Extension of the germinal center stage of B cell development promotes autoantibodies in BXD2 mice.

OBJECTIVE: Regulator of G protein signaling (RGS) proteins inhibit chemokine signaling by desensitizing G protein-coupled receptor signals. This study was undertaken to determine the mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GCs), using BXD2-Rgs13-/- mice. METHODS: Confocal and light microscopy imaging techniques were used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the mouse spleen, and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by real-time quantitative polymerase chain reaction (qPCR). Differential interleukin-17 (IL-17)-mediated expression of RGS13 in GC versus non-GC B cells was analyzed using A20 and 70Z/3 B cells. RESULTS: In the spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 up-regulated RGS13 in A20 GC cells but not 70Z/3 non-GC B cells. BXD2- Rgs13-/- mice exhibited smaller GCs and lower AID levels, suggesting lower somatic hypermutation and affinity maturation. However, GC B cells from BXD2- Rgs13-/- mice showed increased levels of IgMbright plasmablasts, up-regulation of the genes encoding plasma program, including interferon regulatory factor 4, B lymphocyte-induced maturation protein 1, and X-box binding protein 1 and the p-CREB target genes Fosb and Obf1, and down-regulation of the GC program genes Aid, Pax5, and Bach2 compared to BXD2 mice. BXD2-Rgs13-/- mice had lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. CONCLUSION: RGS13 deficiency is associated with a reduction in GC program genes and the exit of fewer pathogenic IgM plasmablasts in BXD2 mice. Our findings indicate that prolonged GC program, mediated by up-regulation of RGS13, enhances AID expression and enables the generation of pathogenic autoantibodies in autoreactive GCs.

Type I interferon-dependent CD86(high) marginal zone precursor B cells are potent T cell costimulators in mice.

OBJECTIVE: To investigate the role of CD86(high) marginal zone (MZ) precursor B cells in type I interferon (IFN)-induced T cell-dependent responses in autoimmune BXD2 mice. METHODS: Confocal microscopic imaging was used to determine the location of plasmacytoid dendritic cells (DCs), MZ precursor B cells, and CD4 T cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Immunohistochemical staining was used to determine IgG(bright) cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Enzyme-linked immunosorbent assay was used to determine serum levels of IFNα and autoantibodies, and 4-hydroxy-3-nitrophenylacetyl hapten (NP)-chicken γ-globulin (CGG) (NP-CGG)- or NP-Ficoll-induced anti-NP2 antibody titers. Real-time quantitative polymerase chain reaction was used to determine the levels of type I IFN transcripts. T cell proliferation was measured using (3) H-thymidine. The expression of CD86 and CD80 was determined by fluorescence-activated cell sorting analysis. RESULTS: The deletion of type I IFN receptor abrogated the development of IgG(bright) cells and suppressed a T cell-dependent antibody response. Type I IFN signaling was associated with the expression of CD86, but not CD80, on follicular, MZ, and MZ precursor B cells. However, MZ precursor B cells demonstrated the highest expression of CD86 and the highest capacity for T cell costimulation with intact type I IFN receptor. This effect was blocked by an antibody that neutralizes CD86. In IFN receptor-intact BXD2 mouse spleens, MZ precursor B cells clustered at the T cell-B cell border. CD86 deletion suppressed germinal center formation, autoantibody production, and development of autoimmune diseases in BXD2 mice. CONCLUSION: Type I IFN can promote autoimmune responses in BXD2 mice through up-regulation of CD86(high) expression on MZ precursor B cells and trafficking of MZ precursor B cells to the T cell-B cell border to provide costimulation of CD4 T cells.

Inhibition of the catalytic function of activation-induced cytidine deaminase promotes apoptosis of germinal center B cells in BXD2 mice.

OBJECTIVE: To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. METHODS: We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN-transgenic mice). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6-8-month-old mice. RESULTS: Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage-related genes in GC B cells of BXD2-Aicda-DN-transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN-transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM- and anti-CD40 plus anti-Ig-induced B cell proliferative responses were decreased in BXD2-Aicda-DN-transgenic mice. CONCLUSION: Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

IL-17 activates the canonical NF-kappaB signaling pathway in autoimmune B cells of BXD2 mice to upregulate the expression of regulators of G-protein signaling 16.

We previously identified that autoreactive B cells from BXD2 mice can be targeted by IL-17, leading to upregulation of the expression of regulators of G-protein signaling (Rgs) genes that facilitated the development of spontaneous germinal centers. Little is known about the signaling pathway used by IL-17 to upregulate RGS. In the current study, we found that IL-17 rapidly activates the canonical NF-kappaB signaling pathway and that BXD2 B cells exhibit higher basal and activated phosphorylated p65 levels than B6 or BXD2-Il17ra(-/-) B cells. Inhibition of p65 phosphorylation downregulated RGS16 expression and abrogated the IL-17-induced chemotactic arrest of B cells in response to CXCL12. Knockdown of TNFR-associated factor 6 or NF-kappaB activator 1 in 70Z/3 pre-B cells led to decreased Rgs16 expression, indicating that both of these two genes are involved in IL-17-mediated activation of NF-kappaB signaling in B cells. These findings identify the signaling pathway regulated by IL-17 to contribute to the development of spontaneous germinal centers in autoimmune BXD2 mice.

Marginal zone precursor B cells as cellular agents for type I IFN-promoted antigen transport in autoimmunity.

The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgM(hi)CD1d(hi)CD21(hi)CD23(hi) in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-alpha, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnalphar(-/-)) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnalphar(-/-) mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN-producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.

Net illumination reduces fisheries bycatch, maintains catch value, and increases operational efficiency.

Small-scale fisheries are vital for food security, nutrition, and livelihoods in coastal areas throughout the world's oceans.1-9 As intricately linked social-ecological systems, small-scale fisheries require management approaches that help ensure both ecological and socioeconomic sustainability.7,10-14 Given their ease of use and lucrative nature, coastal gillnet fisheries are globally ubiquitous.10,15 However, these fisheries often result in high discarded capture of non-target organisms (bycatch) that can lead to significant cascading effects throughout trophic chains16-18 and costly fisheries restrictions that result in important revenue losses in coastal communities with scarce economic alternatives.19,20 Despite these challenges, few solutions have been developed and broadly adopted to decrease bycatch in coastal gillnet fisheries, particularly in developing nations.5,21 Here we used controlled experiments along Mexico's Baja California peninsula to show that illuminating gillnets with green LED lights-an emerging technology originally developed to mitigate sea turtle bycatch-significantly reduced mean rates of total discarded bycatch biomass by 63%, which included significant decreases in elasmobranch (95%), Humboldt squid (81%), and unwanted finfish (48%). Moreover, illuminated nets significantly reduced the mean time required to retrieve and disentangle nets by 57%. In contrast, there were no significant differences in target fish catch or value. These findings advance our understanding of how artificial illumination affects operational efficiency and changes in catch rates in coastal gillnet fisheries, while illustrating the value of assessing broad-scale ecological and socioeconomic effects of species-specific conservation strategies.

Establishment of a definitive protocol for the diagnosis and management of body packers (drug mules).

BACKGROUND: 'Mules' or body packers are people who transport illegal drugs by packet ingestion into the gastrointestinal tract. These people are otherwise healthy and their management should maintain minimal morbidity. In this study, experience with body packers is presented and an algorithm for conservative and surgical management is provided. METHODS: The clinical patient database for all body packer admissions at Mary Immaculate Hospital of the Caritas Health Care Inc. from 1993 to 2005 was interrogated. 56 patients (4.5%) required admission out of a total of 1250 subjects confirmed to be body packers and apprehended by United State Customs officials at JFK International Airport, New York. The retrieved patient data were analysed retrospectively. RESULTS: 70% of the body packers were men, with a male to female ratio of 2.8 to 1. The mean age was 33 years and 52% were from Columbia. Heroin was the most common illegally transported substance (73%). 25 patients (45%) required surgical intervention, whereas 31 patients (55%) were successfully managed conservatively. Indications for intervention included: bowel obstruction, packet rupture/toxicity, and delayed progression of packet transit on conservative management. Multiple intraoperative manoeuvres were used to remove the foreign bodies: gastrotomy, enterotomy and colotomy. Wound infection was the most common complication and is associated with distal enterotomy and colotomy. CONCLUSIONS: Men were more likely to present as body packers than women. Proximal enterotomies are preferred and multiple enterotomies should be avoided. A confirmatory radiological study is needed to demonstrate complete clearance of packets. A systematic protocol for the management of body packers results in minimal morbidity and no mortality.

Screening procedure for airborne pollutants emitted from a high-tech industrial complex in Taiwan.

Despite the modernization of computational techniques, atmospheric dispersion modeling remains a complicated task as it involves the use of large amounts of interrelated data with wide variability. The continuously growing list of regulated air pollutants also increases the difficulty of this task. To address these challenges, this study aimed to develop a screening procedure for a long-term exposure scenario by generating a site-specific lookup table of hourly averaged dispersion factors (χ/Q), which could be evaluated by downwind distance, direction, and effective plume height only. To allow for such simplification, the average plume rise was weighted with the frequency distribution of meteorological data so that the prediction of χ/Q could be decoupled from the meteorological data. To illustrate this procedure, 20 receptors around a high-tech complex in Taiwan were selected. Five consecutive years of hourly meteorological data were acquired to generate a lookup table of χ/Q, as well as two regression formulas of plume rise as functions of downwind distance, buoyancy flux, and stack height. To calculate the concentrations for the selected receptors, a six-step Excel algorithm was programmed with four years of emission records and 10 most critical toxics were screened out. A validation check using Industrial Source Complex (ISC3) model with the same meteorological and emission data showed an acceptable overestimate of 6.7% in the average concentration of 10 nearby receptors. The procedure proposed in this study allows practical and focused emission management for a large industrial complex and can therefore be integrated into an air quality decision-making system.

In vitro evaluation of hemolysis and sublethal blood trauma in a novel subcutaneous vascular access system for hemodialysis.

Hemodialysis requires reliable frequent access to the patients' vasculature, with blood flow rates of > 300 ml/min. Currently in the U.S. market, there are three types of hemodialysis access systems: the native arteriovenous fistula, generally using 15G needles; the synthetic arteriovenous (AV) graft, also generally using 15G needles; and the percutaneous catheter. Some of the problems with current vascular access technologies include insufficient blood flow, blood trauma, thrombosis, infection, cardiac load, and venous stenosis. The LifeSite System (Vasca, Inc.) represents an alternative for vascular access, and consists of a subcutaneous valve and 12F cannula accessed by a standard 14G needle. The LifeSite valve is implanted in the upper torso with the cannula generally entering the right internal jugular vein. The purpose of this study was to compare the LifeSite System with two known vascular access systems: the 10F dialysis catheter (Tesio-Cath, MedComp) and the 15G A.V. Fistula Needle Set (JMS Co., Ltd.) with regard to blood damage produced by these devices in use. Mechanical hemolysis and sublethal blood trauma were evaluated by means of in vitro blood pumping through a circulating loop incorporating a hemodialysis vascular access system. Sublethal blood damage was examined by using a hemorheologic assay that included parameters such as erythrocyte mechanical fragility, plasma total protein and fibrinogen concentrations, and blood viscosity. The tests demonstrated that, at both studied flow rates of 300 ml/min and 450 ml/min, the LifeSite produced lower hemolysis and less sublethal damage to blood than either the Tesio-Cath catheter or the A.V. Fistula Needle Set.

Cytokine regulation of B-cell migratory behavior favors formation of germinal centers in autoimmune disease.

Chemotaxis is essential for shaping immune responses and chemokine-receptor antagonists are now being evaluated as therapies for various inflammatory and autoimmune diseases. However, the dysregulation of chemotaxis in autoimmune disease may involve both promotion and inhibition of B-cell migration. This review focuses on the disparate mechanisms by which two inflammatory cytokines that have been associated with autoimmune disease, namely interferon-alpha (IFN-alpha) and interleukin-17 (IL-17), may regulate B-cell migratory responses. Chemotactic responses play a key role in orchestrating the cell-cell interactions in the germinal centers (GCs). This process involves active shuttling of the antigen-carrying B cells between the marginal zone and the GCs. We have shown that in autoimmune BXD2 mice, the migration of marginal zone precursor B cells is promoted by high levels of IFN-alpha produced by plasmacytoid dendritic cells in the marginal sinus that antagonize the activity of the S1P(1) chemokine receptor. In contrast, within the GCs, interleukin-17A (IL-17A) upregulates the expression of regulators of G protein signaling (RGS) in B cells to desensitize the G protein-coupled receptor (GPCR) signaling pathway of CXCL12 and CXCL13 chemokines. This promotes a prolonged stable interaction of B and T cells in the GC that induces high levels of activation-induced cytidine deaminase (AICDA) thereby enabling development of pathogenic autoantibody-producing B cells.

Immunochemical and electrophysiological analyses of magnetically responsive neurons in the mollusc Tritonia diomedea.

Tritonia diomedea uses the Earth's magnetic field as an orientation cue, but little is known about the neural mechanisms that underlie magnetic orientation behavior in this or other animals. Six large, individually identifiable neurons in the brain of Tritonia (left and right Pd5, Pd6, Pd7) are known to respond with altered electrical activity to changes in earth-strength magnetic fields. In this study we used immunochemical, electrophysiological, and neuroanatomical techniques to investigate the function of the Pd5 neurons, the largest magnetically responsive cells. Immunocytochemical studies localized TPeps, neuropeptides isolated from Pd5, to dense-cored vesicles within the Pd5 somata and within neurites adjacent to ciliated foot epithelial cells. Anatomical analyses revealed that neurites from Pd5 are located within nerves innervating the ipsilateral foot and body wall. These results imply that Pd5 project to the foot and regulate ciliary beating through paracrine release. Electrophysiological recordings indicated that, although both LPd5 and RPd5 responded to the same magnetic stimuli, the pattern of spiking in the two cells differed. Given that TPeps increase ciliary beating and Tritonia locomotes using pedal cilia, our results are consistent with the hypothesis that Pd5 neurons control or modulate the ciliary activity involved in crawling during orientation behavior.

Magnetic orientation and navigation in marine turtles, lobsters, and molluscs: concepts and conundrums.

The Earth's magnetic field provides a pervasive source of directional information used by phylogenetically diverse marine animals. Behavioral experiments with sea turtles, spiny lobsters, and sea slugs have revealed that all have a magnetic compass sense, despite vast differences in the environment each inhabits and the spatial scale over which each moves. For two of these animals, the Earth's field also serves as a source of positional information. Hatchling loggerhead sea turtles from Florida responded to the magnetic fields found in three widely separated regions of the Atlantic Ocean by swimming in directions that would, in each case, facilitate movement along the migratory route. Thus, for young loggerheads, regional magnetic fields function as navigational markers and elicit changes in swimming direction at crucial geographic boundaries. Older turtles, as well as spiny lobsters, apparently acquire a "magnetic map" that enables them to use magnetic topography to determine their position relative to specific goals. Relatively little is known about the neural mechanisms that underlie magnetic orientation and navigation. A promising model system is the marine mollusc Tritonia diomedea, which possesses both a magnetic compass and a relatively simple nervous system. Six neurons in the brain of T. diomedea have been identified that respond to changes in magnetic fields. At least some of these appear to be ciliary motor neurons that generate or modulate the final behavioral output of the orientation circuitry. These findings represent an encouraging step toward a holistic understanding of the cells and circuitry that underlie magnetic orientation behavior in one model organism.

Identifiable neurons inhibited by Earth-strength magnetic stimuli in the mollusc Tritonia diomedea.

Diverse animals use the Earth's magnetic field as an orientation cue, but little is known about the sensory, processing and motor elements of the neural circuitry underlying magnetic orientation behavior. The marine mollusc Tritonia diomedea has both a magnetic compass sense and a simple nervous system accessible to electrophysiological analysis. Previous studies have revealed that four identifiable neurons, known as LPd5, RPd5, LPd6 and RPd6, respond with enhanced electrical activity to changes in Earth-strength magnetic fields. Here we report that two additional neurons, LPd7 and RPd7, are inhibited by magnetic stimuli. Cobalt fills of the Pd7 neurons indicated that two prominent neurites emerge from the soma and project to the periphery through the ipsilateral cerebral nerves CeN6 and CeN3; in some cases, a third neurite was visible in CeN2. The nerves extend to the anterior region of the animal where they innervate the lateral body walls, oral veil and mouth region. Action potentials in the Pd7 neurons propagate from the central ganglia toward the periphery. Thus, the Pd7 cells have characteristics of efferent neurons. The precise function of these cells during magnetic orientation behavior, however, remains to be determined.

Identification of magnetically responsive neurons in the marine mollusc Tritonia diomedea.

Behavioral experiments have demonstrated that the marine mollusc Tritonia diomedea can use the Earth's magnetic field as an orientation cue. Little is known, however, about the neural mechanisms that underlie magnetic orientation behavior in this or any other animal. In previous studies, two neurons in the brain of Tritonia, known as LPd5 and RPd5, were shown to respond with enhanced electrical activity to changes in earth-strength magnetic fields. We report evidence that two additional neurons, known as LPd6 and RPd6, also respond with increases in electrical activity when the magnetic field around the animal is altered. Anatomical analyses revealed that prominent neurites from the Pd6 cells are located within two ipsilateral nerves, pedal nerves 1 and 2. These nerves extend to the periphery of the animal and innervate tissues of the anterior ipsilateral foot and body wall. Electrophysiological recordings demonstrated that action potentials generated by the Pd6 cells propagate from the central ganglia toward the periphery. These results imply that the Pd6 cells play an efferent role in the magnetic orientation circuitry. Given that these cells contain cilio-excitatory peptides and that Tritonia crawls using ciliary locomotion, the Pd6 neurons may control or modulate cilia used in crawling, turning, or both.