Objective

This prospective study aims to address mortality in the context of the early pulmonary immune response to burn and inhalation injury.

Methods

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.

Results

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.

Conclusions

Our study demonstrates that the early pulmonary immune response to burn and smoke inhalation may be attenuated in patients who succumb to their injuries.

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.

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.

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.

Methods

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.

Results

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.

Conclusions

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.

Background

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.

Methods

Thirty-five consecutive patients underwent a combination of esophageal function testing, upper endoscopy, barium swallow, and gastric emptying scan after lung transplantation.

Results

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%.

Conclusion

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.

Introduction

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.

Methods

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.

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.

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.

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.

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.

Objective

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.

Design

Prospective, randomized controlled study.

Setting

Animal laboratory in a university medical center.

Subjects

Young (6–12 week) and aged (20–24 month) FVB/N mice.

Interventions

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.

Conclusions

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.

OBJECTIVE—To determine the mortality rate after diabetes-related lower-extremity amputation (LEA) in an African-descent Caribbean population.

RESEARCH DESIGN AND METHODS—We conducted a prospective case-control study. We recruited case subjects (with diabetes and LEA) and age-matched control subjects (with diabetes and no LEA) between 1999 and 2001. We followed these groups for 5 years to assess mortality risk and causes.

RESULTS—There were 205 amputations (123 minor and 82 major). The 1-year and 5-year survival rates were 69 and 44% among case subjects and 97 and 82% among control subjects (case-control difference, P < 0.001). The mortality rates (per 1,000 person-years) were 273.9 (95% CI 207.1–362.3) after a major amputation, 113.4 (85.2–150.9) after a minor amputation, and 36.4 (25.6–51.8) among control subjects. Sepsis and cardiac disease were the most common causes of death.

CONCLUSIONS—These mortality rates are the highest reported worldwide. Interventions to limit sepsis and complications from cardiac disease offer a huge potential for improving post-LEA survival in this vulnerable group.

Background

Activation of muscarinic M1 receptors is mediated via interaction of orthosteric agonists with the acetylcholine binding site or via interaction of allosteric agonists with different site(s) on the receptor. The focus of the present study was to determine if M1 receptors activated by allosteric agonists undergo the same regulatory fate as M1 receptors activated by orthosteric agonists.

Results

The orthosteric agonists carbachol, oxotremorine-M and pilocarpine were compared to the allosteric agonists AC-42, AC-260584, N-desmethylclozapine and xanomeline. All ligands activated M1 receptors and stimulated interaction of the receptors with β-arrestin-1. All ligands reduced cell surface binding and induced the loss of total receptor binding. Receptor internalization was blocked by treatment with hypertonic sucrose indicating that all ligands induced formation of clathrin coated vesicles. However, internalized receptors recycled to the cell surface following removal of orthosteric, but not allosteric agonists. Whereas all ligands induced loss of cell surface receptor binding, no intracellular vesicles could be observed after treatment with AC-260584 or xanomeline. Brief stimulation of M1 receptors with AC-260584 or xanomeline resulted in persistent activation of M1 receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles.

Conclusion

These results indicate that allosteric agonists differ from orthosteric ligands and among each other in their ability to induce different regulatory pathways. Thus, signaling and regulatory pathways induced by different allosteric ligands are ligand specific.

The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver.

The metabolic syndrome is a complex clustering of metabolic defects associated with physical inactivity, abdominal adiposity, and aging.

Purpose

To examine the effects of exercise training intensity on abdominal visceral fat (AVF) and body composition in obese women with the metabolic syndrome.

Methods

Twenty-seven middle-aged, obese women (mean ± SD; age: 51 ± 9 years and body mass index: 34 ± 6 kg/m2) with the metabolic syndrome completed one-of-three 16-week aerobic exercise interventions: (i) No Exercise Training (Control): Seven participants maintained their existing levels of physical activity, (ii) Low-Intensity Exercise Training (LIET): eleven participants exercised 5 days · week-1 at an intensity ≤ lactate threshold (LT) (iii) High-Intensity Exercise Training (HIET): nine participants exercised 3 days · week-1 at an intensity > LT and 2 days ·week-1 ≤ LT. Exercise time was adjusted to maintain caloric expenditure (400 kcal·session-1). Single-slice computed tomography scans obtained at the L4-L5 disc-space and mid-thigh were used to determine abdominal fat and thigh muscle cross-sectional areas. Percent body fat was assessed by air displacement plethysmography.

Results

HIET significantly reduced total abdominal fat (p<0.001), abdominal subcutaneous fat (p=0.034) and AVF (p=0.010). There were no significant changes observed in any of these parameters within the Control or LIET conditions.

Conclusions

The present data indicate that body composition changes are affected by intensity of exercise training with HIET more effective for reducing total abdominal fat, subcutaneous abdominal fat and AVF in obese women with the metabolic syndrome.

The anti-apoptotic Bcl-2 protein has the remarkable ability to prevent cell death from several noxious stimuli. Intriguingly, Bcl-2 overexpression in one cell type has been reported to protect against cell death in neighboring non-Bcl-2 overexpressing cell types. The mechanism of this “trans” protection has been speculated to be secondary to the release of a cytoprotective factor by Bcl-2 overexpressing cells. We employed a series of adoptive transfer experiments in which lymphocytes that overexpress Bcl-2 were administered to either wild type mice or mice lacking mature T and B cells (Rag-1-/-) to detect the presence or absence of the putative protective factor. We were unable to demonstrate “trans” protection. However, adoptive transfer of apoptotic or necrotic cells exacerbated the degree of apoptotic death in neighboring non-Bcl-2 overexpressing cells (p≤0.05). Therefore, this data suggests that dying cells emit signals triggering cell death in neighboring non-Bcl-2 overexpressing cells, i.e. a “trans” destructive effect.

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNA strands that base pair with mRNA to degrade it or inhibit its translation. Because sleep and sleep loss induce changes in many mRNA species, we hypothesized that sleep loss would also affect miRNA levels in the brain. Rats were sleep-deprived for 8 h then decapitated; hippocampus, prefrontal and somatosensory cortices and hypothalamus tissues were harvested and frozen in liquid nitrogen. MiRNA was extracted and then characterized using microarrays. Several let-7 miRNA microarray results using hippocampus and prefrontal cortex samples were verified by PCR. From the array data it was determined that about fifty miRNA species were affected by sleep loss. For example, in the hippocampus of sleep-deprived rats, miRNA expression increased compared to cage control samples. In contrast, the majority of miRNA species in the somatosensory and prefrontal cortices decreased, while in the hypothalamus miRNA species were both up- and down-regulated after sleep deprivation. The number of miRNA species affected by sleep loss, their differential expression in separate brain structures and their predicted targets suggest that they have a role in site-specific sleep mechanisms. Current results are, to our knowledge, the first demonstration of the homeostatic process, sleep, altering brain miRNA levels.

Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with several CNS physiological and pathophysiological actions including sleep, memory, thermal and appetite regulation. Short interfering RNAs (siRNA) targeting TNFα were incubated with cortical cell cultures and microinjected into the primary somatosensory cortex (SSctx) of rats. The TNFα siRNA treatment specifically reduced TNFα mRNA by 45% in vitro without affecting interleukin-6 or gluR1-4 mRNA levels. In vivo the TNFα siRNAα reduced TNFα mRNA, interleukin-6 mRNA and gluR1 mRNA levels compared to treatment with a scrambled control siRNA. After in vivo microinjection, the density of TNFα-immunoreactive cells in layer V of the SSctx was also reduced. Electroencephalogram (EEG) delta wave power was decreased on days 2 and 3 on the side of the brain that received the TNFα siRNA microinjection relative to the side receiving the control siRNA. These findings support the hypothesis that TNFα siRNA attenuates TNFα mRNA and TNFα protein in the rat cortex and that those reductions reduce cortical EEG delta power. Results also are consistent with the notion that TNFα is involved in CNS physiology including sleep regulation.

Canine parvovirus (CPV), which causes hemorrhagic enteritis in dogs, has 3 antigenic variants: types 2a, 2b, and 2c. Molecular method assessment of the distribution of the CPV variants in Europe showed that the new variant CPV-2c is widespread in Europe and that the viruses are distributed in different countries.

Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expressed sequence tag encoding a previously uncharacterized zinc finger protein belonging to the PRDM (PRDI-BF1 and RIZ homology domain) family of chromatin-remodeling proteins and named it PRISM (PR domain in smooth muscle). PRISM is expressed in a variety of smooth muscle-containing tissues and displays especially robust expression in the cardiac outflow tract and descending aorta during embryogenesis. PRISM is localized to the nucleus and contains an amino-terminal PR domain and four Krüppel-like zinc fingers at the carboxy terminus. We show that PRISM acts as a transcriptional repressor by interacting with class I histone deacetylases and the G9a histone methyltransferase, thereby identifying PRISM as a novel SMC-restricted epigenetic regulator. Overexpression of PRISM in cultured primary SMCs induces genes associated with the proliferative smooth muscle phenotype while repressing regulators of differentiation, including myocardin and GATA-6. Conversely, small interfering RNA-mediated knockdown of PRISM slows cell growth and induces myocardin, GATA-6, and markers of SMC differentiation. We conclude that PRISM acts as a novel epigenetic regulator of SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs.