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Habitat restoration is a key strategy for recovering imperiled species, and planning habitat restoration activities cost effectively can help advance recovery objectives. Habitat restoration planning involves decisions about where and when to undertake restoration, and what type of restoration to undertake. This article focuses on decisions about the amount of restoration to undertake for a given type, location, and time, termed intervention intensity. A return on investment framework is developed for incorporating intervention intensity into habitat restoration planning. The framework is then applied in the context of planning habitat restoration for Pacific salmon recovery as a case study. Results showed that no single intervention type or location dominated, and several returns to scale relationships emerged across the candidate interventions. Scenarios that considered interventions across multiple intensities outperformed single-intensity scenarios in terms of total benefits and cost effectiveness. These findings highlight the usefulness of exploratory return on investment analysis for prioritizing habitat restoration interventions, and underscore the importance of systematically considering how much restoration to undertake, in addition to what to do and where.

Stream temperatures in the Pacific Northwest are projected to increase with climate change, placing additional stress on cold-water salmonids. We modeled the potential impact of increased stream temperatures on four anadromous salmonid populations in the Chehalis River Basin (spring-run and fall-run Chinook salmon Oncorhynchus tshawytscha, coho salmon O. kisutch, and steelhead O. mykiss), as well as the potential for floodplain reconnection and stream shade restoration to offset the effects of future temperature increases. In the Chehalis River Basin, peak summer stream temperatures are predicted to increase by as much as 3°C by late-century, but restoration actions can locally decrease temperatures by as much as 6°C. On average, however, basin-wide average stream temperatures are expected to increase because most reaches have low temperature reduction potential for either restoration action relative to climate change. Results from the life cycle models indicated that, without restoration actions, increased summer temperatures are likely to produce significant declines in spawner abundance by late-century for coho (-29%), steelhead (-34%), and spring-run Chinook salmon (-95%), and smaller decreases for fall-run Chinook salmon (-17%). Restoration actions reduced these declines in all cases, although model results suggest that temperature restoration alone may not fully mitigate effects of future temperature increases. Notably, floodplain reconnection provided a greater benefit than riparian restoration for steelhead and both Chinook salmon populations, but riparian restoration provided a greater benefit for coho. This pattern emerged because coho salmon tend to spawn and rear in smaller streams where shade restoration has a larger effect on stream temperature, whereas Chinook and steelhead tend to occupy larger rivers where temperatures are more influenced by floodplain connectivity. Spring-run Chinook salmon are the only population for which peak temperatures affect adult prespawn survival in addition to rearing survival, making them the most sensitive species to increasing stream temperatures.

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.

Inhibition of triacylglycerol (TAG) biosynthetic enzymes has been suggested as a promising strategy to treat insulin resistance, diabetes, dyslipidemia, and hepatic steatosis. Monoacylglycerol acyltransferase 3 (MGAT3) is an integral membrane enzyme that catalyzes the acylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to generate DAG and TAG, respectively. Herein, we report the discovery and characterization of the first selective small molecule inhibitors of MGAT3. Isoindoline-5-sulfonamide (6f, PF-06471553) selectively inhibits MGAT3 with high in vitro potency and cell efficacy. Because the gene encoding MGAT3 (MOGAT3) is found only in higher mammals and humans, but not in rodents, a transgenic mouse model expressing the complete human MOGAT3 was used to characterize the effects of 6f in vivo. In the presence of a combination of diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) inhibitors, an oral administration of 6f exhibited inhibition of the incorporation of deuterium-labeled glycerol into TAG in this mouse model. The availability of a potent and selective chemical tool and a humanized mouse model described in this report should facilitate further dissection of the physiological function of MGAT3 and its role in lipid homeostasis.

In the western United States, the long-term recovery of many Pacific salmon populations is inextricably linked to freshwater habitat quality. Industrial activities from the past century have left a legacy of pollutants that persist, particularly near working waterfronts. The adverse impacts of these contaminants on salmon health have been studied for decades, but the population-scale consequences of chemical exposure for salmonids are still poorly understood. We estimated acute and delayed mortality rates for seaward migrating juvenile Chinook salmon that feed and grow in a Superfund-designated area in the Lower Willamette River in Portland, Oregon. We combined previous, field-collected exposure data for juvenile Chinook salmon together with reduced growth and disease resistance data from earlier field and laboratory studies. Estimates of mortality were then incorporated into a life cycle model to explore chemical habitat-related fish loss. We found that 54% improved juvenile survival-potentially as a result of future remediation activities-could increase adult Chinook salmon population abundance by more than 20%. This study provides a framework for evaluating pollution remediation as a positive driver for species recovery.

Mesenchymal stem cells (MSCs) are potent immunosuppressive cells that have shown promise in the treatment of rheumatoid arthritis (RA). Deciphering the intrinsic characteristics of MSCs that correlate with their biologic activity will facilitate their clinical use. Recently, the role of glucocorticoid-induced leucine zipper (GILZ) in the development of RA has been documented. The aim of this study was to evaluate whether GILZ expression by MSCs may contribute to their therapeutic effect.MSCs were isolated from GILZ-deficient (GILZ(-/-) ) mice and wild-type mice. MSCs (1 × 10(6) cells) were injected twice via the tail vein into mice with collagen-induced arthritis (CIA).In vitro, we showed that GILZ is a key factor involved in the immunosuppressive potential of MSCs. MSCs derived from GILZ(-/-) mice did not suppress the proliferation of CD4+ T cells and were less efficient than MSCs derived from WT mice in altering Th17 cell polarization. Thus, we investigated the role of GILZ in an experimental model of arthritis and demonstrated that although WT MSCs significantly reduced paw swelling in arthritic mice, GILZ(-/-) MSCs did not. Moreover, the magnitude of the effects of GILZ(-/-) MSCs on Th17 cell frequency was significantly lower than that of WT MSCs. The therapeutic effect of MSCs correlated with the generation of Treg cells bearing the CD4 + RORγt+IL-17(low) IL-10+ signature, and Th17 cell polarization was GILZ dependent.This study demonstrates that GILZ has an essential role in the therapeutic effectiveness of MSCs in arthritis by favoring Th17 cell polarization toward a regulatory phenotype. Therefore, potentiation of GILZ expression in MSCs could represent a means to enhance their therapeutic effect in autoimmune diseases.

IL-10 is a potent anti-inflammatory cytokine that has received growing attention for its therapeutic potential. We examined the efficiency of adenoviral-mediated gene transfer of IL-10 on the incidence and severity of murine collagen-induced arthritis (CIA). Male DBA1 mice immunized with collagen II were treated by systemic administration of 109 plaque-forming units of replication-defective adenoviral vector expressing viral IL-10 (vIL-10) on day 30, when clinical symptoms of arthritis start. The transgene was shown to inhibit the onset of CIA, to decrease severity, and profoundly suppress the overall joint histopathology of the experimental arthritis. Significant IL-10 concentrations were obtained in the serum of injected animals for 7 days. Inhibition of arthritis was enhanced by administration of increasing doses of adenovirus-vIL-10. In addition, the local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by a monoclonal anti-vIL-10 Ab. The CIA symptoms in the group treated with the same construct expressing inactive vIL-10 (vIL-10 mut) were similar to those in untreated animals. Our data indicate that a single systemic administration of an adenoviral vector encoding vIL-10 may be a good candidate to suppress arthritis.

Aquatic species are threatened by climate change but have received comparatively less attention than terrestrial species. We gleaned key strategies for scientists and managers seeking to address climate change in aquatic conservation planning from the literature and existing knowledge. We address 3 categories of conservation effort that rely on scientific analysis and have particular application under the U.S. Endangered Species Act (ESA): assessment of overall risk to a species; long-term recovery planning; and evaluation of effects of specific actions or perturbations. Fewer data are available for aquatic species to support these analyses, and climate effects on aquatic systems are poorly characterized. Thus, we recommend scientists conducting analyses supporting ESA decisions develop a conceptual model that links climate, habitat, ecosystem, and species response to changing conditions and use this model to organize analyses and future research. We recommend that current climate conditions are not appropriate for projections used in ESA analyses and that long-term projections of climate-change effects provide temporal context as a species-wide assessment provides spatial context. In these projections, climate change should not be discounted solely because the magnitude of projected change at a particular time is uncertain when directionality of climate change is clear. Identifying likely future habitat at the species scale will indicate key refuges and potential range shifts. However, the risks and benefits associated with errors in modeling future habitat are not equivalent. The ESA offers mechanisms for increasing the overall resilience and resistance of species to climate changes, including establishing recovery goals requiring increased genetic and phenotypic diversity, specifying critical habitat in areas not currently occupied but likely to become important, and using adaptive management. Incorporación de las Ciencias Climáticas en las Aplicaciones del Acta Estadunidense de Especies en Peligro para Especies Acuáticas.

Significant challenges remain in the ability to estimate habitat change under the combined effects of natural variability, climate change, and human activity. We examined anticipated effects on shallow water over low-sloped beaches to these combined effects in the lower Willamette River, Oregon, an area highly altered by development. A proposal to stabilize some shoreline with large rocks (riprap) would alter shallow water areas, an important habitat for threatened Chinook salmon (Oncorhynchus tshawytscha), and would be subject to U.S. Endangered Species Act-mandated oversight. In the mainstem, subyearling Chinook salmon appear to preferentially occupy these areas, which fluctuate with river stages. We estimated effects with a geospatial model and projections of future river flows. Recent (1999-2009) median river stages during peak subyearling occupancy (April-June) maximized beach shallow water area in the lower mainstem. Upstream shallow water area was maximized at lower river stages than have occurred recently. Higher river stages in April-June, resulting from increased flows predicted for the 2080s, decreased beach shallow water area 17-32%. On the basis of projected 2080s flows, more than 15% of beach shallow water area was displaced by the riprap. Beach shallow water area lost to riprap represented up to 1.6% of the total from the mouth to 12.9 km upstream. Reductions in shallow water area could restrict salmon feeding, resting, and refuge from predators and potentially reduce opportunities for the expression of the full range of life-history strategies. Although climate change analyses provided useful information, detailed analyses are prohibitive at the project scale for the multitude of small projects reviewed annually. The benefits of our approach to resource managers include a wider geographic context for reviewing similar small projects in concert with climate change, an approach to analyze cumulative effects of similar actions, and estimation of the actions' long-term effects. Efectos Combinados del Cambio Climático y la Estabilización de Bordes de Ríos Hábitats de Aguas Poco Profundas del Salmón Chinook.

The fibrinolytic system is often the target for pathogenic bacteria, resulting in increased fibrinolysis, bacterial dissemination, and inflammation. The purpose of this study was to explore whether proteases NprB and InhA secreted by Bacillus anthracis could activate the host's fibrinolytic system. NprB efficiently activated human pro-urokinase plasminogen activator (pro-uPA), a key protein in the fibrinolytic cascade. Conversely, InhA had little effect on pro-uPA. Plasminogen activator inhibitors (PAI)-1, 2 and the uPA receptor were also targets for NprB in vitro. InhA efficiently degraded the thrombin-activatable fibrinolysis inhibitor (TAFI) in vitro. Mice infected with B. anthracis showed a significant decrease in blood TAFI levels. In another mouse experiment, animals infected with isogenic inhA deletion mutants restored TAFI levels, while the levels in the parent strain decreased. We propose that NprB and InhA may contribute to the activation of the fibrinolytic system in anthrax infection.

Anthrax is a zoonotic disease caused byBacillus anthracis. The infection is associated with inflammation and sepsis, but little is known about the acute-phase response during disease and the nature of the bacterial factors causing it. In this study, we examined the levels of the acute-phase proteins (APPs) in comparative experiments using mice challenged with spores and a purifiedB. anthracisprotease InhA as a possible factor mediating the response. A strong increase in the plasma levels of APPs such as haptoglobin and serum amyloid A was observed during infection. Protein and mRNA levels of plasminogen activator inhibitor (PAI)-1 in the liver were also increased concurrently with bacterial dissemination at 72 h post-infection. Similar effects were observed at 6 h post injection with InhA. Induction of hepatic transforming growth factor-β1, a PAI-1 inducer, was also found in the liver of InhA-injected mice. PAI-1 elevation by InhA resulted in an increased level of urokinase-type plasminogen activator complex with PAI-1 and a decreased level of D-dimers indicating inhibition of blood fibrinolysis. These results reveal an acute liver response to anthrax infection and provide a plausible pathophysiological link between the host inflammatory response and the pro-thrombotic haemostatic imbalance in the course of disease through PAI-1 induction in the liver.

Bacillus anthracis infection is associated with severe hemostatic disturbances but their roles and contribution to fatality remain incompletely characterized. We undertook analyses of circulating antithrombin levels during the course of infection using a comparison of lethal and nonlethal murine anthrax models. Plasma samples were obtained from DBA/2 mice challenged intraperitoneally with the spores of either toxigenic B. anthracis Sterne strain or nontoxigenic, avirulent delta Sterne strain. We found that plasma antithrombin levels were rapidly depleted in Sterne spore-challenged mice, concomitant with elevation of neutrophil elastase (NE) and massive syndecan shedding from the liver into circulation. The shed syndecan bound with antithrombin accelerated NE-mediated antithrombin proteolysis. The liver response to infection demonstrated strain-specific compensatory increases of antithrombin and syndecan gene transcription. Both bacterial strains induced changes in blood coagulation parameters consistent with the onset of disseminated intravascular coagulation. We propose that antithrombin depletion proceeding through activation of neutrophils and massive shedding of heparin-like syndecan from the liver into circulation contribute to anthrax coagulopathy.

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37 degrees C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F(2) strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.

Interleukin -1 receptor antagonist ( IL-1Ra ) is a new option among biotherapies against rheumatoid arthritis ( RA ).of this review is to recall the rationale of use of IL-1Ra and to analyse the results available in the current literature.Pathophysiological data of RA give a specific position for IL-1 as a potential target for immunotherapy in this disease, confirmed in animal models. Phase II and III studies with IL-1Ra (Anakinra) demonstrated clinical efficacy versus placebo (42% responders in ACR 20 in Anakinra + methotrexate, and 23% in the placebo + methotrexate group at 24 weeks) and a structural effect (slowing of radiological progression at six months). Anakinra has obtained an European license and is indicated in RA not controlled by methotrexate, in daily subcutaneous administration (100 mg/day), in combination with methotrexate. Tolerance is fair; the most frequent side effect is represented by injection site reactions.This ambulatory biotherapy offers new perspectives in combination with other slow acting drugs as well as biologic agents such as anti-TNF, currently under evaluation.

Gene therapy has the significant advantage of providing targeted and continuous administration of a therapeutic agent within the joint. A number of targets of choice are discussed, including cytokines, metalloproteinase inhibitors and angiogenesis costimulators. Gene transfer can also be used to inhibit intracellular biological cascades (apoptosis, signal transduction, cell differentiation). Preliminary Phase I ex vivo studies have confirmed the feasibility of gene therapy in rheumatoid arthritis. Vectors will have to be improved before in vivo studies are performed.

We used the severe combined immunodeficient (SCID) mouse model to assess the effect of interleukin-4 (IL-4) or IL-10 injection on cartilage degradation and mononuclear cell (MNC) recruitment to human rheumatoid synovium in vivo. Human rheumatoid synovium and cartilage from five rheumatoid arthritis patients, obtained after joint replacement surgery, were engrafted subcutaneously to 6-8-week-old SCID CB17 mice. Synovial tissues were injected with recombinant human IL-4 (rhIL-4, 100 ng; rhIL-10, 100 ng), both cytokines, or tumour necrosis factor-alpha (TNF-alpha) (1000 U), or phosphate-buffered saline twice a week for 4 weeks. The graft was removed and immunochemical analysis was carried out to assess intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression. Moreover, cartilage degradation was assessed through the quantification of the erosion surface on a computerized image of the engrafted cartilage at high power view. MNC recruitment in the synovial tissue was determined by labelling blood MNC with indium-111 before their intraperitoneal injection. The activity obtained in the region of the graft were determined with a gamma camera 72 hr postinjection. The results are expressed as a percentage of initial injected activity. After 4 weeks we observed a decrease of cartilage area in controls (77 +/- 8%), inhibited after injection of IL-4, IL-10, or both cytokines (90 +/- 3%, 89.1 +/- 4%, 89.2 +/- 5% respectively), and 57 +/- 17% after TNF-alpha injection. The % MNC activity in the graft decreased to 77 +/- 81% (NS), 9 +/- 4% (P < 0.003) and 19 +/- 6% (P < 0.007) compared with untreated synovial tissue after treatment with IL-4, IL-10, or both cytokines, respectively. Moreover, IL-10 but not IL-4 decreased the expression of ICAM-1 but not VCAM-1 or E-selectin by synovial cells. These results suggest that IL-10 and IL-4 could have chondroprotective properties, and that IL-10 but not IL-4 inhibits MNC traffic towards the synovial tissue efficiently.

The 65 kDa heat shock protein (HSP) chaperonin is a highly conserved intracellular protein. HSP are involved in the pathogenesis of arthritis, but are not able to induce experimental arthritis. T cell clones recognizing the 180-188 amino acid sequence of 65 kDa HSP are present in inflamed synovium of both adjuvant arthritis and rheumatoid arthritis (RA). Oral administration of bovine collagen II or co-chaperonin 10 kDa HSP has been shown to induce an immune tolerance state to collagen induced arthritis (CIA). We investigate the effect of oral gavage with 65 kDa HSP on CIA.We immunized 6-8-week-old DBA1 male mice with bovine type II collagen. A group of 25 mice were given oral recombinant mycobacterial 65 kDa HSP before immunization (30 microg in 200 microl phosphate buffered saline (PBS) at Days -7, -5, -2) while PBS alone was administered in 27 controls. A 3rd group was fed 65 kDa HSP according to the same protocol but was not immunized with collagen II (n = 8). The clinical arthritis score was recorded 3 times/week until Day 60. Antibodies to collagen II were determined by ELISA.The incidence of arthritis was comparable in the 2 groups (72 vs 70%). The onset of arthritis was not delayed in mice fed HSP. However, the severity of arthritis was lower 10 days after arthritis onset in animals fed 65 kDa HSP (clinical score 1.83 +/- 0.79 vs 2.74 +/- 1.1; p0.0001). No animals in Group 3 had arthritis. Serum IgG anti-type II collagen levels were decreased in HSP treated mice (optical density 0.33 +/- 0.21 vs 0.46 +/- 0.21; p0.0001). However, the ratio of IgG1/IgG2a antitype II collagen antibody response remained unchanged in the mice fed 65 kDa HSP.These results suggest that oral administration of 65 kDa HSP may diminish collagen induced arthritis.

The aim of this study was to assess regulation of mononuclear cell (MNC) traffic to human synovial tissue by TNF-alpha and IL-1 and the involvement of ICAM-1 in MNC retention in rheumatoid synovial tissue. Human rheumatoid arthritis synovium was engrafted subcutaneously in 6-8 week-old SCID/CB17 mice. Three weeks later, we injected 20 x 10(6) human peripheral blood mononuclear cells (PBMC) previously labelled with 111indium intraperitoneally into mice containing control or cytokine-injected grafts. Total body scintigraphy was performed 72 h postinjection. The graft was removed and immunochemical analysis carried out to assess ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression. In some experiments, mice were treated intravenously with 500 micrograms MoAb anti-ICAM-1 (BIRR-1) or an isotype-matched control MoAb before introduction of MNC. TNF-alpha, but not IL-1 alpha, enhanced MNC retention in the rheumatoid synovial graft 72 h post-injection (graft activity 989 +/- 1227 ct/min per 200 pixels or 3.36 +/- 4.16% of initial injected activity versus 411 +/- 157 ct/min per 200 pixels or 1.13 +/- 0.45% in controls; P0.03). TNF-alpha enhanced ICAM-1 expression by synovial cells and endothelial cells, whereas VCAM-1 or E-selectin expression was not enhanced on either cell type. After MoAb treatment of ICAM-1, synovial lymphocyte recruitment of TNF-alpha-treated mice decreased significantly to levels below that of control mice (160 +/- 97 ct/min per 200 pixels, 0.54 +/- 0.33%; P0.01). Mononuclear cell retention in rheumatoid synovial tissue engrafted into SCID mice was up-regulated by TNF-alpha and blocked by MoAb to ICAM-1. These results suggest that ICAM-1 is involved in mononuclear cell retention in rheumatoid synovium.

Integrin-adressin binding is a critical step in lymphocyte attachment to target tissues. The lymphocyte function associated antigen (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) pathway has been shown to be involved in the homing of lymphocytes to arthritic joints in animal models. The mucosal recognition system [alpha E beta 7/E-cadherin, alpha 4 beta 7/mucosal vascular adressin cellular adhesion molecule 1 (MADCAM-1)] has been implicated in the autoimmune process of nonobese diabetic mice and in rheumatoid arthritis (RA). We developed a model for in vivo study of radiolabelled lymphocyte circulation and attachment to human engrafted rheumatoid synovium, and studied the involvement of LFA-1 and alpha E beta 7 integrin.We engrafted human RA or osteoarthritis (OA) synovium subcutaneously in 6-week-old SCID/CB17 mice. Three weeks later, we injected intraperitoneally 20 x 10(6) human peripheral blood lymphocytes (PBL) labelled with 3 mCi 99mtechnetium hexamethyl propylenamine oxime. A mouse total body scintigraphy was obtained 20 h postinjection. The same protocol was performed after pretreatment of the PBL with monoclonal antibodies (Mab) against CD11a (25-3) or against alpha E beta 7 human mucosyl lymphocyte marker 1.PBL migrated in the rheumatoid synovial graft 20 h postinjection (activity in the region of interest of the graft: 7699 +/- 4383 cpm/200 pixel or 4.43 +/- 2.65% of initial activity) versus OA engrafted synovial tissue (1453 +/- 1137 or 0.74 +/- 0.6% of initial activity), p = 0.007. The homing to the engrafted rheumatoid synovial tissue of PBL from healthy subjects was not significantly different from the migration of PBL from patients with RA. A Mab against alpha E beta 7 significantly decreased lymphocyte attachment to rheumatoid synovial tissue (3094 +/- 3808 cpm/200 pixel or 2.65 +/- 2.4% of injected activity), p0.03. The same results were obtained with Mab against CD11a (5007 +/- 4190 cpm/200 pixel or 2.27 +/- 1.2%), p0.01. Our results show increased blood lymphocytes homing to rheumatoid synovial tissue engrafted in SCID mice versus OA tissue.The data suggest that both LFA-1 and mucosal recognition integrin alpha E beta 7 are involved in lymphocyte binding to target tissues in RA.

To study the dynamic response of the hypothalamo-pituitary- adrenal axis and of prolactin (PRL) pituitary secretion in rheumatoid arthritis (RA).We performed a cortisol releasing hormone (CRH) provocation test followed by determination of adrenocorticotropin hormone (ACTH), beta-endorphin, and cortisol concentration, and then a thyrotropin releasing hormone (TRH) provocation test followed by assessment of PRL pituitary secretion in 10 patients with RA and 5 control subjects. All were women under 40 years of age. Hormone concentrations were assessed by radioimmunoassay.Basal PRL cortisol, and ACTH concentrations were similar in patients with RA and controls. We observed a dissociation between the pituitary secretion of beta-endorphin and of ACTH in response to CRH in RA. The ACTH peak and total ACTH production (area under the curve, AUC) were similar in the 2 groups. In contrast, basal beta-endorphin was increased in RA (12.6 +/- 1.41 vs 8.29 +/- 0.144 pg/ml), and the response upregulated (AUC: 83,080 +/- 12,000 vs 54,200 +/- 2400) after CRH compared to controls (p0.05). Cortisol adrenal response curve was blunted, but did not reach statistical significance. In contrast, the PRL response to TRH was increased at 120 and 150 min (3461 +/- 303 vs 1897 +/- 520 muIU/ml)(p0.01) in patients with RA, independent of disease activity.We observed upregulated pituitary PRL secretion in RA, and a dissociation of ACTH stress. The implication concerning the neuroendocrine system in the chronic immune response in RA is discussed.

CB17 SCID mice have severe combined immunodeficiency as a result of a mutation on chromosome 16 responsible for deficient activity of an enzyme involved in DNA repair. Because VDJ rearrangement does not occur, the humoral and cellular immune systems fail to mature. SCID mice do not reject human cells. They have been used to study the development of human tumors, human hematopoiesis and humoral responses in antibody-dependent organ-specific autoimmune diseases and in systemic lupus erythematosus. Studies involving grafting of synovial membrane under the renal capsule or in the subcutaneous tissue of SCID mice have provided information on the cells involved in the pathogenesis of rheumatoid arthritis. SCID mice have also been used to study adhesion molecules that play a role in the recruitment of lymphocytes in rheumatoid synovial tissue. The SCID mouse model provides new means of investigating immunologic treatments for rheumatoid arthritis.

Control points of regulator action during spermatogenesis are not completely known. Using the shark testis model, which facilitates analysis of spermatogenesis stage-by-stage in vivo and in vitro, an early biochemical marker of programmed cell death (PCD) was detected. Nucleosomal oligomers were seen in DNA extracts of testis and isolated spermatocysts (clonal germ cell/ Sertoli cell units) at premeiotic (PrM), but not meiotic (M) or postmeiotic (PoM), stages. Cell nuclei isolated from M stages of development were susceptible to cleavage by micrococcal nuclease, suggesting that developmental control of factors other than a nuclease-insensitive chromatin structure may account for stage specificity. Cytological features of apoptosis were seen in germ cells, but not Sertoli cells, of a subset of isolated PrM spermatocysts and appeared to be all-or-none in affected clones. In culture, DNA fragmentation occurred on schedule with or without various additives, but the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased accumulation of DNA breakdown products. Identification of the apoptotic form of PCD as a major, variable component of normal spermatogenesis and the use of PrM spermatocysts as an in vitro test system will allow further definition of mechanisms and developmental and physiological controls. © 1995 Wiley-Liss, Inc.

We set out to determine whether the ability of synovial fluids (SF) in patients with rheumatoid arthritis (RA) to facilitate the proliferation of synovial tissue-derived fibroblastic cell lines was related to the presence of growth factors and/or cytokines.The growth factor activity of 20 RA SF was measured by their ability to induce anchorage-independent growth of the rat NRK-49F (49F) fibroblastic strain. The presence of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) was also assessed using neutralising anti-TGF-beta or anti-PDGF-AB mAbs. Cytokines were measured by functional assays or ELISA:We observed a correlation between growth factor activity and the IL-6 levels in SF. Both were correlated to the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels in SF and serum. IL-6 (at concentrations above 10(4) U/ml), synergized with growth factors in the induction of the anchorage independent (AI) growth of 49F cells. Pretreatment of SF with a neutralising anti-IL-6 mAb substantially reduced the capacity of these SF to induce AI growth of 49F cells, confirming the growth factor activity of IL-6 in this test. In contrast, IL-6 alone or in association with PDGF, epidermal growth factor (EGF) or TGF-beta had no effect on the anchored growth of synovial tissue-derived fibroblasts, and treatment of SF with a neutralising anti-IL-6 mAb did not affect their ability to increase the growth rate of synovial tissue-derived fibroblasts.These results strongly suggest that IL-6 is responsible for the observed correlation between the growth factor activity of SF and inflammatory indexes such as ESR and CRP. However, neither IL-6 nor PDGF were responsible for the observed positive effect of SF on synovial fibroblastic cell lines.

The neuropeptides are involved in the immune response and in hormonal homeostasis. In this review, we analyse the interactions between the cytokine, the neuropeptide and the hormonal networks in rheumatoid arthritis (RA). We first consider pituitary-adrenal axis dysfunction in RA. An inappropriate response to cortisol in chronic inflammation has been reported, i.e., a decrease of the corticotropin-releasing-hormone (CRH) secretion by the hypothalamus. In contrast, the immunostimulant hormone prolactin (PRL) is upregulated. PRL is released by the pituitary after stimulation by neuropeptides [serotonin, thyroid-releasing-hormone (TRH), or vasoactive-intestinal-peptide (VIP)], and is down-regulated by pro-inflammatory cytokines (IL-1, IL-6). The decreased testosterone concentration observed in male RA patients is associated with HLA B 15. Thus, an altered sex hormone status and a genetic predisposition are related to HLA antigens, and increase the subject's susceptibility to the development of RA. The terminal C fibres release neurotransmitters such as substance P, neurokinin A and calcitonin-gene-related-peptide (CGRP) within the joints, and contribute to local inflammation, synoviocyte proliferation and collagenase production. The parasympathetic system may attenuate the immune response through the neuropeptide VIP. In contrast, the beta 2 adrenergic fibres of the sympathetic nervous system increase joints degradation in RA. This review presents the currently extensive knowledge regarding the immune-neuro-hormonal network, and its implication in the pathogenesis of RA.

The effect of thyroid hormone on maturation of fetal rabbit lung was studied with maternal treatment using 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT), a synthetic analogue of triiodothyronine. To investigate the in vivo kinetics and distribution of DIMIT, we prepared [3H]DIMIT and injected both pregnant rats (18-21 d gestation) and rabbits (25 d gestation). In the rat, maximal concentrations of radioactivity in maternal plasma, fetal plasma, and amniotic fluid occurred within 10 min, 1-2 h, and 4-6 h, respectively, after intramuscular injection. After 7 h the concentration of radioactivity in fetal plasma was 163 and 71% of the maternal level in rats and rabbits, respectively, indicating that DIMIT readily crosses the placenta. We treated pregnant rabbits for 1-2 d with DIMIT in doses of 0.5-3 mg/kg per d and examined the fetuses at 26 and 27 d gestation. Treatment did not affect fetal growth or viability. In fetal liver, DIMIT increased the activity of NADPH cytochromeac reductase by 64% and decreased the glycogen content by 73% compared to controls. The rate of choline incorporation by lung minces increased in dose-dependent manner to a maximum of +104% at 3 mg/kg DIMIT; this does stimulated by 38% the activity of lung phosphatidic acid phosphatase (PAPase), a corticosteroid-responsive enzyme, but there was no increase in tissue PAPase activity at most lower doses of DIMIT that enhanced choline incorporation. Treated lungs had 38% less glycogen tha controls, but there was no effect on tissue levels of DNA, protein, or phospholipid. DIMIT treatment increased the amount of total phospholipid (+163%). saturated phosphatidylcholine (+330%), and PAPase activity (+134%) in lung lavage fluid. The DIMIT effects on both choline incorporation by lung minces and phospholipid content of lavage fluid were substantially greater than what had occurred with an optimal dose of betamethasone. DIMIT also increased corticosteroid binding capacity in fetal plasma and produced a dose-dependent increase (maximal threefold) in total and free corticoids of both maternal and fetal plasma. It is estimated that elevated endogenous corticoids probably account for less than one-third of the increases in phospholipid synthesis and secretion observed at the higher doses of DIMIT. These data indicate that administration of DIMIT to pregnant rabbits accelertes maturation of the surfactant system in fetal lung. The magnitude of the effects on phospholipid synthesis and secretion, along with the minimal effect of PAPase activity in fetal lung tissue, suggest that thyroid hormones affect different biochemical processes from those influenced by glucocorticoids.

Theoretical electronic structure calculations on the thyroid hormones and analogues, as well as model hormone--receptor interactions, have been carried out. These studies (a) support the concept that the 4'-OH group is a H-bond donor to the in vivo nuclear receptor and suggest that at the receptor this OH group is trans to the 3' (distal) substituent; (b) indicate that there is an important intramolecular interaction between 3' and 4' substituents, and those 3' substituents that most favor both 4' OH orientation trans to the 3' group and a more acidic OH group substantially increase binding and biological activity; and (c) support the concept that there is a direct correlation between the conformational free energy of the aromatic rings and biological activity.

The effects of an analog of thyroxine, 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT), on fetal rat hepatocyte ultrastructure and microsomal function were investigated by using the techniques of quantitative electron microscopy and enzyme assays. Rats were injected with DIMIT (10 microgram/100 g BW) or vehicle daily from the 15th through the 19th day of pregnancy. Fetuses were sacrificed on the 20th day of gestation. In comparison with controls, DIMIT-treated livers 1) were devoid of glycogen; 2) contained smaller hepatocytes; 3) contained a greater number of hepatocytes; 4) had an increased volume density of mitochondria; and 5) had increased NADPH-cytochrome c reductase and glucose-6-phosphatase activities. Surface areas of rough and smooth surfaced endoplasmic reticulum were unaffected by the hormone analog, and cytochrome P-450 was not induced. All of the changes that were produced by DIMIT in the 20-day-old fetal rat, as well as smooth endoplasmic reticulum and cytochrome P-450 development, are observed in normal animals within the first 3 days after birth. The data suggest that thyroid hormone may be a physiological stimulus for certain aspects of early hepatic development, but that it acts in combination or in sequence with other factor(s) to produce the full complement of structural and functional changes that occur perinatally in the rat.

The survival of heterotopically transplanted mouse hearts was enhanced in recipients who were sensitized intraperitoneally to give rise to cell-mediated hyperacute rejection, a T-cell-mediated response characterized by very rapid sensitization and rejection of allogeneic skin grafts.

Quantitative structure-activity correlation studies of thyroid hormone analogues have been utilized to examine (1) in vivo rat antigoiter activities; (2) in vitro binding affinities to intact rat hepatic nuclei, solubilized rat hepatic nuclear protein receptors, and the plasma protein thyroxine binding globulin; and (3) correlations between in vivo antigoiter activities and in vitro binding to nuclear receptors. These studies provide a more precise elucidation of the relative importance of the physiochemical factors which influence thyromimetic activities. In particular, they (1) provide the first systematic QSAR examination of drug-receptor interactions and of the dependence of in vivo activity on such interactions; (2) demonstrate the importance of the interactive effects of the 3' and 5' substituents and of the 4'-OH with each other as well as with nuclear receptors in influencing binding affinity; (3) support the hypothesis that binding to nuclear receptors is the first step in initiating the events which lead to subsequent hormonal expression; (4) show that the free energy of binding to nuclear receptors can be factored into the contributing physicochemical properties of the substituents; and (5) suggest factors that need to be considered in designing new analogues.

Chronically catheterized maternal fetal monkey preparations were used to study pituitary suppression by thyroid hormones in the fetus. Baseline maternal T3, T4, and PRL were all significantly higher than fetal levels of these hormones. TRH was given iv (10 μg/kg) to the mother 3, 6, 12, 20, or 44 h after giving varying doses of T3 to the fetus. Amniotic fluid and fetal tracheal aspirate did not contain detectable TSH. Nonpregnant females and pregnant mothers showed complete suppression of the TSH response to TRH after receiving 20 μg T3 iv 24 h before TRH. Two micrograms of T3 iv to the fetus 3 h before TRH failed to suppress the TSH response (fetal δTSH, 26 μU/ml; δT4, 3.6 μg/dl). Six micrograms of T3 iv (three doses of 2 μg) to the fetus over 36 h achieved a basal T3 level of 2500 ng/dl and lowered fetal δTSH to 9.1 μU/ml. Six micrograms of T3 infused iv to the fetus over 24 h achieved fetal T3 blood levels of 5000 ng/dl, which suppressed δTSH to 2.7 μU/ml. 3,5,-Dimethyl-3'-isopropyl-L-thyronine (DIMIT;...

Prior studies have demonstrated that the thyroid hormones L-triiodothyronine and L-thyroxine stimulate the rapid uptake of 1-amino-cyclopentane-1-carboxylic acid into isolated rat thymocytes. In the present study the effects of several groups of thyroid hormones and structurally related compounds were investigated to determine the structure-function relations required for stimulation of this membrane process. Particular attention was given to (a) analogues with modifications at the oxygen bridge, the phenolic hydroxyl group, and the group at the 3' position of the outer ring, and (b) the steric orientation of the thyroid hormones. The following were found to be important for maximal activity: (a) the L-isomer configuration, (b) the presence of a 4'-hydroxyl group, (c) the presence of one substituent in both the inner and outer rings (3 and 3' positions), (d) the distal orientation of the 3' substituent in the outer ring of L-triiodothyronine, and (e) the lipophilic character of the 3' substituent. Of lesser importance was the presence of halogen atoms, or an oxygen atom in the ether position. Since these structure-function relations seen in thymocytes parallel in many respects those relations seen in whole-animal studies, it is believed that thymocytes will be a useful tool for further studies of thyroid hormones and their analogues.

3,5-Dimethyl-3′-isopropyl-1-thyronine (DIMIT) and 3,5-diiodo-3′-isopropylthyroacetic acid (IpTA 2 ), two thyroid hormone analogs, have been tested in genetically obese Zucker rats and their lean littermates, in comparison with thyroxine (T 4 ) and triiodothyronine (T 3 ) for their thyromimetic activities on body weight gain and lipid levels in serum and liver. The compounds were administered for 9 weeks by orogastric tube to 6- to 8-week-old animals. While body weight gain remained practically unchanged in the lean rats, it decreased significantly in the obese individuals, especially with IpTA 2 . The serum lipid concentrations were also decreased in the obese rats in comparison with their lean littermates, especially with DIMIT. The connection observed between the structure of DIMIT and IpTA 2 on one hand and their effects on the other is in good agreement with previous studies. Our results confirm that the iodine substituents are not necessary for thyromimetic activity and demonstrate that the isopropyi substituent in 3′ plays an important role in the serum lipid-lowering effect of the thyroid hormone analogs tested.

Our experiments show that the T3 nonhalogenated analog 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT) exhibits the general activities of thyroid hormones. DIMIT delivered to hypothyroid Rat by constant infusion or sc injections increases bodyweight gain, BMR before or after epinephrine and phosphorylative oxidation of liver mitochondria.

In order to determine the features of the phenolic ring in position 4 of [Asn1,Ile5]angiotensin II that contribute to pressor activity, analogues with selected aromatic substituents were synthesized by the solid-phase method. They showed pressor activities in the rat: [Asn1,Phe(4-NH2)4]AII, 24%; [Asn1,Phe(4-NO2)4AII, 0.1%; [Asn1,Tyr(3,5-Me2)4]AII, 2.2%; [Asn1,D-Tyr(3,5-Me2)4]AII, 1.4%. These results indicate that the activity contributed by the aromatic character of the phenyl ring in the side chain of position 4 is enhanced by a group in the para position that may function as a proton donor in hydrogen-bond formation. Bulky substituents ortho to this hydrogen-bonding group decrease activity by steric interference with hydrogen-bond formation. Bulky groups than cannot act as hydrogen donors in the para position of the aromatic ring drastically decrease the activating effect of the aromatic ring on pressor activity.

Transfer of iodothyronine across the placenta in most species occurs only with difficulty. Recently, biologically active, nonhalogenated thyroid hormone analogs have been synthesized with properties which might favor placental transfer. To test this possibility, we compared the doses of T4, T3, and thyroid hormone analogs necessary to prevent propylthiouracilinduced goiter formation in rat fetuses. T4 and T3) prevented fetal goiter, but in doses that caused maternal hyperthyroidism; in contrast, the thyroid hormone analogs prevented fetal goiter in doses that were not thyrotoxic to the mother. (Endocrinology 102: 1670, 1978)