic vacuolization increases during sepsis and is associated with mitochondrial injury. However, it is not possible to determine whether the increase in autophagic vacuolization is an adaptive response or a harbinger of cell death.
PMCID: PMC3822608  PMID: 19188912
cell death; inflammation; cytokines; electron microscopy; gene expression
6.  Pulmonary immune changes early after laparoscopic antireflux surgery in lung transplant patients with gastroesophageal reflux disease☆ 
The Journal of surgical research  2012;177(2):e65-e73.
The biologic mechanisms by which laparoscopic antireflux surgery (LARS) might influence the inflammatory process leading to bronchiolitis obliterans syndrome are unknown. We hypothesized that LARS alters the pulmonary immune profile in lung transplant patients with gastroesophageal reflux disease.
In 8 lung transplant patients with gastroesophageal reflux disease, we quantified and compared the pulmonary leukocyte differential and the concentration of inflammatory mediators in the bronchoalveolar lavage fluid (BALF) 4 weeks before LARS, 4 weeks after LARS, and 12 months after lung transplantation. Freedom from bronchiolitis obliterans syndrome (graded 1–3 according to the International Society of Heart and Lung Transplantation guidelines), forced expiratory volume in 1 second trends, and survival were also examined.
At 4 weeks after LARS, the percentages of neutrophils and lymphocytes in the BALF were reduced (from 6.6% to 2.8%, P = 0.049, and from 10.4% to 2.4%, P = 0.163, respectively). The percentage of macrophages increased (from 74.8% to 94.6%, P = 0.077). Finally, the BALF concentration of myeloperoxide and interleukin-1β tended to decrease (from 2109 to 1033 U/mg, P = 0.063, and from 4.1 to 0 pg/mg protein, P = 0.031, respectively), and the concentrations of interleukin-13 and interferon-γ tended to increase (from 7.6 to 30.4 pg/mg protein, P = 0.078 and from 0 to 159.5 pg/mg protein, P = 0.031, respectively). These trends were typically similar at 12 months after transplantation. At a mean follow-up of 19.7 months, the survival rate was 75% and the freedom from bronchiolitis obliterans syndrome was 75%. Overall, the forced expiratory volume in 1 second remained stable during the first year after transplantation.
Our preliminary study has demonstrated that LARS can restore the physiologic balance of pulmonary leukocyte populations and that the BALF concentration of pro-inflammatory mediators is altered early after LARS. These results suggest that LARS could modulate the pulmonary inflammatory milieu in lung transplant patients with gastroesophageal reflux disease.
PMCID: PMC3694396  PMID: 22537841
Gastroesophageal reflux disease GERD; Laparoscopic antireflux surgery LARS; Lung transplantation; Bronchiolitis obliterans syndrome BOS; Inflammation
7.  The Prevalence and Extent of Gastroesophageal Reflux Disease Correlates to the Type of Lung Transplantation 
Evidence is increasingly convincing that lung transplantation is a risk factor of gastroesophageal reflux disease (GERD). However, it is still not known if the type of lung transplant (unilateral, bilateral, or retransplant) plays a role in the pathogenesis of GERD.
Study Design
The records of 61 lung transplant patients who underwent esophageal function tests between September 2008 and May 2010, were retrospectively reviewed. These patients were divided into 3 groups based on the type of lung transplant they received: unilateral (n=25); bilateral (n=30), and retransplant (n=6). Among these groups we compared: (1) the demographic characteristics (eg, sex, age, race, and body mass index); (2) the presence of Barrett esophagus, delayed gastric emptying, and hiatal hernia; and (3) the esophageal manometric and pH-metric profile.
Distal and proximal reflux were more prevalent in patients with bilateral transplant or retransplant and less prevalent in patients after unilateral transplant, regardless of the cause of their lung disease. The prevalence of hiatal hernia, Barrett esophagus, and the manometric profile were similar in all groups of patients.
Although our data show a discrepancy in prevalence of GERD in patients with different types of lung transplantation, we cannot determine the exact cause for these findings from this study. We speculate that the extent of dissection during the transplant places the patients at risk for GERD. On the basis of the results of this study, a higher level of suspicion of GERD should be held in patients after bilateral or retransplantation.
PMCID: PMC3709252  PMID: 22318059
gastroesophageal reflux disease (GERD); laparoscopic antireflux surgery; esophageal function testing; lung transplantation
8.  The protective role of laparoscopic antireflux surgery against aspiration of pepsin after lung transplantation 
Surgery  2011;150(4):598-606.
The goal of this study was to determine, in lung transplant patients, if laparoscopic antireflux surgery (LARS) is an effective means to prevent aspiration as defined by the presence of pepsin in the bronchoalveolar lavage fluid (BALF).
Between September 2009 and November 2010, we collected BALF from 64 lung transplant patients at multiple routine surveillance assessments for acute cellular rejection, or when clinically indicated for diagnostic purposes. The BALF was tested for pepsin by enzyme-linked immunosorbent assay (ELISA). We then compared pepsin concentrations in the BALF of healthy controls (n = 11) and lung transplant patients with and without gastroesophageal reflux disease (GERD) on pH-monitoring (n = 8 and n = 12, respectively), and after treatment of GERD by LARS (n = 19). Time to the development of bronchiolitis obliterans syndrome was contrasted between groups based on GERD status or the presence of pepsin in the BALF.
We found that lung transplant patients with GERD had more pepsin in their BALF than lung transplant patients who underwent LARS (P = .029), and that pepsin was undetectable in the BALF of controls. Moreover, those with more pepsin had quicker progression to BOS and more acute rejection episodes.
This study compared pepsin in the BALF from lung transplant patients with and without LARS. Our data show that: (1) the detection of pepsin in the BALF proves aspiration because it is not present in healthy volunteers, and (2) LARS appears effective as a measure to prevent the aspiration of gastroesophageal refluxate in the lung transplant population. We believe that these findings provide a mechanism for those studies suggesting that LARS may prevent nonallogenic injury to the transplanted lungs from aspiration of gastroesophageal contents.
PMCID: PMC3694415  PMID: 22000170
9.  A review of the role of GERD-induced aspiration after lung transplantation 
Surgical endoscopy  2011;26(5):1201-1204.
The increased prevalence of gastroesophageal reflux disease (GERD) in lung transplantation patients has been established; however, many questions persist regarding the relationship of GERD to aspiration and its potential to induce pulmonary allograft failure. Moreover, the biological implications of aspiration in lung transplantation have yet to be fully elucidated. The goal of this review was to assess the relationship between GERD and aspiration, focusing on the role of these events in the development of allograft injury after lung transplantation.
PMCID: PMC3690196  PMID: 22083335
Gastroesophageal reflux disease (GERD); Aspiration; Lung transplantation; Ambulatory pH-monitoring; Esophageal multichannel pH impedance; Laparoscopic antireflux surgery (LARS)
10.  Laparoscopic Antireflux Surgery for Gastroesophageal Reflux Disease After Lung Transplantation 
The Journal of surgical research  2011;170(2):e279-e286.
Although gastroesophageal reflux disease (GERD) is highly prevalent in lung transplantation, the pathophysiology of GERD in these patients is unknown. We hypothesize that the pathophysiology of GERD after lung transplantation differs from that of a control population, and that the 30-d morbidity and mortality of laparoscopic antireflux surgery (LARS) are equivalent in both populations.
We retrospectively compared the pathophysiology of GERD and the 30-d morbidity and mortality of 29 consecutive lung transplant patients with 23 consecutive patients without lung transplantation (control group), all of whom had LARS for GERD between November 2008 and May 2010.
Both groups had a similar prevalence of endoscopic esophagitis and Barrett’s esophagus, comparable manometric profiles, and similar prevalence of abnormal peristalsis. However, hiatal hernia was more common in controls than in lung transplant patients (57% versus 24%; P = 0.04). Lung transplant patients had a higher prevalence and severity of proximal GERD (65% versus 33%; P = 0.04). The 30-d morbidity and mortality following LARS were similar in both groups regardless of the higher surgical risk of lung transplants (median ASA class: 3 versus 2 for controls, P < 0.001).
These results show that despite similar manometric profiles, lung transplant patients are more prone to proximal reflux than the general population with GERD; the prevalence of endoscopic esophagitis and Barrett’s esophagus is the same in both groups of patients; a hiatal hernia is uncommon after lung transplantation; and the morbidity and mortality of LARS are the same for lung transplant patients as the general population with GERD.
PMCID: PMC3686158  PMID: 21816422
gastroesophageal reflux disease (GERD); laparoscopic antireflux surgery; esophageal function testing; lung transplantation
11.  Exhaled Breath Condensate Collection in the Mechanically Ventilated Patient 
Respiratory Medicine  2012;106(5):601-613.
Collection of exhaled breath condensate (EBC) is a non-invasive means of sampling the airway-lining fluid of the lungs. EBC contains numerous measurable mediators, whose analysis could change the management of patients with certain pulmonary diseases.
While initially popularized in investigations involving spontaneously breathing patients, an increasing number of studies have been performed using EBC in association with mechanical ventilation. Collection of EBC in mechanically ventilated patients follows basic principles of condensation, but is influenced by multiple factors. Effective collection requires selection of a collection device, adequate minute ventilation, low cooling temperatures, and sampling times of greater than ten minutes. Condensate can be contaminated by saliva, which needs to be filtered. Dilution of samples occurs secondary to distilled water in vapors and humidification in the ventilator circuit. Dilution factors may need to be employed when investigating non-volatile biomarkers. Storage and analysis should occur promptly at −70° C to −80° C to prevent rapid degradation of samples.
The purpose of this review is to examine and describe methodologies and problems of EBC collection in mechanically ventilated patients. A straightforward and safe framework has been established to investigate disease processes in this population, yet technical aspects of EBC collection still exist that prevent clinical practicality of this technology. These include a lack of standardization of procedure and analysis of biomarkers, and of normal reference ranges for mediators in healthy individuals. Once these procedural aspects have been addressed, EBC could serve as a non-invasive alternative to invasive evaluation of lungs in mechanically ventilated patients.
PMCID: PMC3314159  PMID: 22398157
Exhaled breath condensate; Biomarkers; Inflammation; Mechanical ventilation; Airway-lining fluid
12.  Ubiquitin and stromal cell-derived factor-1α in bronchoalveolar lavage fluid after burn and inhalation injury 
To determine whether the CXC chemokine receptor (CXCR) 4 ligands ubiquitin and stromal cell-derived factor (SDF)-1α are detectable in bronchoalveolar lavage fluid (BALF) after burn and inhalation injury and whether their concentrations in BALF are associated with injury severity, physiological variables or clinical outcomes.
BALF was obtained on hospital admission from 51 patients (48±18 years) with burn (TBSA: 23±24%) and inhalation injury (controls: 10 healthy volunteers, 42±8 years). BALF was analyzed for total protein and for ubiquitin and SDF-1α by ELISA. Ubiquitin/SDF-1α levels were normalized to total BALF protein content. The extent of inhalation injury was determined during bronchoscopy using a standardized scoring system. Percent TBSA, Baux scores, revised Baux scores and clinical variables were documented.
Ubiquitin and SDF-1α were detectable in 40% of normal BALF specimens. After injury, ubiquitin was detectable in 90% (p<0.01 vs. control) and SDF-1α in 10% of the specimens (p<0.05 vs. control), respectively. While SDF-1α levels were reduced in patients (p<0.01), ubiquitin levels were increased (p<0.01). Ubiquitin concentrations correlated inversely with grade of inhalation injury, revised Baux scores and resuscitation fluid requirements (Spearman correlation coefficients (r): -0.3, -0.33 and -0.45, respectively). Ubiquitin levels correlated positively with arterial oxygenation at the time of bronchoscopy (r: 0.35).
BALF levels of CXCR4 agonists are differentially regulated after burn and inhalation injury. Increases in BALF ubiquitin after inhalation injury may maintain CXCR4 mediated lung protection and repair processes. The finding that BALF ubiquitin decreased with higher grades of inhalation injury may provide a biological correlate for an insufficient local inflammatory response after severe inhalation injury.
PMCID: PMC3626094  PMID: 22105097
Extracellular ubiquitin; chemokine (CXC motif) ligand 12; CXC chemokine receptor 4; fusin; CD184; bronchoscopy
13.  The acute pulmonary inflammatory response to the graded severity of smoke inhalation injury 
Critical Care Medicine  2012;40(4):1113-1121.
To determine if the graded severity of smoke inhalation is reflected by the acute pulmonary inflammatory response to injury.
In a prospective observational study we assessed the bronchoalveolar lavage fluid (BALF) for both leukocyte differential and concentration of 28 cytokines, chemokines, and growth factors. Results were then compared to the graded severity of inhalation injury as determined by Abbreviated Injury Score criteria (0: None, 1: Mild, 2: Moderate, 3: Severe, 4: Massive).
All patients were enrolled at a single tertiary burn center.
The BALF was obtained from 60 patients within 14 hours of burn injury who underwent bronchoscopy for suspected smoke inhalation.
Measurements and Main Results
Those who presented with worse grades of inhalation injury had higher plasma levels of carboxyhemoglobin and enhanced airway neutrophilia. Patients with the most severe inhalation injuries also had a greater requirement for tracheostomy, longer time on the ventilator, and a prolonged stay in the intensive care unit. Of the 28 inflammatory mediators assessed in the BALF, 21 were at their highest in those with the worst inhalation injury scores (Grades 3 and 4), the greatest of which was interleukin (IL)-8 (92,940 pg/ml, Grade 4). When compared in terms of low inhalation injury (Grades 1–2) versus high inhalation injury (Grades 3–4), we found significant differences between groups for IL-4, IL-6, IL-9, IL-15, interferon-γ, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 (p<0.05 for all).
These data reveal that the degree of inhalation injury: 1) has basic and profound effects on burn patient morbidity; 2) evokes complex changes of multiple alveolar inflammatory proteins; and 3) is a determinant of the pulmonary inflammatory response to smoke inhalation. Accordingly, future investigations should consider inhalation injury to be a graded phenomenon.
PMCID: PMC3290689  PMID: 22067627
Burn; Inhalation Injury; Inflammation; Cytokine; Chemokine; Growth Factor
14.  Small cell ovarian carcinoma: genomic stability and responsiveness to therapeutics 
The biology of small cell ovarian carcinoma of the hypercalcemic type (SCCOHT), which is a rare and aggressive form of ovarian cancer, is poorly understood. Tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique SCCOHT cell line, BIN-67, to provide further insight in the biology of this rare type of ovarian cancer.
The tumourigenic potential of BIN-67 cells was determined and the tumours formed in a xenograft model was compared to human SCCOHT. DNA sequencing, spectral karyotyping and high density SNP array analysis was performed. The sensitivity of the BIN-67 cells to standard chemotherapeutic agents and to vesicular stomatitis virus (VSV) and the JX-594 vaccinia virus was tested.
BIN-67 cells were capable of forming spheroids in hanging drop cultures. When xenografted into immunodeficient mice, BIN-67 cells developed into tumours that reflected the hypercalcemia and histology of human SCCOHT, notably intense expression of WT-1 and vimentin, and lack of expression of inhibin. Somatic mutations in TP53 and the most common activating mutations in KRAS and BRAF were not found in BIN-67 cells by DNA sequencing. Spectral karyotyping revealed a largely normal diploid karyotype (in greater than 95% of cells) with a visibly shorter chromosome 20 contig. High density SNP array analysis also revealed few genomic anomalies in BIN-67 cells, which included loss of heterozygosity of an estimated 16.7 Mb interval on chromosome 20. SNP array analyses of four SCCOHT samples also indicated a low frequency of genomic anomalies in the majority of cases. Although resistant to platinum chemotherapeutic drugs, BIN-67 cell viability in vitro was reduced by >75% after infection with oncolytic viruses.
These results show that SCCOHT differs from high-grade serous carcinomas by exhibiting few chromosomal anomalies and lacking TP53 mutations. Although BIN-67 cells are resistant to standard chemotherapeutic agents, their sensitivity to oncolytic viruses suggests that their therapeutic use in SCCOHT should be considered.
PMCID: PMC3635907  PMID: 23433318
Small cell ovarian cancer; Cell line; Immunohistochemistry; BIN-67; Genomic anomalies
15.  Early pulmonary immune hyporesponsiveness is associated with mortality after burn and smoke inhalation injury 
This prospective study aims to address mortality in the context of the early pulmonary immune response to burn and inhalation injury.
We collected bronchoalveolar lavage (BAL) fluid from 60 burn patients within 14 hours of their injury when smoke inhalation was suspected. Clinical and laboratory parameters and immune mediator profiles were compared to patient outcomes.
Patients who succumbed to their injuries were older (p=0.005), had a larger % TBSA (total body surface area) burn (p<0.001), and required greater 24-hour resuscitative fluids (p=0.002). Non-survivors had lower BAL fluid concentrations of numerous immunomodulators, including C5a, IL-1β, IL-1RA, IL-8, IL-10, and IL-13 (p<0.05 for all). Comparing only those with the highest Baux scores to account for the effects of age and % TBSA burn on mortality, non-survivors also had reduced levels of IL-2, IL-4, G-CSF, IFN-γ, MIP-1β, and TNF-α (p<0.05 for all). The apparent pulmonary immune hyporesponsiveness in those who died was confirmed by in vitro culture, which revealed that pulmonary leukocytes from non-survivors had a blunted production of numerous immune mediators.
Our study demonstrates that the early pulmonary immune response to burn and smoke inhalation may be attenuated in patients who succumb to their injuries.
PMCID: PMC3253958  PMID: 21979852
Burn; Inhalation Injury; Cytokines; Chemokines; Mortality
16.  Increased Susceptibility to Candida Infection Following Cecal Ligation and Puncture 
Secondary infection following septic insult represents a significant cause of morbidity and mortality in hospitalized patients. Sepsis induced immunosuppression is a major factor in the host's susceptibility to nosocomial infections and C. albicans accounts for a growing number of these. Given the importance of improving our understanding of the immune response to sepsis and the increasing rates of C. albicans infections, we sought to develop a murine model of double injury consisting of primary peritonitis, i.e., cecal ligation and puncture (CLP), followed by a secondary challenge of C. albicans. As observed in previous work, after primary injury the immune profile of the host changes over time. Therefore, while keeping the mortality rates from the respective individual injuries low, we altered the timing of the secondary injury between two post-CLP time points, day two and day four. Mice subjected to C. albicans infection following CLP have significantly different survival rates dependent upon timing of secondary injury. Animals challenged with C. Albicans at two days post CLP had 91% mortality whereas animals challenged at four days had 47% mortality. This improvement in survival at four days was associated with restoration of innate cell populations and as evidenced by stimulated splenocytes, increases in certain inflammatory cytokines. In addition, we show that susceptibility to C. Albicans infection following CLP is dependent upon the depth of immunosuppression. Although at four days post-CLP there is a partial reconstitution of the immune system, these animals remain more susceptible to infection compared to their single injury (C. albicans alone) counterparts. Collectively, these studies demonstrate that immunosuppression following initial septic insult changes over time. This novel, two hit model of CLP followed by Candida provides additional insight into the immune compromised state created by primary peritonitis, and thereby opens up another avenue of investigation into the causes and possible cures of an emerging clinical problem.
PMCID: PMC3195872  PMID: 21939638
immune suppression; secondary injury; Candida; sepsis
17.  Hepatitis C Virus Induces CD81 and Claudin-1 Endocytosis 
Journal of Virology  2012;86(8):4305-4316.
Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.
PMCID: PMC3318669  PMID: 22318146
18.  Heart rate recovery does not predict endothelial function in obese women 
Obesity and metabolism  2007;3(3):101-105.
The purpose of the present study was to examine the relationship between heart rate recovery (HRREC) and endothelial function in obese women with the metabolic syndrome. The metabolic syndrome has been associated with increased cardiometabolic risk including endothelial dysfunction. However, measurement of endothelial function via flow-mediated dilation (FMD) requires expensive equipment and qualified medical personnel, and therefore may be impractical in some healthcare settings. Heart rate recovery (HRREC) has predictive utility with respect to endothelial function in individuals with suspected coronary artery disease and individuals with the metabolic syndrome also have elevated risk for developing coronary artery disease. Thirty-one obese, sedentary women (age: 47.7±11.2 yr, mean±SD) who met the International Diabetes Federation criteria for the metabolic syndrome underwent a brachial artery ultrasound to determine FMD and nitroglycerine-mediated dilation (NTG). HRREC was assessed during 5 min of active recovery following a standardized VO2 Peak treadmill protocol. Results revealed that the %FMD was not correlated to HRREC when examined across all participants (r= 0.067, p=0.72). However, this relationship was significant in participants with impaired FMD (n=16, %FMD<6%) (r=0.71, p=0.002). Although HRREC may be a significant correlate of FMD in women with the metabolic syndrome and with prevalent endothelial dysfunction, it was not a significant correlate across all women with the metabolic syndrome. The present data do not support the use of HRREC as a possible screening tool for endothelial dysfunction in obese women with the metabolic syndrome.
PMCID: PMC3338316  PMID: 22545071
Exercise; fitness; flow-mediated dilation; metabolic syndrome; obesity
19.  Hepatitis C virus infection of neuroepithelioma cell lines 
Gastroenterology  2010;139(4):1365-1374.
Background & Aims
Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication.
We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism.
Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas non-permissive neural cell lines lacked CLDN1 and in some cases SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1 and HCV E2. Furthermore, anti-CD81, interferon and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells.
Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at comparable levels to that of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo.
PMCID: PMC3298458  PMID: 20538002
OCLN; neurotropism; brain; therapy; replicon; Huh-7; VX-950
20.  Gastroesophageal reflux disease after lung transplantation: Pathophysiology and implications for treatment 
Surgery  2010;148(4):737-745.
Gastroesophageal reflux disease (GERD) is thought to be a risk factor for the development or progression of chronic rejection after lung transplantation. However, the prevalence of GERD and its risk factors, including esophageal dysmotility, hiatal hernia and delayed gastric emptying after lung transplantation, are still unknown. In addition, the prevalence of Barrett’s esophagus, a known complication of GERD, has not been determined in these patients. The purpose of this study was to determine the prevalence and extent of GERD, as well as the frequency of these risk factors and complications of GERD in lung transplant patients.
Thirty-five consecutive patients underwent a combination of esophageal function testing, upper endoscopy, barium swallow, and gastric emptying scan after lung transplantation.
In this patient population, the prevalence of GERD was 51% and 22% in those who had been retransplanted. Of patients with GERD,36% had ineffective esophageal motility (IEM), compared with 6% of patients without GERD (P = .037). No patient demonstrated hiatal hernia on barium swallow. The prevalence of delayed gastric emptying was 36%. The prevalence of biopsy-confirmed Barrett’s esophagus was 12%.
Our study shows that, after lung transplantation, more than half of patients had GERD, and that GERD was more common after retransplantation. IEM and delayed gastric emptying are frequent in patients with GERD. Hiatal hernia is rare. The prevalence of Barrett’s esophagus is not negligible. We conclude that GERD is highly prevalent after lung transplantation, and that delayed gastric emptying and Barrett’s esophagus should always be suspected after lung transplantation because they are common risks factors and complications of GERD.
PMCID: PMC3066258  PMID: 20727564
21.  Laparoscopic Fundoplication with or Without Pyloroplasty in Patients with Gastroesophageal Reflux Disease After Lung Transplantation: How I Do It 
Several studies have confirmed that gastroesophageal reflux disease (GERD) in lung transplant patients is a risk factor for the development and progression of bronchiolitis obliterans syndrome (BOS), a form of rejection after lung transplantation. Moreover, numerous reports indicate that surgical correction of GERD may control the decline in lung function characteristic of BOS. Although laparoscopic fundoplication is an accepted treatment option for these patients with GERD, the surgical technique, which often includes a laparoscopic pyloroplasty, has not been standardized.
The purpose of this article is to describe a step-by-step approach to the laparoscopic treatment of GERD in lung transplant patients. We also address specific technical concerns encountered in the surgical management of this high-risk patient population; we provide data on the safety of this operation; and we illustrate the evidence-based rationale for each technical step of the procedure.
PMCID: PMC3066265  PMID: 20499201
Gastroesophageal reflux disease (GERD); Laparoscopic antireflux surgery (LARS); Gastroparesis; Pyloroplasty; Lung transplantation
22.  The metabolic syndrome, hypertriglyceridemic waist, and cardiometabolic risk factor profile in obese women 
Obesity and metabolism  2007;3(2):50-57.
The hypertriglyceridemic waist (HTGW) and metabolic syndrome (MS) are associated with increased cardiometabolic risk. We evaluated the impact of the HTGW on cardiometabolic risk factors in obese women diagnosed with the MS. Thirty-six abdominally obese women with the MS as defined by the International Diabetes Federation (IDF) [(mean (SD); age 49 (11) y, ht 165 (6) cm, wt 95 (16) kg] participated. The HTGW was defined as follows: a waist circumference ≥80 cm and triglycerides ≥1.7 mM. Unpaired t-tests and Analysis of Covariance (ANCOVA) were employed to detect mean differences between women with MS plus or minus HTGW. Women with the MS plus HTGW had higher total cholesterol (16%, p=0.015), VLDL-cholesterol (97%, p<0.001), non-HDL-cholesterol (16%, p=0.002), insulin (40%, p=0.043), and abdominal visceral fat (24%, p=0.100), and lower total HDL-cholesterol (6%, p=0.024), HDL3 (11%, p=0.031) and Quantitative Insulin Sensitivity Check Index (QUICKI) (5%, p=0.068) compared with women with the MS minus HTGW. Thus, the presence of the HTGW was accompanied by a worsened cardiometabolic risk factor profile in these obese women with the MS. In particular, women with the MS plus HTGW were more insulin resistant compared to women with the MS minus HTGW. In conclusion, the presence of the HTGW in obese women with the MS exacerbates insulin resistance and cardiometabolic risk factors.
PMCID: PMC3157146  PMID: 21857883
Adiposity; cardiovascular disease; diabetes; Syndrome X
23.  Binge drinking 
PMCID: PMC3042447  PMID: 21343280
24.  IL-15 Prevents Apoptosis, Reverses Innate and Adaptive Immune Dysfunction, and Improves Survival in Sepsis 
L-15 is a pluripotent antiapoptotic cytokine that signals to cells of both the innate and adaptive immune system and is regarded as a highly promising immunomodulatory agent in cancer therapy. Sepsis is a lethal condition in which apoptosis-induced depletion of immune cells and subsequent immunosuppression are thought to contribute to morbidity and mortality. This study tested the ability of IL-15 to block apoptosis, prevent immunosuppression, and improve survival in sepsis. Mice were made septic using cecal ligation and puncture or Pseudomonas aeruginosa pneumonia. The experiments comprised a 2×2 full factorial design with surgical sepsis versus sham and IL-15 versus vehicle. In addition to survival studies, splenic cellularity, canonical markers of activation and proliferation, intracellular pro- and antiapoptotic Bcl-2 family protein expression, and markers of immune cell apoptosis were evaluated by flow cytometry. Cytokine production was examined both in plasma of treated mice and splenocytes that were stimulated ex vivo. IL-15 blocked sepsis-induced apoptosis of NK cells, dendritic cells, and CD8 T cells. IL-15 also decreased sepsis-induced gut epithelial apoptosis. IL-15 therapy increased the abundance of antiapoptotic Bcl-2 while decreasing proapoptotic Bim and PUMA. IL-15 increased both circulating IFN-γ, as well as the percentage of NK cells that produced IFN-γ. Finally, IL-15 increased survival in both cecal ligation and puncture and P. aeruginosa pneumonia. In conclusion, IL-15 prevents two immunopathologic hallmarks of sepsis, namely, apoptosis and immunosuppression, and improves survival in two different models of sepsis. IL-15 represents a potentially novel therapy of this highly lethal disorder.
PMCID: PMC2937828  PMID: 20026737
25.  Characterization and Modulation of the Immunosuppressive Phase of Sepsis▿  
Infection and Immunity  2010;78(4):1582-1592.
Sepsis continues to cause significant morbidity and mortality in critically ill patients. Studies of patients and animal models have revealed that changes in the immune response during sepsis play a decisive role in the outcome. Using a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the induction of Pseudomonas aeruginosa pneumonia, we characterized the host immune response. Second, AS101 [ammonium trichloro(dioxoethylene-o,o′)tellurate], a compound that blocks interleukin 10 (IL-10), a key mediator of immunosuppression in sepsis, was tested for its ability to reverse immunoparalysis and improve survival. Mice subjected to pneumonia following CLP had different survival rates depending upon the timing of the secondary injury. Animals challenged with P. aeruginosa at 4 days post-CLP had ∼40% survival, whereas animals challenged at 7 days had 85% survival. This improvement in survival was associated with decreased lymphocyte apoptosis, restoration of innate cell populations, increased proinflammatory cytokines, and restoration of gamma interferon (IFN-γ) production by stimulated splenocytes. These animals also showed significantly less P. aeruginosa growth from blood and bronchoalveolar lavage fluid. Importantly, AS101 improved survival after secondary injury 4 days following CLP. This increased survival was associated with many of the same findings observed in the 7-day group, i.e., restoration of IFN-γ production, increased proinflammatory cytokines, and decreased bacterial growth. Collectively, these studies demonstrate that immunosuppression following initial septic insult increases susceptibility to secondary infection. However, by 7 days post-CLP, the host's immune system has recovered sufficiently to mount an effective immune response. Modulation of the immunosuppressive phase of sepsis may aid in the development of new therapeutic strategies.
PMCID: PMC2849407  PMID: 20100863

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