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.
cell death; inflammation; cytokines; electron microscopy; gene expression
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.
Gastroesophageal reflux disease GERD; Laparoscopic antireflux surgery LARS; Lung transplantation; Bronchiolitis obliterans syndrome BOS; Inflammation
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.
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.
gastroesophageal reflux disease (GERD); laparoscopic antireflux surgery; esophageal function testing; lung transplantation
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.
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.
Gastroesophageal reflux disease (GERD); Aspiration; Lung transplantation; Ambulatory pH-monitoring; Esophageal multichannel pH impedance; Laparoscopic antireflux surgery (LARS)
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.
gastroesophageal reflux disease (GERD); laparoscopic antireflux surgery; esophageal function testing; lung transplantation
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.
Exhaled breath condensate; Biomarkers; Inflammation; Mechanical ventilation; Airway-lining fluid
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.
Extracellular ubiquitin; chemokine (CXC motif) ligand 12; CXC chemokine receptor 4; fusin; CD184; bronchoscopy
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.
Burn; Inhalation Injury; Inflammation; Cytokine; Chemokine; Growth Factor
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.
Small cell ovarian cancer; Cell line; Immunohistochemistry; BIN-67; Genomic anomalies
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.
Burn; Inhalation Injury; Cytokines; Chemokines; Mortality
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.
immune suppression; secondary injury; Candida; sepsis
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.
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.
Exercise; fitness; flow-mediated dilation; metabolic syndrome; obesity
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.
OCLN; neurotropism; brain; therapy; replicon; Huh-7; VX-950
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.
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.
Gastroesophageal reflux disease (GERD); Laparoscopic antireflux surgery (LARS); Gastroparesis; Pyloroplasty; Lung transplantation
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.
Adiposity; cardiovascular disease; diabetes; Syndrome X
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.
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.
Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5–10 μM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.
cell penetrating motif; NLS; TAT; Bcl-2; BH4; cell death; apoptosis; necrosis; total body irradiation
Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
Fluorescence Resonance Energy Transfer (FRET); Receptor Structure-Function; Receptors; Tight Junction; Virus Entry
The Epstein-Barr virus (EBV)-encoded RNAs, EBER-1 and EBER-2, are highly abundant noncoding nuclear RNAs expressed during all forms of EBV latency. The EBERs have been shown to impart significant tumorigenic potential upon EBV-negative Burkitt lymphoma (BL) cells and to contribute to the growth potential of other B-cell lymphoma-, gastric carcinoma-, and nasopharyngeal carcinoma-derived cell lines. However, the mechanisms underlying this EBER-dependent enhancement of cell growth potential remain to be elucidated. Here we focused on the known interaction between EBER-1 and the cellular ribosomal protein L22 and the consequences of this interaction with respect to the growth-promoting properties of the EBERs. L22, a component of 60S ribosomal subunits, binds three sites on EBER-1, and a substantial fraction of available L22 is relocalized from nucleoli to the nucleoplasm in EBV-infected cells. To investigate the hypothesis that EBER-1-mediated relocalization of L22 in EBV-infected cells is critical for EBER-dependent functions, we investigated whether EBER-1 expression is necessary and sufficient for nucleoplasmic retention of L22. Following demonstration of this, we utilized RNA-protein binding assays and fluorescence localization studies to demonstrate that mutation of the L22 binding sites on EBER-1 prevents L22 binding and inhibits EBER-1-dependent L22 relocalization. Finally, the in vivo consequence of preventing L22 relocalization in EBER-expressing cells was examined in soft agar colony formation assays. We demonstrate that BL cells expressing mutated EBER-1 RNAs rendered incapable of binding L22 have significantly reduced capacity to enhance cell growth potential relative to BL cells expressing wild-type EBERs.
Aging is associated with increased inflammation following sepsis. The purpose of this study was to determine if this represents a fundamental age-based difference in the host response or is secondary to the increased mortality seen in aged hosts.
Prospective, randomized controlled study.
Animal laboratory in a university medical center.
Young (6–12 week) and aged (20–24 month) FVB/N mice.
Mice were subjected to 2×25 or 1×30 cecal ligation and puncture (CLP).
Measurements and Main Results
Survival was similar in young mice subjected to 2×25 CLP and aged mice subjected to 1×30 CLP (p=0.15). Young mice subjected to 1×30 CLP had improved survival compared to both other groups (p<0.05). When injury was held constant but mortality was greater, both systemic and peritoneal levels of TNF-α, IL-6, IL-10 and MCP-1 were elevated 24 hours after CLP in aged animals compared to young animals (p<0.05). When mortality was similar but injury severity was different, there were no significant differences in systemic cytokines between aged mice and young mice. In contrast, peritoneal levels of TNF-α, IL-6, and IL-10 were higher in aged mice subjected to 1×30 CLP than young mice subjected to 2×25 CLP despite their similar mortalities (p<0.05). There were no significant differences in either bacteremia or peritoneal cultures when animals of different ages sustained similar injuries or had different injuries with similar mortalities.
Aged mice are more likely to die from sepsis than young mice when subjected to an equivalent insult, and this is associated with increases in both systemic and local inflammation. There is an exaggerated local but not systemic inflammatory response in aged mice compared to young mice when mortality is similar. This suggests that systemic processes that culminate in death may be age-independent, but the local inflammatory response may be greater with aging.
Aging; CLP; Sepsis; Apoptosis; Cytokines; Survival