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In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

Telemedicine has been used to support care at a distance through the use of expensive equipment and broadband communication links. In the past, the operating room has been an isolated environment that has been relatively inaccessible for real-time consultation. Recent technological advances have permitted videoconferencing over low-bandwidth, inexpensive Internet connections. If these connections are shown to provide adequate video quality for surgical applications, low-bandwidth telemedicine will revolutionize surgical practice by enabling remote real-time surgical consultation. Many of the regions around the world that have the most need for advanced surgical mentoring can least afford it. Although expensive broadband network links such as integrated services digital network (ISDN), local area networks (LANs), and dedicated T-1 lines have provided ample throughput for acceptable video image quality in recent years, the Internet has opened new doors for inexpensive alternatives. Early work by NASA demonstrated the usefulness of the Internet for telemedicine applications. 1 Similar work was also conducted using plain old telephone system (POTS) from the jungles of Ecuador. 2 In both cases, low bandwidth proved to be very useful for telemedicine applications. Internet-2 has been shown to provide high-resolution images, 3 but Internet-2 is simply another broadband technology, which will not be broadly available in the foreseeable future in most regions of the world. By using the ordinary telephone and a dial-up account to a local or global Internet service provider (ISP), a reliable remote node network can be established easily and affordably. The Internet has been successfully shown to be a useful and effective tool in supporting telemedicine on an international scale. 4,5 Over low-bandwidth connections such as dial-up, many Internet applications such as e-mail or web browsing are completely adequate. However, transmitting video over this type of connection is more difficult because of the synchronous data transfer rate and high degrees of compression. A series of experiments were performed to determine whether the quality of video images transmitted over Internet protocol (IP) connections were adequate to support surgical applications.

Pulmonary hypertension occurs commonly in patients with chronic hypoxic lung disease and is characterized by the remodeling of the pulmonary artery walls. The molecular mechanisms underlying such remodeling are unknown but we have recently shown that the stress-activated (Jnk and p38) mitogen-activated protein (MAP) kinases are activated in pulmonary artery fibroblasts following acute hypoxia. We now show that Erk and p38 MAP kinases are constitutively activated in fibroblasts derived from the remodeled pulmonary, but not the systemic circulation from rats exposed to chronically hypoxic conditions. Moreover, we find that such fibroblasts show sustained enhanced proliferative capacities relative to pulmonary artery fibroblasts derived from normoxic rats or to aortic fibroblasts from either normoxic or hypoxic rats. Finally, abrogation of p38, but not Erk MAP kinase activity by use of specific inhibitors, prevents the enhanced proliferative capacity exhibited by pulmonary artery fibroblasts. Taken together, these data suggest that enhanced p38 MAP kinase activity provides a molecular mechanism to explain the proliferation of pulmonary artery fibroblasts required for remodeling of the pulmonary vasculature.

Phosphorylcholine (PC) is increasingly becoming recognised as a carbohydrate-associated component of a wide variety of procaryotic and eucaryotic pathogens. Studies employing nematode PC-containing molecules indicate that it possesses a plethora of immunomodulatory activities. ES-62 is a PC-containing glycoprotein, which is secreted by the rodent filarial nematode Acanthocheilonema viteae and which provides a model system for the dissection of the mechanisms of immune evasion induced by related PC-containing glycoproteins expressed by human filarial nematodes. At concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitised humans, ES-62 is able to inhibit antigen receptor-stimulated proliferation of B and T lymphocytes in vitro and in vivo. The active component of ES-62 appears to be PC, as PC conjugated to albumin or even PC alone broadly mimic the results obtained with ES-62. PC-induced impaired lymphocyte responsiveness appears to reflect uncoupling of the antigen receptors from key intracellular proliferative signalling events such as the phosphoinositide 3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways. Although PC-ES-62 can desensitise B and T cells, not all cells are affected, and in fact it is still possible to generate an antibody response to the molecule. Dissection of this response indicates that it is of the TH-2 type. This appears to reflect the ability of ES-62 to direct the polarity of the T cell response by suppressing the production of proinflammatory cytokines, inducing the induction of anti-inflammatory cytokines and by driving the maturation of dendritic cells that direct TH-2 T cell responses.

Keywords: Photomedicine group Reference LPAS-ARTICLE-2001-009 Record created on 2007-07-20, modified on 2016-08-08

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+influx and activation of protein kinase Cε (PKCε). PKCε, however, is known to be Ca2+independent. To determine where Ca2+is needed in this PKCε-mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle cells permeabilized by saponin. Ca2+(0.2–1.0 μM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCε (but not by PKCβII or PKCγ antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPβs. Addition of 1 μM Ca2+to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). Ca2+-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by AACOCF3(cytosolic phospholipase A2inhibitor). These data suggest that Ca2+may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCε, which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPγS augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular Ca2+and G protein activation), producing DAG and AA, and activating PKCε-dependent mechanisms to cause contraction.

The objective of this paper is to determine the effect of varying transmission bandwidth on image quality in laparoscopic surgery. Surgeons located in remote operating rooms connected through a telemedicine link must be able to transmit medical images for interaction. Image clarity and color fidelity are of critical importance in telementoring laparoscopic procedures. The clarity of laparoscopic images was measured by assessing visual acuity using a video image of a Snellen eye chart obtained with standard diameter laparoscopes (2, 5, and 10 mm). The clarity of the local image was then compared to that of remote images transmitted using various bandwidths and connection protocols [33.6 Kbps POTS (IP), 128 Kbps ISDN, 384 Kbps ISDN, 10 Mbps LAN (IP)]. The laparoscopes were subsequently used to view standard color placards. These color images were sent via similar transmission bandwidths and connection protocols. The local and remote images of the color placards were compared to determine the effect of the transmission protocols on color fidelity. Use of laparoscopes of different diameter does not significantly affect image clarity or color fidelity as long as the laparoscopes are positioned at their optimal working distance. Decreasing transmission bandwidth does not significantly affect image clarity or color fidelity when sufficient time is allowed for the algorithms to redraw the remote image. Remote telementoring of laparoscopic procedures is feasible. However, low bandwidth connections require slow and/or temporarily stopped camera movements for the quality of the remote video image to approximate that of the local video image.

Memory B-cell development is impaired by in vivo blockade of the CD40-CD40 ligand (CD40L) interaction using human Fc immunoglobulin G1 (IgG1)-mouse CD40 fusion protein (CD40-Ig); however, germinal centre (GC) formation is not. We show here that the block in B-cell differentiation in these mice is at the stage of rescue from apoptosis and exit from the GC. Thus, GC from CD40-Ig-treated mice contain a three- to fourfold higher level of apoptotic cells than found in control mice injected with human IgG1 alone. This increase in apoptosis is not caused by a blockade of the CD40L-mediated rescue signal but is the result of an intrinsic defect of GC B cells in CD40-Ig-treated mice to receive rescue signals via CD40. While anti-CD40 stimulation maintained the viability in culture of GC B cells from control mice, it did not rescue GC B cells from CD40-Ig-treated mice. This data is consistent with the notion that a 'rewiring' of the CD40 molecule is induced by CD40 ligation early in the response and is necessary to allow B-cell rescue from apoptosis when they subsequently enter the GC.

Two-dimensional magnetohydrodynamic simulations of the solar wind interaction with the magnetized regions on the surface of the Moon suggest “mini-magnetospheres” can form around the regions on the Moon when the magnetic anomaly field strength is above 10 nT at 100 km above the surface (for a surface field strength of 290 nT) and when the solar wind ion density is below 40 cm−3, with typical observations placing anomalous magnetic field strengths around 2 nT at 100 km above the surface. The results suggest that not only can a bow shock and magnetopause form around the small anomalies, but their position and shape can change dramatically with changes in the solar wind conditions. A switch from southward to northward interplanetary magnetic field (IMF) causes the size of the mini-magnetosphere to increase by 90% and the magnetic field at various positions inside the bow shock to increase by a factor of 10. In addition to affecting the stand-off distance, changes in the IMF can also cause the mini-magnetosphere to go from very round to flat and elongated. The scale size of the mini-magnetospheres is 100 km for the range of typical solar wind conditions and the surface magnetic field strengths measured by Lunar Prospector. A stagnation point inside the shock region also exists for several solar wind conditions.

Background & Aims: In cat spontaneous lower esophageal sphincter (LES), tone is maintained by the activity of group I secreted phospholipase A2 (sPLA2-I) that produces arachidonic acid. Arachidonic acid metabolites activate G proteins linked to phospholipases, producing second messengers and activation of a protein kinase C–dependent pathway to maintain tone. We examined the role of sPLA2-I in the maintenance of tone in human LES samples obtained from organ donors. Methods: In vitro LES tone and sPLA2-I–induced contraction of enzymatically isolated LES smooth muscle cells were measured in the absence or presence of inhibitors. Cell permeabilization by saponin allowed use of G-protein antibodies. Results: In vitro LES tone was reduced by inhibitors of sPLA2-I, by indomethacin, by the phosphatidylcholine-specific phospholipase C inhibitor D609, and by the protein kinase C inhibitor chelerythrine. sPLA2-I–induced contraction of isolated LES smooth muscle cells was reduced by indomethacin, pertussis toxin, Gi3 antibodies, D609, and by chelerythrine. Conclusions: Human LES tone is maintained by the activity of sPLA2-I that produces arachidonic acid and metabolites and activation of Gi3-linked receptors and of phosphatidylcholine-specific phospholipase C, resulting in production of diacylglycerol, activation of PKC, and maintenance of tone through a protein kinase C–dependent contractile pathway. GASTROENTEROLOGY 2000;119:1243-1252

Advanced wearable biosensors for vital-signs monitoring (physiologic cipher) are available to improve quality of healthcare in hospital, nursing home, and remote environments. The objective of this study was to determine reliability of vital-signs monitoring systems in extreme environments. Three climbers were monitored 24 hours while climbing through Khumbu Icefall. Data were transmitted to Everest Base Camp (elevation 17,800 feet) and retransmitted to Yale University via telemedicine. Main outcome measures (location, heart rate, skin temperature, core body temperature, and activity level) all correlated through time-stamped identification. Two of three location devices functioned 100% of the time, and one device failed after initial acquisition of location 75% of the time. Vital-signs monitors functioned from 95%-100% of the time, with the exception of one climber whose heart-rate monitor functioned 78% of the time. Due to architecture of automatic polling and data acquisition of biosensors, no climber was ever without a full set of data for more than 25 minutes. Climbers were monitored continuously in real-time from Mount Everest to Yale University for more than 45 minutes. Heart rate varied from 76 to 164 beats per minute, skin temperature varied from 5 to 10 degrees C, and core body temperature varied only 1-3 degrees C. No direct correlation was observed among heart rate, activity level, and body temperature, though numerous periods suggested intense and arduous activity. Field testing in the extreme environment of Mount Everest demonstrated an ability to track in real time both vital signs and position of climbers. However, these systems must be more reliable and robust. As technology transitions to commercial products, benefits of remote monitoring will become available for routine healthcare purposes.

Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.

FcgammaRI, the human high-affinity IgG receptor, is responsible for the internalization of immune complexes and their subsequent targetting to the lysosomes for degradation. We show here that aggregation of FcgammaRI by surface immune complexes in interferon-gamma-primed U937 cells causes the transient appearance of swollen vacuolar structures, probably swollen late endosomes, which disappear as the immune complexes are degraded. Wortmannin and LY294002, specific inhibitors of phosphoinositide 3-kinases (PI 3-kinases), delay the disappearance of these structures and also correspondingly inhibit degradation of FcgammaRI-mediated immune complexes. In addition these inhibitors delay the initial phase of FcgammaRI-mediated endocytosis of immune complexes and block the activity of FcgammaRI-stimulated phospholipase D, an enzyme that has previously been implicated in membrane-trafficking events. p85 is the regulatory subunit of PI 3-kinase. A p85-dependent PI 3-kinase was shown to be involved in the initial phase of FcgammaRI-mediated endocytosis, but not in the trafficking of immune complexes for degradation or the activation of phospholipase D. The results presented here show a role for a p85-independent PI 3-kinase in regulating the trafficking of FcgammaRI-mediated immune complexes, either directly or as a result of the activation of phospholipase D, and a distinct role for a p85-dependent PI 3-kinase isoform in the initial phases of FcgammaRI-mediated internalization of immune complexes.

The human high affinity receptor for immunoglobulin G, FcgammaRI, in dibutyryl cyclic AMP (dbcAMP)-differentiated U937 cells, is coupled to the activation of phospholipase C (PLC) and the conventional protein kinase C (PKC) isoforms, alpha, beta, and gamma. Here we demonstrate that aggregation of FcgammaRI activates the tyrosine-kinase regulated form of phosphatidylinositol-3-kinase (PI-3-kinase) and that an increase of phosphatidylinositol trisphosphate (PIP3) is essential for the activation and translocation of PLCgamma1 in these cells. In addition, activation of the PKC isoforms was ablated by specific inhibitors of PI3-kinase or by overexpression of a dominant negative p85 subunit of PI3-kinase. The findings reported here demonstrate that PLCgamma1 and PKC activation by FcgammaRI are downstream of PI3-kinase, and that in contrast to cytokine primed cells, only the tyrosine-kinase activated isoform of PI3-kinase is coupled to FcgammaRI in dbcAMP-differentiated cells.

Aggregation of receptors for the constant region (Fc) of immunoglobulin G on myeloid cells results in endocytosis or phagocytosis and cellular activation. Previous work has shown, using the cell line U937, that the high-affinity immunoglobulin G receptor, FcgammaRI, activates alternate intracellular signalling pathways depending on the cell differentiation state, which results in a marked change in the nature of calcium transients within the cell. Here, we show that protein kinase C (PKC) is activated in both interferon-gamma (IFN-gamma) -primed and dibutyryl cyclic AMP (dbcAMP) -differentiated cells but that the nature of the particular isoenzymes recruited differs. Thus, in IFN-gamma-primed U937 cells, FcgammaRI aggregation results in an increase of PKC activity which is essentially calcium independent resulting from the translocation to the membrane of the novel PKCs, delta and epsilon, together with the atypical PKC zeta. However, in cells differentiated to a more macrophage phenotype, all PKC enzyme activity after receptor aggregation is calcium dependent. Consistent with this finding, the isoenzymes translocated to the nuclear-free membrane fraction are the conventional PKCs alpha, beta and gamma; results consistent with our previous finding that FcgammaRI couples to phospholipase C in such dbcAMP-differentiated cells. Thus, the nature of PKC isoenzyme activated following FcgammaRI aggregation is defined by differentiation. The calcium dependence of the PKC isoenzyme is consistent with the duration of calcium transients previously reported in the two differentiation states.

Phosphorylcholine (PC) is a structural component of a variety of prokaryotic and eukaryotic pathogens. In some cases, PC in infectious agents can benefit the infected host due to its targeting by both the innate and adaptive immune responses. However, as discussed here, PC exhibits a surprising range of immunomodulatory properties that might be to the detriment of the host.

OBJECTIVES Robotic telesurgery has been demonstrated over long distances and offers theoretical benefits to urologic training and the care of patients in remote regions. The multiple arms and three-dimensional vision of the da Vinci robotic system provide a platform conducive to long-distance telementoring and telesurgery. Whereas prior telesurgical efforts have used dedicated lines for information transmission, the public Internet offers a less expensive alternative. It was the intent of this study to test the validity of using the da Vinci system in urologic telesurgery, and to conduct telerobotic nephrectomies using the public Internet. METHODS We performed four right nephrectomies in porcine models using the da Vinci robotic system. Telementoring and telesurgical approaches were used, with resident surgeons operating a console adjacent to the swine, while attending surgeons simultaneously operated a second console at distances of 1300 and 2400 miles from the operating room. RESULTS All four procedures and both telementoring and telesurgical models were successful. Round-trip delays from 450 to 900 ms were demonstrated. Blood loss was minimal, and there were no intraoperative complications. CONCLUSIONS This study represents the first use of the da Vinci Surgical System in urologic telesurgery and the first successful telesurgical nephrectomy in an animal model.

Cross-linking of the Ag receptors on B cells induces DNA synthesis and proliferation. Butanol trap experiments suggest that one or more phospholipase D activities play a key role in this process. Although phosphatidylcholine-phospholipase D has been shown to play a central role in the transduction of proliferative responses for a wide variety of calcium-mobilizing receptors, we show that the Ag receptors are not coupled to this phospholipase. In addition, phosphatidylcholine-phospholipase D is not stimulated under conditions that mimic T cell-dependent B cell activation. In contrast, ATP, which inhibits surface Ig (sIg)-mediated DNA synthesis in murine B cells via P2-purinoceptors, activates phosphatidylcholine-phospholipase D. Phosphatidylcholine-phospholipase D is therefore associated with antiproliferative signal transduction in mature B cells, but it does not transduce early signals associated with sIg-mediated growth arrest or apoptosis in immature B cells. Mitogenic stimulation of sIg is, however, coupled to a novel nonphosphatidylcholine-hydrolyzing phospholipase D activity. The resultant sIg-generated phosphatidic acid, unlike the phosphatidylcholine-derived phosphatidic acid generated via the purinoceptors, is converted to diacylglycerol. These data provide the first evidence that while the novel sIg-coupled phospholipase D and resultant diacylglycerol generation may play a role in B cell survival and proliferation, phosphatidylcholine-phospholipase D may transduce, via phosphatidic acid, negative immunomodulatory signals in mature B lymphocytes.

The signal transduction that mediates CCK-induced contraction of gallbladder muscle was investigated in the cat. Contraction was measured by scanning micrometry in single muscle cells isolated enzymatically with collagenase. Production ofd- myo-inositol 1,4,5-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG) was quantitated using HPLC and TLC, respectively. Protein kinase C (PKC) activity was determined by measuring the phosphorylation of a specific substrate peptide from myelin basic protein, Ac-MBP-(4—14). CCK-induced contraction was blocked by incubation in strontium medium, pertussis toxin (PTx), and antibodies against Giα3or βγ-subunits but was not blocked by Ca2+-free medium or by antibodies against Gq/11α, Giα1–2, or Goα. The contraction induced by CCK was inhibited by the phospholipase C (PLC) inhibitor U-73122, anti-PLC-β3 antibody, and the IP3receptor antagonist heparin but was not inhibited by the the phospholipase D inhibitor propranolol or antibodies against PLC-β1 or PLC-β2. Western blot analysis of gallbladder muscle revealed the presence of PLC-β2 and PLC-β3 but not PLC-β1. CCK caused a 94% increase in IP3generation and an 86% increase in DAG generation. A low dose of CCK caused PKC translocation, and CCK-induced contraction was blocked by the PKC inhibitor H-7. A high dose of CCK, however, caused no PKC translocation, and its contraction was blocked by the calmodulin antagonist CGS9343B. In conclusion, CCK contracts cat gallbladder muscle by stimulating PTx-sensitive Gi 3protein coupled with PLC-β3, producing IP3and DAG. Low doses activate PKC, whereas high doses activate calmodulin.

Filarial nematodes infect more than 100 million people in the tropics, causing elephantiasis, chronic skin lesions, and blindness. The parasites are long-lived as a consequence of being able to evade the host immune system, but an understanding of the molecular mechanisms underlying this evasion remains elusive. In this study, we demonstrate that ES-62 (2 μg/ml), a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B lymphocytes. Although this interaction is insufficient to cause B lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase and Ras mitogen-activating protein kinase pathways, and hence also to proliferation, via the Ag receptor. The active component of ES-62 appears to be PC, a molecule recently shown to act as an intracellular signal transducer, as the results obtained with ES-62 are broadly mimicked by PC alone. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES, by interfering with B cell function, could play a role in prolonging filarial infection in parasitized individuals.

Phosphoinositide 3-kinases (PI3-kinases) play an important role in the generation of lipid second messengers and the transduction of a myriad of biological responses. Distinct isoforms have been shown to be exclusively activated either by tyrosine kinase-coupled or G protein-coupled receptors. We show here, however, that certain nonclassical receptors can couple to both tyrosine kinase- and G protein-dependent isoforms of PI3-kinase: thus, aggregation of FcγRI, the human high affinity IgG receptor, on monocytes unusually leads to activation of both of these types of PI3-kinase. After aggregation of FcγRI, phosphatidylinositol 3,4,5-triphosphate (PIP3) levels rise rapidly in interferon γ-primed cells, reaching a peak within 30 sec. Moreover, and in contrast to the situation observed after stimulation of these cells with either insulin or ATP, which exclusively activate the tyrosine kinase- and G protein-coupled forms of PI3-kinase, respectively, PIP3levels remain elevated up to 15 min after receptor aggregation. We show here that although the initial peak results from transient activation of the p85-dependent p110 isoform of PI-3kinase, presumably through recruitment of tyrosine kinases by the γ chain, the later sustained rise of PIP3results from activation of the G protein βγ subunit-sensitive isoform, p110γ. This finding indicates that receptors lacking an intrinsic signaling motif, such as FcγRI, can recruit both tyrosine kinase and G protein-coupled intracellular signaling molecules and thereby initiate cellular responses.

Theileria parva-infected B cells express Jagged-1 and activate Notch signalling in a parasite-dependent manner. ES-62, a filarial nematode-secreted phosphorylcholine-containing glycoprotein, is able to further stimulate Notch-mediated signalling in parasitized cells. Notch is also activated to a similar extent by addition of exogenous IL-10, and this occurs prior to any increase in proliferation in T. parva-infected B cells.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein secreted by the filarial nematode Acanthocheilonema viteae that acts to modulate the host immune response to promote the establishment of chronic helminth infection. Reflecting its anti-inflammatory actions, we have previously reported that ES-62 protects mice from developing Collagen-Induced Arthritis (CIA): thus, as this helminth-derived product may exhibit therapeutic potential in Rheumatoid Arthritis (RA), it is important to understand the protective immunoregulatory mechanisms triggered by ES-62 in this model in vivo. We have established to date that ES-62 acts by downregulating pathogenic Th17/IL-17-mediated responses and upregulating the regulatory cytokine IL-10. In addition, our studies have identified that IL-22, another member of the IL-10 family of cytokines, exerts dual pathogenic and protective roles in this model of RA with ES-62 harnessing the cytokine's inflammation-resolving and tissue repair properties in the joint during the established phase of disease. Here, we discuss the counter-regulatory roles of IL-22 in the murine model of CIA and present additional novel data showing that ES-62 selectively induces γδ T cells with the capacity to induce IL-22 production and that γδ T cells with the capacity to produce IL-22, but not IL-17, induced during CIA can be identified by their expression of TLR4. Moreover, we also show that treatment of mice undergoing CIA with the active PC moiety of ES-62, in the form of PC conjugated to BSA, is not only sufficient to mimic the ES-62-dependent suppression of pathogenic IL-17 responses shown previously but also that of the IL-22 and IL-10 up-regulation observed with the parasitic worm product during CIA. These findings not only reinforce the potential of IL-22, firstly described as a Th17-related pro-inflammatory cytokine, as a protective factor in arthritis but also suggest that drugs based on the PC moiety found in ES-62 may be able to harness the joint-protecting activities of IL-22 therapeutically.

Filarial nematodes, parasitic worms that cause elephantiasis, chronic skin lesions, and blindness in the tropics, release a number of molecules, some of which appear to be immunomodulatory/suppressive, into the host environment. Here we demonstrate that ES-62, a phosphorylcholine-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, interferes with activation of B lymphocytes by differential modulation of protein kinase C isoform expression. Indeed, while ES-62 selectively down-regulates expression of the alpha, beta, iota/lambda, delta, and zeta isoforms of PKC, it up-regulates expression of PKC-gamma and -epsilon in B cells. Inhibitor studies suggest that ES-62 appears to promote down-regulation of PKC isoforms mainly by stimulating proteolytic degradation. ES-62 also disrupts the normal activation and nuclear translocation patterns of the alpha and iota/lambda isoforms of PKC following ligation of the Ag receptor. The effects of ES-62 on certain PKC isoforms were found to be modified by coculture with IL-4. Of particular interest was the observation that IL-4 prevented down-regulation of PKC alpha and iota/lambda, isotypes considered to be active in transducing mitogenic signals. Phosphorylcholine-containing secreted products (phosphorylcholine-ES) are also released by human filarial parasites; hence we discuss how these findings may relate to the nature of the human B cell response during filarial infections.

Esophageal reflux is a common condition that affects children and 1 in 10 adults, and if untreated may result in chronic esophagitis, aspiration pneumonia, esophageal strictures, and Barrett's esophagus, a premalignant condition. Although esophagitis is a multifactorial disease that may depend on transient lower esophageal sphincter (LES) relaxation, speed of esophageal clearance, mucosal resistance, and other factors, impairment of LES pressure is a common finding in patients complaining of chronic heartburn. Our data suggest that esophageal and LES circular muscle utilize distinct Ca2+ sources, phospholipid pools, and signal transduction pathways to contract in response to acetylcholine (ACh): (1) In esophageal muscle ACh-induced contraction requires influx of extracellular Ca2+ and may be linked to phosphatidylcholine metabolism, production of diacylglycerol (DAG) and arachidonic acid, and activation of a protein kinase C (PKC)-dependent pathway. (2) In LES muscle ACh-induced contraction utilizes intracellular Ca2+ release arising from metabolism of phosphatidylinositol (PI), and a calmodulin-myosin light chain kinase-dependent pathway. Resting LES tone, on the other hand, may be due to relatively low basal PI hydrolysis resulting in submaximal levels of inositol triphosphate (IP3)-induced calcium release and interaction with DAG to activate PKC. (3) After induction of experimental esophagitis, basal levels of PI hydrolysis and intracellular calcium stores are substantially reduced, resulting in a reduction of resting tone. In addition the signal transduction pathway responsible for LES contraction in response to ACh changes from one that depends on IP3 production, calcium release, and calmodulin activation to one that relies on influx of extracellular calcium and activation of PKC.

Objective The parasitic worm–derived immunomodulator ES‐62 protects against disease in the mouse collagen‐induced arthritis (CIA) model of rheumatoid arthritis (RA) by suppressing pathogenic interleukin‐17 (IL‐17) responses. The Th17‐associated cytokine IL‐22 also appears to have a pathogenic role in autoimmune arthritis, particularly in promoting proinflammatory responses by synovial fibroblasts and osteoclastogenesis. The present study was undertaken to investigate whether the protection against joint damage afforded by ES‐62 also reflects suppression of IL‐22. Methods The role(s) of IL‐22 was assessed by investigating the effects of neutralizing anti–IL‐22 antibodies and recombinant IL‐22 (rIL‐22) on proinflammatory cytokine production, synovial fibroblast responses, and joint damage in mice with CIA in the presence or absence of ES‐62. Results Neutralization of IL‐22 during the initiation phase abrogated CIA, while administration of rIL‐22 enhanced synovial fibroblast responses and exacerbated joint pathology. In contrast, after disease onset anti–IL‐22 did not suppress progression, whereas administration of rIL‐22 promoted resolution of inflammation. Consistent with these late antiinflammatory effects, the protection afforded by ES‐62 was associated with elevated levels of IL‐22 in the serum and joints that reflected a desensitization of the synovial fibroblast responses. Moreover, neutralization of IL‐22 during the late effector stage of disease prevented ES‐62–mediated desensitization of synovial fibroblast responses and protection against CIA. Conclusion IL‐22 plays a dual role in CIA, being pathogenic during the initiation phase while acting to resolve inflammation and joint damage during established disease. Harnessing of the tissue repair properties of IL‐22 by ES‐62 highlights the potential for joint‐targeted therapeutic modulation of synovial fibroblast responses and consequent protection against bone damage in RA.

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [alpha-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the beta 1- and/or beta 2-like subunits and alpha-subunits of the Gs, G1, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

Milkfat was extracted quantitatively from dairy foods and the vitamins A, E and beta-carotene in the extract were analysed by normal phase high performance liquid chromatography (HPLC) using a silica column, a fluorescence detector and a UV/visible detector. Products such as butter and anhydrous milkfat were dissolved simply in hexane, whereas milk and milk powders underwent a Rose-Gottlieb fat extraction prior to HPLC analysis. The extraction method used as superior to commonly used saponification procedures in terms of speed as well as reduced loss and isomerization of the vitamin components in the food, because the chemical form of the vitamin is not changed to a less stable form such as retinol during extraction. The individual geometrical isomers of vitamin A, which possess different in vivo vitamin activities, were uniquely identified and quantified. Recovery studies showed satisfactory results for all three vitamins. The simultaneous nature of the assay allowed the estimation of the total vitamin A activity of the food because both vitamin A and pro-vitamin A (beta-carotene) were quantified. The single extraction and single HPLC analysis used for the three vitamins made the method well suited for routine work. (C) 1996 Elsevier Science Limited

The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

This chapter explores the immunomodulatory effects of nematode infections which protect the host against autoimmune and allergic diseases. Included are discussions on specific nematode molecules (i.e., Ascaris suum PAS-1, cystatins, Dirofilaria immitis-derived antigen (DiAg), galectin-9 homologue of Toxascaris leonina (Tl-GAL), ES-62) that exert immunomodulatory properties in animal studies and their probable mechanisms of action.

Human gallbladders were used to investigate the mechanisms of the impaired contraction induced by cholecystokinin (CCK) associated with cholesterol stones. Single muscle cells were isolated enzymatically with collagenase. Inositol 1,4,5-trisphosphate was measured by high-performance liquid chromatography. Diacylglycerol was assayed by thin-layer chromatography. CCK stimulation showed decreased muscle contraction and production of inositol 1,4,5-trisphosphate and diacylglycerol in gallbladders with cholesterol stones compared with those with pigment stones. Exogenous calmodulin induced maximal contraction of 22.4 +/- 0.5 and 21.0 +/- 0.6% in gallbladders with cholesterol and pigment stones, respectively. Similar findings were observed with a synthetic diacylglycerol analogue. Two G protein activators, aluminum fluoride and guanosine 5'-O-(3-thiotriphosphate), evoked similar responses in these two types of gallbladders, with maximal contractions of 21.3 +/- 0.4 and 23.3 +/- 0.5%, respectively, in those with cholesterol stones and 20.9 +/- 0.8 and 22.6 +/- 0.4%, respectively, in those with pigment stones. These results suggest that receptor-dependent ligands like CCK cannot fully activate the intracellular pathways, which, however, can be fully stimulated by circumventing receptors with G protein activators or second messengers. After G protein activation, the pathways appear to be functionally intact. The defect might then reside in the receptor or in the interaction between receptors and G proteins.

Preservation of high-risk families and prevention of foster-home placement has become a primary focus of child welfare services. Drawing from current research on the needs of high-risk families and on volunteerism, the authors explore the use of volunteers to help agencies providing family-preservation services meet the dual goals of prevention of abuse and neglect and preserving vulnerable families.

In many locations throughout the world, the optimal process is non-existent or has broken down; the United States is no exception as explained from a national, (Sarfaty, 2010) as well as an international perspective by Zwar (2010). The situation has become inefficient because of poorly coordinated, acute-focused, episodic care. The solution lies in the most basic role of the healthcare continuum; primary care. However, to achieve maximum effectiveness and efficiency, adoption of various technologies need to be embraced. While it is referenced by different terms, the concept is often termed patient centered medicine.

Among many survival strategies, parasitic worms secrete molecules that modulate host immune responses. One such product, ES-62, is protective against collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Since interleukin-17 (IL-17) has been reported to play a pathogenic role in the development of RA, this study was undertaken to investigate whether targeting of IL-17 may explain the protection against CIA afforded by ES-62.DBA/1 mice progressively display arthritis following immunization with type II collagen. The protective effects of ES-62 were assessed by determination of cytokine levels, flow cytometric analysis of relevant cell populations, and in situ analysis of joint inflammation in mice with CIA.ES-62 was found to down-regulate IL-17 responses in mice with CIA. First, it acted to inhibit priming and polarization of IL-17 responses by targeting a complex IL-17-producing network, involving signaling between dendritic cells and γ/δ or CD4+ T cells. In addition, ES-62 directly targeted Th17 cells by down-regulating myeloid differentiation factor 88 expression to suppress responses mediated by IL-1 and Toll-like receptor ligands. Moreover, ES-62 modulated the migration of γ/δ T cells and this was reflected by direct suppression of CD44 up-regulation and, as evidenced by in situ analysis, dramatically reduced levels of IL-17-producing cells, including lymphocytes, infiltrating the joint. Finally, there was strong suppression of IL-17 production by cells resident in the joint, such as osteoclasts within the bone areas.Our findings indicate that ES-62 treatment of mice with CIA leads to unique multisite manipulation of the initiation and effector phases of the IL-17 inflammatory network. ES-62 could be exploited in the development of novel therapeutics for RA.

The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1- O-alkyl- sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (Emax) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-([(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122). Basal Ins(1,4,5)P3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by Emax of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P3 concentrations activated a PKC-dependent pathway, whereas contraction induced by Emax of Ins(1,4,5)P3 was calmodulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

[1] Multifluid/multiscale simulations are used to examine the influence of ionospheric outflows on two substorms that occurred on August 13, 2001. Both substorms had well defined onsets in Cluster ion spectrometer (CIS) data of ion composition of the plasma sheet. It is shown that the model is able to account for two orders of magnitude variation in the H+ density and one order of magnitude change in the O+ density in the plasma sheet in association with the ionosphere generating peak outflows of 4 × 1025 H+ ions/s and 2 × 1025 O+ ions/s. The model shows that the growth phase is associated with the venting of solar wind plasma in association with the formation of an X-line and the ejection of a plasmoid. This reconnection occurs in a proton-dominated plasma and occurs well before onset. After this venting, a Y-line configuration develops with the ionospheric plasma being the dominant source of the plasma sheet. Lobe reconnection involves heavy enriched O+ lobe field lines produced by enhanced outflows that start at the beginning of the growth phase. This O+ produces enhanced dissipation and localized formation of flux ropes prior to substorm onset. For the isolated substorms considered here, the acceleration of O+ in the plasma sheet keeps the density low in this region but leads to the buildup of both number and density at the inner edge of the plasma sheet. O+ can contribute nearly 50% of the total energy density in this region just prior to onset. This results in over pressurization and dipolarization in conjunction with onset and associated increases in the nightside auroral currents.

In esophageal mucosa, HCl causes TRPV1-mediated release of calcitonin gene-related peptide (CGRP) and substance P (SP) from submucosal neurons and of platelet-activating factor (PAF) from epithelial cells. CGRP and SP release was unaffected by PAF antagonists but reduced by the purinergic antagonist suramin. ATP caused CGRP and SP release from esophageal mucosa, confirming a role of ATP in the release. The human esophageal epithelial cell line HET-1A was used to identify epithelial cells as the site of ATP release. HCl caused ATP release from HET-1A, which was reduced by the TRPV1 antagonist 5-iodoresiniferatoxin. Real-time PCR demonstrated the presence of mRNA for several P2X and P2Y purinergic receptors in epithelial cells. HCl also increased activity of lyso-PAF acetyl-CoA transferase (lyso-PAF AT), the enzyme responsible for production of PAF. The increase was blocked by suramin. ATP caused a similar increase, confirming ATP as a mediator for the TRPV1-induced increase in enzyme activity. Repeated exposure of HET-1A cells to HCl over 2 days caused upregulation of mRNA and protein expression for lyso-PAF AT. Suramin blocked this response. Repeated exposure to ATP caused a similar mRNA increase, confirming ATP as a mediator for upregulation of the enzyme. Thus, HCl-induced activation of TRPV1 causes ATP release from esophageal epithelial cells that causes release of CGRP and SP from esophageal submucosal neurons and activation of lyso-PAF AT, the enzyme responsible for the production of PAF in epithelial cells. Repeated application of HCl or of ATP causes upregulation of lyso-PAF AT in epithelial cells.

The following on esophageal disease provides updated information the mucosal defense against acid and acid-pepsin injury; the roles of platelet activating factor, mast cells, proinflammatory cytokines, and chemokines in inflammation; differences and similarities in erosive and nonerosive esophagitis; acid and vanilloid receptors in esophageal mucosa; and bile acid receptors in esophageal epithelium.

Until recently, it has not been possible to image and functionally correlate the key molecular and cellular events underpinning immunity and tolerance in the intact immune system. Certainly, the field has been revolutionized by the advent of tetramers to identify physiologically relevant specificities of T cells, and the introduction of models in which transgenic T-cell receptor and/or B-cell receptor-bearing lymphocytes are adoptively transferred into normal mice and can then be identified by clonotype-specific antibodies using flow cytometry in vitro, or immunohistochemistry ex vivo. However, these approaches do not allow for quantitative analysis of the precise anatomical, phenotypic, signaling, and functional parameters required for dissecting the development of immune responses in health and disease in vivo. Traditionally, assessment of signal transduction pathways has required biochemical or molecular biological analysis of isolated and highly purified subsets of immune system cells. Inevitably, this creates potential artifacts and does not allow identification of the key signaling events for individual cells present in their microenvironment in situ. These difficulties have now been overcome by new methodologies in cell signaling analysis that are sufficiently sensitive to detect signaling events occurring in individual cells in situ and the development of technologies such as laser scanning cytometry that provide the tools to analyze physiologically relevant interactions between molecules and cells of the innate and the adaptive immune system within their natural environmental niche in vivo.

We aimed to investigate the effects of progesterone on gene expression and function of both myometrium and circulating leukocytes. We recruited women participating in a randomized clinical trial of progesterone to prevent preterm delivery. These participants had a twin pregnancy and were managed in 1 of 2 tertiary referral centers. Participants were treated with progesterone (90 mg vaginally) or placebo from 24 to 34 weeks of pregnancy. The outcome measures were myometrial and leukocyte gene expression and expression of cell surface markers in circulating leukocytes, all quantified ex vivo. Prolonged in vivo administration of progesterone inhibited myometrial expression of connexins 26 and 43, endothelial nitric acid synthase (eNOS), and the prostaglandin receptor EP2 ex vivo. Administration of progesterone also increased numbers of circulating neutrophils while decreasing lymphocyte proportions and decreasing neutrophil CD11b expression. The observed effects of prolonged in vivo administration of progesterone will minimize the ability of the uterus to contract as a synctium and the ability of peripheral blood leukocytes to migrate into the myometrium during parturition. We suggest that these are putative mechanisms by which progesterone might prevent preterm birth in women at high risk.

[1] Analysis of spectra from the Clementine ultraviolet-visible and near-infrared cameras of small, immature craters and surface soils both on and adjacent to the lunar swirls at Mare Ingenii has yielded the following conclusions about space weathering at a magnetic anomaly. (1) Despite having spectral characteristics of immaturity, the lunar swirls are not freshly exposed surfaces. (2) The swirl surfaces are regions of retarded weathering, while immediately adjacent regions experience accelerated weathering. (3) Weathering in the off-swirl regions darkens and flattens the spectrum with little to no reddening, which suggests that the production of larger (>40 nm) nanophase iron dominates in these locations as a result of charged particle sorting by the magnetic field. Preliminary analysis of two other lunar swirl regions, Reiner Gamma and Mare Marginis, is consistent with our observations at Mare Ingenii. Our results indicate that sputtering/vapor deposition, implanted solar wind hydrogen, and agglutination share responsibility for creating the range in npFe0 particle sizes responsible for the spectral effects of space weathering.

Until recently, it has not been possible to image and functionally correlate the key molecular and cellular events underpinning immunity and tolerance in the intact immune system. Certainly, the field has been revolutionized by the advent of tetramers to identify physiologically relevant specificities of T cells, and the introduction of models in which transgenic T-cell receptor and/or B-cell receptor-bearing lymphocytes are adoptively transferred into normal mice and can then be identified by clonotype-specific antibodies using flow cytometry in vitro, or immunohistochemistry ex vivo. However, these approaches do not allow for quantitative analysis of the precise anatomical, phenotypic, signaling, and functional parameters required for dissecting the development of immune responses in health and disease in vivo. Traditionally, assessment of signal transduction pathways has required biochemical or molecular biological analysis of isolated and highly purified subsets of immune system cells. Inevitably, this creates potential artifacts and does not allow identification of the key signaling events for individual cells present in their microenvironment in situ. These difficulties have now been overcome by new methodologies in cell signaling analysis that are sufficiently sensitive to detect signaling events occurring in individual cells in situ and the development of technologies such as laser scanning cytometry that provide the tools to analyze physiologically relevant interactions between molecules and cells of the innate and the adaptive immune system within their natural environmental niche in vivo.

Muscle strips were used to study the mechanisms that generate cat gallbladder tone. Strontium substitution for calcium and the protein kinase C (PKC) inhibitor H-7 abolished the tone, whereas the calmodulin antagonist W-7 had no effect, suggesting that tone depends on intracellular calcium release and the PKC pathway. Basal levels of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3) were higher in gallbladder muscle than in esophageal muscle, which does not maintain tone. These data suggest that IP3 might interact with DAG to activate PKC during tonic contraction. This interaction was demonstrated in single cells in which a low dose of IP3 potentiated DAG and the potentiation was blocked by H-7. Furthermore, low doses of IP3 induced contraction, which was blocked by H-7 and unaffected by the calmodulin antagonist CGS-9343B; high doses of IP3 were unaffected by H-7 but were blocked by CGS-9343B; DAG-induced contraction was blocked by activated calmodulin. We conclude that 1) the synergistic action of DAG and IP3-calcium release, which further activates PKC, might be responsible for gallbladder tone and 2) activated calmodulin appears to inhibit the effect of PKC.

E-S 62, a major excretory-secretory product of the rodent filarial parasite, Acanthocheilonema viteae, inhibits the polyclonal activation (DNA synthesis) of mouse B cells by mitogenic anti-Ig antibodies. This effect appears to be due at least in part to the phosphorylcholine (PC) moiety of the molecule, because it can be mimicked by PC-BSA or PC-chloride. Activation of the B cells by LPS is not influenced by the presence of E-S 62/PC, suggesting that they may target some aspect of signaling via the Ag receptor. E-S 62/PC failed to inhibit surface Ig-mediated generation of the second messenger, inositol triphosphate, indicating that their target may not be the early biochemical events associated with activation. Exposure to E-S 62/PC was found to lead to a reduction in the level of protein kinase C, an important downstream regulatory enzyme, in anti-Ig-treated cells. This protein-kinase C down-regulation may be the biochemical mechanism underlying E-S 62/PC-mediated inhibition of surface Ig-activated B cells.

[1] Using single-particle tracking with time-dependent global magnetic and electric fields from multifluid simulations, we model the 10 March 1998 storm and investigate storm time acceleration, injection, and trapping mechanisms associated with the formation of the ring current. We examine the contribution from various ionospheric source regions to the storm time ring current and the effect interplanetary magnetic field Bz has on producing an asymmetric and symmetric ring current; we provide the first maps for the relative importance of ionospheric outflow (H+ and O+) regions as a function of all magnetic local times (MLTs) and latitudes between 60° and 80°. During the early part of the storm, high-latitude outflow regions between 00 and 06 MLT are the most efficient sectors at contributing particle density to the ring current, whereas during the main phase of the storm, there is more even contribution from all MLTs. The sectors that contribute the majority of the energy are consistently the high-latitude regions between 03 and 09 MLT. An increase in the contribution of O+ to the current density is observed from the predawn high-latitude region during each of two decreases in Dst examined for the 10 March 1998 storm, supporting the central role oxygen plays in storm development. Asymmetries are observed between H+ and O+ contributions. The dominant ionospheric species contributing to the ring current energy density is shown to vary during the course of the storm with a significant increase in ionospheric O+ contribution to the ring current associated with large decreases in Dst.

[1] Multiscale-multifluid simulations are used to provide high-resolution (∼470 km) simulations of the 26 February 2008 substorm that was well observed by the THEMIS spacecraft to investigate whether the substorm was internally or externally triggered. This substorm occurred during an extended (1 h 45 min) period of weak (−2.5 to −1.5 nT) southward interplanetary magnetic field (IMF). Simulation results show that this substorm was internally triggered well before the IMF start to become more northerly. The southward IMF leads to an enhancement in the cross-polar cap potential, strong thinning of the current sheet, and pseudo-breakup in association with patchy reconnection. The southward IMF also produces an enhancement in the outflow of ionospheric oxygen into the current sheet. Because of the tilt of the magnetic field the strongest outflows originate from the southern polar cap. The arrival of these heavy ions in the tail occurs about 10 min prior to the observed substorm onset and leads to enhanced tail reconnection, including flux rope development and fast earthward flows. The interaction of these fast flows at the inner edge of the plasma sheet is closely associated with auroral onset, indicating that the substorm is internally triggered. Comparison with THEMIS data suggests that reconnection occurred earlier than THEMIS observations. The results show onset is associated with processes at the inner edge of the plasma sheet and is not directly related to the onset of reconnection. Comparison with THEMIS data indicates that processes at scale lengths less than 500 km are occurring.

Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition.

[1] Single-particle tracking with time-dependent global magnetic and electric fields is used to investigate the generation of the ring current from ionospheric outflows during an internally driven substorm. We show that the energization of the ions is not correlated with the time that the ions leave the ionosphere; instead energization is correlated with the formation of an injection front driven by an earthward moving flux rope at onset. Because of the large gyroradius of the O+ ions, they experience strong dawn-dusk acceleration in the vicinity of the injection front. The acceleration is strongly influenced by small-scale structures including the Hall electric field and the development of kinks across the tail. H+ is mainly energized by betatron acceleration as it is injected into the inner magnetosphere with less average energy than the O+ ions. In this paper we investigate the conditions that lead to the formation of the injection front and small-scale structures (∼1 RE) in the current sheet, such as tail kinking and flux ropes, that are correlated with particle convection and energization at substorm onset. High-resolution capabilities allow us to resolve these small-scale processes within a thin (