A phase III,randomized, placebo‐controlled study of belimumab,a monoclonal antibody that inhibits B lymphocyte stimulator,in patients with systemic lupus erythematosus

Objective

To assess the efficacy/safety of the B lymphocyte stimulator inhibitor belimumab plus standard therapy compared with placebo plus standard therapy in active systemic lupus erythematosus (SLE).

Methods

In a phase III, multicenter, randomized, placebo‐controlled trial, 819 antinuclear antibody–positive or anti–double‐stranded DNA–positive SLE patients with scores ≥6 on the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) version of the SLE Disease Activity Index (SLEDAI) were randomized in a 1:1:1 ratio to receive 1 mg/kg belimumab, 10 mg/kg belimumab, or placebo intravenously on days 0, 14, and 28 and then every 28 days for 72 weeks. The primary efficacy end point was the SLE Responder Index (SRI) response rate at week 52 (an SRI response was defined as a ≥4‐point reduction in SELENA–SLEDAI score, no new British Isles Lupus Assessment Group [BILAG] A organ domain score and no more than 1 new BILAG B score, and no worsening in physician's global assessment score versus baseline).

Results

Belimumab at 10 mg/kg plus standard therapy met the primary efficacy end point, generating a significantly greater SRI response at week 52 compared with placebo (43.2% versus 33.5%; P = 0.017). The rate with 1 mg/kg belimumab was 40.6% (P = 0.089). Response rates at week 76 were 32.4%, 39.1%, and 38.5% with placebo, 1 mg/kg belimumab, and 10 mg/kg belimumab, respectively. In post hoc sensitivity analyses evaluating higher SELENA–SLEDAI score thresholds, 10 mg/kg belimumab achieved better discrimination at weeks 52 and 76. Risk of severe flares over 76 weeks (based on the modified SLE Flare Index) was reduced with 1 mg/kg belimumab (34%) (P = 0.023) and 10 mg/kg belimumab (23%) (P = 0.13). Serious and severe adverse events, including infections, laboratory abnormalities, malignancies, and deaths, were comparable across groups.

Conclusion

Belimumab plus standard therapy significantly improved SRI response rate, reduced SLE disease activity and severe flares, and was generally well tolerated in SLE.

     

A tolC Mutant of Francisella tularensis Is Hypercytotoxic Compared to the Wild Type and Elicits Increased Proinflammatory Responses from Host Cells

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS ΔtolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the ΔtolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The ΔtolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.Francisella tularensis is the etiological agent of tularemia. F. tularensis is classified as a category A agent of bioterrorism by the U.S. Centers for Disease Control and Prevention (http://emergency.cdc.gov/agent/agentlist-category.asp) due to its low infectious dose, ease of aerosol dissemination, and capacity to cause high morbidity and mortality (19). There are two clinically relevant subspecies of F. tularensis: subsp. tularensis, which is extremely pathogenic in humans, and subsp. holarctica, which causes a less severe clinical presentation (48). The most severe form of the disease is pneumonic tularemia caused by the inhalation of aerosolized F. tularensis subsp. tularensis (19). The F. tularensis subsp. holarctica-derived live vaccine strain (LVS) was used for many years as the vaccination against tularemia. However, the basis for its attenuation is unknown, and it is no longer in use as a vaccine (46). The LVS is highly virulent in mice, where it causes a disease closely resembling human tularemia (30). These features make the LVS an important model for the study of tularemia. An additional Francisella species, F. novicida, causes disease only in immunocompromised individuals. F. novicida, like the LVS, is highly virulent in mice and widely used as a model of tularemia (20).F. tularensis is a Gram-negative, facultative intracellular pathogen (50). Although factors important for the virulence of F. tularensis are beginning to be identified, the molecular mechanisms behind the extreme pathogenicity of this organism still are largely unknown. In vivo, F. tularensis is a stealth pathogen, evading host cell defenses and dampening host proinflammatory responses. F. tularensis produces an unusual lipopolysaccharide that has low toxicity and does not activate host cells in a TLR4-dependent manner (4, 22). A critical aspect of the pathogenesis of F. tularensis is its ability to escape the phagosome and replicate within the cytosol of a variety of host cells, including both murine and human macrophages and dendritic cells (2, 3, 16, 25, 49). Although F. tularensis does have an extracellular phase (24), it is thought that cytosolic replication allows the bacteria to grow to large numbers while avoiding detection by the host immune system.Host cells respond to F. tularensis invasion by inducing cell death pathways, including apoptosis and pyroptosis (32, 38). In the intrinsic apoptotic pathway, cytochrome c is released from mitochondria into the cytosol, leading to caspase-9 activation and ultimately to the activation of effector caspases such as caspase-3 and -7 (10). In pyroptosis, caspase-1 is activated through the inflammasome complex, resulting in the release of proinflammatory cytokines such as interleukin-1ß (IL-1ß) (6, 32). Lai and coworkers demonstrated that the infection of murine J774 macrophage-like cells with the LVS activated the intrinsic apoptotic pathway as early as 12 h postinfection. Activated caspase-3, but not caspase-1, was detected in the infected cells (38). In contrast, Mariathasan et al. found that the infection of preactivated murine peritoneal macrophages by either the LVS or strain U112 (F. novicida) triggered pyroptosis and the release of IL-1ß (42). In both studies, the induction of cell death was dependent upon the bacteria escaping the phagosome and initiating cytosolic replication. Weiss and colleagues isolated mutants of strain U112 that were attenuated in vivo and caused increased cell death in tissue culture compared to that caused by wild-type U112 (53). This suggests that although host cells initiate death pathways in response to F. tularensis infection, the bacteria has the ability to actively reduce cell death, and this is important for virulence.In addition to triggering death pathways, host cells respond to invading bacteria by mounting a proinflammatory response to alert neighboring cells of the impending bacterial threat (17). However, F. tularensis has been shown to actively suppress these innate host responses. Telepnev and coworkers showed that the LVS disrupted toll-like receptor signaling and blocked the secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1ß by murine and human macrophages (51, 52). Similarly, Bosio and colleagues showed that the LVS inhibited the innate immune response of murine pulmonary dendritic cells to bacterial ligands, and the infection of mice with the fully virulent Schu4 strain (F. tularensis subsp. tularensis) caused an overall state of immunosuppression in the lungs (8, 9).The genome analysis of F. tularensis identified only a few potential virulence factors, suggesting that the bacterium uses novel factors to achieve its high level of pathogenicity (40). Unique to F. tularensis is a 33.9-kb region of DNA termed the Francisella pathogenicity island (FPI) (29, 40, 45). The FPI encodes genes that are essential for intracellular survival and virulence, including iglABCD and pdpABCD (45). F. tularensis lacks type III and IV secretion systems, which is surprising considering its intracellular nature. These secretion systems commonly are used by intracellular pathogens to deliver effector proteins inside host cells to manipulate host cell responses (14, 26). F. tularensis does contain genes encoding a type IV pilus biogenesis system that also functions in the secretion of soluble proteins by a type II-like mechanism and that are important for virulence (12, 31, 54). Finally, F. tularensis appears to contain a functioning type I secretion system that is critical for pathogenesis (28).Type I secretion systems function in the secretion of a variety of toxins and other virulence factors directly from the cytoplasm to the extracellular milieu in a single energized step (33, 37). The type I system consists of three separate components: an outer membrane channel-forming protein, a periplasmic adaptor or membrane fusion protein, and an inner membrane pump that typically belongs to the ATP-binding cassette family. The TolC protein of Escherichia coli, which functions in hemolysin secretion, is the prototypical outer membrane channel component (37). In addition to protein secretion, TolC functions in the efflux of small noxious molecules, conferring multidrug resistance (37). F. tularensis contains three TolC paralogs, TolC, FtlC, and SilC, with TolC and FtlC exhibiting significant homology to the E. coli TolC protein (28, 35). In a previous study we created tolC and ftlC deletion mutants in the F. tularensis LVS (28). We found that both TolC and FtlC participate in multidrug resistance in F. tularensis, but only the ΔtolC mutant was attenuated for virulence in mice by the intradermal route. Thus, tolC is a critical virulence factor of F. tularensis and likely functions in type I secretion in addition to multidrug efflux.Here, we delineate the molecular mechanisms behind the attenuation of the LVS ΔtolC mutant in mice infected by both the intradermal and intranasal routes. In vivo organ burden assays revealed that the ΔtolC strain is decreased for the bacterial colonization of liver, spleen, and most prominently, lungs. In vitro experiments revealed that the ΔtolC mutant is hypercytotoxic to murine macrophages, causing increased apoptosis via a mechanism involving caspase-3 but not caspase-1. In addition, the LVS ΔtolC mutant was hypercytotoxic toward human macrophages and elicited the significantly increased secretion of the proinflammatory chemokines CXCL8 (also known as IL-8) and CCL2 (also known as monocyte chemoattractant protein [MCP-1]). Taken together, these data demonstrate a critical role for TolC, likely via a TolC-secreted toxin(s), in the successful intracellular lifestyle of F. tularensis, its ability to evade host innate immune responses, and its overall virulence.     

Vulnerable plaque detection by 3.0 tesla magnetic resonance imaging

RATIONALE AND OBJECTIVES: A clinical case report is presented on a 76-year-old man who volunteered for a 3.0 T magnetic resonance (MR) carotid protocol. The subject was referred for carotid endarterectomy and histology was performed on the ex vivo specimen and compared with the in vivo images. METHODS: The 3.0 and 1.5 T (obtained for comparison) MR protocol consisted of 2-dimensional (2D) and 3-dimensional (3D) multicontrast bright and black blood imaging for detecting the lumen and vessel wall. RESULTS: The combination of multicontrast black blood transverse images and the 3D time of flight transverse images provided visualization of a narrowed internal carotid artery lumen 4 mm above of the bifurcation and the presence of a complex atherosclerotic plaque containing a large lipid pool, calcification, and intact fibrous cap. Quantitative comparisons including vessel lumen and plaque area, signal-to-noise (SNR) and contrast-to-noise (CNR) ratios were obtained for 1.5 and 3.0 T image data. Plaque composition was verified with histology. Macrophages were also detected in the shoulders of the plaque as demonstrated by CD68 staining and corresponded with a small hyperintense area in the T2W images at 3.0 T, but not observed in comparable 1.5 T images. CONCLUSIONS: High field 3.0 T multicontrast MRI of atherosclerotic plaque has been validated with histology comparison and provides improved detection of complex atherosclerotic plaque with increased SNR and CNR compared with 1.5 T. Further studies validating contrast mechanisms of plaque at 3.0 T are required, but atherosclerotic plaque imaging has clear benefit from application at the higher magnetic field strength.     

REVIEW ARTICLEThrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy

Summary.  We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser-induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P-selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three-dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo .     

In vivo 18F-fluorodeoxyglucose positron emission tomography imaging provides a noninvasive measure of carotid plaque inflammation in patients.

OBJECTIVES: Given the importance of inflammation in atherosclerosis, we sought to determine if atherosclerotic plaque inflammation could be measured noninvasively in humans using positron emission tomography (PET). BACKGROUND: Earlier PET studies using fluorodeoxyglucose (FDG) demonstrated increased FDG uptake in atherosclerotic plaques. Here we tested the ability of FDG-PET to measure carotid plaque inflammation in patients who subsequently underwent carotid endarterectomy (CEA). METHODS: Seventeen patients with severe carotid stenoses underwent FDG-PET imaging 3 h after FDG administration (13 to 25 mCi), after which carotid plaque FDG uptake was determined as the ratio of plaque to blood activity (target to background ratio, TBR). Less than 1 month after imaging, subjects underwent CEA, after which carotid specimens were processed to identify macrophages (staining with anti-CD68 antibodies). RESULTS: There was a significant correlation between the PET signal from the carotid plaques and the macrophage staining from the corresponding histologic sections (r = 0.70; p < 0.0001). When mean FDG uptake (mean TBR) was compared with mean inflammation (mean percentage CD68 staining) for each of the 17 patients, the correlation was even stronger (r = 0.85; p < 0.0001). Fluorodeoxyglucose uptake did not correlate with plaque area, plaque thickness, or area of smooth muscle cell staining. CONCLUSIONS: We established that FDG-PET imaging can be used to assess the severity of inflammation in carotid plaques in patients. If subsequent natural history studies link increased FDG-PET activity in carotid arteries with clinical events, this noninvasive measure could be used to identify a subset of patients with carotid atherosclerosis in need of intensified medical therapy or carotid artery intervention to prevent stroke.