Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.

Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon.

Crohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARgamma functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of beta-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Ppargamma mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARgamma-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn's colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARgamma-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.

Immune activation alters cellular and humoral responses to yellow fever 17D vaccine.

Background. Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. Methods. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. Results. We showed that YF-17D-induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Conclusion. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. Trial registration. Registration is not required for observational studies. Funding. This study was funded by Canada's Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

Feasibility Study of Strengthening Existing Single Span Steel Beam Concrete Deck Bridges, June 1961

Iowa has the same problem that confronts most states in the United States: many bridges constructed more than 20 years ago either have deteriorated to the point that they are inadequate for original design loads or have been rendered inadequate by changes in design/maintenance standards or design loads. Inadequate bridges require either strengthening or posting for reduced loads. A sizeable number of single span, composite concrete deck - steel I beam bridges in Iowa currently cannot be rated to carry today's design loads. Various methods for strengthening the unsafe bridges have been proposed and some methods have been tried. No method appears to be as economical and promising as strengthening by post-tensioning of the steel beams. At the time this research study was begun, the feasibility of posttensioning existing composite bridges was unknown. As one would expect, the design of a bridge-strengthening scheme utilizing post-tensioning is quite complex. The design involves composite construction stressed in an abnormal manner (possible tension in the deck slab), consideration of different sizes of exterior and interior beams, cover-plated beams already designed for maximum moment at midspan and at plate cut-off points, complex live load distribution, and distribution of post-tensioningforces and moments among the bridge beams. Although information is available on many of these topics, there is miminal information on several of them and no information available on the total design problem. This study, therefore, is an effort to gather some of the missing information, primarily through testing a half-size bridge model and thus determining the feasibility of strengthening composite bridges by post-tensioning. Based on the results of this study, the authors anticipate that a second phase of the study will be undertaken and directed toward strengthening of one or more prototype bridges in Iowa.

The transcription factor NFATc2 controls IL-6-dependent T cell activation in experimental colitis.

The nuclear factor of activated T cells (NFAT) family of transcription factors controls calcium signaling in T lymphocytes. In this study, we have identified a crucial regulatory role of the transcription factor NFATc2 in T cell-dependent experimental colitis. Similar to ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-induced chronic intestinal inflammation. Furthermore, NFATc2 deficiency suppressed colitis induced by oxazolone administration. This finding was associated with enhanced T cell apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by mucosal T lymphocytes. Further studies using knockout mice showed that IL-6, rather than IL-23 and -17, are essential for oxazolone colitis induction. Administration of hyper-IL-6 blocked the protective effects of NFATc2 deficiency in experimental colitis, suggesting that IL-6 signal transduction plays a major pathogenic role in vivo. Finally, adoptive transfer of IL-6 and wild-type T cells demonstrated that oxazolone colitis is critically dependent on IL-6 production by T cells. Collectively, these results define a unique regulatory role for NFATc2 in colitis by controlling mucosal T cell activation in an IL-6-dependent manner. NFATc2 in T cells thus emerges as a potentially new therapeutic target for inflammatory bowel diseases.

Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.

Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.

Increased titers of anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients with reduced H-ficolin levels but normal MASP-2 activity.

BACKGROUND AND AIMS: Mannan-binding lectin (MBL) and ficolins are microbial pattern recognition molecules that activate the lectin pathway of complement. We previously reported the association of MBL deficiency with anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with Crohn's disease (CD). However, ASCA are also frequently found in MBL-proficient CD patients. Here we addressed expression/function of ficolins and MBL-associated serine protease-2 (MASP-2) regarding potential association with ASCA. METHODS: ASCA titers and MBL, ficolin and MASP-2 concentrations were determined by ELISA in the serum of patients with CD, ulcerative colitis (UC), and in healthy controls. MASP-2 activity was determined by measuring complement C4b-fixation. Anti-MBL autoantibodies were detected by ELISA. RESULTS: In CD and UC patients, L-ficolin concentrations were significantly higher compared to healthy controls (p<0.001 and p=0.029). In contrast, H-ficolin concentrations were slightly reduced in CD and UC compared to healthy controls (p=0.037 for UC vs. hc). CD patients with high ASCA titers had significantly lower H-ficolin concentrations compared to ASCA-low/negative CD patients (p=0.009). However, MASP-2 activity was not different in ASCA-negative and ASCA-positive CD patients upon both, ficolin- or MBL-mediated MASP-2 activation. Finally, anti-MBL autoantibodies were not over-represented in MBL-proficient ASCA-positive CD patients. CONCLUSIONS: Our results suggest that low expression of H-ficolin may promote elevated ASCA titers in the ASCA-positive subgroup of CD patients. However, unlike MBL deficiency, we found no evidence for low expression of serum ficolins or reduced MASP-2 activity that may predispose to ASCA development.

Effects of Heat Straightening Structural Steel, MLR-91-3, 1992

Heat straightening of steel beams on bridges struck by over height trucks has become common practice in recent years in Iowa. A study of the effects of this heat straightening on the steel beams thus straightened is needed. Appropriate samples for mechanical and metallurgical tests were cut from the same rolled beam from the end which was heated and the end which was not heated and the test results were compared. The test results showed beyond doubt that the steel was being heated beyond the permitted temperature and that the impact properties are being drastically reduced by the current method of heat straightening.

Longevity differs among sexes but is not affected by repeated immune activation in voles (Microtus arvalis)

Investment of resources in immune defences, despite obvious short-term benefits, may be detrimental to long-term maintenance and thus decrease longevity in absence of parasites. In addition, females and males may differ in immune investment and intrinsic longevity because they are subjected to different degrees of sexual competition and extrinsic mortality. In order to test if sex-specific investment in mounting an immune response reduced longevity, we compared the longevity of captive male and female common voles Microtus arvalis regularly challenged with keyhole limpet haemocyanin, an antigen which elicits the production of antibodies, to the longevity of voles injected with the corresponding antigen-free buffer (phosphate-buffered saline). Injections were repeated every 28 days to mimic a chronic infection. The magnitude of immune response did not vary between males and females and did not affect longevity. Overall, females lived longer than males, independently of the immune challenge. Thus, the long-term costs of immunity seem small in voles. The longevity pattern is consistent with the prediction that male-biased predation or parasitism in the wild causes reduced intrinsic lifespan, but this reduction is not mediated by a decrease in male immunity

Strengthening of an Existing Continuous-Span, Steel-Beam, Concrete-Deck Bridge by Post-Tensioning, HR-308, 1990

The need to upgrade a large number of understrength and obsolete bridges in the U.S. has been well documented in the literature. Through several Iowa DOT projects, the concept of strengthening simple-span bridges by post-tensioning has been developed. The purpose of the project described in this report was to investigate the use of post-tensioning for strengthening continuous composite bridges. In a previous, successfully completed investigation, the feasibility of strengthening continuous, composite bridges by post-tensioning was demonstrated on a laboratory 1/3-scale-model bridge (3 spans: 41 ft 11 in. x 8 ft 8 in.). This project can thus be considered the implementation phase. The bridge selected for strengthening was in Pocahontas County near Fonda, Iowa, on County Road N28. With finite element analysis, a post-tensioning system was developed that required post-tensioning of the positive moment regions of both the interior and exterior beams. During the summer of 1988, the strengthening system was installed along with instrumentation to determine the bridge's response and behavior. Before and after post-tensioning, the bridge was subjected to truck loading (1 or 2 trucks at various predetermined critical locations) to determine the effectiveness of the strengthening system. The bridge, with the strengthening system in place, was inspected approximately every three months to determine any changes in its appearance or behavior. In 1989, approximately one year after the initial strengthening, the bridge was retested to identify any changes in its behavior. Post-tensioning forces were removed to reveal any losses over the one-year period. Post-tensioning was reapplied to the bridge, and the bridge was tested using the same loading program used in 1988. Except for at a few locations, stresses were reduced in the bridge the desired amount. At a few locations flexural stresses in the steel beams are still above 18 ksi, the allowable inventory stress for A7 steel. Although maximum stresses are above the inventory stress by about 2 ksi, they are about 5 ksi below the allowable operating stress; therefore, the bridge no longer needs to be load-posted.

Strengthening of Existing Single Span Steel Beam and Concrete Deck Bridges, HR-238, Part 1, 1983

The Phase I research, Iowa Department of Transportation (IDOT) Project HR-214, "Feasibility Study of Strengthening Existing Single Span Steel Beam Concrete Deck Bridges," verified that post-tensioning can be used to provide strengthening of the composite bridges under investigation. Phase II research, reported here, involved the strengthening of two full-scale prototype bridges - one a prototype of the model bridge tested during Phase I and the other larger and skewed. In addition to the field work, Phase II also involved a considerable amount of laboratory work. A literature search revealed that only minimal data existed on the angle-plus-bar shear connectors. Thus, several specimens utilizing angle-plus-bar, as well as channels, studs and high strength bolts as shear connectors were fabricated and tested. To obtain additional shear connector information, the bridge model of Phase I was sawed into four composite concrete slab and steel beam specimens. Two of the resulting specimens were tested with the original shear connection, while the other two specimens had additional shear connectors added before testing. Although orthotropic plate theory was shown in Phase I to predict vertical load distribution in bridge decks and to predict approximate distribution of post-tensioning for right-angle bridges, it was questioned whether the theory could also be used on skewed bridges. Thus, a small plexiglas model was constructed and used in vertical load distribution tests and post-tensioning force distribution tests for verification of the theory. Conclusions of this research are as follows: (1) The capacity of existing shear connectors must be checked as part of a bridge strengthening program. Determination of the concrete deck strength in advance of bridge strengthening is also recommended. (2) The ultimate capacity of angle-plus-bar shear connectors can be computed on the basis of a modified AASHTO channel connector formula and an angle-to-beam weld capacity check. (3) Existing shear connector capacity can be augmented by means of double-nut high strength bolt connectors. (4) Post-tensioning did not significantly affect truck load distribution for right angle or skewed bridges. (5) Approximate post-tensioning and truck load distribution for actual bridges can be predicted by orthotropic plate theory for vertical load; however, the agreement between actual distribution and theoretical distribution is not as close as that measured for the laboratory model in Phase I. (6) The right angle bridge exhibited considerable end restraint at what would be assumed to be simple support. The construction details at bridge abutments seem to be the reason for the restraint. (7) The skewed bridge exhibited more end restraint than the right angle bridge. Both skew effects and construction details at the abutments accounted for the restraint. (8) End restraint in the right angle and skewed bridges reduced tension strains in the steel bridge beams due to truck loading, but also reduced the compression strains caused by post-tensioning.

Strengthening of Existing Single Span Steel Beam and Concrete Deck Bridges, HR-238, Part II, 1985

The unifying objective of Phases I and II of this study was to determine the feasibility of the post-tensioning strengthening method and to implement the technique on two composite bridges in Iowa. Following completion of these two phases, Phase III was undertaken and is documented in this report. The basic objectives of Phase III were further monitoring bridge behavior (both during and after post-tensioning) and developing a practical design methodology for designing the strengthening system under investigation. Specific objectives were: to develop strain and force transducers to facilitate the collection of field data; to investigate further the existence and effects of the end restraint on the post-tensioning process; to determine the amount of post-tensioning force loss that occurred during the time between the initial testing and the retesting of the existing bridges; to determine the significance of any temporary temperature-induced post-tensioning force change; and to develop a simplified design methodology that would incorporate various variables such as span length, angle-of-skew, beam spacing, and concrete strength. Experimental field results obtained during Phases II and III were compared to the theoretical results and to each other. Conclusions from this research are as follows: (1) Strengthening single-span composite bridges by post-tensioning is a viable, economical strengthening technique. (2) Behavior of both bridges was similar to the behavior observed from the bridges during field tests conducted under Phase II. (3) The strain transducers were very accurate at measuring mid-span strain. (4) The force transducers gave excellent results under laboratory conditions, but were found to be less effective when used in actual bridge tests. (5) Loss of post-tensioning force due to temperature effects in any particular steel beam post-tensioning tendon system were found to be small. (6) Loss of post-tensioning force over a two-year period was minimal. (7) Significant end restraint was measured in both bridges, caused primarily by reinforcing steel being continuous from the deck into the abutments. This end restraint reduced the effectiveness of the post-tensioning but also reduced midspan strains due to truck loadings. (8) The SAP IV finite element model is capable of accurately modeling the behavior of a post-tensioned bridge, if guardrails and end restraints are included in the model. (9) Post-tensioning distribution should be separated into distributions for the axial force and moment components of an eccentric post-tensioning force. (10) Skews of 45 deg or less have a minor influence on post-tensioning distribution. (11) For typical Iowa three-beam and four-beam composite bridges, simple regression-derived formulas for force and moment fractions can be used to estimate post-tensioning distribution at midspan. At other locations, a simple linear interpolation gives approximately correct results. (12) A simple analytical model can accurately estimate the flexural strength of an isolated post-tensioned composite beam.

Preventing Cracking at Diaphragm/Plate Girder Connections in Steel Bridges, HR-393, 1998

Some of the Iowa Department of Transportation (Iowa DOT) continuous, steel, welded plate girder bridges have developed web cracking in the negative moment regions at the diaphragm connection plates. The cracks are due to out-of-plane bending of the web near the top flange of the girder. The out-of-plane bending occurs in the "web-gap", which is the portion of the girder web between (1) the top of the fillet welds attaching the diaphragm connection plate to the web and (2) the fillet welds attaching the flange to the web. A literature search indicated that four retrofit techniques have been suggested by other researchers to prevent or control this type of cracking. To eliminate the problem in new bridges, AASHTO specifications require a positive attachment between the connection plate and the top (tension) flange. Applying this requirement to existing bridges is expensive and difficult. The Iowa DOT has relied primarily on the hole-drilling technique to prevent crack extension once cracking has occurred; however, the literature indicates that hole-drilling alone may not be entirely effective in preventing crack extension. The objective of this research was to investigate experimentally a method proposed by the Iowa DOT to prevent cracking at the diaphragm/plate girder connection in steel bridges with X-type or K-type diaphragms. The method consists of loosening the bolts at some connections between the diaphragm diagonals and the connection plates. The investigation included selecting and testing five bridges: three with X-type diaphragms and two with K-type diaphragms. During 1996 and 1997, these bridges were instrumented using strain gages and displacement transducers to obtain the response at various locations before and after implementing the method. Bridges were subjected to loaded test trucks traveling in different lanes with speeds varying from crawl speed to 65 mph (104 km/h) to determine the effectiveness of the proposed method. The results of the study show that the effect of out-of-plane loading was confined to widths of approximately 4 in. (100 mm) on either side of the connection plates. Further, they demonstrate that the stresses in gaps with drilled holes were higher than those in gaps without cracks, implying that the drilling hole technique is not sufficient to prevent crack extension. The behavior of the web gaps in X-type diaphragm bridges was greatly enhanced by the proposed method as the stress range and out-of-plane distortion were reduced by at least 42% at the exterior girders. For bridges with K-type diaphragms, a similar trend was obtained. However, the stress range increased in one of the web gaps after implementing the proposed method. Other design aspects (wind, stability of compression flange, and lateral distribution of loads) must be considered when deciding whether to adopt the proposed method. Considering the results of this investigation, the proposed method can be implemented for X-type diaphragm bridges. Further research is recommended for K-type diaphragm bridges.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.